Fundamental Techniques in Cell Culture

Index of Contents
Section Page No
1.0 Introduction 3
2.0 Design and Equipment for the Cell Culture Laboratory 6
2.1 Laboratory Design 6
2.2 Microbiological Safety Cabinets 6
2.3 Centrifuges 8
2.4 Incubators 8
2.5 Work Surfaces and Flooring 8
2.6 Plasticware and Consumables 8
2.7 Care and Maintenance of Laboratory Areas 9
3.0 Safety Aspects of Cell Culture 10
3.1 Risk Assessment 10
3.2 Disinfection 11
3.3 Waste Disposal 12
4.0 Sourcing of Cell Lines 13
5.0 Main Types of Cell Culture 14
5.1 Primary Cultures 14
5.2 Continuous Cultures 14
5.3 Culture Morphology 14
6.0 The Cell Environment 17
6.1 Basic Constituents of Media 18
6.2 Inorganic Salts 18
6.3 Buffering Systems 18
6.4 Carbohydrates 19
6.5 Vitamins 19
6.6 Proteins and Peptides 19
6.7 Fatty Acids and Lipids 19
6.8 Trace Elements 19
6.9 Serum 20
6.10 Guidelines for Serum Use 20
6.11 Origin of Serum 21
7.0 Cryopreservation and Storage of Cell Lines 22
7.1 Cryopreservation 22
7.2 Ultra-low Temperature Storage 22
7.3 Safety Considerations 24
8.0 Good Cell Banking Practices 25
9.0 Quality Control Considerations 28
9.1 Reagents and Materials 28
9.2 Provenance and Integrity of Cell Lines 28
9.3 Avoidance of Microbial Contamination 28
9.4 Environmental Monitoring 30
9.5 What to do in the event of Contamination 30
10.0 Authentication of Cell Lines 32
10.1 Authentication Techniques 32
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Fundamental Techniques in Cell Culture

Section Page No
10.2 Iso-enzyme Analysis 32
10.3 DNA Fingerprinting 32
10.4 Multi Locus DNA Fingerprinting 33
10.5 Multiplex PCR DNA Profiling 33
11.0 Alternative Cell Culture Systems 34
11.1 Cell Culture Scale-up Systems 34
11.2 Scale-up Solutions 34
11.3 Roller Bottle Culture 36
11.4 Spinner Flask Culture 37
11.5 Other Scale-up Options 37
12.0 Cell Culture Protocols 38
12.1 Basic Do’s and Don’ts of Cell Culture 38
12.2 Protocol 1 – Aseptic Technique and Good Cell Culture Practice 40
12.3 Protocol 2 – Resuscitation of Frozen Cell Lines 42
12.4 Protocol 3 – Subculture of Adherent Cell Lines 44
12.5 Protocol 4 – Subculture of Semi-Adherent Cell Lines 46
12.6 Protocol 5 – Subculture of Suspension Cell Lines 48
12.7 Protocol 6 – Cell Quantification 50
12.8 Protocol 7 – Cryopreservation of Cell Lines 54
12.9 Protocol 8 – Testing for Bacteria and Fungi 56
12.10 Protocol 9 – Detection of Mycoplasma by Culture 58
12.11 Protocol 10 – Testing for Mycoplasma by Indirect DNA Stain 62
Table 1 – Commonly used cell lines of each culture type 16
Table 2 – Different types of culture medium and their uses 17
Table 3 – Comparasion of ultra-low temperature storage methods for cell lines 23
Table 4 – “Half-Way House” Solutions to Scale-up 35
Figure 1 – Diagram of microbiological safety cabinet airflow patterns 7
Figure 2 – Examples of Cell Morphology 15
Figure 3 – Schematic Representation of a Tiered Cell Banking System 26
Figure 4 – Multi Locus DNA Fingerprinting 33
Figure 5 – PCR DNA Profiling 33
Figure 6 – Triple Flask 34
Figure 7 – Bioreactor 34
Figure 8 – Shake Flasks 36
Figure 9 – Roller Deck 36
Figure 10 – Roller Bottles 37
Figure 11 – Spinner Flasks 37
Figure 12 – Flow Scheme for Bacteria and Fungi Testing 57
Figure 13 – Flow Scheme for Detection of Mycoplasma by Culture 59
Figure 14 – Typical ‘fried egg’ colonies, Mycoplasma pneumoniae 61
Figure 15 – Testing for Mycoplasma by Indirect DNA Stain 63
Figure 16 – Hoechst Positive Culture 65
Figure 17 – Hoechst Negative Culture 65
Glossary of Terms 66
Notes 67
Who to Contact 68
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Fundamental Techniques in Cell Culture

1.0 Introduction
This laboratory handbook has been produced as a guide for scientists who have recently
been introduced to the discipline of cell culture.The book has been written and published
by the European Collection of Cell Cultures (ECACC) and Sigma-Aldrich. ECACC is a
universally recognised authority on Cell Culture, providing authenticated Cell Lines and
Cell Culture services to the scientific community and a wide range of commercial
customers. Sigma Aldrich has operations in 33 countries worldwide and manufactures Cell
Culture Products in several locations throughout the world.

The handbook contains essential information about the fundamentals of cell culture ranging
from the sourcing of cell lines and safety, to aspects of cryopreservation and quality
control, which give the reader the essential facts needed when embarking on cell culture
for the first time. Additionally there is a series of 10 detailed protocols, which are
routinely used in the ECACC laboratories.This handbook is designed to provide scientists
with a quick reference guide to cell culture by detailing the more commonly used
protocols and methods. However, it is not intended to be an in-depth text book of cell
culture and you are encouraged to consult relevant specialised literature to obtain more
detailed information.

We hope you like the design of the handbook and that is small enough to fit in a lab coat
pocket, with water resistant paper and not too big to open out on the lab bench. Should
your colleagues require their own personal copy then they can complete the business reply
card at the back of the handbook to obtain their copy.

ECACC and Human Genetic Research

The European Collection of Cell Cultures (ECACC) was first established within CAMR,
Salisbury, UK in 1984 and has grown to be one of the largest animal cell culture collections
in the world. ECACC now supplies authenticated cell lines worldwide and provides a wide
range of cell culture services.

Whereas the ECACC core business is cryopreservation, cell banking and the supply of
authenticated cell lines the operation has progressively diversified and now offers a
comprehensive range of services to cell scientists both in the commercial and academic
sectors.These services include Patent and Safe deposits, screening for microbiological
contaminants, authentication by DNA profiling and a comprehensive programme of training
courses. ECACC also undertakes contract cell culture process upscale development and
the manufacture of cell products for research use.

The Human Genetic Collection

The fastest-growing service within the ECACC range is its Human Genetic Collection.This is
based on processes introduced into, and subsequently developed by ECACC in the late 1980’s,
for the immortalisation of human lymphocytes.This process essentially comprises the

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Fundamental Techniques in Cell Culture

separation of lymphocytes from samples of human peripheral blood and their transformation
into lymphoblastoid cell lines by treatment with the tumourigenic Epstein Barr Virus (EBV).
EBV transformation of human lymphocytes to form a cell line that can be continuously
propagated provides an indispensable means of expanding the limited amount of cellular
material that can be separated from a small volume blood specimen.This expanded cell stock
can be banked and cryopreserved as a source of single donor cells and / or extracted DNA
that can be made available indefinitely.

Since 1989 a number of major human genetic research programmes, such as the British
Diabetes Association-funded Warren I and II studies and the long-running, MRC-funded Human
Genome Mapping Project, have depended upon the ECACC EBV transformation service.A cell
line manufactured from a phenotypically characterised donor, who possibly has a rare genetic
disorder, can be used to provide an almost limitless supply of DNA for distribution to research
groups worldwide. Furthermore a cryopreserved bank of these cells can be resuscitated many
years later so that investigation may be resumed with the benefit of improved technologies and
scientific knowledge.

The ability to expand the amount of cellular DNA available from a small donor sample means
that scientific investigations will not be materials limited. It is theoretically possible to achieve
this expansion by biochemical means such as "whole genome PCR", but to date this has not
proved practicable. Cell line DNA is typically of high quality, and a viable cell offers the
opportunity to examine other types of molecule, such as RNA or cellular proteins, if required
at a later date.

The ECACC Human Genetic Collection now comprises more than 40,000 single donor
lymphocyte stabilates, more than 50% of which are transformed cell lines.This collection
represents more than 700 clinical disorders such as breast cancer, Alzheimers disease,
schizophrenia and psoriasis.

The ECACC General Collection

This collection comprises approximately 1000 different cell lines originating from over 45
different species and a variety of different tissues. It incorporates over 400 human cell lines
with many different cancer cell lines including drug resistant cell lines.

The cells can be shipped either as frozen or growing cultures, and are supplied complete
with a specific data sheet together with a technical advisory pamphlet. Batch specific
Certificates of Analysis and Authentication Records can be provided on request. ECACC
can also supply cell lines on a standing order basis.

All cell lines added to the collection undergo full quality control and authentication procedures.
These include testing for mycoplasma by a combination of culture isolation, Hoechst DNA
staining and PCR, together with culture for containment bacteria, yeast and fungi.
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Fundamental Techniques in Cell Culture

Isoenzyme analysis is used to verify the species of each cell line deposit while DNA
fingerprinting identifies cell line variation and/or cross contamination. ECACC routinely
carries out two methods of DNA fingerprinting. Multilocus, applicable to most common
species and STR DNA profiling for human cell lines only.The latter has the benefit of
producing a digital ‘fingerprint’ which can be compared to ECACC’s extensive range of
archived profiles. All Hela contaminant cell lines are clearly identified in the full catalogue
description.

Hybridoma Collection
This collection comprises over 400 monoclonal antiboby-secreting hybridomas.The
specification of the secreted antibodies covers a wide range of antigens including bacterial and
viral; major histocompatability complex differentiation; oncogenes; plant and immunoglobulin.

ECACC can supply frozen or growing cultures of the hybridomas in the collection. In
addition ECACC may also supply, assuming no restrictions are in place, purified antibody on
a customised basis.The cost of providing purified antibody is dependent on the antibody
production levels of the hybridoma, level of purification and quantity required.

All cell lines added to the collection undergo full quality control and authentication procedures.
These include testing for mycoplasma by a combination of culture isolation, Hoechst DNA
staining and PCR, together with culture for contaminant bacteria, yeast and fungi.

HLA Defined Human B-Lymphoblastoid Collection

This is a specialised collection of over 350 reference cell lines, originating from laboratories
all over the world, to be used in the investigation of the HLA system.The collection was
expanded in 1996 with cell lines from the 12th International Histocompatability Workshop
(IHW) being added.A further 60 cell lines from the 13th IHW collection are due to be
added later this year, along with a number of recently banked lines that were originally part
of the 10th IHW collection.

Both serology and DNA typing data is available for the HLA Class I and II antigens
together with information on non-HLA genes within the HLA region.This resource is
available both as cell lines or DNA.

Human Random Control Collection

This is a collection of 500 lymphoblastoid cell lines derived from randomly selected
Caucasian blood donors whose parents and grandparents were born in the UK or Ireland.
DNA from these controls has already been extensively used in genome screening
programmes.This resource will be available as extracted DNA in a 96 well array format.

Full information on all the ECACC collections are available from the ECACC Website or in
a CDrom format which can be requested by completing the enclosed reply card.
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Fundamental Techniques in Cell Culture

2.0 Design and Equipment

for the Cell Culture Laboratory
2.1 Laboratory Design
Perhaps one of the most under-rated aspects of tissue culture is the need to design the
facility to ensure that good quality material is produced in a safe and efficient manner. Most
tissue culture is undertaken in laboratories that have been adapted for the purpose and in
conditions that are not ideal. However, as long as a few basic guidelines are adopted this
should not compromise the work.

There are several aspects to the design of good tissue culture facilities. Ideally work should
be conducted in a single use facility which, if at all possible, should be separated into an area
reserved for handling newly received material (quarantine area) and an area for material
which is known to be free of contaminants (main tissue culture facility). If this is not possible
work should be separated by time with all manipulations on clean material being completed
prior to manipulations involving the ‘quarantine material’. Different incubators should also be
designated. In addition, the work surfaces should be thoroughly cleaned between activities.
All new material should be handled as ‘quarantine material’ until it has been shown to be free
of contaminants such as bacteria, fungi and particularly mycoplasma. Conducting tissue
culture in a shared facility requires considerable planning and it is essential that a good
technique is used throughout to minimise the risk of contamination occurring.

For most cell lines the laboratory should be designated to at least Category 2 based on the
Advisory Committee on Dangerous Pathogens (ACDP) guidelines (ACDP, 1985)C. However,
the precise category required is dependent upon the cell line and the nature of the work
proposed.The guidelines make recommendations regarding the laboratory environment
including lighting, heating, the type of work surfaces and flooring and provision of hand
washing facilities. In addition it is recommended that laboratories should be run at air
pressures that are negative to corridors to contain any risks within the laboratory.

C AdvisoryCommittee on Dangerous Pathogens (1985) Categorisation of Biological

Agents According to Hazard and Categories of Containment, 4th edition, HSE books,
Sudbury, UK.

2.2 Microbiological Safety Cabinets

A microbiological safety cabinet is probably the most important piece of equipment since,
when operated correctly, it will provide a clean working environment for the product, whilst
protecting the operator from aerosols. In these cabinets operator and/or product protection
is provided through the use of HEPA (high efficiency particulate air) filters.The level of
containment provided varies according to the class of cabinet used. Cabinets may be ducted
to atmosphere or re-circulated through a second HEPA filter before passing to atmosphere.

