Chapter 4 - Cell Lines, Cloning, Selection Flashcards Preview

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Flashcards in Chapter 4 - Cell Lines, Cloning, Selection Deck (9)
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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