JADWAL S2 MINAT HISTOLOGI DAN BIOLOGI SEL T.A.

2019/2020

Koord
Kegiatan (SKS) inator Februari Maret April Mei Juni Juli Agst Oktober Okt-Des
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Seminar Lokakary
Lokakarya PAAI a menulis
kapasitas Temu dalam publikasi
komunikasi ilmiah rangka Libur Idul ilmiah
Praktikum akademik alumni & purnatug Fitri (21 mahasis
TDRB (27-31 dan saintis mahasiswa as DR. Mei-10 wa minat
Lain-lain Jan) (7-8 Feb.) (27-28 Feb) Herwiyati Juni) Histologi
Kultur Sel dan
Sitogenetika
(1/1) DKP 03-Feb 28-Feb
Mikroteknik (2/1) RS 03-Feb 16-Mar
Histologi lanjut
(2/0) RS 16-Mar 20-Apr
Imunologi Lanjut
(2/1)
JF 16-Mar 20-Apr
Biologi Molekuler
lanjut (2/1)
DP ### ####
Biologi Tumor
(2/0) DP ### ####
Neurobiologi
(2/0) RS ### ##
Seminar 1 (1/0) JF 3 Agts 31 Agst
Seminar 2 (1/0) JF Okt-Des
 NILAI ABSOLUT
Nilai Rentang skor Nilai Rentang skor
A 75 - 100 C- 52,5-54,99
A- 72,5–74,99 C/D 50-52,49
A/B 70-72,49 D+ 47,5-49,99
B+ 67,5-69,99 D 45-47,49
B 65-67,49 E <45
B- 62,5-64,99
B/C 60-62,49
C+ 57,5-59,99
C 55-57,49
BASIC CELL CULTURE
Jajah Fachiroh

Department Histology and Cell Biology

Faculty of Medicine UGM
[email protected]
CONTENTS
1. Introduction
2. 2D and 3D culture
3. Cell culturing: types and how to keep it going
4. Requirements for culture lab
5. Contamination and toxication
Introduction
 Cellculture is the process by which prokaryotic, eukaryotic or plant cells
are grown under controlled conditions (in vitro)

This lecture: refers to the culturing of cells derived from animal cells

 Roux in 1885 for the first time maintained embryonic chick cells in a salt
solution for several days

 Cell culture was first successfully undertaken by Ross Harrison in 1907

(cultured frog neuroblast in lymph medium)
 Major developments
in cell culture technology

1. the use of antibiotics which inhibits the growth of contaminants.

2. the use of trypsin to remove adherent cells to subculture further
from the culture vessel
3. the use of chemically defined culture medium.
 Areas where cell culture technology
is currently playing a major role

Model systems
Studying basic cell biology, interactions between disease causing agents and
cells, effects of drugs on cells, process and triggering of aging & nutritional
studies

Toxicity testing
Study the effects of new drugs

Cancer research
Study the function of various chemicals, virus & radiation to convert normal
cultured cells to cancerous cells
 Virology
Cultivation of virus for vaccine production, also used to study there infectious cycle.
Genetic Engineering
Production of commercial proteins, large scale production of viruses for use in vaccine
production e.g. polio, rabies, chicken pox, hepatitis B & measles
Gene therapy
Cells having a functional gene can be replaced to cells which are having non-functional
gene

Whatever your aim in using cell culture as part of research method, appropriate type of cells
and cultures are substantial
Types of cell and culturing cells
 Common cell lines
Human cell lines
• HeLa (Henrietta Lacks); first human cancer cell line
• MCF-7 breast cancer
• HL 60 Leukemia
• HEK-293 Human embryonic kidney

• Primate cell lines

• Vero African green monkey kidney epithelial cells
• Cos-7 African green monkey kidney cells
And others such as CHO from hamster, sf9 & sf21 from insect
cells
List of breast cancer cell lines (example)
 Primary cell line
Cells when surgically or enzymatically removed from an organism and placed in
suitable culture environment will attach and grow

(Novacovic & Presney, 2006)

 Primary cell line

1. Primary cells have a finite life span

2. Primary culture contains a very heterogeneous population of cells
3. Sub culturing of primary cells leads to the generation of cell lines (one type of cell)
4. Cell lines have limited life span, they passage several times before they become senescent
5. Lineage of cells originating from the primary culture is called a cell strain
 Continuous cell line (i.e. myeloma cell line)
• Cell lines which either occur spontaneously (e.g. HeLa cells) or induced virally (i.e. infected by
EBV) or chemically transformed, and able to grow indefinitely (under appropriate condition)
• Characteristics :
1. Smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio
2. Fast growth and have aneuploid chromosome number
3. Reduced serum and anchorage dependence and grow more in suspension conditions
4. Ability to grow up to higher cell density
5. Different in phenotypes from donor tissue
6. Stop expressing tissue specific genes
Types of Culture
Different culture methods
can be used in primary
and or continuous cell line
Organ culture is essentially a technique for
studying the behaviour of integrated tissues
by using whole or section of organ

