Introduction to Animal Cell Culture
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The document provides a comprehensive overview of animal cell culture, including its definition, essential equipment, media types, and culture methods. It outlines the necessary safety protocols, storage techniques for cells, and the importance of cell characterization and the various applications of cell culture in research and industry. Additionally, it includes multiple-choice questions to test understanding of the material.
Introduction to Animal Cell Culture
- 1. Animal cell science & Technology 1. Introduction to Cell Culture Shailendra Singh Shera, Ph.D
- 2. 1. Introduction to cell culture Outline: 1. What is cell culture? 2. Cell culture equipment 3. Media and supplements for cell culture 4. Cell culture environment 5. Cell culture methods 6. Morphology of cells 7. Storage 8. Cell culture safety 9. Applications 10. MCQs for practice
- 3. What is cell culture? The observation by Jolly ( 1903) that animal cell can be cultivated outside animal body in an artificial environment is a significant discovery with respect to cell culture. The The actual beginning of animal cell culture and tissue culture was made by Harrison (1907) and later by Carrel (1912). For cultivation of animal cells in laboratory cells are firstly removed from the body through excision and disaggregated using mechanical or enzymatic meddum. The disaggregated tissues are then cultivated in suitable medium and favorable growth media. Definition: “Animal cell culture is defined as the cultivation and maintenance of animal cells in an artificial and controlled environment.”
- 4. Cell culture equipments Animal cell culture is a sophisticated techniques and requires high level of technical skills. Given the complexity involved in animal cells, culturing requires some advanced and sophisticated equipments. Further, the choice of environment depends upon the types of work performed--- routine or high value research or teaching. But, all cell culture laboratory should have following basic equipment to start with: Auto clave Deep freezer ( -80° C) Cell culture micro-well tubes Cell counter Serological Pippetors Cell culture grade tubes and other labware Cell culture hood / Biosafety cabinet Media, sera, cell media additives Sodium Hypochlorite for routine disinfection CO2 incubator Centrifuges pH meter Gloves Inverted and Fluorescence microscope Cryo preservation facility Chemical balance Liquid Nitrogen Freezer ( -20° C) Pasteur pippetes
- 5. System of cell culture •Various methods are available for culturing animal or mammalian cells in artificial environment. •Cells can be either cultured as monolayer or suspension culture. Monolayer culture are best suited for growing anchorage dependent cells while suspension culture are best suited for anchorage independent cells. Various methods of culturing cells are: •Coverslip or slide culture method ( single coverslip with plasma clot and double coverslip method) •Hanging drop methods •Flask culture methods ( Used for establishing strains from fresh explant of tissues)
- 6. Media and supplements of cell culture The purpose of media is to provide favorable condition for growth, expansion and survival of cultivated cell. The media provides nutrient and energy, maintains required pH ( 7.2-7.4), osmotic factors for cellular growth and division. Common components of animal cell culture media includes: amino acids, vitamins, carbohydrate, Inorganic salts, buffering system, antibiotics. Animal cell culture media can be divided into following category: i. Natural Media ii. Synthetic media (i) Natural media: Natural media consist solely of naturally occurring biological fluids e.g. Plasma clot, serum, aminiotic fluid (ii) Synthetic media: Artificial or synthetic media are prepared by adding nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors. Eg. Ham's F-10, RPMI-1640, DMEM ( Dulbecco Modified Eagle Medium), MEM (Minimum Essential Media).
- 7. Synthetic media Serum containing media Serum Free media Chemically defined media Protein free media Balanced salt solution Balance salt solution is used to keep cell alive only. Types of synthetic media
- 8. Media Supplements Media Supplements refers to thing, molecules or substance added in addition to enhance the quality of media. Supplements helps animal cells to produce proteins and biomolecules required in high Concentration for growth, maintenance and survival. The advantages of supplements are: i. Helps in customization of growth condition of cells ii. Improves cell viability and growth iii. Keeps cell healthier longer Some examples of media supplements are: •Amino Acid Solution * Fibroblast growth factors •Bovine Serum Albumin * Cytokines •Transferrin •Insulin •Lipid supplements •Sodium pyruvate •Yeast Solution
- 9. Cell culture environment The invitro culture of animal cells outside body requires exactly same condition as available inside body. The invitro culture has required tools to maintain and control all physiochemical and physiological factors such as nutrient requirements, pH, temperature , buffering and contamination. Media Contains nutrients, lipids, vitamins, antibiotics, trace elements, pH indicators, hormones pH 7.2-7.4 ; should be around neutral Temperature Depends upon the animal cells of hosts Mammalian cells/ human : 37°C Insect cells: 27°C-30°C CO2 Required for buffering 5-10 % is common; Very minute amount of O2 is also required Phenolphthalein red •pH indicator; Changes color when pH of medium changed The change of pH may be due to accumulation of toxic growth or •Due to bacterial, yeast and fungal contamination Antibiotic •Used to porevent contamination of cells in culture •Frequent use in cell culture not recommended •Penicillin-Streptomycin solution = 50-100 µg/ml •amphotericin B = 2.5 µg/ml
- 10. Morphology & Characterization Cells in culture attains basically three types of morphology . The categorization of cells are based on their shape and appearance under microscope. Fibroblastic cells Epithelial cells Lymphoblast Adherent cells Adherent cells Suspension Spindle shaped Polygonal shaped and appearance Round, spherical shaped Adapted from: https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/introduction-to-cell- culture.html Fibroblast Epithelial Lymphoblast
- 11. Characterization Once the cells are cultured in vitro, the cells should be identified for their known properties and behavior. Characterization of cell line is the first indispensible step after each cell line is generated for determining its functionality, authenticity, contamination, origin etc According to current regulation, following aspects of cell line characterization should be considered •Origin and history of cell line •Cellular morphology and growth characteristics •Purity of cell lines •Tumorigenicity & oncogenecity •Stability •It guarantees against the advertent contamination of cell line during cell culturing work
- 12. Methods of cell characterization Various methods of cell line for identification of and cross contaminations and misidentifications are outlined below: •Morphological characteristics •Karyotyping ( Chromosome analysis) •Viral susceptibility •Specific antigenecity •DNA fingerprinting •MHC ( Major Histocompatibility Complex) •Flow Cytometry •Confoccal Microscopy •Staining ( Giemsa Staining)
- 13. Storage •Storage facility in animal tissue laboratory is of utmost importance. Storage is required for safe keeping of media, reagents, cell lines, culture dishes and preserving cells and cell lines . •Proper storage saves time, expenses and makes working with culture stress less and easy. •Labwares, chemicals, reagents and spares can be safely kept in properly labelled dedicated cupboard. •Biological and other labile substances can be stored in refrigerator ( 4° and -20°C). •Cell and cell lines should be cryo-preserved and stored in liquid N2 . Cryopreservation: Surplus cells and cells marked for future use are treated with suitable cryoprotectant ( Dimethyl sulphoxide, DMSO or glyccerol ) and stored at temperatures below –130°C (cryopreservation) until they are needed Usually for long term storage , cells are stored in liquid N2 at -196°C.