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Figure 1. Diagram of microbiological safety cabinet airflow patterns

Class I Class II

Class III

Environmental monitoring with Tryptose Soya Broth agar settle plates inside the cabinet for a
minimum of four hours should be a good indicator of how clean a cabinet is. There should
be no growth of bacteria or fungi on such plates.
In most cases a class II cabinet is adequate for animal cell culture. However each study must
be assessed for its hazard risk and it is possible that additional factors, such as a known virus
infection or an uncertain provenance, may require a higher level of containment.
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Fundamental Techniques in Cell Culture

2.3 Centrifuges
Centrifuges are used routinely in tissue culture as part of the subculture routine for most
cell lines and for the preparation of cells for cryopreservation. By their very nature
centrifuges produce aerosols and thus it is necessary to minimise this risk. This can be
achieved by purchasing models that have sealed buckets. Ideally the centrifuge should have a
clear lid so that the condition of the load can be observed without opening the lid. This will
reduce the risk of the operator being exposed to hazardous material if a centrifuge tube has
broken during centrifugation. Care should always be taken not to over-fill the tubes and to
balance them carefully. These simple steps will reduce the risk of aerosols being generated.
The centrifuge should be situated where it can be easily accessed for cleaning and
maintenance. Centrifuges should be checked frequently for signs of corrosion.

2.4 Incubators
Cell cultures require a strictly controlled environment in which to grow. Specialist incubators
are used routinely to provide the correct growth conditions, such as temperature, degree of
humidity and CO2 levels in a controlled and stable manner. Generally they can be set to run at
temperatures in the range 28°C (for insect cell lines) to 37°C (for mammalian cell lines) and
set to provide CO2 at the required level (e.g. 5-10%). Some incubators also have the facility to
control the O2 levels. Copper-coated incubators are also now available. These are reported to
reduce the risk of microbial contamination within the incubator due to the microbial inhibitory
activity of copper. The inclusion of water bath treatment fluid (Prod. No. S5525) in the
incubator water trays will also reduce the risk of bacterial and fungal growth in the water trays.
However, there is no substitute for regular cleaning. (Note “Sigma Clean” Prod. No. S5525 is
harmful by inhalation, contact with skin or if swallowed and is also a severe irritant.)

2.5 Work Surfaces and Flooring

In order to maintain a clean working environment the laboratory surfaces including bench-
tops, walls and flooring should be smooth and easy to clean.They should also be waterproof
and resistant to a variety of chemicals (such as acids, alkalis, solvents and disinfectants). In
areas used for the storage of materials in liquid nitrogen, the floors should be resistant to
cracking if any liquid nitrogen is spilt. Refer to Section 7.3 for safety considerations on the
use of liquid nitrogen. In addition, the floors and walls should be continuous with a coved
skirting area to make cleaning easier and reduce the potential for dust to accumulate.
Windows should be sealed. Work surfaces should be positioned at a comfortable working
height.

2.6 Plasticware and Consumables

Almost every type of cell culture vessel, together with support consumables such as tubes
and pipettes, are commercially available as single use, sterile packed, plasticware. Suppliers
include Sigma-Aldrich, Nunc, Greiner, Bibby Sterilin and Corning.The use of such plasticware
is more cost effective than recycling glassware, enables a higher level of quality assurance
and removes the need for validation of cleaning and sterilisation procedures. Plastic tissue
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culture flasks are usually treated to provide a hydrophilic surface to facilitate attachment of
anchorage dependent cells.

2.7 Care and Maintenance of Laboratory Areas

In order to maintain a clean and safe working environment tidiness and cleanliness are key.
Obviously all spills should be dealt with immediately. Routine cleaning should also be
undertaken involving the cleaning of all work surfaces both inside and outside of the
microbiological safety cabinet, the floors and all other pieces of equipment e.g. centrifuges.
Humidified incubators are a particular area for concern due to the potential for fungal and
bacterial growth in the water trays. This will create a contamination risk that can only be
avoided by regular cleaning of the incubator. All major pieces of equipment should be
regularly maintained and serviced by qualified engineers.

For example:

Microbiological safety cabinets should be checked six monthly to ensure that they are
safe to use in terms of product and user protection.These tests confirm that the
airflow is correct and that the HEPA filters are functioning properly
The temperature of an incubator should be regularly checked with a NAMAS (National
Accreditation of Measurement and Sampling, UK), or equivilent calibrated
thermometer and the temperature adjusted as necessary
Incubator CO2 and O2 levels should also be regularly checked to ensure the levels are
being correctly maintained

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 Fundamental Techniques in Cell Culture

3.0 Safety Aspects of Cell Culture

3.1 Risk Assessment
The main aim of risk assessment is to prevent injury, protect property and avoid harm to
individuals and the environment.The performance of risk assessment is a legal requirement
under the Health and Safety at Work Act, UK.There are other EC directives covering
Health and Safety at Work, you can visit the European Agency for Safety and Health at Work
website www.europe.osha.eu.int for information on legislation and standards, or you should
contact your on-site representative. Consequently risk assessments must be undertaken
prior to starting any activity.The assessment consists of 2 elements:

1. Identifying and evaluating the risks.

2. Defining ways of minimising or avoiding the risk.

For animal cell culture the level of risk is dependent upon the cell line to be used and is
based on whether the cell line is likely to cause harm to humans. The different
classifications are given below:

Low risk Non human/non primate continuous cell lines and some well
characterised human diploid lines of finite lifespan (e.g. MRC-5).

Medium risk Poorly characterised mammalian cell lines.

High risk Cell lines derived from human/primate tissue or blood.

Cell lines with endogenous pathogens (the precise categorisation is

dependent upon the pathogen) – refer to ACDP guidelines, 1985, for
detailsC.

Cell lines used following experimental infection where the

categorisation is dependent upon the infecting agent - refer to ACDP
guidelines, 1985, for details.

CAdvisory Committee on Dangerous Pathogens (1985) Categorisation of Biological

Agents According to Hazard and Categories of Containment, 4th edition, HSE books,
Sudbury, UK

A culture collection, such as ECACC will recommend a minimum the containment level
required for a given cell line based upon its risk assessment. For most cell lines the
appropriate level of containment is Category 2. However, this may need to be increased to
Category 3 depending upon the type of manipulations to be carried out and whether large
culture volumes are envisaged. For cell lines derived from patients with HIV or HTLV
Category 3 containment is required.
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Fundamental Techniques in Cell Culture

Containment is the most obvious means of reducing risk. Other less obvious measures
include restricting the movement of staff and equipment into and out of laboratories.
Good laboratory practise and good bench techniques such as ensuring work areas are
uncluttered, reagents are correctly labelled and stored, are also important for reducing risk
and making the laboratory a safe environment in which to work. Staff training and the use
of written standard operating procedures and risk assessments will also reduce the
potential for harm. Training courses covering the basics of tissue culture safety are offered
by ECACC.

3.2 Disinfection
Methods designed for the disinfection/decontamination of culture waste, work surfaces and
equipment represent important means for minimising the risk of harm.

The major disinfectants fall into four groups and their relative merits can be summarised as
follows:

Hypochlorites (e.g. Chloros, Presept)

Good general purpose disinfectant
Active against viruses
Corrosive against metals and therefore should not be used on metal surfaces e.g.
centrifuges
Readily inactivated by organic matter and therefore should be made fresh daily
Should be used at 1000ppm for general use surface disinfection, 2500ppm in discard
waste pots for washing pipettes, and 10,000ppm for tissue culture waste and spillages

NB: When fumigating a cabinet or room using formaldehyde all the hypochlorites must first
be removed as the two chemicals react together to produce carcinogenic products.

Phenolics (e.g. Sudol, Hycolin)

Not active against viruses
Remains active in the presence of organic matter

Alcohol (e.g. ethanol, isopropanol)

Effective concentrations 70% for ethanol, 60-70% for isopropanol
Their mode of activity is by dehydration and fixation
Effective against bacteria. Ethanol is effective against most viruses but not non-
enveloped viruses
Isopropanol is not effective against viruses

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 Fundamental Techniques in Cell Culture

Aldehydes (e.g. glutaraldehyde, formaldehyde)

Aldehydes are irritants and their use should be limited due to problems of sensitisation
Glutaraldehyde may be used in situations where the use of hypochlorites is not suitable
e.g. cleaning of centrifuge bowls or materials constructed of stainless steel that may be
attacked or corroded by using hypochlorite solutions.

3.3 Waste Disposal

Any employer has a ‘duty of care’ to dispose of all biological waste safely in accordance with
national legislative requirements. Given below is a list of ways in which tissue culture waste
can be decontaminated and disposed of safely. One of the most important aspects of the
management of all laboratory-generated waste is to dispose of waste regularly and not to
allow the amounts to build up.The best approach is ‘little and often’. Different forms of
waste require different treatment.

Tissue culture waste (culture medium) - Inactivate overnight in a solution of

hypochlorite (10,000ppm) prior to disposal to drain with an excess of water
Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight
before disposal by autoclaving and incineration
Solid waste such as flasks, centrifuge tubes, contaminated gloves, tissues etc. should be
placed inside heavy duty sacks for contaminated waste and autoclaved prior to
incineration. These bags are available from Bibby Sterilin and Greiner
If at all possible waste should be incinerated rather than autoclaved

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4.0 Sourcing of Cell Lines

Large numbers of cell lines look identical. Cell lines with very different origins and
biological characteristics typically cannot be separated on grounds of morphology or
culture characteristics. Infection or contamination of a cell line with an adventitious virus
or mycoplasma may significantly change the characteristics of the cells but again such
contamination will be inapparent. Cell lines will also change with time in culture, and to add
to all these natural hazards it is all too easy to mis-label or cross-contaminate different cell
lines in a busy cell culture laboratory.

The opportunities for inadvertently introducing error into a cell line are limitless and ever
present. It is in the nature of the science that, once introduced, an error will be propagated,
compounded, consolidated and disseminated.

The integrity and biological characteristics of a cell line have to be actively maintained by a
well organised system of “husbandry” based on systematic cell banking supported by
testing regimens in a structured quality assured environment. Such a controlled
environment will only prevail in a dedicated professionally organised cell culture laboratory
or cell bank.A small research laboratory with a high throughput of short-term research
students, a minimum of permanent laboratory staff and no formal quality management
programme will find it difficult to maintain its cell lines unchanged over many years.

For all these reasons it is strongly recommended that new cell lines should only be
acquired from a specialist, reputable culture collection such as ECACC. Moreover, if a
laboratory believes it already has a certain cell line in its liquid nitrogen store, the identity
and purity of such a cell line should be questioned in the absence of a well recorded
culture history and recent test data. If there is a doubt, it is straightforward and cost
effective to replace such cell stocks with authenticated material from a Culture Collection.

When a Cell Culture Collection “accessions” a new cell line it will characterise the cell line
using techniques such as isoenzyme analysis and DNA profiling so that the identity of the
cell line can subsequently be verified.The Collection will then establish a hierarchy of
Master and Working cell banks, cryopreserved in liquid nitrogen, that are demonstrated
free from microbial contamination including mycoplasma. Customers are supplied from
these authenticated Working Cell Banks (WCB). Replacement WCB's are manufactured
from the original Master Cell Bank (MCB) and the new WCB will again be fully tested.

ECACC supplies its cell lines together with advice on how to maintain the line.A Technical
Support team will subsequently assist with any difficulties and can often provide additional
technical information about the cell line. Culture Collections exist to ensure that animal
cell research is conducted using standardised, authenticated material that ensures the work
can be reproduced.An authenticated cell line of known provenance is the very “bed rock”
of any cell based project.

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5.0 Main Types of Cell Culture

5.1 Primary Cultures
Primary cultures are derived directly from excised, normal animal tissue and cultured either
as an explant culture or following dissociation into a single cell suspension by enzyme
digestion. Such cultures are initially heterogeneous but later become dominated by
fibroblasts. The preparation of primary cultures is labour intensive and they can be
maintained in vitro only for a limited period of time. During their relatively limited life span
primary cells usually retain many of the differentiated characteristics of the cell in vivo.

5.2 Continuous Cultures

Continuous cultures are comprised of a single cell type that can be serially propagated in
culture either for a limited number of cell divisions (approximately thirty) or otherwise
indefinitely. Cell lines of a finite life are usually diploid and maintain some degree of
differentiation.The fact that such cell lines senesce after approximately thirty cycles of
division means it is essential to establish a system of Master and Working banks in order to
maintain such lines for long periods.

Continuous cell lines that can be propagated indefinitely generally have this ability because
they have been transformed into tumour cells.Tumour cell lines are often derived from
actual clinical tumours, but transformation may also be induced using viral oncogenes or by
chemical treatments.Transformed cell lines present the advantage of almost limitless
availability, but the disadvantage of having retained very little of the original in vivo
characteristics.

5.3 Culture Morphology

Morphologically cell cultures take one of two forms, growing either in suspension (as single
cells or small free floating clumps) or as a monolayer that is attached to the tissue culture
flask.The form taken by a cell line reflects the tissue from which it was derived e.g. cell lines
derived from blood (leukaemia, lymphoma) tend to grow in suspension whereas cells
derived from solid tissue (lungs, kidney) tend to grow as monolayers. Attached cell lines can
be classified as endothelial such as BAE-1 (Prod. No. 88031149-1v1), epithelial such as HeLa
(Prod. No. 93021013-1v1), neuronal such as SH-SY5Y(Prod. No. 94030304-1v1) or
fibroblasts such as MRC-5 (Prod. No. 84101801-1v1) and their morphology reflects the area
within the tissue of origin.

