What type of cryoprotectant is used during cryopreservation of animal cells?
- 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
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?
- 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
- 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
- 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.
- Unprogramed death
- No changes, no condensation, cleavage or enzyme cascade. All of these are apoptic
- Plasma membrane disrupted, and cellular contents expelled into EC Material and can cause
Look at graph. – length of each phase varies on nutrient factors.
- How do u tell cell lines if they transformed? Transformed cells can grow in suspension, normal
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.
- 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