Lecture 10 Flashcards
list reasons for cryopreservation of cells
minimize change over time (eg genetic drift)
keeps cell line properties constant across experiments
avoids transformation
keeps cells available without continuous culture, saves cost and avoids senescence
good to have for replacing contaminated cells
distribution to other labs
describe consideration prior to freezing cells
finite vs continuous: freeze early passage vs clone and characterize
media, serum and substrate requirements
validate: contaminated, transformed, identity, provenance
consider cell quality at start: growth condition should be end of log phase, physiological state should be healthy, number of cells should be healthy, cell handling and harvesting should be good quality
describe how we can avoid damage due to freezing and thawing.
cellular damage is induced by intracellular ice crystals and osmotic effects. solution is dehydration, pH changes, and protein denaturation.
Preventative steps:
hydrophilic cryoprotectant
slow cooling (let water move out of cell)
appropriate storage (-135°C)
rapid thawing
vitrification for sensitive cells
explain why controlled cooling times are important. what is best rate? devices?
slow rate allows cells enough time to dehydrate and prevent intracellular ice crystals. Best rate is 1-3°C/min. Can use Mr Frosty (most common) or electronic programmable cooling units
describe temperature changes during cell freezing. define latent heat, eutectic point, and how freezing profiles vary between method of freezing
see slides 11-13
vial in -70°C freezer profile begins freezing faster than 1°C/min, has a rise at eutectic point, then has too slow rate at end
vial in controlled 1°C/min freezer profile has good consistent lower in temperature but still has temp increase at eutectic point
vial in optimized cooling profile has large temp drop to compensate for eutectic point so whole profile is consistently lowering temp 1°C/min
how does vitrification differ from regular cell freezing?
involves snap freezing in small straws/glass tubes. media often has more than 1 cryoprotectant and liquid is cooled so fast that it forms a glass. this reduces crystal formation. Good for very few cells as large #s won’t fit in the straw. For sensitive cells (embryo)
how are cells stored long term? what is the temp range and practical options for storage and shipping?
lower storage temp = longer viable storage period. should be stored at -135°C or lower for long times, any higher leaves some water unfrozen and allows reactions and death.
liquid nitrogen is best option, should be in vapor but is more often in liquid b/c easier to maintain
describe the individual steps of the cell freezing
- trypsinize cells
- centrifuge and remove supernatant
- resuspend in freezing medium (w DMSO)
- aliquot into freezing vials (10^6 to 10^7, high conc)
- place in freezing device and freeze at appropriate rate
- move to N2Liq storage
explain cryoprotectants and optimal concentrations
they slow cooling rates (allow water to move out of cell before freezing, prevent membrane damage) and have low toxicity, ability to penetrate membranes, capacity to dissolve electrolyte, strongly binds water
DMSO: 10%. standard, higher efficacy
Glycerol: 5-10%. less toxic
First dilute cryoprotectant, then resuspend! use high serum (30%-90%)
describe the individual steps of the thawing procedure
- thaw rapidly in warm water/hand
- disinfect vial and agitate
- transfer content into flask
- dilute cells slowly
- incubate several hours
- change media to remove DMSO
- sensitive cells should be centrifuged before culturing
- evaluate and verify cells
describe shipping of cells. two options
frozen cells: use thick walled container and dry ice/liquid nitrogen to keep cold
living cultures: mid to log phase, fill flask completely with medium and package with absorbent material. Do not freeze while transporting!