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Figure 2. Examples of Cell Morphology

Hela - epithelial
(Prod. No. 93021013-1v1)

BAE-1 - endothelial
(Prod. No. 88031149-1v1)

MRC-5 - fibroblast
(Prod. No. 84101801-1v1)

SH-SY5Y - neuronal
(Prod. No. 94030304-1v1)

(magnification x400) 15

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 Fundamental Techniques in Cell Culture

The cell lines most commonly ordered from ECACC are listed in the table below
(Table 1). The lines are classified by cell type.

Attached Cell Lines

Name Species and tissue of origin Morphology
MRC-5 (Prod. No. 84101801) Human lung Fibroblast
HELA (Prod. No. 93021013) Human cervix Epithelial
VERO (Prod. No. 84113001) African Green Monkey Kidney Epithelial
NIH 3T3 (Prod. No. 93061524) Mouse embryo Fibroblast
L929 (Prod. No. 85011425) Mouse connective tissue Fibroblast
CHO (Prod. No. 85050302) Chinese Hamster Ovary Fibroblast
BHK-21 (Prod. No. 85011433) Syrian Hamster Kidney Fibroblast
HEK 293 (Prod. No. 85120602) Human Kidney Epithelial
HEPG2 (Prod. No. 85011430) Human Liver Epithelial
BAE-1 (Prod. No. 88031149) Bovine aorta Endothelial
Suspension Cell Lines
Name Species and tissue of origin Morphology
NSO (Prod. No. 85110503) Mouse myeloma Lymphoblastoid-like
U937 (Prod. No. 85011440) Human Hystiocytic Lymphoma Lymphoblastoid
Namalwa (Prod. No. 87060801) Human Lymphoma Lymphoblastoid
HL60 (Prod. No. 98070106) Human Leukaemia Lymphoblastoid-like
WEHI 231(Prod. No. 85022107) Mouse B-cell Lymphoma Lymphoblastoid
YAC 1 (Prod. No. 86022801) Mouse Lymphoma Lymphoblastoid
U 266B1 (Prod. No. 85051003) Human Myeloma Lymphoblastoid
SH-SY5Y (Prod. No. 94030304) Human neuroblastoma Neuroblast
Table 1. Commonly used cell lines of each culture type

There are some instances when cell cultures may grow as semi-adherent cells e.g. B95-8
where there appears to be a mixed population of attached and suspension cells. For these
cell lines it is essential that both cell types are subcultured to maintain the heterogeneous
nature of the culture.

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6.0 The Cell Environment

(including types of culture medium)
In general terms cultured cells require a sterile environment and a supply of nutrients for
growth. In addition the culture environment should be stable in terms of pH and temperature.
Over the last thirty years various defined basal media types have been developed and are now
available commercially. Originally balanced salt solutions were used to maintain contractility of
mammalian heart tissue and Tyrode’s salt solution (Prod. No.T2397) was designed for use in
work with primary mammalian cells.These have since been modified and enriched with amino
acids, vitamins, fatty acids and lipids. Consequently media suitable for supporting the growth of
a wide range of cell types are now available.The precise media formulations have often been
derived by optimising the concentrations of every constituent. Examples of the different
media and their uses are given in the table below (Table 2).

Media type Examples Uses

Balanced salt solutions PBS, Hanks BSS, Earles salts Form the basis of many complex
DPBS (Prod. No. D8537/D8662) media
HBSS (Prod. No. H9269/H9394)
EBSS (Prod. No. E2888)
Basal media MEM (Prod. No. M2279) Primary and diploid cultures.
DMEM (Prod. No. D5671) Modification of MEM containing
increased level of amino acids and
vitamins. Supports a wide range of
cell types including hybridomas.
GMEM (Prod. No. G5154) Glasgows modified MEM was
defined for BHK-21 cells
Complex media RPMI 1640 Originally derived for human
(Prod. No. R0883) leukaemic cells. It supports a wide
range of mammalian cells including
hybridomas
Iscoves DMEM Further enriched modification of
(Prod. No. 13390) DMEM which supports high density
growth
Leibovitz L-15 Designed for CO2 free
(Prod. No. L5520, liquid) environments
TC 100 (Prod. No.T3160) Designed for culturing insect cells
Graces Insect medium
(Prod. No. G8142)
Schneiders Insect
medium (Prod. No. S0146)
Serum Free Media CHO (Prod. No. C5467) For use in serum free applications.
HEK293 (Prod. No. G0791)
Ham F10 and derivatives NOTE these media must be
Ham F12 (Prod. No. N4888) supplemented with other factors
DMEM/F12 (Prod. No. D8062) such as insulin, transferrin and
epidermal growth factor. These
media are usually HEPES buffered
Insect cells Sf-900 II SFM, SF Insect- Specifically designed for use with Sf9
Medium-Z (Prod. No. S3902) insect cells
Table 2. Different types of culture medium and their uses
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6.1 Basic Constituents of media

Inorganic salts
Carbohydrates
Amino Acids
Vitamins
Fatty acids and lipids
Proteins and peptides
Serum

Each type of constituent performs a specific function as outlined below:

6.2 Inorganic Salts

The inclusion of inorganic salts in media performs several functions. Primarily they help to
retain the osmotic balance of the cells and help regulate membrane potential by provision
of sodium, potassium and calcium ions.All of these are required in the cell matrix for cell
attachment and as enzyme cofactors.

6.3 Buffering Systems

Most cells require pH conditions in the range 7.2 - 7.4 and close control of pH is essential
for optimum culture conditions.There are major variations to this optimum. Fibroblasts
prefer a higher pH (7.4 - 7.7) whereas, continuous transformed cell lines require more acid
conditions pH (7.0 - 7.4).

Regulation of pH is particularly important immediately following cell seeding when a new

culture is establishing and is usually achieved by one of two buffering systems; (i) a "natural"
buffering system where gaseous CO2 balances with the CO3 / HCO3 content of the culture
medium and (ii) chemical buffering using a zwitterion called HEPES (Prod. No. H4034).

Cultures using natural bicarbonate/CO2 buffering systems need to be maintained in an

atmosphere of 5-10% CO2 in air usually supplied in a CO2 incubator. Bicarbonate/CO2 is
low cost, non-toxic and also provides other chemical benefits to the cells.

HEPES (Prod. No. H4034) has superior buffering capacity in the pH range 7.2 - 7.4 but is
relatively expensive and can be toxic to some cell types at higher concentrations. HEPES
(Prod. No. H4034) buffered cultures do not require a controlled gaseous atmosphere.

Most commercial culture media include phenol red (Prod. No. P3532/P0290)as a pH
indicator so that the pH status of the medium is constantly indicated by the colour. Usually
the culture medium should be changed / replenished if the colour turns yellow (acid) or
purple (alkali).

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6.4 Carbohydrates
The main source of energy is derived from carbohydrates generally in the form of sugars.
The major sugars used are glucose and galactose however some media contain maltose or
fructose.The concentration of sugar varies from basal media containing 1g/l to 4.5g/l in
some more complex media. Media containing the higher concentration of sugars are able
to support the growth of a wider range of cell types.

6.5 Vitamins
Serum is an important source of vitamins in cell culture. However, many media are also
enriched with vitamins making them consistently more suitable for a wider range of cell
lines.Vitamins are precursors for numerous co-factors. Many vitamins especially B group
vitamins are necessary for cell growth and proliferation and for some lines the presence of
B12 is essential. Some media also have increased levels of vitamins A and E.The vitamins
commonly used in media include riboflavin, thiamine and biotin.

6.6 Proteins and Peptides

These are particularly important in serum free media.The most common proteins and
peptides include albumin, transferrin, fibronectin and fetuin and are used to replace those
normally present through the addition of serum to the medium.

6.7 Fatty Acids and Lipids

Like proteins and peptides these are important in serum free media since they are normally
present in serum. e.g. cholesterol and steroids essential for specialised cells.

6.8 Trace Elements

These include trace elements such as zinc, copper, selenium and tricarboxylic acid
intermediates. Selenium is a detoxifier and helps remove oxygen free radicals.

Whilst all media may be made from the basic ingredients this is time consuming and may
predispose to contamination. For convenience most media are available as ready mixed
powders or as 10x and 1x liquid media. All commonly used media are listed in the Sigma-
Aldrich Life Science Catalogue. If powder or 10x media are purchased it is essential that
the water used to reconstitute the powder or dilute the concentrated liquid is free from
mineral, organic and microbial contaminants. It must also be pyrogen free (Prod. No.
W3500, water, tissue culture grade). In most cases water prepared by reverse osmosis and
resin cartridge purification with a final resistance of 16-18Mø is suitable. Once prepared
the media should be filter sterilised before use. Obviously purchasing 1x liquid media direct
from Sigma eliminates the need for this.

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6.9 Serum
Serum is a complex mix of albumins, growth factors and growth inhibitors and is probably
one of the most important components of cell culture medium.The most commonly used
serum is foetal bovine serum. Other types of serum are available including newborn calf
serum and horse serum. The quality, type and concentration of serum can all affect the
growth of cells and it is therefore important to screen batches of serum for their ability to
support the growth of cells. In addition there are other tests that may be used to aid the
selection of a batch of serum including cloning efficiency, plating efficiency and the
preservation of cell characteristics.

Serum is also able to increase the buffering capacity of cultures that can be important for
slow growing cells or where the seeding density is low (e.g. cell cloning experiments). It also
helps to protect against mechanical damage which may occur in stirred cultures or whilst
using a cell scraper. A further advantage of serum is the wide range cell types with which it
can be used despite the varying requirements of different cultures in terms of growth
factors. In addition serum is able to bind and neutralise toxins. However, serum is subject to
batch-batch variation that makes standardisation of production protocols difficult.There is
also a risk of contamination associated with the use of serum.These risks can be minimised
by obtaining serum from a reputable source since suppliers of large quantities of serum
perform a battery of quality control tests and supply a certificate of analysis with the serum.
In particular serum is screened for the presence of bovine viral diarrhoea virus (BVDV) and
mycoplasma. Heat inactivation of serum (incubation at 56°C for 30 minutes) can help to
reduce the risk of contamination since some viruses are inactivated by this process.
However the routine use of heat inactivated serum is not an absolute requirement for cell
culture.The use of serum also has a cost implication not only in terms of medium
formulation but also in downstream processing. A 10% FBS supplement contributes 4.8mg
of protein per millilitre of culture fluid which complicates downstream processing
procedures.

6.10 Guidelines for serum use

Foetal bovine serum (FBS) has been used to prepare a number of biologicals and has an
excellent record of safety.The recognition of Bovine spongiform encepalopathy (BSE) in 1986
and it’s subsequent spread into continental Europe along side the announcement of the
probable link between BSE and a new variant of Creutzfeldt Jacob disease in Humans,
stimulated an increased concern about safe sourcing of all bovine materials. In 1993 the Food
and Drug Administration (FDA) "recommended against the use of bovine derived materials
from cattle which have resided in, or originated from countries where BSE has been diagnosed.
The current (European Union) EU guidelines on viral safety focus on sourcing, testing and
paying particular attention to the potential risk of cross contamination during slaughtering or
collection of the starting tissue.As far as BSE is concerned, the EU guidelines on minimising the
risk of BSE transmission via medicinal products, CPMP/BWP/877/96, recommends the main
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measures to be implemented in order to establish the safety of bovine material verus the BSE
risk.Again, similarly the focus is on geographical origin, the age of the animals, the breeding and
slaughtering conditions, the tissue to be used and the conditions of it’s processing.

The use of FBS in production processes of medicinal products is acceptable provided good
documentation on sourcing, age of the animals and testing for the absence of adventitious
agents is submitted.All responsible suppliers of FBS for bio-pharmaceutical applications will
provide such documentation.

Recent regulatory requirements in Europe stress the importance of justifying the use of
material of bovine, caprine or ovine origin in the production of pharmaceutical products.Thus,
although FBS has been used for many years in the production process of many medicinal
products such as viral vaccines and recombinant DNA products, at present there is a justified
trend to remove all material of animal origin from manufacturing processes. Sigma-Aldrich has
recognised this growing trend and works closely with customers to optimise animal free media
formulations to meet each customer’s cell culture requirements.

Similarly the FDA has similar guidelines when accepting regulatory submissions.The FDA
regulates all medicinal products for Human use, such as therapeutics, vaccines and diagnostics,
and, usually, the United States Department Agriculture (USDA) are not involved.

The USDA regulates all medicinal products for veterinary use or for agricultural use. Similarly,
the USDA regulates all products that contain a primary component of animal origin.

It is important that you are aware of the use and restrictions when using serum, for further
information contact you local Sigma-Aldrich sales office for specific information on the serum
being used (contact information is at the back of this handbook).

With specific reference to serum the USDA has declared that for materials which fall under
their jurisdiction, only biological products manufactured using serum from approved countries
of origin be allowed into the USA.

6.11 Origin of Serum

USA/Canada, New Zealand, Finland and Denmark -No safety testing required.
Australia, Mexico, Central America - Safety testing may be required, depending on the
geographical region where the serum was collected.

Sigma-Aldrich carries out all safety test requirements stipulated by the USDA in USA
laboratories. If necessary Sigma-Aldrich will assist customers in obtaining approval from the
USDA, on any batch of serum of Australian origin supplied by Sigma-Aldrich.

Sigma-Aldrich sources serum from the states of Victoria and Tasmania.Tasmania will shortly
be considered a separate geographical area within Australia eliminating the requirement for
any safety testing.

