Cryopreservation

Cryopreservation is a technique in which low temperature is used to preserve the

living cells and tissue. In this technique, tissues can be preserved for a very long time.
Cryopreservation is a process that preserves organelles, cells, tissues, or any other
biological constructs by cooling the samples to very low temperatures. The purpose of
cryopreservation is to store cells indefinitely by halting the cell’s metabolism with
ultralow temperatures. The freeze-thaw process is stressful to all cells and tissues. Therefore,
effective techniques were developed to prevent cell death and damage. The science that deals
with cryopreservation is known as “cryobiology”. It can be done over the following
temperature:

i. Solid carbon dioxide (at -79 oC)

ii. Low-temperature deep freezer (at -80 oC)
iii. In vapor phase nitrogen (at -150 oC)
iv. In liquid nitrogen (at -196 oC)

Organelles, cells, tissues, extracellular matrix, organs, and other biological

structures that are vulnerable to harm from uncontrolled chemical kinetics are
maintained by cooling to extremely low temperatures. Cryo-preservation or cryo-
conservation is the term for this process. The temperature which is normally used is
−80 oC (using solid carbon dioxide) or −196 oC (using liquid nitrogen). The main aim of
the Cryopreservation technique is to achieve low temperatures without incurring further
harm due to ice crystal formation during freezing. Cryopreservation is a process that
maintains biological samples in a state of suspended animation at cryogenic temperature
for any considerable period and is used to preserve the fine structure of cells. The
freezing behavior of the cells can be altered in the presence of a cryoprotective agent
(CPA; also called cryoprotectants), which affects the rates of water transport, nucleation,
and ice crystal growth.

Steps of Cryopreservation
The technique followed by the regeneration of plants involves the following steps:

a) Selection of Material: For cryopreservation, the selection of proper plant

material is important. Two important factors depend on it such as nature and
density. Any tissue can be selected for this purpose, for example embryo,
meristem, ovules seeds, etc. The density should be high.
b) Addition of Cryoprotectant: The chemical material is important as it prevents
cryo destruction. Some examples of cryoprotectants are alcohol, some amino
acids like proline, and dimethyl sulfoxide (DMSO). Mainly two cryoprotectants
should be used together instead of a single one as they are considered to be more
effective. DMSO being more common for mammalian cells may be used at a
concentration of up to 10% w/v (1.28M) and can be filtered for cell culture use. If
single addition of DMSO media causes toxicity then multi-step addition may be
used to gradually increase DMSO concentration and decrease osmotic damage.
Glycerol may act as a non-electrolyte to decrease electrolyte concentration in cell
freezing solutions and is most commonly used for preservation of
microorganisms, red blood cells and spermatozoa.

While CPA protects cells during the slow freezing process they can also
cause cell toxicity. The use of serum may help to protect the cells but this is not
recommended for clinical or commercial uses.
c) Freezing: Different species of plants show different types of sensitivity to low
temperatures. They are different types of methods:

i. Slow Freezing Method: In this process, the tissue or plant material is

slowly frozen at a slow cooling rate. The major advantage is that the
plant cells are partially hydrated and serve in a better manner. Slow
freezing first substitutes the water within the cytoplasm with CPA,
which reduces cell damage and adjusts the cooling rate in accordance
with the permeability of the cell membrane. Slow-cooling protocols
involve a typical cooling rate of about 1 °C/min in the presence of less
than 1.0 M of CPA, with use of a high-cost controlled-rate freezer or a
benchtop portable freezing container. The advantages of slow freezing
are that it has a low risk of contamination during the procedures and
does not demand high manipulation skills.

ii. Rapid Freezing Method: The vials are plunged in liquid nitrogen. In
this process, the temperature decreases from -300 to - 1000 degrees
rapidly. Vitrification is a process by which cell suspensions are
transformed directly from the aqueous phase to a gasseous state, after
direct exposure to liquid nitrogen. The process requires cooling of the
cells or tissues to deep cryogenic temperatures (i.e., with liquid
nitrogen) after their exposure to high concentrations of CPA (in the
ratio of 40 ‒ 60%, w/v), with subsequent rapid cooling to avoid ice
nucleation. A major advantage of vitrification is the low risk of freeze
 injury, thereby ensuring a sufficiently high cell survival rate.
iii. Dry Freezing Method: In this method hydrated cells and seeds are
stored.
d) Storage in Liquid Nitrogen: It is also important for the maintenance of the
sale or material at a specific temperature. In general, the temperature is kept –
70 °C to – 196 °C. Prolonged storage is done at the temperature of -196 °C in
liquid nitrogen. A continuous supply of nitrogen is needed to prevent
damage.
e) Thawing: The thawing process is usually carried out by plunging the vials
into a warm water bath with vigorous swirling. It also causes the vials to get
transferred or move to another bath at 0 °C
f) Washing & Reculturing: The preserved material is washed to remove the
cryoprotectant. Furthermore, the material is recultured in a fresh medium.
g) Measurement of Viability: Due to storage stress, there is a possibility of cell
death. The presence of viability can be seen in most cases. It is calculated by
the formula: No. of cells growing / No. of cells thawed ×100
h) Regeneration of Plants: After that, the viable seeds are cultured on a non-
specific growth medium. Suitable environmental conditions are maintained.

