Chapter 4 - Cell Lines, Cloning, Selection Flashcards
What parameters need to be considered in selecting a cell line to work with?
Specific functional requirements
Cell line type - Continuous vs Finite, Normal vs Transformed
Growth characteristics - impacts how fast the cells grownadn the product yield for biotechnology purposes
Species - human cell lines require more biohazard precautions than cell lines from other animals
What are the phases of a cell culture?
Lag phase - initial phase in which no increase to cell density is observed. At this moment growth factors are still synthesized and need to reach critical concentration in culture.
**culture inoculated at low cell densities have longer lag phases (vice-versa)
Growth phase - observe an exponential increase to cell numbers during this phase; cells are going through the cell cycle during this phase
Stationary phase - no further increase in cell density; growth rate = death rate. Growth is limited by depletion of nutrients, accumulation of metabolic by-products and confluent monolayer of the culture
Decline phase - phase of cell death and low cell viability in culture. Leads to apoptosis or necrosis.
What is apoptosis?
Programmed cell death in which DNA is cleaved into 180bp fragments. The cell membranes are blebbed and may cause depletion of nutrients.
What is necrosis?
Un-programmed cell death in which the DNA is not cleaved and membranes are not blebbed. Plasma membrane is disrupted causing the cellular contents to spill into the extracellular environment.
**Usually caused by severe stress
Rank the various types of culture types in decrease order of clone-ability.
Continuous > Finite > Primary Cultures
Explain dilution cloning
Start out with a confluent monolayer, or whatever cell culture. Trypsonize and dilute into a single-cell suspension. Seed cells into individual wells and the goal here is to acheive one cell per well. Trypsonize a well and seed into new culture flask.
How can cloning efficiency be improved?
Use a nutrient rich medium
Add serum to basal medium (FBS is better than calf or horse)
Add hormones or intermediary metabolites (ie. insulin or pyruvate)
Pre-treat the substrate (ie. coat plastic ware with polystyrene or fibronectin to improve playing efficiency
What treatments can be done to select for a particular cell type?
Selective adhesion - different cell types adhere more readily than others to the culture ware
Selective detachment - trypsinization may remove different cell types more rapidly than others
Culture substrate - culture ware can be coated to enhance attachment or growth of specific cell types
Feeder layers - cells that are arrested in growth (innactivated) foudn on the bottom of culture dish
Explain Feeder layer selection treatment
Irradiate feeder cells (this stops the feeder cells from proliferating but does not kill them)
Trypsonize the feeder cells to create a subconfluent monoalyer
Add diluate cell suspension to feeder layer and incubate
