Basic techniques of mammalian cell culture

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Basic techniques of mammalian cell culture

  1. 1. Basic Techniques ofmammalian cell culture Presented By: Kiran N.Birje M.Sc.II
  2. 2. Introduction• In order to realize production potential of cell culture, the provision of culture system which would allow optimum expression of desirable genetic trait is necessary.• Culture system could generally be categorised as closed system and open system.• In closed culture system, some essential components of system ( e.g. culture and products) cannot be supplied or withdrawn from the bioreactor during the process.• Whereas in open culture system, further addition of culture medium can be made.
  3. 3. Closed culture system• Based on their culture homogeneity, closed culture system can be classified as homogenously mixed batch culture and plug- flow culture, where localized mixing of whole culture is allowed.• Batch culture: A batch-wise culture system where culture medium and inoculum( in case of ATC cells) are added in culture vessel at beginning of process and then incubated at suitable temperature and gaseous environment for an appropriate period of time is called a Batch culture.
  4. 4. Batch culture system
  5. 5. Batch culture systemBatch culture is still widely used in some industrial process e.g. brewery industry,for its ease of operation; its less stringnent sterilization; and its easy management of feed stocks.These advantages allow the use of unskilled labour and low risk of financial loss.Disadvantage:Product formation is confined to a certain period of cultivation. e.g. Antibiotic production.
  6. 6. Open culture system• To maintain cultures at steady state, i.e. with its physiology fully adjusted to culture conditions, concentration of substrate need to be maintained and end product need to be removed simultaneously.• Thus, a fresh sterile culture medium is added to bioreactor continuously to replenish the substrate that are consumed, and simultaneously diluting the end products that are accumulating in the system.• This is often called as continuous flow culture system.
  7. 7. Open culture system• Three main types of open culture system are distinguished:1. Chemostat culture2. Turbidostat culture3. Fed-batch cultureIn chemostat, where dilution rate (D=F/V) is constant.Advantage: To understand physiological function of culture. To study effect of culture environment without interfering the growth rate. To maintain maximum output of biomass or product.
  8. 8. Open culture system - Chemostat
  9. 9. Open culture system- Turbidostat• In turbidostat , biomass concentration in culture vessel is maintained at constant by monitoring the turbidity of culture through the use of photoelectric cell and regulating the feeding rate of nutrient solution.• Advantage: Useful in case of slow growing cells and microorganism and those with complex cell cycles. To maintain constant biomass concentration at unstable envinoment.
  10. 10. Open culture system- Turbidostat
  11. 11. Primary cell culture• Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture.• Primary cells have a finite life span• Primary culture contains a very heterogeneous population of cells.• Sub culturing of primary cells leads to the generation of cell lines.• Cell lines have limited life span, they passage several times before they become senescent.• Cells such as macrophages and neurons do not divide in vitro so can be used as primary cultures.• Lineage of cells originating from the primary culture is called a cell strain.
  12. 12. Continuous cell lines• Most cell lines grow for a limited number of generations after which they ceases• Cell lines which either occur spontaneously or induced virally or chemically transformed into Continuous cell lines• Characteristics of continuous 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 up to higher cell density -different in phenotypes from donor tissue
  13. 13. 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
  14. 14. Primary cell culture• How can we obtain Primary Cell Culture?• Stages:1. Acquisition of sample2. Isolation of tissue3. Dissection and/or disaggregation4. Culture after seeding into culture vessel
  15. 15. Isolation of cell lines for in vitro culture Primary cell culture Resected Tissue Cell or tissue culture in vitro Primary culture Sub-culture Secondary culture Sub-culture Cell LineSingle cell isolation Successive sub-culture Immortalization Loss of controlClonal cell line of cell growth Senescence Transformed cell line Immortalised cell line
  16. 16. Isolation of cells• Fine dissection to obtain explant• Mechanical disaggregation involves dissection with or without maceration• Enzymatic disaggregation
  17. 17. Primary Explant technique• Developed originally by Harrison in 1907• A fragment of tissue embedded in blood plasma or lymph + embryo extract and serum• Placed on a coverslip inverted over a concavity slide• Clotted plasma held tissue together• Outgrowth was subcultured or explant transferred• It also termed as mechanical disaggregation.