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7.0 Cryopreservation and Storage

of Cell Lines
7.1 Cryopreservation of Cell Lines
The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need
to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited
life span. The other main advantages of cryopreservation are:

Reduced risk of microbial contamination

Reduced risk of cross contamination with other cell lines
Reduced risk of genetic drift and morphological changes
Work conducted using cells at a consistent passage number (refer to cell banking
section below)
Reduced costs (consumables and staff time)

There has been a large amount of developmental work undertaken to ensure successful
cryopreservation and resuscitation of a wide variety of cell lines of different cell types. The
basic principle of successful cryopreservation is a slow freeze and quick thaw. Although the
precise requirement may vary with different cell lines as a general guide cells should be
cooled at a rate of –1°C to –3°C per minute and thawed quickly by incubation in a 37°C
waterbath for 3-5 minutes. If this and the additional points given below are followed then
most cell lines should be cryopreserved successfully.

1. Cultures should be healthy with a viability of >90% and no signs of microbial

contamination.
2. Cultures should be in log phase of growth (this can be achieved by using pre-confluent
cultures i.e. cultures that are below their maximum cell density and by changing the
culture medium 24 hours before freezing).
3. A high concentration of serum/protein (>20%) should be used. In many cases serum is
used at 90%.
4. Use a cryoprotectant such as dimethyl sulphoxide (DMSO Prod. No. D2650) or glycerol
(Prod. No. G2025) to help protect the cells from rupture by the formation of ice
crystals. The most commonly used cryoprotectant is DMSO at a final concentration of
10%, however, this is not appropriate for all cell lines e.g. HL60 (Prod. No. 98070106-1v1)
where DMSO is used to induce differentiation. In such cases an alternative such as
glycerol (Prod. No. G2025) should be used (refer to ECACC data sheet for details of
the correct cryoprotectant). Sigma also offers ready made cell freezing media
containing DMSO (Prod. No. C6164), glycerol (Prod. No. C6039) and a serum-free
formulation containing DMSO (Prod. No. C6295).

7.2 Ultra-low Temperature Storage of Cell Lines

Following controlled rate freezing in the presence of cryoprotectants, cell lines can be
cryopreserved in a suspended state for indefinite periods provided a temperature of less
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than -135°C is maintained. Such ultra-low temperatures can only be attained by specialised
electric freezers or more usually by immersion in liquid or vapour phase nitrogen.
The advantages and disadvantages can be summarised as follows:

Method Advantages Disadvantages

Electric (-135°C) Freezer Ease of maintenance Requires liquid nitrogen back-up
Steady temperature Mechanically complex
Low running costs Storage temperatures high
relative to liquid nitrogen
Liquid Phase Nitrogen Steady ultra-low Requires regular supply of liquid
(-196°C) temperature nitrogen
Simplicity and High running costs
mechanical reliability Risk of cross-contamination
via the liquid nitrogen
Vapour Phase Nitrogen No risk of cross- Requires regular supply of liquid
contamination from nitrogen
liquid nitrogen High running costs
Low temperatures Temperature fluctuations between
achieved 135°C and -190°C
Simplicity and reliability
Table 3. Comparison of ultra-low temperature storage methods for cell lines.

Storage in liquid phase nitrogen allows the lowest possible storage temperature to be
maintained with absolute consistency, but requires the use of large volumes (depth) of
liquid nitrogen and sealed glass ampoules. Both of these requirements create potential
hazards.There have also been documented cases of cross contamination by virus pathogens
via the liquid nitrogen medium. For these reasons ultra-low temperature storage is most
commonly in vapour phase nitrogen.

For vapour phase nitrogen storage, the ampoules are positioned above a shallow reservoir
of liquid nitrogen, the depth of which has to be carefully maintained.A vertical temperature
gradient will exist through the vapour phase, the extremes of which will depend on the
liquid levels maintained, the design of the vessel, and the frequency with which it is opened.
Temperature variations in the upper regions of a vapour phase storage vessel can be
extreme if regular maintenance is not carried out.

All liquid nitrogen storage vessels should include alarms that at least warn of low liquid
nitrogen levels.This is particularly true of vapour phase storage systems.The bulk liquid
nitrogen storage vessel should not be allowed to become less than half full before it is re-
supplied.This will ensure that at least one delivery can be missed without catastrophic
consequences.

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Inventory Control
All ultra-low temperature storage vessels will include a racking / inventory system designed
to organise the contents for ease of location and retrieval.This should be supported by
accurate record keeping and inventory control incorporating the following:

Each ampoule should be individually labelled, using “wrap around”, liquid nitrogen
resistant labels with identity, lot number and date of freezing
The location of each ampoule should be recorded ideally on an electronic database or
spreadsheet, but also on a paper storage plan
There should be a control system to ensure that no ampoule can be deposited or
withdrawn without updating the records

7.3 Safety Considerations

General safety issues

It is important that staff are trained in the use of liquid nitrogen and associated equipment
including the storage vessels which need to be vented safely and containers which may need
to be filled.As with all laboratory procedures personal protective equipment should be
worn at all times whilst handling nitrogen, including a full-face visor and thermally insulated
gloves in addition to a laboratory coat. Proper training and the use of protective equipment
will minimise the risk of frostbite and other minor incidents.

Risk of asphyxiation
The single most important safety consideration is the potential risk of asphyxiation due to
the high levels of nitrogen that can lead to oxygen depletion.This is critical since oxygen
depletion can very rapidly cause loss of consciousness, without warning.

Consequently liquid nitrogen refrigerators should be placed in well-ventilated areas in order

to minimise this risk. Large volume stores should have low oxygen alarm systems.

Preventative measures
Use oxygen alarms set to 18% oxygen (v/v)
Staff training – staff should be trained to evacuate the area immediately on hearing the
alarm and not return until the oxygen is back to normal (~ 20% v/v)
Staff should work in pairs when handling liquid nitrogen
Prohibit the use of nitrogen outside of normal working hours
Mechanical ventilation systems should be installed if at all possible

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8.0 Good Cell Banking Practices

It is bad practice to maintain a cell line in continuous or extended culture for the following
reasons:

Risk of microbial contamination

Loss of characteristics of interest (i.e. surface antigen or monoclonal antibody
expression)
Genetic drift particularly in cells known to have an unstable karyotype
(i.e. CHO Prod. No. 85050302-1v1, BHK 21 Prod. No. 85011433-1v1)
Loss of cell line due to exceeding finite life-span e.g. human diploid cells such as MRC-5
(Prod. No. 84101801-1v1)
Risk of cross contamination with other cell lines
Increased consumables and staff costs

All of these potential risk factors may be minimised by the implementation of a cell banking
system as described below.This type of system is known as a tiered banking system or
Master Cell Banking system. On initial arrival into the laboratory a new cell culture should
be regarded as a potential source of contamination from bacteria, fungi and mycoplasma
and should be handled under quarantine conditions until proven negative for such microbial
contaminants. Following initial expansion 3-5 ampoules should be frozen as a token stock
before a Master Bank is prepared. One of the token stock ampoules should then be
thawed and expanded to produce a Master Bank of 10-20 ampoules depending upon the
anticipated level of use.

Ampoules of this bank (2-3) should be allocated for quality control comprising
confirmation that the cell count and viability of the bank is acceptable and that the bank is
free of bacteria/fungi and mycoplasma.Additional tests (such as viral screening and
authenticity testing) may also be required. Once these tests have been completed
satisfactorily an ampoule from the Master Bank should be thawed and cultured to produce
a Working Bank. The size of this bank will again depend on the envisaged level of demand.
Quality control tests (cell count and viability and the absence of microbial contaminants)
are again required prior to using the cultures for routine experimentation or production. It
is also important at this stage to confirm that the Master and Working Banks are
genetically identical by DNA profiling techniques.

Implementation of this banking system ensures:

Material is of a consistent quality

Experiments are performed using cultures in the same range of passage numbers
Cells are only in culture when required
The original cell line characteristics are retained

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Figure 3. Schematic Representation of a Tiered Cell Banking System

New cell line (Handled under quarantine conditions)

Token freeze of 3-5 ampoules and initial mycoplasma test

FAIL PASS QUARANTINE FACILITY

Abandon banking procedure Resuscitate one ampoule and expand in culture MAIN CELL
CULTURE FACILITY

Cryo-preservation of Master Bank

(10-100 ampoules)

Mandatory Quality Control Tests

Cell count and viability
Microbial QC including mycoplasma
Authentication

FAIL PASS

Repeat Banking Resuscitate one ampoule and expand in culture

Cryopreservation of Working Bank (20-200 ampoules)

Quality control tests

Cell count and viability
Microbial QC including mycoplasma
Authentication

FAIL PASS

Repeat Banking released for use Released for use

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Notes
1. The number of ampoules prepared for Master and Working Banks depends upon the
forecast demand for their use.
2. The number of ampoules sampled for quality control is dependent upon the size of
bank. Ideally 5-10% of the bank should be tested before use.
3. Ampoules from the Working Cell Bank should be used sequentially keeping cells in
culture for not more than a predetermined number of cell doublings. This number will
be least in the case of cell lines having a finite life-span (e.g. diploid lines).
4. The Working Bank should be replenished from an ampoule of the Master Bank. This
should be done in sufficient time to allow the quality control to be completed.
5. A new Master Bank should be prepared before the number of original Master stock
drops below five ampoules.
6. The panel of quality control tests performed depends upon the use intended
e.g. regulatory authorities may require additional tests such as viral screening and
karyotypic studies.

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9.0 Quality Control Considerations

Introduction
Quality is important in all aspects of tissue culture since the quality of materials used (i.e.
media and other reagents) will affect the quality of the cultures and products derived from
them. The main areas of quality control that are of concern for tissue culture are:

The quality of the reagents and materials

The provenance and integrity of the cell lines
The avoidance of microbial contamination

9.1 Reagents and Materials

A potential source of contamination is reagents and materials, in particular bovine serum
which has been identified as a source of bovine viral diarrhoea virus (BVDV). Porcine
trypsin is also a potential source of Mycoplasma hyorhinis. Good quality reagents and
materials are available from numerous manufacturers of tissue culture media and
supplements. In addition manufacturers including Sigma will carry out a range of quality
control tests including screening for mycoplasma and BVDV and supply a Certificate of
Analysis with their products.These state the product and lot numbers and forms a vital part
of record keeping and tracking of reagents used in the production of cell stocks. It is
advisable to further test key reagents such as FBS to ensure that they are ‘fit for purpose’
due to batch-to-batch variation.

Manufacturers of sterile plastic ware (flasks, centrifuge tubes, pipettes) designed for tissue
culture use are also supplied with Certificates of Analysis for each batch produced, which
should be kept for future reference.

9.2 Provenance and Integrity of Cell Lines

The sourcing of cell lines can have an important effect on quality since freshly imported cell
lines are a major source of contamination.The advantages of obtaining cell lines from a
recognised source such as a culture collection are:
Contaminant free
Fully characterised and authenticated in terms of DNA profile and species of origin
Supplied with a detailed data sheet

Once cell lines have been obtained from a reputable source it is important to implement
master and working cell banking procedures and the associated quality control steps such as
routine testing for microbial contaminants and confirming the identity of cultures.

9.3 Avoidance of Microbial Contamination

Potential sources of contamination include other cell lines, laboratory conditions and staff
poorly trained in core areas such as aseptic techniques and good laboratory practice.Thus
the use of cells and reagents of known origin and quality alone is not sufficient to guarantee
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quality of product (cell stock or culture products); it is necessary to demonstrate quality

throughout the production process and also in the final product. Routine screening aids the
early detection of contamination since all manipulations are a potential source of
contamination.

The 3 main types of microbial contaminants in tissue culture are:

Bacteria and Fungi

Mycoplasma
Viruses

Bacterial and Fungal Contamination

Bacterial contamination is generally visible to the naked eye and detected by a sudden
increase in turbidity and colour change of the culture medium as the result of a change in
pH. The cell culture may survive for a short time but the cells will eventually die. Daily
microscopic observation of cultures will ensure early detection of contamination and
enable appropriate action to be taken as soon as the first signs of contamination become
apparent (see below). In addition specific tests for the detection of bacteria and fungi
should be used as part of a routine and regular quality control screening procedure (see
Protocol 8).

Mycoplasma Contamination
Mycoplasmas are the smallest free-living self-replicating prokaryotes.They lack a cell wall
and lack the ability to synthesize one. They are 0.3µm in diameter and can be observed as
filamentous or coccal forms.There are 5 major species that are tissue culture
contaminants, namely M. hyorhinis, M. arginini, M. orale, M. fermentans and Acholeplasma
laidlawii.

The effects of mycoplasma infection are more insidious than those of bacteria and fungi
inducing several long term effects.These include:
Reduced growth rate
Morphological changes
Chromosome aberrations
Alterations in amino acid and nucleic acid metabolism

However, despite these well-documented effects the presence of mycoplasma is often not
tested for with the consequence that in such laboratories the majority of cell lines are
positive for mycoplasma. Mycoplasma contamination is difficult to detect requiring the use
of specialist techniques (see Protocol 9 - Isolation by culture and Protocol 10 – Detection
by DNA staining). In the past only specialist laboratories, such as culture collections, have
performed these tests. However a variety of commercial kits are now available although
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the performance characteristics of these kits can be extremely variable. A combination of

these should be used as part of a routine and regular quality control screening procedure.
Culture collections such as ECACC are able to test cultures if required. Mycoplasma
testing products are available from Sigma, refer to pages 489-490 of Life Sciences Catalogue.

