Cryopreservation

Question 1

Background

Answer 1

started in 1949 by freezing down spermatozoa

Question 2

Reasons for freezing cells down

Answer 2

prevent genetic drift
provides baseline for cellular changes
assures homogeneity of cell lines
backup

Question 3

Happenings to cells between room temp and 0

Answer 3

cellular metabolism slows

Question 4

Happenings to cells between 0 and -20

Answer 4

ice crystals form outside the cell
solute concentration of the media increases
water moves out of the cell
cellular dehydration and shrinkage

Question 5

Optimum cooling rate between 0 and -20

Answer 5

-1 to -2 degrees/min

Question 6

Happenings to cells between -20 and -130

Answer 6

cooling rate increases to -4 to -5 degrees/min

no detectable genetic changes

Question 7

Cryoprotectants definition

Answer 7

little effect on the damage caused by fast freezing (intracellular ice crystal formation), but rather prevent or lessen the damage caused by slow freezing (dehydration and shrinkage)

Question 8

Types of cryoprotectants

Answer 8

DMSO for mammalian cells and glycerol for prokaryotes

Question 9

Cryoprotectants purpose

Answer 9

lower the freezing point
allows a slower cooling rate
prevents or lessens cellular damage due to dehydration
doesn’t need to enter the cell to work

Question 10

DMSO

Answer 10

use at a final concentration of 5-15%

can kill the cells

Question 11

Glycerol

Answer 11

use at a final concentration of 2-20%

less to the cells than DMSO but cause more osmotic problems

Question 12

Control 1 of freezing controls

cells are at 2x10^6 cells/ml in DMEM80/FBS20 and 0.5 mls are transferred to a T75 and put in the incubator

Answer 12

this is a positive control, you expect growth and you are testing the viability

Question 13

Control 2 of freezing controls

cells in the freezing medium are stored at 4 C for 60 minutes and then inoculated into a T75 and put in the incubator

Answer 13

looking at cytotoxicity of DMSO

Question 14

Control 3 of freezing controls

a vial of cryopreserved cells was thawed and inoculated into a T75 and put in the incubator

Answer 14

this is testing the viability after thawing

Question 15

Factors that affect successful freezes

Answer 15

end of log-phase growth
1-2x10^6 cells/vial
appropriate cryoprotectant and concentration
storage vessels
cooling rates
cryogenic storage
adequate record keeping

Question 16

Items that need to be noted in log

Answer 16

cell type (and who you got them from)
date frozen
number of cells frozen
canister
cane number
number of vials frozen
freezing media
additional comments

Question 17

Transformations (not related to mammalian cells)

Answer 17

DNA/RNA transfer into bacteria, simple eukaryotes and plant cells

Question 18

Transformations (related to mammalian cells)

Answer 18

permanent alteration of the cell phenotype, presumed to occur via an irreversible genetic change, may be spontaneous

Question 19

Transfection definition

Answer 19

to transfer, by artificial means, of genetic material from one cell to another, when less than the whole nucleus of the donor cell is transferred, usually achieved by transferring isolated chromosomes, DNA, or cloned genes (this affects mammalian cells)