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Principles of cell culture

Cell culture e publication for students teaching

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Principles of cell culture

  1. 1. PrinciPles of cell culture By Megha Kadam Bedekar & Pooja Kanyal Central Institute of Fisheries Education (ICAR) Mumbai
  2. 2. introduction • Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells. • Cell culture was first successfully undertaken by Ross Harrison in 1907 • Roux in 1885 for the first time maintained embryonic chick cells in a cell culture
  3. 3. cell culture in vitro - A brief history • 1885: Roux maintained embryonic chick cells alive in saline solution for short lengths of time • 1912: Alexis Carrel cultured connective tissue and showed heart muscle tissue contractility over 2-3 months • 1943: Earle et al. produced continuous rat cell line • 1962: Buonassisi et al. Published methods for maintaining differentiated cells (of tumour origin)
  4. 4. • 1970s: Gordon Sato et al. published the specific growth factor and media requirements for many cell types • 1979: Bottenstein and Sato defined a serum- free medium for neural cells • 1980 to date: Tissue culture becomes less of an experimental research field, and more of a widely accepted research tool
  5. 5. Why is cell culture used for? AreAs Where cell culture technology is currently PlAying A mAjor role. • Model systems for 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.
  6. 6. • Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. • Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells. • 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
  7. 7. • Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene • Tissue culture In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells.
  8. 8. tyPes of cells On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into three. • Epithelial like-attached to a substrate and appears flattened and polygonal in shape • Lymphoblast like- cells do not attach remain in suspension with a spherical shape • Fibroblast like- cells attached to an substrate appears elongated and bipolar
  9. 9. cell morPhologies vAry dePending on cell tyPe Fibroblastic Endothelial Epithelial Neuronal
  10. 10. isolAtion of cell lines for in -vitro culture Resected Tissue Cell or tissue culture in vitro Primary culture Secondary culture Sub-culture Cell Line Sub-culture Immortalization Successive sub-cultureSingle cell isolation Clonal cell line Senescence Transformed cell line Immortalised cell line Loss of control of cell growth
  11. 11. Primary cultures • Derived directly from animal tissue embryo or adult? Normal or neoplastic? • Cultured either as tissue explants or single cells. • Initially heterogeneous – become overpopulated with fibroblasts. • Finite life span in vitro. • Retain differentiated phenotype. • Mainly anchorage dependant. • Exhibit contact inhibition. tyPes of cell cultured in vitro
  12. 12. Secondary cultures • 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.
  13. 13. Continuous cultures • Derived from a primary or secondary culture • Immortalised: • Spontaneously (e.g.: spontaneous genetic mutation) • By transformation vectors (e.g.: viruses &/or plasmids) • Serially propagated in culture showing an increased growth rate • Homogeneous cell population • Loss of anchorage dependency and contact inhibition • Infinite life span in vitro
  14. 14. Cont.. • Genetically unstable • Characteristics of continous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and have aneuploid chromosome number -reduced serum and anchorage dependence and grow more in suspension conditions -ability to grow upto higher cell density -different in phenotypes from donar tissue -stop expressing tissue specific genes
  15. 15. Basic equipments used in cell culture • Laminar cabinet-Vertical are preferable. • Incubation facilities- Temperature of 25-30 C for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38.5 C
  16. 16. • Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C • Microscope- An inverted microscope with 10x to 100x magnification • Tissue culture ware- Culture plastic ware treated by polystyrene
  17. 17. rules for working with cell culture Never use contaminated material within a sterile area Use the correct sequence when working with more than one cell lines. • Diploid cells (Primary cultures, lines for the production of vaccines etc.) • Diploid cells (Laboratory lines) • Continous, slow growing line • Continous, rapidly growing lines • Lines which may be contaminated • Virus producing lines
  18. 18. Basic aseptic conditions • 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
  19. 19. safety aspect in cell culture • Possibly keep cultures free of antibiotics in order to be able to recognize the contamination • 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 secs at a temperature of 200 C • Switch on the laminar flow cabinet 20 mts prior to start working • Cell cultures which are frequently used should be subcultered & stored as duplicate strains
  20. 20. other key facts…….? • Use actively growing cells that are in their log phase of growth, which are 80-90% viable • Keep exposure to trypsin at a minimum • 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 • A lower concentration of 104cells/ml to initiate subculture of rapidly growing cells & a higher concentration of 105cells/mlfor slowing growing cells
  21. 21. cell culture environment (in vitro) Requirements- • Substrate or liquid (cell culture flask or scaffold material) chemically modified plastic or coated with ECM proteins. • Nutrients (culture media) • Environment (CO2, temperature 37o C, humidity) Oxygen tension maintained at atmospheric but can be varied. • Sterility (aseptic technique, antibiotics and antimycotics) Mycoplasma tested.