Viral Contamination
Some cell lines contain endogenous viruses and secrete virus particles or express viral
antigens on their surface (e.g. EBV transformed lines).These cell lines are not considered
contaminated. However, bovine serum is a potential source of bovine viral diarrhoea virus
(BVDV) contamination. Use of infected serum will lead to contamination of cell lines with
the virus. Contamination of cell lines with BVDV may cause slight changes in growth rate
but since this virus is non-cytopathic macroscopic and microscopic changes in the culture
will not be detected. Suppliers of bovine serum are aware of this and screen sera
accordingly and generally serum is sold as BVDV tested.

9.4 Environmental Monitoring

It is good practice to monitor the laboratory environment where cell cultures and their
products are prepared. Class II microbiology safety cabinets should be checked every 6
months to ensure that they are working efficiently. However it is also advisable to monitor
the number of contaminants within the cabinet by periodically placing open settle plates
(blood agar bacteriological culture plates) on the cabinet work surfaces. In addition settle
plates should be used to assess airborne microbial burden at selected points around the
laboratory. Plates should be left open for a period of 4 hours. After this time they should
be covered, placed in sealed boxes and incubated at 32°C and 22°C for up to 7 days.At the
end of this period the plates should be examined for the presence of microbial growth.The
position of each plate in the cabinet should be recorded and results stored for trend
analysis.

Acceptable limits should be defined in terms of “alert” levels and “action” levels, the actual
values being dependent on the criticality of the work and the levels of cleanness that can be
achieved under normal operating conditions.

9.5 What to do in the event of contamination

One hugely under-estimated problem in tissue culture is the routine use of antibiotics.
Continuous use of antibiotics is unnecessary and can lead to the development of resistant
strains that are difficult to eradicate and may require the use of more exotic antibiotics that
may be toxic to the cell cultures. In addition the use of antibiotics may mask a low level of
contamination.

Once a contamination has been detected, whether is it due to bacteria, fungi or

mycoplasma, the recommended course of action is to discard the culture and continue the
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work with earlier stocks that are known to be free of contaminants or obtain fresh stocks
from a recognised source. However if this is not possible eradication of the contaminant
may be attempted with the use of antibiotics. In addition culture collections such as
ECACC will attempt to eradicate any contaminants if required. Please contact ECACC for
further details.

Viral infections are virtually impossible to remove from cultures since they do not respond
to antibiotic treatment. Also, since they are intra cellular parasites it is not possible to
remove them by centrifugation or other separation techniques. If virus free stocks or a
virus free alternative is not available then a thorough risk assessment should be undertaken
prior to continuing work with the infected cell line.

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10.0 Authentication of Cell Lines

10.1 Authentication Techniques
Whatever the scope of work to be carried out it is important to know that the work is
being conducted using the correct reagents.This is no less important for cell cultures, since
if cell cultures are not what they are reported to be then work can be invalidated and
resources wasted.There is now considerable evidence of gross cross-contamination of cell
lines, in particular with HeLa (Prod. No. 93021013-1v1) where up to 16 lines were offered
to ECACC with DNA profiles identical to HeLa.These include Hep 2,WISH, INT 407,
Chang liver and Giradi heart.To minimise the risk of working with contaminated cell lines it
is advisable to obtain cells from a recognised source such as a culture collection that will
have confirmed the identity of the cells as part of the banking process.

Tests used to authenticate cell cultures include iso-enzyme analysis, karyotyping/ cytogenetic
analysis and more recently molecular techniques of DNA profiling.Whilst most of the
techniques above are generalised tests and are applicable to all cell lines additional specific
tests may also be required to confirm the presence of a product or antigen of interest.

10.2 Iso-Enzyme Analysis

Iso-enzymes are a series of enzymes present in different species that have similar catalytic
properties but differ in their structure. By studying the iso-enzymes present in cell lines it is
possible to identify the species from which the cell line was derived.The technique is also
used as a means of excluding the possibility of gross cross-contamination of the cell line
with another culture of a different species.

The principles upon which iso-enzyme analysis is based are:

Each iso-enzyme has multiple gene loci coding for different polypeptides with identical
enzyme activity (e.g. lactate dehydrogenase, LD)
Electrophoretic migration rates change dependent on sub-unit composition e.g. LD has
five possible iso-forms (LD 1-5)
Different species have different combinations of these iso-forms
Using a typical panel of 4 iso-enzymes a composite picture is built up enabling the
species of origin to be determined by the use of reference tables

10.3 DNA Fingerprinting

DNA fingerprinting enables the following:
Identification of individual cell lines from the same species
Confirmation of the identity of cell banks compared to reference master stocks
Detection of cross-contamination

Multi locus DNA fingerprinting and multiplex - PCR DNA profiling are the methods used
routinely as part of ECACC's routine cell banking procedures.

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10.4 Multi Locus DNA Fingerprinting

Uses multi locus Jeffrey’s probes 33.15 or 33.6, along with Southern blotting
technology producing a complex banding pattern.
Probes cross-hybridise with most common species
Has the disadvantage that the profiles require visual interpretation and comparison
with other samples can be subjective

Figure 4. Multi Locus DNA Fingerprinting

Each lane represents the

genotype for one cell line
using a series of primers.

10.5 Multiplex - PCR (STR) DNA profiling

Uses a set of primers (9 used at ECACC) recognising micro-satellites using PCR and
automated DNA sequencing techniques
Primers are species specific and are used only for human cell lines
Produces a colour-coded banding pattern, that translates into a digital code that can
easily be stored on a database and compared to other stored profiles

Figure 5. PCR DNA profiling

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11.0 Alternative Culture Systems

11.1 Cell Culture Scale-up Systems
Most tissue culture is performed on a small scale where relatively small numbers of cells are
required for experiments.At this scale cells are usually grown in T flasks ranging from 25cm2 to
175cm2.Typical cell yields from a T175 flask range from 1x107 for an attached line to 1x108 for a
suspension line. However exact yields will vary depending on the cell line. It is not practicable to
produce much larger quantities of cells using standard T flasks, due to the amount of time required
for repeated passaging of the cells, demand on incubator space and cost.

When considering scaling up a cell culture process there are a whole range of parameters to
consider which will need to be developed and optimised if scale-up is to be successful.These
include problems associated with nutrient depletion, gaseous exchange particularly oxygen
depletion and the build up of toxic by-products such as ammonia and lactic acid.To optimise such
a process for quantities beyond 1L volumes is best left to expert process development scientists.

However there are many commercially available systems that attempt to provide a "half-way
house" solution to scale-up which do not necessarily require expert process development
services. A selected list of some of the systems available along with a brief summary of their
potential yields, advantages and disadvantages is provided in Table 4.

11.2 Scale-up Solutions

Figure 6.Triple Flask

(Prod. No. F8542)

Figure 7. Bioreactor

The exmple shown is a Cell-Pharm 2000

hollow fibre based bioreactor from Biovest
International Inc. and produces grams scale of
antibody per month. These reactors are in
routine use under cGMP manufacturing
conditions at Sigma Aldrich Immunochemical
Production facility in Rehovot, Israel.
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 Fundamental Techniques in
Technology Suspension Attached Max Vol Max S/A Max cells Max cells Advantages Disadvantages
(ml) (cm2) (susp) (att)
T Flask 150 225 1.5x108 ~107 Cheap, disposable, no Small scale. Multiples required
cleaning / sterilisation required for larger batches.
Triple Flask ( ) 150 525 1.5x108 3x107 Cheap, disposable, no cleaning Difficult to harvest attached cells.
/ sterilisation required Multiples required for larger batches
sigma-aldrich.com

Cell Factory N/A 8000 25280 N/A 1.5 x 1010 Disposable – Single batch Require additional equipment
manufacture (vessels etc which may require

Cell Culture
cleaning). Difficult to harvest cells
Roller Bottles 1000 1700 1x109 1x108 Cheap, disposable, no cleaning Require "decks" to turn. Multiples
/ sterilisation required.Versatile. required for larger batches.
Automated systems available. Automation very costly
Expanded Roller N/A (1000) 3400 N/A 2x108 As above As above (no advantage for
Bottles suspension cells)
Shake Flasks N/A 1000 N/A 1 x 109 N/A Some disposables available. Suspension Only*. Glass vessels to
be cleaned & sterilised. Requires
Shaker incubator
Spinner Flasks N/A 1000 N/A 1 x 109 N/A Some semi-disposables Suspension Only* Glass vessels to
available be cleaned & sterilised. Requires
Stirrer-base + incubator

Table 4.“Half-Way House” Solutions to Scale-up - without attempting to adapt cells or the process

A word of caution – although the systems listed in Table 4 are often described as off-the-shelf solutions to scale-up they are not universally applicable to all
cell types and often require a period for the user to adapt to the system as well as the cells!
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 Fundamental Techniques in Cell Culture

Figure 8. Shake Flasks

Figure 9. Roller Deck

11.3 Roller Bottle Culture

This is the method most commonly used for initial scale-up of attached cells also known as
anchorage dependent cell lines. Roller bottles are cylindrical vessels that revolve slowly
(between 5 and 60 revolutions per hour) which bathes the cells that are attached to the
inner surface with medium. Roller bottles are available typically with surface areas of
1050cm2 (Prod. No. Z35, 296-9).The size of some of the roller bottles presents problems
since they are difficult to handle in the confined space of a microbiological safety cabinet.
Recently roller bottles with expanded inner surfaces have become available which has made
handling large surface area bottles more manageable, but repeated manipulations and
subculture with roller bottles should be avoided if possible.A further problem with roller
bottles is with the attachment of cells since as some cells lines do not attach evenly.This is a
particular problem with epithelial cells.This may be partially overcome a little by optimising
the speed of rotation, generally by decreasing the speed, during the period of attachment for
cells with low attachment efficiency.
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Figure 10. Roller Bottle

11.4 Spinner Flask Culture

This is the method of choice for suspension lines including hybridomas and attached lines
that have been adapted to growth in suspension e.g. HeLa S3. Spinner flasks are either
plastic or glass bottles with a central magnetic stirrer shaft and side arms for the addition
and removal of cells and medium, and gassing with CO2 enriched air. Inoculated spinner
flasks are placed on a stirrer and incubated under the culture conditions appropriate for the
cell line. Cultures should be stirred at 100-250 revolutions per minute. Spinner flask
systems designed to handle culture volumes of 1-12 litres are available from Techne, Sigma,
and Bellco, e.g. (Prod. No’s. Z380482-3L capacity and Z380474-1L capacity).

Figure 11. Spinner Flasks

(Prod. No. Z380431)

11.5 Other Scale up Options
The next stage of scale up for both suspension and attached cell lines is the bioreactor that
is used for large culture volumes (in the range 100-10,000 litres). For suspension cell lines
the cells are kept in suspension by either a propeller in the base of the chamber vessel or by
air bubbling through the culture vessel (Prod. No. C4853 (220v) or C4728 (110v)). However
both of these methods of agitation give rise to mechanical stresses. A further problem with
suspension lines is that the density obtained is relatively low; in the order of 2x106 cells/ml.

For attached cell lines the cell densities obtained are increased by the addition of micro-carrier
beads.These small beads are 30-100µm in diameter and can be made of dextran, cellulose,
gelatin, glass or silica, and increase the surface area available for cell attachment considerably. The
range of micro-carriers available means that it is possible to grow most cell types in this system.

A recent advance has been the development of porous micro-carriers which has increased
the surface area available for cell attachment by a further 10-100 fold.The surface area on
2g of beads is equivalent to 15 small roller bottles, refer to page 356 of Sigma Life Science
catalogue for further details.
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12.0 Cell Culture Protocols

12.1 Basic Techniques - The “Do’s and Don’ts” of Cell Culture
Given below are a few of the essential "do’s and don’ts" of cell culture. Some of these are
mandatory e.g. use of personal protective equipment (PPE). Many of them are common
sense and apply to all laboratory areas. However some of them are specific to tissue
culture.

The Do’s
1. Use personal protective equipment, (laboratory coat/gown, gloves and eye protection)
at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron
should be worn when handling liquid nitrogen.
2. Always use disposable caps to cover hair.
3. Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the
general laboratory environment.The use of different coloured gowns or laboratory
coats makes this easier to enforce.
4. Keep all work surfaces free of clutter.
5. Correctly label reagents including flasks, medium and ampoules with contents and date
of preparation.
6. Only handle one cell line at a time.This common-sense point will reduce the possibility
of cross contamination by mislabelling etc. It will also reduce the spread of bacteria and
mycoplasma by the generation of aerosols across numerous opened media bottles and
flasks in the cabinet.
7. Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between
operations and allow a minimum of 15 minutes between handling different cell lines.
8. Wherever possible maintain separate bottles of media for each cell line in cultivation.
9. Examine cultures and media daily for evidence of gross bacterial or fungal
contamination. This includes medium that has been purchased commercially.
10. Quality Control all media and reagents prior to use.
11. Keep cardboard packaging to a minimum in all cell culture areas.
12. Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced
at regular intervals.
13. Test cells for mycoplasma on a regular basis.

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The Don’ts
1. Do not continuously use antibiotics in culture medium as this will inevitably lead to the
appearance of antibiotic resistant strains and may render a cell line useless for
commercial purposes.
2. Don’t allow waste to accumulate particularly within the microbiological safety cabinet
or in the incubators.
3. Don't have too many people in the lab at any one time.
4. Don't handle cells from unauthenticated sources in the main cell culture suite.They
should be handled in quarantine until quality control checks are complete.
5. Avoid keeping cell lines continually in culture without returning to
frozen stock.
6. Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or
as advised on ECACC's cell culture data sheet.
7. Do not allow media to go out of date. Shelf life is only 6 weeks at +4°C once
glutamine and serum is added.
8. Avoid water baths from becoming dirty by using Sigma Clean (Prod. No. S5525).
9. Don’t allow essential equipment to become out of calibration. Ensure microbiological
safety cabinets are tested regularly.

