CRYOPRESERVATION

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Presented by Joshua Samuel (11MBT04) I M.Sc. Biotechnology DR.G.R.D.C.S

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Cryopreservation is the storage of living cells at ultra-low temperatures, usually in liquid nitrogen. Cryopreservation is a four-step process: Adding cryoprotective agents to cells before cooling Cooling the cells to the low temperature at which the cells are stored Warming the cells Removing the cryoprotective agents from the cells after thawing

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Weighing cells: collect cells and inoculate a certain mass of these cells into a certain volume of fresh medium. This technique has the advantage that the cell density is similar for every subculture, and thus results in better reproducibility; however, the weighing step increases the chance of contamination. Pipetting cells: transfer by pipetting/pouring a determined volume (ml) of cell suspension into a certain volume of fresh medium. This approach is not as accurate as weighing cells because of the problem of nonhomogeneous cultures. It requires continuous gentle shaking of the flask with one hand while pipetting with the other in order to have a representative group of cells from the different sizes of cell aggregates present in the culture.

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Pouring cell culture: add a certain volume of fresh medium into the cell suspension culture and distribute this among more flasks. Compared with weighing or pipetting, the pouring technique is simpler (fewer tools used), faster and has less chance of contamination, but low reproducibility.

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Cryopreservation of plant shoots

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Risk involved in cryopreservation Solution effects As ice crystals grow in freezing water, solutes are excluded, causing them to become concentrated in the remaining liquid water. High concentrations of some solutes can be very damaging. Extracellular ice formation When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing. Dehydration The migration of water causing extracellular ice formation can also cause cellular dehydration. The associated stresses on the cell can cause damage directly. Intracellular ice formation While some organisms and tissues can tolerate some extracellular ice, any appreciable intracellular ice is almost always fatal to cells.

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Preventing risks during cryopreservation

1. 2.

Slow programmable freezing Vitrification

Cryoprotectants used are(commonly) 1.Glycerol 2.Dimethyl sulphoxide 3. Polyethylene Glycol Monomethylether (350, 550, 750, 2000,
5000) PEG 1,500 PEG 6,000 PEG 10,000 PEG 20,000

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References 1. http://www.uoguelph.ca/plant/research/embryo/synseeds.htm 2. http:// www.fisicabiologica.com.ar/curso-criopreservacion-de-gametas-2009/papers/Cryopr 3. http:// www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg17395.html 4. http:// www.nature.com/nprot/journal/v6/n6/full/nprot.2010.144.html 5. http://en.wikipedia.org/wiki/Cryopreservation 6. http://revistas.inia.es/index.php/sjar/article/viewFile/88/85

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THANK YOU
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