  22. 22. cell culture environment (in vitro) Basal Media • Maintain pH and osmolarity (260-320 mOsm/L). • Provide nutrients and energy source. Components of Basal Media Inorganic Salts • Maintain osmolarity • Regulate membrane potential (Na+ , K+ , Ca2+ ) • Ions for cell attachment and enzyme cofactors
  23. 23. • pH Indicator – Phenol Red • Optimum cell growth approx. pH 7.4 • Buffers (Bicarbonate and HEPES) • Bicarbonate buffered media requires CO2 atmosphere • HEPES Strong chemical buffer range pH 7.2 – 7.6 (does not require CO2) • Glucose • Energy Source
  24. 24. Components of Basal Media 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 (1 g/L) and high (4.5 g/L) concentrations of sugars in basal media Cell Culture environment (in vitro)
  25. 25. Vitamins • Precursors for numerous co-factors • B group vitamins necessary for cell growth and proliferation • Common vitamins found in basal media is riboflavin, thiamine and biotin Trace Elements • Zinc, copper, selenium and tricarboxylic acid intermediates
  26. 26. Supplements L-glutamine • Essential amino acid (not synthesised by the cell) • Energy source (citric acid cycle), used in protein synthesis • Unstable in liquid media - added as a supplement Non-essential amino acids (NEAA) • Usually added to basic media compositions • Energy source, used in protein synthesis • May reduce metabolic burden on cells
  27. 27. Growth Factors and Hormones (e.g.: insulin) • Stimulate glucose transport and utilisation • Uptake of amino acids • Maintenance of differentiation Antibiotics and Antimycotics • Penicillin, streptomycin, gentamicin, amphotericin B • Reduce the risk of bacterial and fungal contamination • Cells can become antibiotic resistant – changing phenotype • Preferably avoided in long term culture
  28. 28. Foetal Calf/Bovine Serum (FCS & FBS) • Growth factors and hormones • Aids cell attachment • Binds and neutralise toxins • Long history of use • Infectious agents (prions) • Variable composition • Expensive • Regulatory issues (to minimise risk) Heat Inactivation (56o C for 30 mins) – why? • Destruction of complement and immunoglobulins • Destruction of some viruses (also gamma irradiated serum).
  29. 29. How do we Culture Cells in tHe laboratory? Revive frozen cell population Isolate from tissue Maintain in culture (aseptic technique) Sub-culture (passaging) Cryopreservation Count cells Containment level 2 cell culture laboratory Typical cell culture flask ‘Mr Frosty’ Used to freeze cells
  30. 30. Check confluency of cells Remove spent medium Wash with PBS Resuspend in serum containing media Incubate with trypsin/EDTA Transfer to culture flask Passaging Cells
  31. 31. Why passage cells? • To maintain cells in culture (i.e. don’t overgrow) • To increase cell number for experiments/storage How? • 70-80% confluency • Wash in PBS to remove dead cells and serum • Trypsin digests protein-surface interaction to release cells (collagenase also useful) • EDTA enhances trypsin activity • Resuspend in serum (inactivates trypsin) • Transfer dilute cell suspension to new flask (fresh media) • Most cell lines will adhere in approx. 3-4 hours
  32. 32. Passage cells Resuspend cells in serum containing media Centrifuge & Aspirate supernatant Transfer to cryovial Freeze at -80o C Resuspend cells in 10% DMSO in FCS Transfer to liquid nitrogen storage tank CryoPreservation of Cells
  33. 33. • Why cryopreserve cells? • Reduced risk of microbial contamination. • Reduced risk of cross contamination with other cell lines. • Reduced risk of genetic drift and morphological changes. • Research conducted using cells at consistent low passage. • How? • Log phase of growth and >90% viability • Passage cells & pellet for media exchange • Cryopreservant (DMSO) – precise mechanism unknown but prevents ice crystal formation • Freeze at -80o C – rapid yet ‘slow’ freezing • Liquid nitrogen -196o C
  34. 34. Cell viability • Cell viability is determined by staining the cells with trypan blue • As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye • Stain the cells with trypan dye and load to haemocytometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 100 total nu. of cells
  35. 35. Manual Cell Count (HeMoCytoMeter) Diagram represent cell count using hemocytometer.
  36. 36. autoMated Cell Count Cellometer lets you: • View cell morphology, for visual confirmation after cell counting • Take advantage of 300+ cell types and easy, wizard-based parameter set-up • Save sample images with results securely on your computer, plus autosave results on the network for added convenience and data protection
  37. 37. CoMMon Cell lines • Human cell lines • -MCF-7 breast cancer • HL 60 Leukemia • HEK-293 Human embryonic kidney • HeLa Henrietta lacks • 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
  38. 38. tHe ideal growtH Curve for Cells in Culture
  39. 39. 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
  40. 40. effeCts of biologiCal ContaMination’s • They competes for nutrients with host cells. • Secreted acidic or alkaline by-products ceses the growth of the host cells. • Degraded arginine & purine inhibits the synthesis of histone and nucleic acid. • They also produces H2O2 which is directly toxic to cells.
  41. 41. deteCtion of ContaMinants • In general indicators of contamination are turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolization, inclusion bodies and cell lysis • Yeast, bacteria & fungi usually shows visible effect on the culture (changes in medium turbidity or pH) • Mycoplasma detected by direct DNA staining with intercalating fluorescent substances e.g. Hoechst 33258
  42. 42. • Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA. • The best and the oldest way to eliminate contamination is to discard the infected cell lines directly.
  43. 43. ContaMination A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use. 1-Chemical Contamination Media Incubator Serum water 2-Biological Contamination Bacteria and yeast Viruses Mycoplasmas Cross-contamination by other cell culture
  44. 44. How Can Cell Culture ContaMination be Controlled?