Cryopreservation and Reconstitution of Preserved Cell Lines

by:Abdulrahman Mohammed

L-2012-V-21-D

Introduction

Cryopreservation: Storage of live material at temperatures so low, that all biological processes are suspended (-196°C) and material does not decompose.

Cryopreservation allows virtually indefinite storage of biological material without deterioration over a time scale of at least several thousands of years.

Techniques are available for the preservation of microorganisms, isolated tissue cells, small multicellular organisms, and even more complex organisms such as embryos.

Introduction cont.….

Cell lines

finite life, senesce after approximately thirty cycles of division

usually diploid and maintain some degree of differentiation.

it is essential to establish a system to maintain such lines for long periods

Benefits Of Freezing Cells

Cryopreservation minimizes:

genetic change

Senescence leading to extinction of cell line

Transformation to tumor related properties

Contamination

Distribution to others

Saving reagents, time

Equipment failure such as incubator

Cross-contamination by other cell lines

CRYOPROTECTANT:

Cryoprotectants (CPs) are macromolecules

added to the freezing medium to protect thecells from the detrimental effects ofintracellular ice crystal formation or from thesolution effects, during the process of freezingand thawing.

Solution effects : Forms of injury to cells, cooled slowly enough to prevent damaging intracellular ice crystal formation .

CHARACTERISTICS OF CRYOPROTECTANT:

Higher degree of cell survival during freezing.

TYPES OF CRYOPROTECTANTS

Permeating CPs: ( INTRACELLULAR)

Non permeating CPs: (EXTRACELLULAR)

MECHANISM OF CRYOPROTECTANTS

Cryoprotectants acts by salt buffering action

(bind water and less ice crystal formed), gives

more time to cell for de-hydrate and interact

with cell membrane to make it less brittle

during freezing.

PERMEATING CPS :

Lower the freezing point of the solution, increase the viscosity and thus reduce diffusion in solutions.

Replace intracellular water to prevent the formation of large ice crystals intracellularly.

Ability to reduce the amount of water which freezes as ice.

Reduce exposure of cell to elevated salt and solute concentration.

Glycerol (GLY), Dimethyl Sulfoxide (DMSO),Propylene glycol (PG), and Ethylene glycol(EG).

NON- PERMEATING CPS: (EXTRACELLULAR)

LOW MOLECULAR WEIGHT

It changes the water balance of cells by shrinking cells before freezing.

Sucrose, Maltose, Trehalose, Sorbitol and Acetamide.

HIGH MOLECULAR WEIGHT

Closing the cell membrane defect.

Repair damaged cell membrane post thawing.

Polyvinyl Pyrorolidone (PVP), Dextran, Serum,BSA

Ethylene glycol is preferred cryoprotectant for cryopreservation of llama embryo.

CRYOPROTECTANT MOLECULAR WEIGHT

Ethylene glycol 62.07

Propylene glycol 76.10

DMSO 78.13

Glycerol 92.10

Theoretical Background Of Cell Freezing

Optimal Cell Freezing Is Characterized By

Maximum number of viable cells upon thawing

Minimum intracellular crystal formation

Minimum formation of foci of high solute concentration

Optimal Freezing Is Accomplished By

Cooling slowly so water escapes

Cooling fast enough to avoid crystal formation

Use hydrophylic cryoprotectant

Storing at lowest possible temperatures

minimize negative effect of solute foci on proteins

Thaw rapidly

minimize crystal growth and solute gradients

High Cell Concentration Seems To Enhance Survival Possibly due to “leakiness” effect from cryogenic damage

Centrifugation is avoided since dilution of cryoprotectant is high when reseeding Ex. 1 mL of 1x107 cells diluted to 20 mL volume giving a 5x105 cells/mL. If

cryoprotectant was 10% it will become 0.5%

Toxicity is unlikely at 0.5%

Residual cryoprotectant can be removed as soon as cells start growing

Freezing Medium DMSO, Glycerol

DMSO used at 5-15%, 10% is common

DMSO should be stored in glass or polypropyleneCan dissolve rubber and some plastics leading to impurities

Many laboratories increase FBS concentration to 40, 50 or 90%

Cell Concentration And Freezing Medium

Optimal Cooling Rate: 1°C/minute

Compromise between fast freezing minimizing crystal formation and slow allowing for extracellular water migration

