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 the cells from the detrimental effects of intracellular ice crystal formation or from the solution effects, during the process of freezing and 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.

Lower the freezing point .

Protect cell membrane from freeze-related injury.

High solubility.

Low toxicity at high concentrations .

Low molecular weight . Ability to interact with water via hydrogen

bonding.

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 polypropylene

Can 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.

THANK YOU FOR LISTENINGTHANK YOU FOR LISTENINGTHANK YOU FOR LISTENING