dr siti suri lecture 10 cyropreservation

8/14/2019 Dr Siti Suri Lecture 10 Cyropreservation

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CRYOPRESERVATION

Purpose of cell preservation:Genotypic drift due to genetic instabilitySenescence and the resultant extinction of the cell lineTransformation of growth characteristics and acquisitionof malignant-associated properties

Phenotypic instability due to selection anddedifferentiationContamination by microorganismCross-contamination by other cell lines

Misidentification due to careless handlingIncubators failuresSaving time and materials maintaining lines not inimmediate useNeed for distribution to other users


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Requirement before cell freezing:

Acquisition

Finite cell line ; early passage


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Requirement before cell freezing: continue

Validation

Provenance; record the origin, history , propertiesAuthentication; check characteristic against origin

and provenanceTransformation; determine transform status(growth, genetic, structural, neoplastic)Contamination; microbial, mycoplasmaCross-contamination and misidentification; criteria toconfirm identity.


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Principle of cryopreservation

Optimal freezing of cells for maximum viable cell recovery on

thawing depends on minimizing intracellular ice crystalformation and reducing damage from foci of high concentratesolutes formed when intracellular water freezes.

Factors:

Freezing slowlyHydrophilic cryoprotectant to sequester water

Storing the cells at lowest temperatureThawing rapidly


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Frozen the cell at high cell concentration sufficient toallow dilution 1:10 or 1:20 to dilute the cryoprotectant;Normal subculture 1x 105/ml, freeze 1x 107/ml.

Choices of freezing medium (cryoprotectant); Glycerol,dimethyl sulfoxide (DMSO), polyvinylpyrrolidone (PVP),polyethylene glycol (PEG), hydroxyethyl starch (HES)

and serum.Optimal Cooling rate is at 1oC/minA control cooling is achieved by insulating the ampouleand kept at -70oC or -90oC for at least 4 hours before

transferring to the liquid nitrogen freezer (-196oC ).


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Freezing

Cells


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Ampoules on Cane

Plastic ampoules are clipped onto an aluminum cane, enclosed

in a cardboard tube, and placed inside an insulating foam tube.

The insulating tube is plugged at either end with cotton or

another suitable insulating material.

Aluminum

Cane

Cardboard

Tube

Insulating Foam Tube


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o

C

Freezing CurveRecord of the

fall in

temperature inan ampoule

containing

medium, clipped

with five other

ampoules on an

aluminum cane,

enclosed in a

cardboard tube,

placed within apolyurea-foam

tube and placed

in a freezer at

70

o

C


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Cryofreezeers

Narrow neck; reduce rate of evaporation, wide neck

Storage system; cane systems uses ampoules,rectangular drawerAmpoules; plastic and glass

Little cell deterioration found at -196oC but significantdeterioration (5-10% per annum) may occur at -70oC

Freezer records contains:

InventoryFree storage spaceA cell strain index (origin and history)


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Nitrogen Freezer Design


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Thawing stored ampoules

Thawed rapidly by using warm water.Dilute the cells slowlyCentrifugation (optional)Incubate in incubatorChange media on the following day


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ThawingCells


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CELL BANK

Function for storage and distribution of validated cell lines.

ATCC; American Type Culture Collection (ATCC)ECACC; European Collection of cell culturesCCR, Coriell Cell RepositoriesDSMZ; German Resource Centre for biological MaterialsHSRRB;Health Science Research Resources Bank (Japan)JCRB, Japanese Collection of Research BioresourcesRiken


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TRANSPORTING CELLSCultures is transfer from one lab to another lab as frozenampoules or as living cultures.

Shipping frozen ampoulesSolid CO2; 5kg up to 3 days.Thick walled polystyrene foam containerPolypropylene Centrifuge tube

Shipping living culturesmid- to late log phase as confluent or post confluent willexhaust the medium rapidlypolyethylene baginflated platic bagLabel Fragile DO NOT FREEZE


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Transportation Container for Cells

dr siti suri lecture 10 cyropreservation

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