The major steps in Cryopreservation

1. The process of combining CPAs with cells or tissues before cooling

2. The freezing of cells or tissues at a low temperature, followed by their storage
3. The process in which cells or tissues are being warmed up
4. After freezing, the process of removal of CPAs from cells or tissues

Applications:
i. In Medical sciences
Cryopreservation gained prominence in human medicine after its use in
infertility treatment. Since then, gamete cryopreservation has been developed to combat
infertility.
Sperm was the first successfully frozen reproductive cell and remains the easiest
to freeze due to its tiny cytoplasm and thus low water content. Also, sperm nuclear
material is compressed and protected from damage. For these reasons, cryopreservation
of sperm cells is frequently used in human medicine today.
Live births from assisted reproductive cycles employing frozen semen or
embryos have been observed in recent years. Human oocytes and ovarian tissues have
also been cryopreserved. Studies and research on immunological memory lymphoid
cells, aortic root allografts, and osteoblasts for bone banking are still going on.
Human medicine is also now commonly performing cryopreservation of cornea,
umbilical cord, and hematopoietic cells, as well as sperm banking.
Cryopreservation of bull semen has been used to reproduce rare and threatened
species. Every year, more than 25 million bovine calves are artificially impregnated with
frozen-thawed bull sperm. Tissues, cell lines, DNA, and serum samples can also now be
kept in cryogenic banks.

ii. In Biological sciences

Cryopreservation is one of the most reliable strategies for preserving plant

genetic resources for the long term.
In agriculture, germplasm cryopreservation is used to improve domestic varieties'
genetics and adaptability to environmental changes. While the practice of preserving
plant germplasm in cryogenic temperatures is relatively new, scientists have been
developing cryopreservation procedures for plant cells and tissues for over 40 years
now.
It is an ideal method for long term conservation of material. Disease-free plants
can be conserved and propagated and recalcitrant seed can be maintained for a long
time. Endangered species can be maintained. Pollen can be maintained to increase
longevity.
Aquatic biotechnologies rely on cryopreservation of gametes, embryos, and embryonic
cells to propagate economically significant species, safeguard endangered species, and
maintain genetic variety.
The results of studies show that marine fish sperm cryopreservation is more successful
than freshwater fish cryopreservation and that fertilization rates are similar to
mammalian species.

Advantages of Cryopreservation
i. Cryopreservation boosts the efficiency of assisted reproductive treatments by
allowing all extracted and/or fertilized cells to be kept for future use.
ii. By freezing embryos between cycles, ovarian stimulation is not required each
time, and if the woman's ovaries are overstimulated, implantation can be
postponed without squandering retrieved oocytes.
iii. Cryopreservation allows couples who conceive in their first treatment cycle
to contribute their unused frozen embryos to research.
iv. It is currently common to implant only one or two embryos, with any
remaining embryos being cryopreserved for future treatment cycles.
 v. Cryopreservation allows people who are losing their fertility to keep their
reproductive cells and maybe conceive via aided methods in the future.
Women who want to delay childbearing or have a family history of early
menopause may use it.
vi. Cryopreservation is a powerful tool for preserving endangered species'
germplasm. It can also help to maintain plant fertility.
vii. Once the material is successfully conserved at a particular temperature, it can
be preserved identifiably.
viii. No change or contamination of fungus or bacteria takes place after the
storage process is completed and material is preserved.
ix. Minimal space is required for the purpose of cryopreservation.
x. Minimal labor is required for the purpose of cryopreservation.

Cryopreservation of Animal Cells

The development of animal cell lines is expensive, time-consuming, and labor-
intensive. The continuous cell line has several advantages of over fertilizers cell lines
such as:
i. They survive indefinitely.
ii. They grow more rapidly.
iii. They can clone more easily.

Cryopreservation of Plant Cell

Due to the gradual disappearance of economic and rare species the necessity for
storage of genetic resources increases. The convent journal method of the storage fails to
prevent losses caused by:

i. Attack of pathogen and pest

ii. Climatic disorders
iii. Natural disorder
iv. Political and economic causes

The material to be preserved is stored at low temperatures due to which growth

rate of cells retards. Consequently, biological activities are reserved for a long period of
time.