Dr Siti Suri Lecture 10 Cyropreservation

CRYOPRESERVATION   Purpose of cell preservation:  •Genotypic drift due to genetic instability •Senescence and the resultant extinction of the cell line •Transformation of growth characteristics and acquisition of malignant-associated properties •Phenotypic instability due to selection and dedifferentiation •Contamination by microorganism •Cross-contamination by other cell lines •Misidentification due to careless handling •Incubators failures •Saving time and materials maintaining lines not in immediate use •Need for distribution to other users

Requirement before cell freezing: Acquisition •Finite cell line ; early passage <5 subcultures •Continuous cell line; clone, select , characterize

  Standardization

•Medium; optimal medium •Serum (if used); batch number •Substrate; type and supplier

Requirement before cell freezing: continue Validation •Provenance; record the origin, history , properties •Authentication; check characteristic against origin and provenance •Transformation; determine transform status (growth, genetic, structural, neoplastic) •Contamination; microbial, mycoplasma •Cross-contamination and misidentification; criteria to confirm identity.

Principle of cryopreservation Optimal freezing of cells for maximum viable cell recovery on thawing depends on minimizing intracellular ice crystal formation and reducing damage from foci of high concentrate solutes formed when intracellular water freezes. Factors: •Freezing slowly •Hydrophilic cryoprotectant to sequester water •Storing the cells at lowest temperature •Thawing rapidly

Frozen the cell at high cell concentration sufficient to allow 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/min A control cooling is achieved by insulating the ampoule and kept at -70oC or -90oC for at least 4 hours before transferring to the liquid nitrogen freezer (-196oC ).

Freezing Cells

Ampoules on Cane Insulating Foam Tube

Cardboard Tube

Aluminum 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.

Record of the fall in temperature in an ampoule containing medium, clipped with five other ampoules on an aluminum cane, enclosed in a cardboard tube, placed within a polyurea-foam tube and placed in a freezer at – 70oC

Freezing Curve

C

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Cryofreezeers •Narrow neck; reduce rate of evaporation, wide neck •Storage system; cane systems uses ampoules, rectangular drawer •Ampoules; plastic and glass Little cell deterioration found at -196oC but significant deterioration (5-10% per annum) may occur at -70oC Freezer records contains: •Inventory •Free storage space •A cell strain index (origin and history)

Nitrogen Freezer Design

Thawing stored ampoules •Thawed rapidly by using warm water. •Dilute the cells slowly •Centrifugation (optional) •Incubate in incubator •Change media on the following day

Thawing Cells

CELL BANK Function for storage and distribution of validated cell lines. •ATCC; American Type Culture Collection (ATCC) •ECACC; European Collection of cell cultures •CCR, Coriell Cell Repositories •DSMZ; German Resource Centre for biological Materials •HSRRB; Health Science Research Resources Bank (Japan) •JCRB, Japanese Collection of Research Bioresources •Riken

TRANSPORTING CELLS Cultures is transfer from one lab to another lab as frozen ampoules or as living cultures. Shipping frozen ampoules •Solid CO2; 5kg up to 3 days. •Thick –walled polystyrene foam container •Polypropylene Centrifuge tube Shipping living cultures •mid- to late log phase as confluent or post confluent will exhaust the medium rapidly •polyethylene bag •inflated platic bag •Label “Fragile” DO NOT FREEZE

Transportation Container for Cells