Cell culture techniques

Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture

1. CELL CULTURE TECHNIQUES Presented by S. Nivedhitha M.Pharmacy 1 st year Pharmacology Madras Medical College

2. INTRODUCTION Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been already established. Cell culture was first successfully undertaken by Ross Harrison in 1907 ---- Growth of frog embryo nerve fiber invitro. Roux in 1885 for the first time maintained embryonic chick cells in a cell culture. 2

3. Major developments in cell culture technology First development was the use of antibiotics which inhibits the growth of contaminants. Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel. Third was the use of chemically defined culture medium. 3

4. Terminologies Organ culture: Culture of native tissue (undisaggregated tissue) that retains most of the in vivo histological features. – whole organs/ intact organ fragments. Cell culture: Culture of dispersed (disaggregated) cells obtained from original tissue. Histotypic culture: Culturing of cells for their reaggregation to form a tissue-like structure. Primary culture: Culture produced by the freshly isolated cells taken from an organism. Cells- heterogeneous, slow growing & represent the tissue of origin with regard to their properties 4

5. Cell line: The subculturing of the primary culture gives rise to cell lines. Passage/subculture: The process of transfer of cells from one culture vessel to another culture vessel (fresh media). Passage number: No. of times a culture has been subcultured. Finite cell lines: The cell lines with limited /finite culture life span. Continuous cell lines: Indefinite growth of cells in the subsequent subcultures. 5

6. TISSUE CULTURE LABORATORY DESIGN 6

7. Equipments used in cell culture lab The specific requirements of a cell culture lab depends mainly on the type of research conducted. All culture lab should be free from pathogenic microbes (aseptic) and share some basic equipments. Cell culture hood -biosafety cabinet/laminar flow hood Horizontal LAF : parallel to work surface, not recirculating. Vertical LAF : From top of cabinet & drawn through work surface, recirculated. Fitted with HEPA filters and routine maintenance checks –every 3-6 months. 7

8. US centers for disease control and prevention (CDC) classifies BSC into 3 classes in two ways : The level of – personnel & environmental protection provided - product protection provided Class I 8

9. CLASS II TYPES: A1, A2, B1, B2, C1 –based on diff in minimum inflow velocities & exhaust systems. 90% all BSC installed are type A2 cabinets . Type A2 Type B2 9

10. Class III 10

11. Incubator : Temp 37 C , CO 2 2-5%, 95% Air at 99% relative humidity. Refrigerators : Liquid media at 4 c, enzymes (trypsin) & media components (glutamine, serum) at -20 c,-80 c Microscopes : Inverted & dissecting microscope. 11

12. Centrifuge : Separate the particles from a solution according to their sizes, shapes, density, viscosity of the medium by using the principle of centrifugal force. Liquid N 2 container: long term storage with low liq. N 2 evaporation. Vapour phase and liquid phase. Haemocytometer : To estimate the cell number and viability. 12

13. Water bath : Temp 5-100 c. For thawing of cells taken out from the freezer or liq. N 2 tank to prevent any mechanical damage to the cells . Flow cytometry : Analyze the characteristics of the cells. Culture vessels : Multiwell plates, petri dishes, flasks, stirrer bottles. Anchorage dependent cells attach to the surface of vessel & grow. While, some cells undergo transformation and become anchorage independent. 13

14. Culture media Provides the required nutrients and components for the cells to grow and divide. Choice of media depends on– type of cells to be cultured and –purpose of culture (growth, differentiation, production of desired products). Natural media- media components are from the natural origin that provide sufficient nutritional support for proliferation of animal cells. E.g. clots, biological fluids, tissue extracts. Artificial media: They are chemically synthesized. Serum containing media Serum free media Chemically defined media Protein free media 14

15. Types of culture media 15

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18. Chemically defined media These media contain contamination-free ultra pure inorganic and organic ingredients, and may also contain pure protein additives, like growth factors. All the chemical components are known. Protein free media Do not contain any protein and only contain non-protein constituents. Formulations like MEM, RPMI-1640 are protein free and protein supplement is provided when required. 18

19. Properties and requirements of media 19 • pH • 7.4 • indicator-phenol red.

20. 20 • Buffers (Bicarbonate and HEPES) • Bicarbonate buffered media requires CO 2 atmosphere • HEPES strong chemical buffer range pH 7.2 – 7.6 • Keto acids (oxalacetate and pyruvate) • Intermediate in glycolysis/krebs cycle • Keto acids added to the media as additional energy source • Maintain maximum cell metabolism • Carbohydrates • Energy source • Glucose and galactose • Low (1g/L) and high(4.5 g/L) concentrations of sugars used in basal media.