  18. 18. Modified technique• Tissue is chopped• Washed with PBS or Hanks BSS• Pieces seeded onto culture surface• Medium supplemented with serum• Adhere to surface and proliferate
  19. 19. Enzymatic disaggregation• Enzymes used for disaggregation – trypsin + EDTA or only trypsin• Warm trypsin - @ 37°C + removed by centrifugation + neutralized with serum and medium• Cold trypsin – Soak tissue in trypsin at 4°C for 6 – 18 hrs followed by incubation at 37°C for 20-30 minutes for disaggregation
  20. 20. Effectiveness of different enzymesTrypsin and Pronase give Trypsin and Pronase maycomplete disaggregation damage the cellsCollagenase and dispase Collagenase and dispasegive incomplete are less harmfuldisaggregation Hyaluronidase + Collagenase digests intracellular matrix DNase – disperses DNA released from lysed cells
  21. 21. Differences between warm and cold trypsin treatmentWarm trypsin Cold trypsinShorter time period Gives higher yield ofLesser yield viable cells with improved survival after 24 h cultureCentrifugation is No stirring orrequired centrifugation is required Incubation at 4 C can be done overnight Embryonic organs
  22. 22. What are the set of conditions required commonly by most of the cultures?• Fat and necrotic tissue are removed during dissection• Chopping should be fine with sharp instruments• Enzymes used for disaggregation should be removed• Concentration of cells – primary > subculture• Choice of Rich medium• Choice of Embryonic tissue
  23. 23. Mechanical disaggregationCollecting cells that spill outwhen tissue is sliced –Scraping/SpillagePressing the dissected tissuethrough series of sieves -SievingForcing the tissue fragmentsthrough a SyringePipetting repeatedlySoft tissues – Spleen,embryonic liver, embryonic andadult brain and some human andanimal soft tumors
  24. 24. Cell separation• There are two popular methods by which cells may be selected on basis of size, density, charge and surface area.1. Flow cytometry : measures the light scattering properties of cells, which are proportional to surface area.2. Flow cytofluorimetry: specific fluorochromes attached to cells and the cells are separated on basis of fluorescence emission.Both methods can be performed using Fluorescenes Activated Cell Sorter (FACS)
  25. 25. Cell separationPhysical methods of cell separation:1. Separation based on cell size: Centrifugation by v= r2 /42. Separation based on cell density: Suitable density gradient used. Mostly in ATC percoll, Metrazamide.3. Separation based on surface charge: Electrophoresis.4. Separation based on affinity: chromatography. Antibodies or lectins used.
  26. 26. Viable cell countHemacytometer:The hemocytometer has an exact volume under the coverslip, one can determine the concentration (cells/ml) of live and dead cells in the chamber. The cell concentration of the original cell suspension will be the same as that of the chamber – except for any dilutions made.Dead cells take up the dye, trypan blue, and appear blue under the microscope. Living cells exclude tyrpan blue, and appear white. Thus, the percentage of viable cells can be calculated.
  27. 27. • Determine the viable cell density of the original mixture according to the followingFormula:• %Viability = Total number of viable cells /Total number of viable and nonviable cells X 100• Cells/ml = Total viable cells counted in 4 squares/4 X 10,000 or• Cells/ml = Total viable cells counted in 5 squares/5 X 10,000
  28. 28. Daily culture maintenance• Love your cells!• Observe daily !!!!!– Morphology– pH of the medium– Contamination Check the incubator setting and levels of• – CO2level both on the incubator and in the tank– Temperature– Water level– Sing them a lullaby before you go home
  29. 29. Maintenance of stock culture Consistency and reproducibility within successive experiments requires that stock cultures be maintained by routine protocol. Establish and maintain culture stocks for given experimental series under identical seeding density, media and incubation condition.Replenish the medium on routine schedule and before nutrients are severely depleted. Frequency of feeding is dependent on population density. Passage stock cultures on a routine schedule.
