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Lecture 10

BIOL483- Lecture 10 - Oct. 1.docx

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BIOL 483
Vivian Dayeh

What type of cryoprotectant is used during cryopreservation of animal cells? - DMSO - Glycerol - Liquid Nitrogen Naming a cell line - Designation is ideal, want it to be short - Number = how many times it took u to create cell line (NML1, NML2) o Sometimes a subset, cloned and created a set - You would explain the number or designation to avoid ambiguity. - You also want to have something that’s unique because you could have two HLs. Human liver or human lung? If you have a slow growing cell line – its harder to do research, fast growing = easier. All primate cell lines need a vertical flow hood. Animal cells in culture exhibit the following DON’T CONFUSE THESE TWO GRAPHS = PHASES OF A CULTURE vs. EVOLUTION OF A CELL LINE!!! Evolution of cell line = starting with primary culture up to difference with continuous vs finite cell line This is if you look at culture at one time, what do u see? First phase - Lag phase o Initial phase, you seeded cell in tissue culture flask o New growth, new nutrient media o Cells are specific density o Don’t see apparent change in cell density, haven’t started to grow exponentially yet. Second phase - Exponential growth, pop doubles Third phase - Stationary phase o No further increase in cell density o Growth rate = death rate o Limitations for growth and limitations of nutrients start to occur o Toxic products = metabolic byproducts may be a reason as well to inhibit growth o Depdning on if cells are contact inhibited, confluent layer may be achieved and cells cant grow no more, only true for contact inhibited cells Fourth phase - Phase of cell death - Apoptosis = neat way of dying, necrosis = ugly way of dying - Blebbing of cell membrane *** (pinches off or blebs off) - Apoptic bodies form and complete defragmentation of cell. Necrosis - Unprogramed death - No changes, no condensation, cleavage or enzyme cascade. All of these are apoptic characteristics - Plasma membrane disrupted, and cellular contents expelled into EC Material and can cause inflammation Look at graph. – length of each phase varies on nutrient factors. Cloning - How do u tell cell lines if they transformed? Transformed cells can grow in suspension, normal cells wont! Most widely used technique to clone a cell line = DILUTION CLONING In a tissue culture cell flask u have 1 mil cells, you want ot clone only one of them and devlop a cell line. U can do this thru dilution cloning. Must clone at low density. If cells growing at low density it will form discrete small cell colonies. Dilution cloning = mechanism to create a homogenious mixture from a single cell. Exponential culture - Happy healthy, most likely confluent layer of cells Start process of passaging Trypsinize to single cell suspension – take cells take media out and wash cells with PBS or ADTA, add tryspin, till cells are detached
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