Dr Siti Suri Lecture 10 Cyropreservation
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Transcript of 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