  30. 30. Antibiotic free stock cultures• The interrupted use of antibiotics in the medium can mask low- level contamination by resistant microorganisms.• Some laboratories carries two sets of stocks for each cell line ..one in complete medium and one in antibiotic-antimycotic free medium.• The antibiotic free culture medium is a necessity.• Simple Test used: Replica plating• Mycoplasma contamination can be more difficult to detect.• These organisms can cause numerous problems such as acute cell lysis and can also lead to alteration in cell structure and function, karyotypes, metabolism and growth characteristics that may not be overtly apparent for many cell generation.
  31. 31. Types of cell culturesA. Monolayer Culture:In cell culture a monolayer refers to a layer of cells in which no cell is growing on top of another, but all are growing side by side and often touching each other on the same growth surface.Most animal cell lines and primary cultures grow as a single thickness cell layer orsheet attached to a plastic or glass substrate.Once the available substrate surface is covered by cells(a confluent culture), growth slows and then ceases. Thus, in order to keep the cells healthy and actively growing, it is necessary to subculture them at regular intervals
  32. 32. Monolayer Culture
  33. 33. Suspension culture• The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e.,tissue-culture treated). However, many cell lines can also be adapted for suspension culture. Similarly, most of the commercially available insect cell lines grow well in monolayer or suspension culture.• Cells that are cultured in suspension can be maintained in culture flasks that are not tissue-culture treated, but as the culture volume to surface area is increased beyond which adequate gas exchange is hindered (usually 0.2 – 0.5 mL/cm2), the medium requires agitation. This agitation is usually achieved with a magnetic stirrer or rotating spinner flasks.
  34. 34. Suspension culture
  35. 35. Suspension cultureAdvantage over Monolayer cell culture: Easier to passage, but requires daily cell counts and viability. Does not require enzymatic or mechanical dissociation. Growth is limited by concentration of cells in the medium, which allows easy scale-up. Used for bulk protein production, batch harvesting, and many research applications.
  36. 36. Micro carrier culture• A microcarrier is a support matrix allowing for the growth of adherent cells in bireactors.• Microcarriers can be made from a number of different materials including DEAE-dextran, glass, polystyrene plastic, acrylamide, and collagen, and these microcarrier materials, along with different surface chemistries, can influence cellular behavior, including morphology and proliferation.• Several types of microcarriers are available commercially including dextran-based (Cytodex, GE Healthcare), collagen- based (Cultispher, Percell), and polystyrene based (Solo Hill Engineering) microcarriers. They differ in their porosity, specific gravity, optical properties, presence of animal components, and surface chemistries.
  37. 37. Micro carrier culture
  38. 38. Micro carrier culture• Microcarrier cell cultureis typically carried out in spinner flasks, although other vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also support microcarrier-based cultures.• Microcarrier culture introduces new possibilities and for the first time makes possible the practical high yield culture of anchorage-dependent cells.• In microcarrier culture cells grow as monolayers on the surface of small spheres.• By using microcarriers in simple suspension culture systems it is possible to achieve yields of several million cells per milliliter.
  39. 39. Micro carrier cultureThe advantages of microcarrier technology in the vaccine industry include(a) ease of scale-up,(b) ability to precisely control cell growth conditions in sophisticated, computer-controlled bioreactors,(c) an overall reduction in the floor space and incubator volume required for a given-sized manufacturing operation, and(d) a drastic reduction in technician labor.
  40. 40. spinner flasks Fluidized bed bioreactors
  41. 41. Stem cell culture• Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth.• Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity.Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions.scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non- embryonic "somatic" or "adult" stem cells.
  42. 42. • Stem Cells grown on feeder layers of, for example, SCO mouse fibroblasts.,will proliferate, but not differentiate.• Leukemia inhibitory factor (LIF) appears to be one of the main regulatory factors that hold cells within the stem cell compartment, whereas retinoids, vitamin D3, and planar polar compounds induce lineage specific differentiation in mouse and human in case of human teratoma cell culture.• Most Human ES cell line traditionally established on mouse embryonic feeder (MEF) layer in high conc. Of non human serum, and most available hES cell lines have required coculture with MEFs to maintain pluripotency and cellular expansion and to inhibit differentiation.• Major limitation of Human ES culture is its propagation is still highly laborious and technically demanding.
  43. 43. Stem cell cultures (ESC)
  44. 44. Stem cell cultures (ESC)
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