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Tissue Culture overview for dbt jrf net.pptx
- 1. BioScholars Target DBT, CSIR,ICMR 2025 Topic:- Animal Biotechnology Sub Topic- Tissue Culture By: Subhadeep Mandal MSc. Calcutta University JRF, IICB Kolkata
- 5. Type Primary cultureSecondary culture Cell line Cell strain Clonal culture Origin Derived from the growing cell or tissue Derived from primary culture Re culturing of primary culture Specific cell culture Copy from single strain Lifespan Finite Indefinite Can be finite or infinite Can be finite or infinite Can be finite or infinite Contamination Contamination high Contamination low Contamination low Contamination low Contamination low Advantages •Used for production of vaccine and therapeutic development. •Serve as in vivo model Used for the production of hormones and antibodies. •Serve as invitro model Development of a cell line over several generations. •Homogenity of cells. Cost Effective •Easy to use Cost Effective •Easy to use
- 7. Isolation of celllines for in vitro culture Tissue of interest Cell or tissue culture in vitro Primary culture Secondary culture Cell Line Immortalization Transformed cell line (cancerous cells) Immortalised cell line Successive sub-culture Senescence Single cell isolation Clonal cell line Loss of control of cell growth Sub-culture Sub-culture
- 11. Normal cells usuallydivide only a limited number of times before losing their ability to proliferate, which is a genetically determined even to known as senescence; these cell lines are known as finite. However, some cell lines be come immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line. Finite vs Continuous Cell Line
- 12. Continuous cultures Derived froma primary or secondary culture. Immortalised: Spontaneously (e.g.: spontaneous genetic mutation). -By transformation vectors (e.g.: viruses &/or plasmids). Serially propagated culture shows an increased growth rate and shows homogeneous cell population. Loss of anchorage dependency and contact inhibition. Infinite life span in vitro. Differentiated phenotype: very little retained with transformed cell lines (cancerous cells).
- 13. Primarycellculturesorcelllines Primary culture refersto the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate(i.e.,reach confluence) and retains differentiated phenotype. Atthisstage,thecellshavetobesub- cultured(i.e.,passaged)bytransferringthemtoanewvesselwithfreshgrowthmediumtoprovidemoreroomforcontinued growth. Celllinesderivedfromprimarycultureshavealimitedlifespan(i. e.,theyarefinite)andastheyarepassaged, cellswiththehighestgrowthcapacitypredominate,resultingin adegreeofgenotypicandphenotypicuniformityinthepopulatio n.
- 15. Derived from aprimary cell culture and 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. Contact inhibition is a growth mechanism. In most cases when two cells collide they attempt to move in a different direction to avoid future collisions Secondary cultures
- 16. Normal somatic cells Normalsomatic cells when grown in culture will become growth inhibited when they encounter another cell. The cells in our bodies are governed by growth control mechanisms and cellular senescence(aging). Cell aging puts a limit on the number of time sacell can divide: the more a cell has divided,the less likely it will be to divide again. Growth mechanisms are in place to stop or continue cell growth depending on the conditions. Cancerous Cells Cancerous cells typically lose this property and thus grow in an uncontrolled manner even when in contact with neighbouring cells. They aren’t motivated to change direction upon contact, so they pile up and grow over each other.
- 20. Primary culture Anchorage dependentAnchorage Independent Anchorage dependent Anchorage dependent Most of the animal cells Cells present in body fluid Propagate as monolayer attached to the cell culture Propagate in the Suspension culture (RBC, WBC) Need subculturing Need subculturing Confluency
- 22. 1. Anchorage dependant -Mostanimal derived cells. -Adhere to bottom of a flask and form a monolayer. -Eventually cover entire surface of substratum. -Proliferation then stops. -Need to subculture cells to fresh medium. -Proliferation can begin again. 2. Anchorage independent -Cells associated with body fluid (eg. blood cells). -Grown in suspension. -Will eventually need sub-culturing Cell lines used for in vitro culture are two types
- 24. Microculture Macroculture Carrier Adherentcell Support matrix Size 150-250μm >250μm Example DEAE-Dextran, Alginate gel, polystyrene, collagen, polyacrylamide Alginate gels in lowconcentration Advantages Ease of scaleup Efficient oxygenation High and precise control Efficient nutrient exchange Surface/volume ratio higher High yield Reduction in technical labour Microculture Macroculture Adherent cell Support matrix 150-250μm >250μm DEAE-Dextran, Alginate gel, polystyrene, collagen, polyacrylamide Alginate gels in low concentration Ease of scaleup Efficient oxygenation High and precise control Efficient nutrient exchange Surface/volume ratio higher High yield Reduction in technical labour
- 26. Cell line Speciesof origin Tissue of origin Cell morphology 3T3 Mouse Connective Fibroblast CHO Chinese Hamster Ovary Epithelial BHK21 Syrian Hamster Kidney Fibroblast HeLa Human Cervical Carcinoma Epithelial MDCK Dog Kidney Epithelial MRC-S Human Lung Fibroblast, Finite
- 28. Animal tissue culture •Culturingof animal cell, tissues and organs in the artificial environment (In vitro). •Culturing of whole organism is not possible only cells, tissues and organs of organism can be cultured . Cell culture mediaCarbon source Glucose Energy generation Nitrogen source Amino acid Nucleotideand protein synthesis Growthfactor EGF, PDGF, FGF Stimulate cell proliferation and differentiation Hormones Cortisone Cell attachment Steroid Mitogenic Thyroid Increase BMR Protease inhibitor Alpha-antitrypsin Alpha2 macroglobulin Trace elements
- 29. SerumFetal bovine serum(FBS) Albumin Mechanical protection Transporter Detoxifier Buffer Transferrin Transfer of Iron Attachment Protein Fibronectin Laminin Growth factors Many growth factors
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- 33. 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) -Energy source, used in protein synthesis -May reduce metabolic burden on cells Growth Factors and Hormones (e.g.: insulin) -Stimulate glucose transport and utilization -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
- 34. Foetal Calf/Bovine Serum(FCS & FBS) -Growth factors and hormones -Aids cell attachment -Binds and neutralise toxins -Long history of use Supplements to basal media (contd.) Heat Inactivation (560C for 30 mins) -why? -Destruction of immunoglobulins -Destruction of some viruses (also gamma irradiated serum) Care! Overdoing heat inactivation can damage growth factors, hormones & vitamins and affect cell growth.
- 35. How do weculture 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 Liquid nitrogen is also used
- 36. Check confluency ofcells Remove spent medium Wash with PBS Resuspend in serum containing media Incubate with trypsin/EDTA Transfer to culture flask
- 37. Why passage cells? Tomaintain 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 is also useful) EDTA enhances trypsin activity Resuspendin serum (inactivates trypsin) Transfer dilute cell suspension to new flask (fresh media) Most cell lines will adhere in approx. 3-4 hours Check
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