21. • Vitamins • Precursors of numerous co-factors • Vit B necessary for cell growth and proliferation • Commonly found- riboflavin, thiamine, biotin • Trace elements • Zinc, copper, selenium & tricarboxylic acid intermediates. • Supplements • L-glutamine • Non-essential amino acids(NEAA) • Growth factors and hormones 21

22. Morphology of Cells in culture Fibroblastic(or fibroblast-like) Epithelial-like cells Lymphoblast-like cells Neuronal cells 22

23. Types of cell culture Cell culture is classified into three: Primary cell culture Adherent cell culture Suspension cell culture Secondary cell culture Cell line Finite cell line Continuous cell line 23

24. Culturing cells in the laboratory 24

25. Primary cultures 25

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29. SUB CULTURING or PASSAGING CELLS Check confluency of cells Remove spent medium Wash with PBS Incubate with trypsin/EDTA Resuspend in serum containing media Transfer to culture flask Why passage cells? To maintain cells in culture (i.e. don’t over grow) To increase cell number for experiments / storage 29

30. Standard growth curve of cells 30

31. Adherent cell culture • Those cell which attach to the surface of the culture vessel/flask. • Forms a monolayer • They have to detached from the surface before they get sub cultured. • Growth limited to the surface area Suspension culture • Those cell doesn’t get attached to the surface of culture flask. • They are free floating. • Cells in blood stream. • Growth is limited to concentration of cells 31

32. Subculture of monolayer 32

33. SECONDARY CELL CULTURE Derived from a primary cell culture. Isolated by selection or cloning. Becoming a more homogeneous cell population. Finite life span in vitro. Retain differentiated phenotype. Mainly anchorage dependant. Exhibit contact inhibition. 33

34. CELL LINE Finite cell line Limited life span Go through a limited number of cell generation. Properties: • Contact inhibition • Density limitation • Anchorage dependence Less growth rate Doubling time (24-92 hrs) Continuous cell line Grow indefinitely Grows either in monolayer or in suspension Properties: • Absence contact inhibition • Absence anchorage dependence High growth rate Doubling time(12-24hrs) 34

35. 35 COMMON CELL LINES Vero, Cos 7 – African green monkey kidney cells CHO – Hamster Sf 9, Sf 21 – Insect cells

36. CRYOPRESEVATION OF CELLS Passage cells Resuspend cells in serum containing media Centrifuge & aspirate supernatant Resuspend cells in 10% DMSO in FCS Transfer to cryovial freeze at -80 C Transfer to liquid Nitrogen tank Why cryopreserve cells? Reduced risk of microbial contamination Reduced risk of cross contamination with other cells Reduced risk of genetic drift and morphological changes Research conducted using cells at consistent low passage 36

37. 37 Cryoprotectants Cell membrane permeating (eg: dimethyl sulfoxide(DMSO), glycerol, 1,2 propanediol) Cell membrane non –permeating (eg: 2-metyl-2,4-pentanediol, polymers –polyvinyl pyrrolidone, hydroxyethyl starch)

38. Freezing methods Slow freezing methods- cooling rate 1 c/min,< 1.0M CPA Vitrification – Aq. State to glassy state in liqN 2 ,high conc. CPA(40-60% weight/volume) Application of cryopreservation Cryopreservation of cell or organs Cryosurgery Biochemistry and molecular biology Food sciences Ecology and plant physiology Medical applications such as, blood transfusion, bone marrow transplantation, artificial insemination and IVF 38

39. Advantages of cell culture Controlled physiological environment (pH, temp., osmotic pressure, o 2 ) Regulation of physiological conditions Homogeneity of cell types (achieved through serial passages) Easy to characterize cells for cytological and immunological studies Storage of cultured cells for several years Legal, moral and ethical questions of animal experimentation are avoided. Limitations Expertise is needed Expensive – 10 times higher than direct use of animal tissue Unstable aneuploid chromosome constitution Control of environmental factors not easy. 39

40. APPLICATION OF CELL CULTURE Excellent model system for studying • N ormal physiology, cell biology and biochemistry of cells • The effects of drugs, radiation and toxic compounds on the cell • The interaction between disease causing agents and cells ( mutagenesis, carcinogenesis) • N utritional studies • T he process and triggers of aging. Toxicity testing Genetic engineering and gene therapy Studies dealing with genetics (eg: gene transfer, genetic analysis, transformation, immortalization, senescence) Cell-based manufacturing Laboratory production of medical and pharmaceutical compounds for wide range of applications. 40

41. vaccines, insulin, interferon, hormones, other therapeutic protein Genetic counselling (amniocentesis) Virology •Cultivation of virus for vaccine production, also used to study the infectious cycle . Cancer research •Study the functions of various chemicals, virus and radiation to convert normal cultured cells to cancerous cells. Drug screening and development 41

42. References Biotechnology by Satyanarayanan. Butler. M (2005). Animal cell culture: recent achievements and perspectives in the production of biopharmaceuticals. Applied microbiology and biotechnology,68(3),283-291 https://www.slideshare.net/mobile/meghabedekar/principle-of-cell-culture https://www.ncbi.nlm.nih.gov/pmc/articles www.biotechnologynotes.com/animals/animal-cell-culture-history-types-and-applications/671 Animal cell culture practical approach by John R.W.Masters 42

43. THANK YOU 43