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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