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12.2 Protocol 1 - Aseptic Technique and Good Cell Culture Practice

Aim
To ensure all cell culture procedures are
performed to a standard that will prevent
contamination from bacteria, fungi and
mycoplasma and cross contamination with
other cell lines.

Materials
Chloros / Presept solution (2.5g/l)
1% formaldehyde based disinfectant
e.g.Virkon,Tegador
70% ethanol in water (Prod. No. R8382)

Equipment
Personal protective equipment (sterile
gloves, laboratory coat, safety visor)
Microbiological safety cabinet at
appropriate containment level

Procedure
1. Sanitise the cabinet using 70% ethanol before commencing work.
2. Sanitise gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds
before commencing work.
3. Put all materials and equipment into the cabinet prior to starting work after sanitising
the exterior surfaces with 70% ethanol.
4. Whilst working do not contaminate gloves by touching anything outside the cabinet
(especially face and hair). If gloves become contaminated re-sanitise with 70% ethanol
as above before proceeding.
5. Discard gloves after handling contaminated cultures and at the end of all cell culture
procedures.
6. Equipment in the cabinet or that which will be taken into the cabinet during cell
culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped
with tissue soaked with 70% ethanol prior to use.
7. Movement within and immediately outside the cabinet must not be rapid. Slow
movement will allow the air within the cabinet to circulate properly.
8. Speech, sneezing and coughing must be directed away from the cabinet so as not to
disrupt the airflows.
9. After completing work disinfect all equipment and material before removing from the
cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry
with tissue. Dispose of tissue by autoclaving.
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Fundamental Techniques in Cell Culture

10. Cell culture discard in chloros (10,000) ppm must be kept in the cabinet for a
minimum of two hours (preferably overnight) prior to discarding down the sink with
copious amounts of water.
11. Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or
Virkon or fumigate the cabinet according to the manufacturers instructions. However
you must ensure that it is safe to fumigate your own laboratory environment due to
the generation of gaseous formaldehyde, consult your on-site Health and Safety
Advisor.

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12.3 Protocol 2 - Resuscitation of Frozen Cell Lines

Check technical data sheet Aim

Many cultures obtained from a culture
collection, such as ECACC, will arrive frozen
Prepare flasks
and in order to use them the cells must be
thawed and put into culture. It is vital to
Collect cells thaw cells correctly in order to maintain the
viability of the culture and enable the culture
to recover more quickly. Some
Allow to thaw
cryoprotectants, such as DMSO (Prod. No.
D2650), are toxic above 4°C therefore it is
Pipette cells into pre-warmed growth medium, essential that cultures are thawed quickly and
dilute if required
diluted in culture medium to minimise the
toxic efects.
Incubate at appropriate temperature
Materials
Examine cells after 24 hours Media– pre-warmed to the appropriate
temperature (refer to the ECACC Cell
Line Data Sheet for the correct medium
and size of flask to resuscitation into.)
70% ethanol in water (Prod. No. R8382)
DMSO (Prod. No. D2650)

Equipment
Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
Waterbath set to appropriate temperature
Microbiological safety cabinet at appropriate containment level
CO2 incubator
Pre labelled flasks
Marker Pen
Pipettes
Ampoule Rack
Tissue

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Fundamental Techniques in Cell Culture

Procedure
1. Read Technical data sheet to establish specific requirements for your cell line.
2. Prepare the flasks as appropriate (information on technical data sheet). Label with cell
line name, passage number and date.
3. Collect ampoule of cells from liquid nitrogen storage wearing appropriate protective
equipment and transfer to laboratory in a sealed container.
4. Still wearing protective clothing, remove ampoule from container and place in a
waterbath at an appropriate temperature for your cell line e.g. 37°C for mammalian
cells. Submerge only the lower half of the ampoule. Allow to thaw until a small
amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety
cabinet.
5. Wipe the outside of the ampoule with a tissue moistened (not excessively) with 70%
alcohol hold tissue over ampoule to loosen lid.
6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the
DMSO (Prod. No. D2650) (flasks prepared in Step 2).
7. Incubate at the appropriate temperature for species and appropriate concentration of
CO2 in atmosphere.
8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as
necessary.

Key Points
1. Most text books recommend washing the thawed cells in media to remove the
cryoprotectant. This is only necessary if the cryoprotectant is known to have an
adverse effect on the cells. In such cases the cells should be washed in media before
being added to their final culture flasks. See Protocol 7 for further details.
2. Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too
slow resulting in a loss of viability.
3. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95%
air filtered through a 0.2µm filter.
4. For some cultures it is necessary to subculture before confluence is reached in order
to maintain their characteristics e.g. the contact inhibition of NIH 3T3
(Prod. No. 93061524) cells is lost if they are allowed to reach confluence
repeatedly.

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 Fundamental Techniques in Cell Culture

12.4 Protocol 3 - Subculture of Adherent Cell Lines

Assess cultures Aim

Adherent cell lines will grow in vitro until they
have covered the surface area available or the
Remove spent medium medium is depleted of nutrients. At this point
the cell lines should be sub-cultured in order
Wash cells with PBS without Ca2+/Mg2+. to prevent the culture dying.To subculture the
Repeat if required. cells they need to be brought into suspension.
The degree of adhesion varies from cell line to
cell line but in the majority of cases proteases,
Incubate for 2-10 minutes
e.g. trypsin, are used to release the cells from
the flask. However, this may not be appropriate
Examine cells for some lines where exposure to proteases is
harmful or where the enzymes used remove
membrane markers/receptors of interest. In
Resuspend cells in fresh medium
these cases cells should be brought into
suspension into a small volume of medium
Transfer cells into fresh, warmed, new media mechanically with the aid of cell scrapers.

Materials
Incubate as required
Media– pre-warmed to 37°C (refer to
the ECACC Cell Line Data Sheet for the
Repeat as necessary correct medium)
70% ethanol in water (Prod. No. R8382)
PBS without Ca2+/Mg2+
(Prod. No. D8537)
0.25% trypsin/EDTA in HBSS, without
Ca2+/Mg2+ (Prod. No.T4049)
Trypsin (Prod. No.T4424)
Soyabean trypsin Inhibitor
(Prod. No.T6414)
Equipment
Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
Waterbath set to appropriate temperature
Microbiological safety cabinet at appropriate containment level
CO2 incubator
Pre-labelled flasks
Marker Pen
Pipettes
Ampoule Rack
Tissue

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Fundamental Techniques in Cell Culture

Procedure
1. View cultures using an inverted microscope to assess the degree of confluency and
confirm the absence of bacterial and fungal contaminants.
2. Remove spent medium.
3. Wash the cell monolayer with PBS without Ca2+/Mg2+ (Prod. No. D8537) using a
volume equivalent to half the volume of culture medium. Repeat this wash step if the
cells are known to adhere strongly.
4. Pipette trypsin/EDTA (Prod. No.T4049) onto the washed cell monolayer using 1ml per
25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the
excess trypsin.
5. Return flask to the incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are
detached and floating.The side of the flasks may be gently tapped to release any
remaining attached cells.
7. Resuspend the cells in a small volume of fresh serum-containing medium to inactivate
the trypsin. Remove 100-200µl and perform a cell count (Protocol 6- Cell
Quantification).
8. Transfer the required number of cells to a new labelled flask containing pre-warmed
medium (refer to ECACC Cell Line Data Sheet for the required seeding density).
9. Incubate as appropriate for the cell line.
10. Repeat this process as demanded by the growth characteristics of the cell line.

Key Points
1. Some cultures whilst growing as attached lines adhere only lightly to the flask, thus it
is important to ensure that the culture medium is retained and the flasks are handled
with care to prevent the cells detaching prematurely.
2. Although most cells will detach in the presence of trypsin alone the EDTA is added to
enhance the activity of the enzyme.
3. Trypsin is inactivated in the presence of serum.Therefore, it is essential to remove all
traces of serum from the culture medium by washing the monolayer of cells with PBS
without Ca2+/Mg2+ (Prod. No. D8537).
4. Cells should only be exposed to trypsin/EDTA (Prod. No.T4049) long enough to
detach cells. Prolonged exposure could damage surface receptors.
5. Trypsin should be neutralised with serum prior to seeding cells into new flasks
otherwise cells will not attach.
6. Trypsin may also be neutralised by the addition of soyabean trypsin inhibitor
(Prod. No.T6414), where an equal volume of inhibitor at a concentration of 1mg/ml is
added to the trypsinised cells.The cells are then centrifuged, resuspended in fresh culture
medium and counted as above. This is especially necessary for serum-free cell culture.
7. If a CO2 incubator is not available gas the flasks for 1-2min with 5% CO2 in 95% air
filtered through a 0.2µm filter.
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12.5 Protocol 4 - Subculture of Semi-Adherent Cell Lines

Assess cultures Aim
Some cultures grow as a mixed population
Decant spent medium
(e.g. B95-8 - marmoset) where a proportion
of cells do not attach to the tissue culture
flask and remain in suspension. Therefore to
Wash cells with 1-2mls PBS without Ca2+/Mg2+ maintain this heterogeneity both the attached
cells and the cells in suspension must be
Pipette trypsin/EDTA onto cell monolayer, rotate subcultured.
flask and decant excess
Materials
Incubate for 2-10 minutes
Media– pre-warmed to 37°C (refer to
the ECACC Cell Line Data Sheet for the
correct medium)
Examine cells 70% ethanol in water (Prod. No. R8382)
PBS without Ca2+/Mg2+
Centrifuge (Prod. No. D8537)
0.25% trypsin/EDTA in HBSS, without
Ca2+/Mg2+ (Prod. No.T4049)
Decant and re-suspend cells
Trypsin (Prod. No.T4424)
Soyabean trypsin Inhibitor
Pipette into fresh media (Prod. No.T6414)

Repeat as necessary

Equipment
Personal protective equipment (sterile gloves, laboratory coat, safety visor)
Waterbath set to 37°C
Microbiological safety cabinet at the appropriate containment level
Centrifuge
Inverted phase contrast microscope
CO2 incubator
Haemocytometer (Bright-line, Prod. No. Z35, 962-9, Improved Neubauer Grid,
Camlab CCH.AC1)
Pre-labelled flasks
Tissues

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Fundamental Techniques in Cell Culture

Procedure
1. View cultures using an inverted phase contrast microscope to assess the degree of
confluency and confirm the absence of bacterial and fungal contaminants. Give the flask
a gentle knock first, this may dislodge the cells from the flask and remove the need for
a trypsinisation step with the subsequent loss of some cells due to the washings.
2. Decant spent medium into a sterile centrifuge tube and retain.
3. Wash any remaining attached cells with PBS without Ca2+/Mg2+ (Prod. No. D8537)
using 1-2ml for each 25cm2 of surface area. Retain the washings.
4. Pipette trypsin/EDTA (Prod. No.T4049) onto the washed cell monolayer using 1ml per
25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the
excess trypsin.
5. Return flask to incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are
detached and floating.The side of the flasks may be gently tapped to release any
remaining attached cells.
7. Transfer the cells into the centrifuge tube containing the retained spent medium and cells.
8. Centrifuge the remaining cell suspension at 150g for 5 minutes.Also centrifuge the
washings from Number 3 above if they contain significant numbers of cells.
9. Decant the supernatants and resuspend the cell pellets in a small volume (10-20mls) of
fresh culture medium. Pool the cell suspensions. Count the cells.
10. Pipette the required number of cells to a new labelled flask and dilute to the required
volume using fresh medium (refer to ECACC Cell Line Data Sheet for the required
seeding density).
11. Repeat this process every 2-3 days as necessary.

Key Points
1. Although most cells will detach in the presence of trypsin alone the inclusion of EDTA
is used to enhance the activity of the enzyme.
2. Trypsin is inactivated in the presence of serum.Therefore, it is essential to remove all
traces of serum from the culture medium by washing the monolayer of cells with PBS
without Ca2+/Mg2+ (Prod. No. D8537). Repeated warming to 37°C also inactivates
trypsin.
3. Cells should only be exposed to trypsin/EDTA (Prod. No.T4049) long enough to
detach cells. Prolonged exposure could damage surface receptors. In general a
shorter time of exposure to trypsin is required for semi adherent cell lines.
4. Trypsin should be neutralised with serum prior to seeding cells into new flasks
otherwise cells will not attach.
5. Trypsin may also be neutralised by the addition of Soyabean trypsin Inhibitor (Prod. No.
T6414), where an equal volume of inhibitor at a concentration of 1mg/ml is added to
the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture
medium and counted as above.
6. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95%
air filtered through a 0.2µm filter.
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12.6 Protocol 5 - Subculture of Suspension Cell Lines

View cultures and assess Aim

In general terms cultures derived from blood
(e.g. lymphocytes) grow in suspension. Cells
Centrifuge 150g for 5 minutes
(pH of medium must be acidic)
may grow as single cells or in clumps (e.g.
EBV transformed lymphoblastoid cell lines).
For these types of lines subculture by dilution
Add 10-20% conditioned media to fresh media is relatively easy. But for lines that grow in
clumps it may be necessary to bring the cells
Take sample of cells into a single cell suspension by centrifugation
and resuspension by pipetting in a smaller
volume before counting.
Calculate cells/ml and reseed according to
recommended density
Materials
Media– pre-warmed to 37°C (refer to
Repeat every 2-3 days the ECACC Cell Line Data Sheet for the
correct medium)
70% Ethanol in water (Prod. No. R8382)

Equipment
Personal protective equipment (sterile gloves, laboratory coat, safety visor)
Waterbath set to 37°C
Microbiological safety cabinet at appropriate containment level
Centrifuge
CO2 incubator
Inverted phase contrast microscope
Haemocytometer (Bright-line, Prod. No. Z35, 962-9, Improved Neubauer,
Camlab CCH.AC1)
Pre-labelled flasks

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Fundamental Techniques in Cell Culture

Procedure
1. View cultures using an inverted phase contrast microscope. Cells growing in
exponential growth phase should be bright, round and refractile. Hybridomas may be
very sticky and require a gentle knock to the flask to detach the cells. EBV
transformed cells can grow in very large clumps that are very difficult to count and the
centre of the large clumps may be non-viable.
2. Do not centrifuge to subculture unless the pH of the medium is acidic (phenol red =
yellow) which indicates the cells have overgrown and may not recover. If this is so,
centrifuge at 150g for 5 minutes, re-seed at a slightly higher cell density and add 10-
20% of conditioned medium (supernatant) to the fresh media.
3. Take a small sample of the cells from the cell suspension (100-200µl - Protocol 6 -
Cell Quantification). Calculate cells/ml and re-seed the desired number of cells into
freshly prepared flasks without centrifugation just by diluting the cells.The data sheet
will give the recommended seeding densities
4. Repeat this every 2-3 days.

