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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1

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

2

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.

3

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.

4

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

5

Rank the various types of culture types in decrease order of clone-ability.

Continuous > Finite > Primary Cultures 

6

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.

7

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

8

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

9

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