Tuesday, 13 December 2016

Technique of Cryopreservation (Biotechnology)



Introduction : To ensure reproducible results and continuity in research and biomedical processes, today's scientists are faced with the task of genetically stabilizing replicable materials such as living cells and organisms, and ensuring sub-cellular components such as nucleic acids and proteins are preserved unchanged. Serial subculturing result in contamination or genetic drift. Improper storage and handling of non-replicable materials can lead to divergent and irreproducible research results. However, a population of living cells or a suspen­sion of subcellular components can be stabilized by subjecting them to cryogenic temperatures which, for all practical purposes, stops time. The process of stabilizing biological materials at ultra low (-196oC) temperature is called cryopreservation. Techniques are available for the preserva­tion of microorganisms, tissues, primary cells, established cell lines, small multicellular organisms, complex cellular structures such as embryos, as well as nucleic acid and proteins.

     Mechanism/Process involved in Cryopreservation.
The cryopreservation process involves complex phenomena that, even after decades of research, are not fully understood. Cryobiological studies have led to speculation on what occurs during the freez­ing of living cells and how adverse phenomena can be overcome. Since water is the major component of all living cells and must be available for the chemical processes of life to occur, cellular metabolism stops when all water in the system is converted to ice. Ice forms at different rates during the cooling process. Slow cool­ing leads to freezing external to the cell before intracellular ice be­gins to form. As ice forms external to the cell, water is removed from the extracellular environment and an osmotic imbalance occurs across the cell membrane leading to water migration out of the cell. The increase in solute concentration outside the cell, as well as intracellularly as water leaves the cell, can be detri­mental to cell survival. if too much water remains inside the cell, damage due to ice crystal formation and recrystallization during warming can occur and is usually lethal.

     Cryoprotective Used in Cryopreservation.
Many compounds have been tried as Cryoprotective agents, either alone or in combination, including sugars, solvents and even serum. Although there are no absolute rules in cryopreservation, glycerol and DMSO have been widely used and traditionally have been demonstrated to be the most effective agents for preserving living cells and organisms. Other cryoprotectants that have been used occasionally, either alone or in combination include: poly­ethylene glycol, propylene glycol, glycerine, polyvinylpyrolidone, sorbitol, dextran and trehalose.
Cryoprotective agents serve several functions during the freezing process. Freezing point depression is observed when DMSO is used which serves to encourage greater dehydration of the cells prior to intracellular freezing. Cryoprotective agents also seem to be most effective when they can penetrate the cell, delay intracellular freezing and minimize the solution effects. The choice of a Cryoprotective agent is dependent upon the type of cell to be preserved. For most cells, glycerol is the agent of choice because it is usually less toxic than DMSO. However, DMSO is more penetrating and is usually the agent of choice for larger, more complex cells such as protists. The Cryoprotective agent should be diluted to the desired concentration in fresh growth medium prior to adding it to the cell suspension. This minimizes the potentially deleterious effects of chemical reactions, and assures a more uniform exposure to the Cryoprotective agent when it is added to the cell suspension, reducing potential toxic effects. DMSO and glycerol are generally used in concentrations ranging from 5-10% (v/v), and are not usually used together in the same suspension with the exception of plant cells.

Preservation of Some Cell Types/ Genetic Material
Plant Cells: Plant cells respond to cryopreservation in a manner similar to other cells. The stage in the growth cycle from which they are harvested can affect their recovery, most optimum being late log phase. Also, cell density may play a role in recovery, the optimum cell density depending on the species being preserved. Slow warming is just as effective in some cases. Vitrification can also be used to preserve plant cells by using concentrated cell suspensions and rapid rates of cooling. Hardening of plants leads to greater tolerance of stressful condi­tions, such as experienced during the freezing process. Plants produce increased quantities of some compounds such as sugars and even glycerol which contribute to protecting the cells from osmotic stress during freezing. Undifferentiated callus tissue is often preserved in an effort to stabilize characteristics that can be affected by continued cultivation. Preservation of seeds is also an acceptable method of stabilizing plant germplasm, and the most common method is storage at low humidity and cool temperatures. However some seeds are tolerant of the increased desiccation associated with freezing and cryogenic storage, and can be stored at liquid nitrogen temperatures.

Viruses: Most viruses can be frozen as cell-free preparations without dif­ficulty and do not require controlled cooling. The exceptions are those viruses cultured in viable infected cells which require controlled cooling. For cell-adapted viruses the preservation pro­cess should be applicable to survival of the host cell. When viruses are harvested from eggs, the high protein content of the allantoic fluid or yolk sac provides protection during the freezing process. Plant viruses can be preserved either in infected plant tissue or as purified virus preparations. The virus preparations are suspended in DMSO or another cryoprotectant prior to freezing. Recovery is generally best when the cooling rate is controlled, although most plant viruses will tolerate a rapid freezing procedure. Recovery of plant viruses simply involves thawing in a warm bath, followed by inoculation into the appropriate plant host.

Embryo: Embryos have been preserved both by controlled cooling and vitrification. Recovery depends on the stage of embryonic development, and is measured by successful implantation leading to fetal development.

Step-by-Step procedure for Cultured Cells
1.     Harvest cells from late log or early stationary growth. Scrape cells from the growth surface if they are anchorage dependent. Centrifuge broth or anchorage independent cultures to obtain a cell pellet, if desired.
2.     Prepare presterilized DMSO or glycerol in the concentration desired in fresh growth medium. When mixing with a suspension of cells, prepare the Cryoprotective agents in twice the desired final concentration.
3.     Add the cryoprotectant solution to the cell pellet or mix the solution with the cell suspension. Begin timing the equilibration period.
4.     Gently dispense the cell suspension into vials.
5.     Begin cooling the cells after the appropriate equilibration time.
a. Uncontrolled cooling-- places the vials on the bottom of a -60°C freezer for 90 minutes.
b. Semi-controlled cooling--use Mr. Frosty freezing container to freeze the vials in a 70°C freezer.
c. Controlled cooling--use a programmable cooling unit to cool the cells at 1°C per minute to -40°C.
6.  Remove the cells from the cooling unit and place them at the appropriate storage temperature.
7.    To reconstitute, remove a vial from storage and place into a water bath at 37°C. When completely thawed, gently transfer the entire contents to fresh growth medium.
References
Baust, J.M. 2002. Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell Preservation Technology 1:17-31
Mazur, P. 1984. Freezing of living cells: mechanisms and implications. Am J. Physiol. 247: 125-142.
Mazur, P., S.P. Leibo and E.H.Y. Chu. 1972. A two factor hypothesis of freezing injury. Experimental Cell Research71:345-355.
Simione, F.P. Cryopreservation: Storage and Documentation Systems, In: Biotechnology: Quality Assurance and Validation, Drug Manufacturing Technology Series, Vol. 4, Interpharm Press, Buffalo Grove, Illinois, 1999, pgs. 7-31.
Withers, L.A. 1985. Cryopreservation of cultured plant cells and protoplasts. In: K.K. Kartha, Ed. Cryopreservation of Plant Cells and Organs, CRC Press, Inc., Boca Raton, Florida.

Article compiled by
Pravin B. Berad (Ph.D. Scholar)  
Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola (M.S.)


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