• Maintain histology of the tissue

• Optimized gas exchange (avoid central necrosis) and
minimize cell migration
• Does not last long (~3wks), does not propagated
 • A piece of tissue, maintained the
original structure
• Planted on agar
• Short lived (2-3 weeks)

Primary explant outgrowth

(Sango et al., Current Diabetes Reviews, 2006, Vol.2, No.2, pp.169-183)
The most common used system
(2D system)

Cells do not attached to the

neighboring cells
 On the basis of morphology (shape & appearance) or on their functional characteristics.
They are divided into three:
2 Dimensional culture

1. Lymphoblast like- cells do not attach remain in suspension

with a spherical shape

2. Fibroblast like- cells attached to an substrate appears

elongated and bipolar
3. Epithelial like-attached to a substrate and appears
flattened and polygonal in shape

2,3. Attached = Adherent/ monolayer cells

Suspension and adherent cells type treated differently during culture

3 Dimensional culture
to provide structure for the cells
3 Dimensional culture
(Ankrum et al., 2017)
3D culture enables the formation
of natural-like cellular growth
Polarization is required to ensure
biological correctness
3D culture enables the formation
of natural-like cellular growth
(Langhans, 2018)
(Huroau-Vechot et al., 2018)
Figure 1. Common 3D techniques used for the creation of spheroids. (A) Hanging drop
methods. Cells are deposited on a petri dish lid, which is flipped over a petri dish
containing PBS; (B) ultra low attachment plates. Cells are seeded in an ultra-low
attachment plate which prevents them from adhering; (C) suspension cultures. Cells are
placed in spinner flasks (left) or bioreactors (right) and put under gravitational forces; (D)
scaffold based-models. Cells are either seeded on the top of a hydrogel (left) or
embedded in it (right); (E) magnetic levitation. Cells are magnetized in culture and
attracted to a magnet located on the top
Real-time observation in 3D culture
Challenges in 3D culture:
1. large cellular aggregates require a careful control of the gas exchange as well as the
diffusion of soluble nutrients and chemical agents
2. Biochemical analysis is complicated by the lower cell density and the additional steps
required to separate the cells from the matrix
(available ECM gels are extracted from animals or cultured cells
→ quality control is difficult.
→ non-animal based ECM are available now (adjustable components, pH, etc
https://www.raft3dcellculture.com
Basic requirements for cell culture
Basic equipments

1. Laminar cabinet-
with UV and air-filter

2. Incubator
25-30 C (for insect), 37 C (for mammalian cells)—cut out ~ 38.5 C
with CO2 regulator 2-5%
95% air at 99% relative humidity (use water tray)
 3. inverted microscope
microscope with its light source and condenser on the
top, above the stage pointing down, while the objectives
and turret are below the stage pointing up. (invented in
1850 by J. Lawrence Smith)

Inverted microscopes are useful for observing living cells

or organisms at the bottom of a large container (e.g. a
tissue culture flask) under more natural conditions than
on a glass slide

5. plasticwares
4. refrigerator
Culture media

• Choice of media depends on the type of cell being cultured

• Commonly used Medium are GMEM, EMEM,DMEM etc.
• Media is supplemented with antibiotics viz. penicillin,
streptomycin etc.
• Prepared media is filtered and incubated at 4 C

Keep the media sterile

 Basic aseptic conditions

Keep out contaminants!!!!

Keep the cell in the culture happy
• If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen
• Swab all bottle tops & necks with 70% ethanol
• Flame all bottle necks & pipette by passing very quickly through the hottest part of the
flame
• Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the
little finger
• Work either left to right or vice versa, so that all material goes to one side, once finished
• Clean up spills immediately & always leave the work place neat & tidy
 Safety aspect in cell culture
• Possibly keep cultures free of antibiotics in order to be able to recognize the
contamination (kinda difficult in Indonesia)
• Never use the same media bottle for different cell lines. If caps are dropped or bottles
touched unconditionally touched, replace them with new ones
• Necks of glass bottles prefer heat at least for 60” at a temperature of > 100 C
• Switch on the laminar flow cabinet 20’ prior to start working
• Cell cultures which are frequently used should be subcultured & stored as duplicate
strains
 Other key factors for success culturing:
make the cells happy

• Use actively growing cells that are in their log phase of growth, which are 80-90% viable
• A lower concentration of 10E4cells/ml to initiate subculture of rapidly growing cells & a
higher concentration of 10E5cells/ml for slowing growing cells
• Handle the cells gently. Do not centrifuge cells at high speed or roughly re-suspend the cells
• Feeding & sub culturing the cells at more frequent intervals then used with serum containing
conditions may be necessary
 Cell viability
• Cell viability is determined by staining
the cells with trypan blue
• trypan blue dye is permeable
to dead cells, but not to viable cells

