1. Cryopreservation and Reconstitution of
Preserved Cell Lines
Abdulrahman Mohammed
L-2012-V-21-D

2. 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.

3. 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

4. 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

5. 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 .

6. 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.

7. TYPES OF CRYOPROTECTANTS
 Permeating CPs: ( INTRACELLULAR)
 Non permeating CPs: (EXTRACELLULAR)

8. 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.

9. 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).

10. 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

11.  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

14. 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

15. Cell Concentration And Freezing Medium
 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%

16. Cooling Rate
 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

18. 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

19. Ampoules
 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

20. 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.

21. 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.

22. Thawing Stored Ampoules
 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

23. 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.

24. Reconstitution (thawing) of preserved cell lines
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.

25. 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.

26. 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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