CRYOPRESERVATION

• Water is the major component in biological specimens,

usually by more than 50 %.
• In ordinary ice all of the water molecules are connected by
hydrogen bonds, six molecules forming a hexagonal ring
resembling that of cyclohexane.
• By means of hydrogen bonding the water molecules interact
with each other and with almost all molecules of biological
interest.
• The hydrogen bond energy contributes substantially to
solvation enthalpies.
• Liquid water is the most mobile component in biological
systems.
• The main task of cryofixation is to keep the water at the same
place it occupied at room temperature.
Fig. A) Lattice structure of hexagonal ice, showing the 6-
membered rings of water molecules. B) A structural element in
liquid water.
• Cryopreservation is the use of very low temperatures to
preserve structurally intact living cells and tissues.
• Unprotected freezing is normally lethal.
• Freezing injury-ice crystals pierce or tease the cells.
• It destroyes them by direct mechanical action, or that damage
is from secondary effects via changes in the composition of
the liquid phase.
• In the specific case of cryofixation, cooling of the specimen
gives rise to ultrastructural changes by destabilizing the
original structure in the sense of thermodynamics.
• At the same time, cooling tends to slow down any reaction
rates which might lead to artefication, and increases the
lifetime of the original structure by depriving the specimen of
the activation energy.
• Spermatozoa were the first mammalian cells to be
cryopreserved successfully.
• Cryoprotectants, simply by increasing the total concentration
of all solutes in the system, reduce the amount of ice formed
at any given temperature.
• Cryoprotectants should be biologically acceptable they must
be able to penetrate into the cells and have low toxicity.
• Many compounds have such properties like glycerol, dimethyl
sulfoxide, ethanediol, and propanediol.
• If the water permeability of the cell membrane is known it is
possible to predict the effect of cooling rate on cell survival.
• Ice can be avoided by vitrification - the production of a glassy
state that is defined by the viscosity reaching a sufficiently
high value to behave like a solid, but without any
crystallization.
• Cryopreservation is based on the ability of certain small
molecules to enter cells and prevent dehydration and
formation of intracellular ice crystals, which can cause cell
death and destruction of cell organelles during the freezing
process.
• Glycerol is used primarily for cryoprotection of red blood cells.
• DMSO is used for protection of most other cells and tissues.
• A sugar called trehalose, which occurs in organisms capable of
surviving extreme dehydration, is used for freeze-drying
methods of cryopreservation.
• Trehalose stabilizes cell membranes and it is particularly
useful for the preservation of sperm, stem cells and blood
cells.
• When cells are brought into a hypertonic glycerol medium,
water will leave the cells because of the osmotic pressure
difference.
• At the same time, glycerol will enter the cells, after a short
period of equilibration: cells will regain their original volume.
• The amount of ice formed is lower, the unfrozen fraction
remains larger, the degree of shrinkage of the cells is limited.
• The electrolyte concentration in the unfrozen solution and in
the cells will be relatively small.
• Most systems of cellular cryopreservation use a controlled-
rate freezer.
• This freezing system delivers liquid nitrogen into a closed
chamber into which the cell suspension is placed.
• Careful monitoring of the rate of freezing helps to prevent
rapid cellular dehydration and ice-crystal formation.
• In general, the cells are taken from room temperature to
approximately −90 °C (−130 °F) in a controlled-rate freezer.
• The frozen cell suspension is then transferred into a liquid-
nitrogen freezer maintained at extremely cold temperatures
with nitrogen in either the vapour or the liquid phase.
• Hematopoietic stem cells are collected from a patient’s bone
marrow prior to treatment with high-dose chemotherapy.
• Patient’s cryopreserved cells are thawed and infused back into
the body.
• The ability to cryopreserve hematopoietic stem cells has
greatly enhanced the outcome for the treatment of certain
lymphomas and solid tumour malignancies.
• In the case of patients with leukemia, their blood cells are
cancerous and cannot be used for autologous bone-marrow
rescue.
• So these patients rely on cryopreserved blood collected from
the umbilical cords of newborn infants or on cryopreserved
hematopoietic stem cells obtained from donors.
• Today there is intense interest in the growth of the cells in
tissue culture systems, as well as in the cryopreservation of
the cells for future therapy for a wide variety of disorders of
the nervous and muscle systems and diseases of the liver and
heart.
• Cells can live more than a decade if properly frozen.
• Certain tissues such as parathyroid glands, veins, cardiac
valves, and aortic tissue, can be successfully cryopreserved.
Freezing is also used to store and maintain long-term viability
of early human embryos, ova (eggs), and sperm.
• Research has shown that whole animals frozen in the absence
of cryoprotective agents can yield viable cells containing intact
DNA upon thawing.
• Nuclei of brain cells from whole mice stored at −20 °C (−4 °F)
for more than 15 years have been used to generate lines of
embryonic stem cells.
