Lecture 6

Question 1

explain substrate consideration for anchorage dependent and anchorage independent cells

Answer 1

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anchorage dependent (monolayer) will have yield proportional to surface area of vessel.

Question 2

describe surface treatments and coating options

Answer 2

1. conditioning: reuse a dish or pretreat with spent medium
2. polymers, photoetching, patterning: Polt-L-Lys and Poly-D-Lys polymers. photoetching by UV
3. collagen and gelatin: collagen for epithelial cells and gelatin for muscle or endothelial cells. addition of fibronectin or laminin to medium
4. matrix components: commercial products from natural sources or combinations of laminin, fibronectin, enacting, heparin sulfate, collagen
5. extracellular matrix: growing cells produce matrix, remove the original cells and seed new cells on matrix

Question 3

explain how feeder layers and preconditioned media work

Answer 3

Feeder layers are the use of living cells as support. They supplement medium, produce growth factors, and condition substrate. Can be problematic due to contamination. You must use a specific feeder layer for each specific project
preconditioned media?

Question 4

describe considerations regarding cell yield and surface area

Answer 4

monolayer cell lines have a yield that is proportional to the surface area. larger flasks give larger cell yields.

Question 5

describe specialized vessels for certain purposes

Answer 5

multilayer flasks and roller bottles can be used to increase surface area for monolayers. stirrer flasks can increase yield for suspension cells
multiwell plates allow for multiple replicates of small volumes
filter well inserts for histotypic or organ-typically cultures
hollow fibers for large scale bioreactors
3D matrices for tissue engineering

Question 6

explain sterilization options and considerations for choosing the appropriate method

Answer 6

Dry heat: good for heat stable (metal, glass, etc). can have some charring
steam: heat stable liquids (water, salt solutions, media etc). some packaging is steam permeable, not good
gamma radiation: plastics, heat sensitive reagents. can cause chemical alterations of plastics
short wave UV: flat surfaces, circulating air. Leaves shadow areas, spores are resistant, and can be unsafe in lab (UV hurts skin and such)
hypochlorite (bleach): good for contaminated solutions. leaves residues
Ethanol: good for dissecting instruments and plastics. is a fire risk and spores are resistant
filtration: only for aqueous solutions, not for solvents

Question 7

explain how filter sterilization works, describe difference between positive and negative pressure systems

Answer 7

can be done with a vacuum pump or in line filter (syringes or peristaltic pump). vacuum is a negative pressure system, most commonly used in small labs. The filter is airtight to the bottle below and the vacuum is created which pulls the media down through the filter
In line filter pushes media through filter by positive pressure. peristaltic pump is good for large volumes. doesn’t have to be airtight.