Key Points
1. If the cell line is a hybridoma or other cell line that produces a substance (e.g.
recombinant protein or growth factor) of interest retain the spent media for analysis.

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12.7 Protocol 6 - Cell Quantification

Suspend cells (as per protocols 3 & 4) Aim

For the majority of manipulations using cell
cultures, such as transfections, cell fusion
Resuspend cells in fresh medium
techniques, cryopreservation and subculture
routines it is necessary to quantify the
Remove 100-200µl of cell suspension number of cells prior to use. Using a
consistent number of cells will maintain
Add Trypan Blue (dilution factor x 2)
optimum growth and also help to standardise
procedures using cell cultures.This in turn
gives results with better reproducibility.
Prepare cover strip
Materials
Fill chamber with cell suspension Media– pre-warmed to appropriate
temperature (refer to the ECACC Cell
Count cells
Line Data Sheet for the correct medium
and temperature)
70% ethanol in water (Prod. No. R8382)
Calculate concentration (refer to procedure) 0.4% Trypan Blue Solution
(Prod. No.T8154)
Trypsin/EDTA (Prod. No.T4049)

Equipment
Personal protective equipment (sterile gloves, laboratory coat, safety visor)
Waterbath set to appropriate temperature
Microbiological safety cabinet at appropriate containment level
Centrifuge
CO2 incubator
Haemocytometer (Bright-line, Prod. No. Z35, 962-9, Improved Neubauer,
Camlab CCH.AC1)
Inverted phase contrast microscope
Pre-labelled flasks

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Fundamental Techniques in Cell Culture

Procedure
1. Bring adherent and semi adherent cells into suspension using trypsin/EDTA
(Prod. No.T4049) as above (Protocol 3 and 4) and resuspend in a volume of fresh
medium at least equivalent to the volume of trypsin. For cells that grow in clumps
centrifuge and resuspend in a small volume and gently pipette to break up clumps.
2. Under sterile conditions remove 100-200µl of cell suspension.
3. Add an equal volume of Trypan Blue (Prod. No.T8154) (dilution factor =2) and mix by
gentle pipetting.
4. Clean the haemocytometer.
5. Moisten the coverslip with water or exhaled breath. Slide the cover-slip over the
chamber back and forth using slight pressure until Newton’s refraction rings appear
(Newton’s refraction rings are seen as rainbow-like rings under the cover-slip).
6. Fill both sides of the chamber (approx 5-10µl) with cell suspension and view under a
light microscope using x20 magnification.
7. Count the number of viable (seen as bright cells) and non-viable cells (stained blue) -
(see below). Ideally >100 cells should be counted in order to increase the accuracy of
the cell count (see notes below). Note the number of squares counted to obtain your
count of >100.
8. Calculate the concentration of viable and non-viable cells and the percentage of viable
cells using the equations below.
Where:
A is the mean number of viable cells counted, i.e. Total viable cells counted
Number of squares
B is the mean number of non-viable cell counted, i.e. Total non-viable cells counted
Number of squares
C is the dilution factor and
D is the correction factor (this is provided by the haemocytometer manufacturer).

Concentration of viable cells (cells/ml) = A x C x D

Concentration of non-viable cells (cells/ml) = B x C x D

Total number of viable cells = concentration of viable cells x volume

Total number of cells = number of viable + number of dead cells

Percentage Viability = No of viable cells x 100

Total No of cells

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 Fundamental Techniques in Cell Culture

Key Points
1. Trypan Blue (Prod. No.T8154) is toxic and is a potential carcinogen. Protective clothing,
gloves and face/eye protection should be worn. Do not breathe the vapour.
2. The central area of the counting chamber is 1mm2.This area is subdivided into 25
smaller squares (1/25mm2). Each of these is surrounded by triple lines and is then
further divided into 16 (1/400mm2).The depth of the chamber is 0.1mm.
3. The correction factor of 104 converts 0.1mm3 to 1ml (0.1mm3 = 1mm2 x 0.1mm)
4. There are several sources of inaccuracy:
The presence of air bubbles and debris in the chamber.
Overfilling the chamber such that sample runs into the channels or the other
chamber
Incomplete filling of the chamber.
Cells not evenly distributed throughout the chamber.
Too few cells to count.This can be overcome by centrifuging the cells, resuspending
in a smaller volume and recounting.
Too many cells to count. This can be overcome by using a higher dilution factor in
trypan blue e.g. 1:10

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sigma-aldrich.com
 Fundamental Techniques in Cell Culture

12.8 Protocol 7 - Cryopreservation of Cell Lines

Assess cells Aim

The protocol below describes the use of
passive methods involving an electric -80°C
Suspend cells in trypsin (protocols 3 & 4)
freezer for the cryopreservation of cell
cultures. ECACC routinely use a
Resuspend cells in fresh media programmable rate controlled freezer (Planer
Series Two) from Planer Products.This is the
Remove and count cells (protocol 6) most reliable and reproducible way to freeze
cells but as the cost of such equipment is
beyond the majority of research laboratories
Centrifuge remaining culture 150g for 5 minutes
the methods below are described in detail. If
large numbers of cell cultures are regularly
Resuspend cells in freeze medium being frozen then a programmable rate
controlled freezer is recommended.
Pipette 1ml aliquots of cells
Materials
Freeze medium (commonly 70% basal
Place ampoules in -80°C freezer overnight
medium, 20% FCS, 10% DMSO
(Prod. No. D2650) or glycerol, check
Transfer to liquid nitrogen storage vessel ECACC data sheets for details).
70% ethanol in water (Prod. No. R8382)
PBS without Ca2+/Mg2+
(Prod. No. D8537)
0.25% trypsin/EDTA in HBSS, without
Ca2+/Mg2+ (Prod. No.T4049)
DMSO (Prod. No. D2650)
Trypsin/EDTA (Prod. No.T4049)
HL60 (Prod. No. 98070106-1v1)

Equipment
Personal protective equipment (sterile gloves, Laboratory coat)
Full-face protective mask/visor
Waterbath set to 37°C
Microbiological safety cabinet at appropriate containment level
Centrifuge
Haemocytometer (Sigma Bright- line Prod. No. Z35, 962-9, Improved Neubauer –
Camlab CCH.AC1)
Pre labelled ampoules/cryotubes
Cell Freezing Device (e.g. Nalgene Mr Frosty Prod. No. C1562)

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Fundamental Techniques in Cell Culture

Procedure
1. View cultures using an inverted microscope to assess the degree of cell density and
confirm the absence of bacterial and fungal contaminants.
2. Bring adherent and semi adherent cells into suspension using trypsin/EDTA (Prod. No.
T4049) as above (Protocol 3 and 4 – Subculture of adherent/attached and semi-adherent
cell lines) and re-suspend in a volume of fresh medium at least equivalent to the volume of
trypsin. Suspension cell lines can be used directly.
3. Remove a small aliquot of cells (100-200µl) and perform a cell count (Protocol 6 –
Cell Quantification). Ideally the cell viability should be in excess of 90% in order to
achieve a good recovery after freezing.
4. Centrifuge the remaining culture at 150g for 5 minutes.
5. Re-suspend cells at a concentration of 2-4x106 cells per ml in freeze medium.
6. Pipette 1ml aliquots of cells into cyroprotective ampoules that have been labelled with
the cell line name, passage number, cell concentration and date.
7. Place ampoules inside a passive freezer e.g. Nalgene Mr Frosty (Prod. No. C1562). Fill
freezer with isopropyl alcohol and place at –80°C overnight.
8. Frozen ampoules should be transferred to the vapour phase of a liquid nitrogen
storage vessel and the locations recorded.

Key Points
1. The most commonly used cryoprotectant is dimethyl sulphoxide (DMSO Prod. No.
D2650), however, this is not appropriate for all cell lines e.g. HL60 (Prod. No.
98070106-1v1)where DMSO is used to induce differentiation. In such cases an alternative
such as glycerol should be used (refer to ECACC data sheet for details of the correct
cryoprotectant).
2. ECACC freeze medium recommended above has been shown to be a good universal
medium for most cell types. Another commonly used freeze medium formulation is: 70%
basal medium, 20% FCS, 10% DMSO but this may not be suitable for all cell types. Check
it works for your cells before using on a regular basis (Prod. No. C6164).
3. It is essential that cultures are healthy and in the log phase of growth.This can be
achieved by using pre-confluent cultures (cultures that are below their maximum cell
density) and by changing the culture medium 24 hours before freezing.
4. The rate of cooling may vary but as a general guide a rate of between –1°C and –3°C
per minute will prove suitable for the majority of cell cultures.
5. An alternative to the Mr Frosty system is the Taylor Wharton passive freezer where
ampoules are held in liquid nitrogen vapour in the neck of Dewar.The system allows the
ampoules to be gradually lowered thereby reducing the temperature. Rate controlled
freezers are also available and are particularly useful if large numbers of ampoules are
frozen on a regular basis.
6. As a last resort if no other devices are available ampoules may be placed inside a well-
insulated box (such as a polystyrene box with sides that are at least 1cm thick) and placed
at –80°C overnight. It is important to ensure that the box remains upright throughout the
freezing process. Once frozen, ampoules should be transferred to the vapour phase of a
liquid nitrogen storage vessel and the locations recorded.
7. If using a freezing method involving a -80°C freezer it is important to have an allocated
section for cell line freezing so that samples are not inadvertently removed. If this happens at a
crucial part of the freezing process then viability and recovery rates will be adversely affected.
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12.9 Protocol 8 - Testing for Bacteria and Fungi

Aim
In cases of gross contamination the naked eye may identify the presence of bacteria and fungi.
However, it is necessary to detect low-level infections by incubation of cell cultures and/or
their products in microbiological broth. Equally these sterility tests can be used to confirm the
absence of bacteria and fungi from the preparation which is important when preparing cell
banks or cell culture products.

Materials
Soyabean Casein Digest (Tryptone Soya Broth,TSB) (15ml aliquots) (Prod. No. S1674)
TSB Powder (Prod. No.T8907)
Fluid Thioglycollate Medium (20ml aliquots) (TGM) (Prod. No. F4797)
Bacillus subtilis NCTC*
Candida albicans NCTC*
Clostridium sporogenes NCTC*

Equipment
Personal protective equipment (latex medical gloves, laboratory coat, safety glasses)
Waterbath set to 37°C
Microbiological safety cabinet at appropriate containment level
Centrifuge
Incubator set at 32°C
Incubator set at 22°C

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Fundamental Techniques in Cell Culture

Figure 12. Flow Scheme for Bacteria and Fungi Testing

Fluid Thioglycollate Medium Tryptone Soya Broth
Inoculate 2 broths with 1.5ml sample Inoculate 2 broths with 1.5ml sample
Inoculate 1 broth with 100cfu of positive control organism Inoculate 1 broth with 100cfu of positive control organism
Leave 2 broths uninoculated as negative controls Leave 2 broths uninoculated as negative controls

Incubate half the broths of each type at 32°C for 14 days and the other half at 22°C for 14 days

Examine the broths for turbidity on day 14

Procedure
1. Culture cell line in the absence of antibiotics for 2 passages prior to testing.
2. Bring attached cells into suspension with the use of a cell scraper. Suspension cell lines may
be tested directly.
3. Inoculate 2 x Thioglycollate Medium (TGM) (Prod. No. F4797) and 2 x Tryptone Soya
broth (TSB) (Prod. No.T8907) with 1.5ml test sample.
4. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml C.albicans (containing 100 colony forming
units, cfu).
5. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml B. subtilis (containing 100cfu).
6. Inoculate 1 TGM with 0.1ml C. sporogenes (containing 100cfu).
7. Leave 2 (TGM) and 2 (TSB) un-inoculated as negative controls.
8. Incubate broths as follows:
For TSB, incubate one broth of each pair at 32°C the other at 22°C for 14 days
For TGM, incubate one broth of each pair at 32°C the other at 22°C for 14 days
For the TGM inoculated with C.sporogenes incubate at 32°C for 14 days
9. Examine Test and Control broths for turbidity after 14 days.
Criteria for a Valid Result
All positive control broths show evidence of bacteria and fungi within 14 days of incubation and
the negative control broths show no evidence of bacteria and fungi.
Criteria for a Positive Result
Test broths containing bacteria or fungi show turbidity.
Criteria for a Negative Result
Test broths should be clear and show no evidence of turbidity.