• With cryopreservation, water is made unavailable to the
bacteria by freezing and the dehydrated cells are stored at low
temperatures.
• Methods can be broadly classed according to the storage
temperature;
• -20 to -30°C is achievable with standard laboratory freezers.
• -70°C with ultra-low temperature freezers
• -140 to -196°C with liquid nitrogen.
• Storage of cells in the nitrogen vapor phase (-140°C) or the
liquid nitrogen phase (-196°C) is increasingly being used.
• Storage of cultures in the range of -60 to -80°C will often
result in good viability and may be used when liquid nitrogen
is not available or in noncritical applications where some loss
of culture viability can be tolerated.
• Temp. above -30°C give poor results because of the formation
of eutectic mixtures and hence the exposure of cells to high
salt concentrations.
Cryoprotectants
• Good solubility in water and low toxicity are two basic
requirements for cryoprotectant additives.
• Toxicity varies with temperature, concentration, time of
exposure, as well as with the particular cell being frozen.
• Toxicity of 10 percent DMSO on human lymphocytes at room
temperature for 30 min is minimal.
• DMSO and glycerol are used in the empirically determined
concentration range of 5 percent to 10 percent.
• The optimal concentration will depend on the cell, the
composition of the freezing mixture, and the cooling rate.
• Higher concentrations of DMSO and glycerol were more
effective with slower cooling rates.
Cryoprotectant removal
• In most cases, adequate dilution of cyroprotectant is a better
choice than complete removal at culture initiation.
• Adverse effects of DMSO on the recovery of cells were absent
at dilutions of tenfold or more when 5% DMSO was used in
the freeze mixture.
• DMSO will also evaporate from the medium at 37° C.
• Removal of the cryoprotectant the day after thawing by
changing the medium may yield poor results in some
circumstances.
• Changing the medium too soon could cause a substantial
reduction in the number of viable cells needed to start the
culture.
Thawing
• The thawing process is the reverse of freezing.
• In general, thawing may be as difficult to control in large
volumes as freezing.
• Thermal thawing may be conducted using convection heat
transfer from air or liquid, steam at a pressure below
atmospheric, heat transfer by conduction (heated plates or
blocks), heat pipes, or infrared radiation.
• Electrical thawing: resistance or dielectric or microwave
heating.
• The thawing process can be divided into the following steps:
• Heating the solid to a thawing temperature plateau
• Thawing (melting of ice)
• Heating the liquid to a desire temperature
Cryotechniques for microscopy
• Radiation damage structural (e.g. loss of crystallinity) or
compositional (e.g. mass loss) changes by the electron beam.
• It’s a serious factor in limiting the amount of microstructural
information that can be collected from an electron
microscope specimen.
• Damage may be reduced or minimized if the physical factors
that affect it are well understood.
• The photographs taken using a cryomicroscope show at –40°C
a uniform structure both in the already dried and in the frozen
part.
• The development of cryoelectron microscopy techniques has
added a new dimension to the problem of radiation damage
to organic and biological specimens.
• Cryomicroscope studies have the advantage of showing
pictures of the structural changes and the frozen product can
be freeze-dried in most instruments.
Fig. - Scheme of a cryomicroscope. 1 = Objective of the microscope; 2 = cover-glass;
3 = sample; 4 = sample support; 5 = electrical heating; 6 = cooling chamber with LN2
connection; 7 = vacuum connection.
• At the present time, low temperature scanning electron
microscopy for morphological purposes may be considered to
be well established and routine.
• Frozen-hydrated bulk samples prepared for SEM can show
details of fluid-filled and air-filled spaces which are not readily
apparent in fixed and dried specimens.
Freeze-drying
• Freeze-drying is a process in which frozen material is dried
through the sublimation of ice.
• Freezing and freeze-drying techniques have become standard
methods for the long-term maintenance of bacterial cultures.
• The procedure consists of the three stages:
• Freezing - Initially the material is frozen, causing a physical
separation of the water as ice from the solids.
• Sublimation - The ice is removed from the product by direct
conversion to vapor (sublimation), energy is required in the
form of heat.
• Desorption - Following the removal of ice crystals, what
remains of the product is a concentrated solute phase which
will become, at the end of the process, the freeze-dried
material.
• Storage of freeze-dried biological material is extremely cost
efficient as no expensive and bulky liquid nitrogen containers
are necessary.
• The material may be stored at ambient temperature and
unlike cryogenic storage, there is no risk of equipment
malfunction or of personal injury from liquid nitrogen.
• Freeze drying is useful for gene banking of genetic resources
with the objective of regenerating live animals and recovering
lost breeds.
• However, freeze drying generally reduces cell viability.
• Therefore, standard insemination procedures generally
cannot be used for freeze-dried sperm.
• However, freeze-dried sperm have been successfully used to
produce live offspring using ICSI in mice and rabbits.