Cooling Curve Is Affected By

Ambient temperature

Insulation

Specific heat of ampoule contents, volume of ampoule

latent heat absorption during freezing

Cooling Rate

Insulation During Freezing Use 2 Polystyrene Foam Boxes To Store Vials

This set up provides insulation for 1°C/minute cooling

Place In A –80°C Freezer

Transfer To Liquid Nitrogen After 24 Hrs Or -150°C Freezer

Liquid Nitrogen Is Widely Used To Store Cells Long Term

-196 °C

Several types of liquid nitrogen cryofreezers are available

Use polypropylene cryovials

Resistant to cracking

Some Repositories Prefer Glass

Better properties for long term storage

Labeling Is Very Important

Stored cells can outlive you! Proper labeling is essential

In your label include

Cell type, date and cell number

Use an alcohol resistant marker

Ampoules

Protocol-Suspension Cultures

1. Count the number of viable cells to be cryopreserved. Cells should be in log phase.

• Centrifuge the cells at ~200 to 400 × g for 5 min to pellet cells. Using a

pipette, remove the supernate down to the smallest volume without disturbing the cells.

2. Resuspend cells in freezing medium at 1 × 107 to 5 × 107 cells/ml for serum-containing medium, or 0.5 × 107 to 1 × 107 cells/ml for serum-free medium.

3. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.

4. Cells are frozen slowly at 1°C/min. This can be done by programmable coolers or by placing vials in an insulated box in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.

Protocol-Adherent Cultures

1. Detach cells from the substrate with dissociating agents.

Detach as gently as possible to minimize damage to the cells (see Dissociation of Cells from Culture Vessels).

2. Resuspend the detached cells in complete growth medium and establish the viable cell count.

3. Centrifuge at ~200 × g for 5 min to pellet cells. Using a pipette, withdraw the supernate down to the smallest volume without disturbing the cells.

4. Resuspend the cells in freezing medium at 5 × 106 to 1 × 107 cells/ml.

5. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.

6. Cells are frozen slowly at 1°C/min. This can be done by programmable

coolers or by placing vials in an insulated box in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.

Thawing Of Cells Should Be Rapid

Water bath @ 37°C

Reason: minimize crystal formation

Spray Vial With Alcohol To Avoid Contamination

Dilution Should Be Done Slowly

DMSO will cause severe osmotic damage if done fast

Most Cells Do Not Require Centrifugation

Some Do Require Centrifugation

Ex. suspension growing cells

Thawing Stored Ampoules

Reconstitution (thawing) of preserved cell lines

Cryopreserved cells are fragile and require gentle

handling. Cryopreserved cells are thawed quickly

and plated directly into complete growth medium.

If cells are particularly sensitive to cryopreservative

(DMSO or glycerol), they are centrifuged to remove

cryopreservative and then plated into complete

growth medium.

The following are suggested procedures for thawing

cryopreserved cells.

A) Direct Plating Method:

1. Remove cells from storage and thaw quickly in a 37°C water bath.

2. Plate cells directly with complete growth medium.

Use 10 to 20 ml of complete growth medium per 1 ml of frozen cells. Perform a viable cell count.

Cell inoculum should be at least 3 × 105 viable cells/ml.

3. Culture cells for 12 to 24 h. Replace medium with fresh complete growth medium to remove the cryopreservative.

Reconstitution (thawing) of preserved cell lines

Reconstitution (thawing) of preserved cell lines

B) Centrifugation Method

1. Remove cells from storage and thaw quickly in a

37°C water bath.

2. Place 1 to 2 ml of frozen cells in ~25 ml of complete

growth medium. Mix very gently.

3. Centrifuge the cells at ~80 × g for 2 to 3 min.

4. Discard supernatant.

5. Gently resuspend the cells in complete growth

medium and perform a viable cell count.

6. Plate the cells. Cell inoculum should be at least 3 ×

105 viable cells/ml.

Reference

o 1. Freshney, R. I., 1994. Culture of Animal Cells: A Manual of Basic Technique, pages 254-263. (3rd edition); Wiley-Liss, New York.

o 2. Hay, R. J., 1978. Preservation of Cell Culture Stocks in Liquid Nitrogen, pages 787-790. TCA Manual 4.

o 3. Schroy, C. B., and P. Todd, 1976. A Simple Method for Freezing and Thawing Cultured Cells, pages 309-310. TCA Manual 2, Procedure Number 76035.

o 4. Shannon, J. E. and M. L. Macy, 1973. Freezing, Storage, and recovery of Cell Stocks, pages 712-718. In Tissue Culture: Methods and Applications. P. F. Kruse and M. K. Patterson Jr. Eds. (Academic Press, New York).

o 5. Waymouth, C. and D. S. Varnum, 1976. Simple Freezing Procedure for Storage in Serum-free Media of Cultured and Tumor Cells of Mouse, pages 311-313. TCA Manual 2, Procedure Number 76165.

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