Notes
1. The positive controls should be handled in a laboratory remote from the main tissue
culture laboratory.
2. Control organisms (Bacillus subtilis, Clostridium sporogenes and Candida albicans) are also
available from the National Collection of Type Cultures (NCTC), UK*.
3. This test procedure should be carried out in a microbiology laboratory away from the cell
culture laboratory.

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 Fundamental Techniques in Cell Culture

12.10 Protocol 9 - Detection of Mycoplasma by Culture

Aim
Detection of mycoplasma by culture is the reference method of detection and has a
theoretical level of detection of 1 colony-forming unit (cfu). However there are some strains
of mycoplasma that are non-cultivable (certain strains of Mycoplasma hyorhinis).The method is
suitable for the detection of mycoplasma in both cell cultures and cell culture reagents and
results are obtained within 4 weeks. Mycoplasma colonies observed on agar plates have a
‘fried egg’ appearance (see figure 14).

Materials
70% ethanol in water (Prod. No. R8382)
Mycoplasma Pig Agar plates (in 5cm petri dishes)
Mycoplasma Pig Agar broths (in 1.8ml aliquots)
M. orale NCTC* 10112
M. pneumoniae NCTC* 10119

Equipment
Personal protective equipment (sterile gloves, laboratory coat, safety visor)
Waterbath set to 37°C
Microbiological safety cabinet at appropriate containment level
CO2 Incubator set at 32°C
Gas Jar (Gallenkamp)
Gas Pak Anaerobic System (Gallenkamp)
Gas Pak Catalyst (Gallenkamp)
Gas Pak Anaerobic Indicator (Gallenkamp)

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Fundamental Techniques in Cell Culture

Figure 13. Flow Scheme for Detection of Mycoplasma by Culture

Label plates and broths

Agar plates Broths

Inoculate 2 plates with 0.1ml test sample Inoculate 2 broths with 0.2ml test sample
Inoculate 2 plates with 100cfu M. Pneumoniae Inoculate 2 broths with 100cfu M. orale
Leave 1 plate uninoculated as negative control Inoculate 2 broths with 100cfu M. pneumoniae
Leave 1 plate uninoculated as negative control

Incubate anaerobically Incubate aerobically

14 days at 37°C 14 days at 37°C

Observe all plates for Subculture 0.1ml of broth onto Agar

Mycoplasma colonies between 3, 7, 10 and 14 days

Incubate anaerobically
14 days at 37°C

Observe all plates for

Mycoplasma colonies

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 Fundamental Techniques in Cell Culture

Procedure
1. Inoculate 2 agar plates with 0.1ml of test sample.
2. Inoculate 1 agar plate with 100cfu M. pneumoniae.
3. Inoculate 1 agar plate with 100cfu M. orale.
4. Leave 1 agar plate un-inoculated as a negative control.
5. Inoculate 1 broth with 0.2 ml of test sample.
6. Inoculate 1 broth with 100cfu M. pneumoniae.
7. Inoculate 1 broth with 100cfu M. orale.
8. Leave 1 agar plate un-inoculated as a negative control.
9. Incubate agar plates anaerobically for 14 days at 37°C using a gas jar with anaerobic gas
pak and catalyst.
10. Incubate broths aerobically for 14 days at 37°C.
11. Between 3 and 7 days and 10 and 14 days incubation, subculture 0.1 ml of test broth onto
an agar plate and incubate plate anaerobically as above.
12. Observe agar plates after 14 days incubation at x300 magnification using an inverted
microscope for the presence of mycoplasma colonies (see Figure 14).

Criteria for a Valid Result

All positive control agar plates and broths show evidence of mycoplasma by typical colony
formation on agar plates and usually a colour change in broths.
All negative control agar plates and broths show no evidence of mycoplasma.

Criteria for a Positive Result

Test agar plates infected with mycoplasma show typical colony formation.

Criteria for a Negative Result

The test agar plates show no evidence of mycoplasma.

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Fundamental Techniques in Cell Culture

Notes
1. Mycoplasma colonies have a typical colony formation commonly described as “fried egg”
(See Figure 8) due to the opaque granular central zone of growth penetrating the agar
surrounded by a flat translucent peripheral zone on the surface. However in many cases
only the control zone will be visible.
2. Positive controls may be included at a concentration to give 100 colony-forming units.
These controls should obviously be handled in a laboratory remote from the main tissue
culture laboratory.
3. Control organisms (M. pneumoniae, and M. orale) are available from National Collection of
Type Cultures (UK).
4. Mycoplasma pneumoniae is a potential pathogen and must be handled in a class II
microbiological safety cabinet operating to ACDP Category 2 Conditions.
5. This test procedure should be carried out in a microbiology laboratory away from the cell
culture laboratory.

Figure 14 - Typical “fried egg colonies” Mycoplasma pneumoniae

(magnification x400)

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12.11 Protocol 10 - Testing for Mycoplasma by Indirect DNA Stain

(Hoechst 33258 stain)
Aim
DNA staining methods such as Hoechst staining techniques are quick with results available within
24 hours which compares favourably with 4 weeks for detection by culture. However the staining
of cultures directly with a DNA stain, results in a much-reduced sensitivity (~106cfu/ml). This may
be improved by co-culturing the test cell line in the presence of an indicator cell line such as Vero
(Prod. No. 84113001-1v1).This enrichment step results in a sensitivity of 104 cfu/ml of culture.This
step also improves sensitivity by increasing the surface area upon which mycoplasma can adhere.
Like detection by culture, DNA staining methods are suitable for the detection of mycoplasma
from cell cultures or cell culture reagents.

Materials
Media– pre-warmed to 37°C (refer to the ECACC Cell Line Data Sheet for the
correct medium)
70% ethanol in water (Prod. No. R8382)
Methanol (Prod. No. 175)
Acetic Acid Glacial (Prod. No.A6283)
Hoechst 33258 stain solution (Prod. No. H6024)
Vero cells (Prod. No. 84113001-1v1)
Mountant (Autoclave 22.2ml 0.2M citric acid with 27.8ml 0.2M disodium
phosphate. Add 50ml glycerol. Filter sterilise and store at 4°C) (Prod. No. M1289)
Mycoplasma hyorhinis NCTC* 10112

Equipment
Personal protective equipment (sterile gloves, laboratory coat, safety visor)
Waterbath set to 37°C
Microbiological safety cabinet of appropriate containment level
Centrifuge
CO2 Incubator set at 37°C
Microscope (uv Epi-Fluorescent.)
35mm plastic tissue culture dishes (Prod. No. C6296)
Multidish 24 well (Prod. No. M9655)
Cell scraper
Microscope slides and 22mm cover slips
Aluminium foil (Prod. No. Z18514-0)

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Fundamental Techniques in Cell Culture

Figure 15.Testing for Mycoplasma by Indirect DNA Stain

Prepare culture dishes

Prepare indicator cells

Incubate at 37°C for 2 to 24 hours

Inoculate 2 dishes of indicator cells with 1ml of sample

Inoculate 2 dishes of indicator cells with 100cfu of positive control organisms
Leave 2 dishes un-inoculated as negative controls

Incubate for 3-5 days at 37°C in 5% CO2

Observe plates for bacterial and/or fungal contamination. Discard if microbially contaminated

Fix samples and allow to dry for 30-120 mins

Add Hoechst stain for 5 mins

Mount

Observe using UV Epi-fluorescence microscope (x1000)

Procedure
1. For each sample and control sterilise 2 cover slips in a hot oven at 180°C for 2 hours or
by immersing in 70% ethanol (Prod. No. R8382) and flaming in a blue Bunsen flame until
the ethanol has evaporated. Also sterilise 2 cover slips to use as a negative control.
2. Place the cover slips in 35mm culture dishes (Prod. No. C6296) (1 per dish).
3. Store until needed.
4. To prepare the Vero (Prod. No. 84113001-1v1) indicator cells add 2x104 cells in 2ml of
antibiotic-free growth medium to each tissue culture dish.
5. Incubate at 37°C in 5% CO2 for 2 – 24 hrs to allow the cells to adhere to the cover slips.
6. Bring attached test cell lines into suspension using a cell scraper. Suspension cell lines may
be tested directly.
7. Remove 1ml of culture supernatant from duplicate dishes and add 1ml of test sample to
each. Inoculate 2 dishes with 100cfu M. hyorhinis and 2 with 100cfu M. orale.
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 Fundamental Techniques in Cell Culture

8. Leave duplicate tissue culture dishes un-inoculated as negative controls.

9. Incubate dishes at 37°C in 5% CO2 for 1-3 days.
10. After 1 day observe one dish from each pair for bacterial or fungal infection. If
contaminated discard immediately. Leave the remaining dish of each pair for a further 2 days.
11. Fix cells to cover-slip by adding a minimum of 2ml of freshly prepared fixative (1:3 glacial
acetic acid: absolute methanol) to the tissue culture dish and leave for 3 to 5 minutes.
12. Decant used fixative to toxic waste bottle.Add another 2ml aliquot of fixative to cover-slip
and leave for a further 3 to 5 min. Decant used fixative to toxic waste.
13. Air dry cover-slip by resting it against the tissue culture dish for 30-120 min.
14. Replace cover-slip in dish and add a minimum of 2ml Hoechst stain (Prod. No. H6024).
Leave for 5 minutes shielded from direct light by aluminium foil (Prod. No. Z18514-0).
15. Decant used and unused stain to toxic waste.
16. Add 1 drop of mountant to a pre-labelled microscope slide and place cover-slip (cell side
down) onto slide.
17. Keep slide covered with aluminium foil (Prod. No. Z18514-0), allowing it to set for at least
15 min at 37°C or for 30 min at room temperature.
18. Observe slide under uv Epi-Fluorescence at x1000.

Criteria for a Valid Result

Negative controls show no evidence of mycoplasma infection
Positive controls show evidence of mycoplasma infection
Vero cells clearly seen as fluorescing nuclei.

Criteria for a Positive Result

Samples infected with mycoplasma are seen as flourescing nuclei plus extra-nuclear
fluorescence of mycoplasma DNA (small cocci or filaments).

Criteria for a Negative Result

Uninfected samples are seen as fluorescing nuclei against a dark background. There should be
no evidence of mycoplasma.

Notes
1. DNA stains such as Hoechst stain (Prod. No. H6024) bind specifically to DNA. In all
cultures cell nuclei will fluoresce. Uncontaminated cultures will show only fluorescent
nuclei whereas mycoplasma positive cultures contain small cocci or filaments which may
or may not be adsorbed onto the cells (see figure 16).
2. Hoechst stain is toxic and should be handled and discarded with care.
3. Culture dishes should be placed in a sealed box or cultured in large petri dishes to reduce
evaporation.
4. Positives should obviously be handled in a laboratory remote from the main tissue culture
laboratory.
5. Control organisms (M. hyorhinis) are available from the National Collection of Type
Cultures (UK).

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Fundamental Techniques in Cell Culture

6. In some instances results may be difficult to interpret for the following reasons:
Bacterial/yeast/fungal contamination
Too much debris in the background
Broken nuclei as cells are all dead
Too few or no live cells
7. Although this procedure recommends the setting up of positive controls, this may not
necessarily be feasible nor desireable in a cell culture facility with limited resources. If
positive controls are to be set up they should be done so in a separate laboratory from
the main tissue culture facility. If this is not possible then positive slides can be purchased
from ECACC. If positive controls are not being used then it is strongly recommended that
you get an independent testing laboratory to periodically test your cell lines.

Figure 16. Hoechst Positive Culture

(magnification x400)

Figure 17. Hoechst Negative Culture

(magnification x400)

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 Fundamental Techniques in Cell Culture

Glossary of Terms
Abbreviations
ACDP Advisory Committee on Dangerous Pathogens
BSA Bovine Serum albumin
BSE Bovine spongiform encepalopathy
BVDV Bovine viral diarrhoea virus
FBS Foetal bovine serum
EC European Community
ECACC European Collection of Cell Cultures
FDA Food and Drug Administration
FCS Foetal Calf Serum
HEPA High Efficiency Particulate Air
MCB Master Cell Bank
NAMAS National Accreditation of Measurement and Sampling
NCTC National Collection of Type Cultures
PPE Personal Protective Equipment
USDA United States Department of Agriculture
WCB Working Cell Banks

Chemical Symbols
Ca2+ calcium ion
CO2 carbon dioxide
DMSO dimethyl sulphoxide
Mg2+ magnesium ion
O2 oxygen
PBS phosphate buffered saline
TGM Thioglycollate medium

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Fundamental Techniques in Cell Culture

Notes

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 Fundamental Techniques in Cell Culture

Who to Contact
For further details on any of these culture systems please contact ECACC.

ECACC Contact Information

ECACC
CAMR
Salisbury
Wiltshire, SP4 0JG, UK

Tel +44 (0) 1980 612512

Fax +44 (0) 1980 611315

E-Mail [email protected]
Web www.ecacc.org.uk
The ECACC Cell Line Collections contain over 35,000 Cell Lines, delivered to any
destination in the world.

40 Species
50 Tissue Types
350 HLA Defined
450 Hybridomas
500 Human Control
750 Human Genetic Disorders

Contact us for your FREE Cell Line and Services Catalogue, available as Printed Hardcopy and
CD-ROM, by completing the business reply card at the back of the book.

Sigma-Aldrich and ECACC would like to thank Dr Alison Stacey, Cambridge, UK and
Labcaire, Clevedon, UK for their help in compiling this laboratory handbook.

Front Cover Image:Artist’s impression of a nuclear pore. © Moyra Campbell 2001.

[email protected].
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