- 14. Cell culture safety •Ensuring safety of yourself, your colleagues, family and society at large is of immense importance while working in cell culture laboratory. People working in cell culture laboratory are continuously at risk of exposure to toxic, mutagenic and infectious chemicals and biological agents respectively. •While dealing with animal and human tissues, extra precautions are needed as they contain viruses and other dangerous biological agent. •By following good laboratory practices, we can avoid healthy hazard while working Some of the many guidelines that should be followed are outlined below: Wear safety clothes (gloves, closed shoes, lab coat). If working with highly infectious and contagious virus such as Coronvirus-2 ,wear personal protective equipments. No or low aerosol creation. Decontaminate all surfaces before and after the experiment Avoid using sharp objects. Wash hands when entering and before leaving the laboratory. Work in accordance with the facility guidelines.
- 15. Applications •Animal cell culture finds applications in basic, applied research and commercial and Industrial scale. •In basic research cells in culture are used to study basics about intracellular activity such as DNA transcription, protein synthesis, cell cycle etc.; Intracellular flux; Genomic ; Proteomics, cell-cell and cell-material interactions. •In applied research , cell cultures are used for production of high value therapeutic compounds such as mono clonal antibodies, vaccines etc; Toxicology studies; Immunology, pharmacology and Tissue engineering. •As a model system for diseases and drug screening. •Helps to study normal cell homeostasis, cell biochemistry, metabolism, • mutagenesis, diseases, and compound effects. Cell culture are preferred for high value products due to batch to batch consistency and reproducibility.
- 16. Testing your understanding ( MCQs)* 1. Animal cells grown in culture generally retain their functions even though they are growing in vitro. ( True / False) 2. When we grow cells in culture a. we can only grow one cell type at a time b. cells with a growth advantage can outgrow a cell culture given enough time c. cells supply other cells everything they need to grow d. serum must always be heat-inactivated 3. L-glutamine is an important amino acid because a. It is an amino acid b. Cells get much of their energy from the catabolism of L-glutamine c. It is not important since it is made by some cells d. It is an amine donor 4.When growing animal cells it is important to a. use continuous vigilance to guard against contamination b. wear sturdy work shoes c. have lots of computer memory d. drink plenty of water
- 17. 5. Antibiotics should be a. always be used b. used, only when necessary c. never be used d. only during a cross-contamination event 6. Which benchmark cell line verification is not recommended by ATCC a. STR analysis b. growth curve analysis c. DNA sequencing d. mycoplasma detection 7. Characterization of cell line is necessary for a. Identification of species b. Detection of contamination c. To check the stability of cells in culture d. All the above 8. Cell culture are used to produce high value therapeutic product due to a. Reproducibility b. Batch to batch consistency c. Both (a) and (b) d. Only (a) 9. Cross-contamination is a. not to much of a problem and never happens b. the result of working with multiple cells at one time c. can be controlled with antibiotics d. cannot really happen if you follow good protocols *Questions adapted from : Practice and Learn Animal cell Science and Technology: Multiple choice question for learning. Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
- 18. 1. https://www.labmanager.com/laboratory-technology/cell-line-characterization-methods-reviewed- 20877 2. https://www.thermofisher.com/in/en/home/life-science/cell-culture/mammalian-cell-culture/media- supplements.html 3. https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/introduction-to-cell- culture.html 4. https://www.ptglab.com/support/cell-culture-protocol/introduction-to-cell-culture/ References & Further reading Further readings 1. Watson, J.D., Gilman, M., Witowski J.and Zoller, M. Recombinant DNA, 2nd ed., Scientific American Books, 1983 2. Glick, B.R. and Pasternack, J.J. Molecular Biotechnology, 3rd ed., ASM Press, 2003 3. Davis J.M. Basic Cell Culture: A Practical Approach, IRL Press, 1998 4. Freshney R.I. Animal Cell Culture a practical approach, 1987 5. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning. Author: Shailendra Singh Shera . Publisher: Amazon Kindle.