• Stain the cells with trypan dye and load to

haemocytometer and calculate % of viable cells
- % of viable cells= # of unstained cells x 10.0000
total # of cells
 Subculturing (Passage)
• Subculturing requires to keep the cells at their growing
phase.
• When to subculture??
1. Cell density (80-90% confluency); adherent cell >
suspension cells
2. Drop in pH (may relates also with cell density); marked by
change color of medium
→ Regular subculturing maintain “regular cell growth;
deviation may means disturbance of culture

For monolayer culture, enzyme such as trypsin,

dipase, collagenase in combination with EDTA
breaks the cellular glue that attached the cells to
the surface
 Adherent cells

• Cells which are anchorage dependent

• Cells are washed with PBS (free of ca & mg ) solution.
• Add enough trypsin/EDTA to cover the monolayer
• Incubate the plate at 37 C for 1-2 min.
• Tap the vessel from the sides to dislodge the cells
• Add complete medium to dissociate and dislodge the cells
• with the help of pipette which are remained to be adherent
• Add complete medium depends on the subculture
 Suspension cells
• Easier to passage as no need to detach them
• As the suspension cells reach to confluency
→ Asceptically remove 1/3rd of medium
→Replaced with the same amount of pre-warmed medium
(“refreshing culture ”)
 Cell toxication
and biological contamination
 Cell toxicity
• Cytotoxicity causes inhibition of cell growth
• Observed effect on the morphological alteration in the cell layer or cell shape
• Characteristics of abnormal morphology is the giant cells, multinucleated cells, a
granular bumpy appearance, vacuoles in the cytoplasm or nucleus
• Cytotoxicity is determined by substituting materials such as medium, serum,
supplements flasks etc. at a time
Contaminant’s of cell culture
Cell culture contaminants of two types:
• Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or
traces of disinfectants that are invisible
• Biological-cause visible effects on the culture they are mycoplasma, yeast,
bacteria or fungus or also from cross-contamination of cells from other cell lines
Effects of Biological Contamination’s

• They competes for nutrients with host cells

• Secreted acidic or alkaline by-products ceases the growth of the host cells
• They also produces H2O2 which is directly toxic to cells
 Detection of contaminants
• Bacterial contamination: turbid culture media change in growth rates, abnormally high
pH, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolization,
inclusion bodies and cell lysis
 Detection of contaminants
Yeast, bacteria & fungi usually shows visible effect on the culture
(changes in medium turbidity or pH)

http://www.unclineberger.org/tissueculture/contamin
ant
Detection of contaminants
Mycoplasma contamination does not cause cell death, but
slowing down the culture growth.
Mycoplasma cant be observed under microscope.
Routine mycoplasma test should be done, by direct DNA staining
with intercalating fluorescent substances e.g. Hoechst 33258, or
by PCR amplification of mycoplasmal RNA

Fixed cells culture

(-) (+)

Example of fluorescence method for detecting mycoplasma

contamination

http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/biological-contamination/mycoplasma-
contamination.html
 The best and the oldest way to eliminate contamination is to
discard the infected cell lines directly

However, antibiotics, antimycotic can be used, but needs concentration test prior (also
checked that all equipments and materials are contaminats-free)
→ This doesn’t eliminates mycoplasma contamination.
What to do when your experiment is over and you have
your culture growing?

Store your cells, using cryopreservation

(concentrated cells in freezing medium, and stored
in liquid nitrogen

And when needed, the ampule can be thawed, and

recultured
 Freezing cells for storage
• Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration
• Dislodge the cells by trypsin treatment
• Dilute the cells with growth medium
• Transfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 mts at RT
and remove the growth medium by aspiration
• Resuspend the cells in 1-2ml of freezing medium (contains DMSO)
• Transfer the cells to cryovials, incubate the cryovials at -80 C overnight
• Next day transfer the cryovials to Liquid nitrogen
 Working with cryopreserved cells
• Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously
until medium is thawed
• Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with 70%
ethanol and discard the supernatant
• Resuspend the cell pellet in 1 ml of complete medium with 20% FBS and
transfer to properly labeled culture plate containing the appropriate amount of
medium
• Check the cultures after 24 hrs to ensure that they are attached to the plate
• Change medium as the colour changes, use 20% FBS until the cells are
established
 SUMMARY

1. Cell culture is a way to grow cells in vitro

2. Primary culture is contains same type of cell derived from original
organ
3. Two types of cells for culture : suspension and confluent, determine
way to keep them
4. Keep the cells happy determine the long lasting culture. Happy
means adequate environmental condition (medium, pH, temp, etc,
no contaminants)
5. Cells can be preserved by freezing them under certain medium