Biological Techniques 1

Contents
Topic: Cryopreservation
Cryo-Preservation .................................................................................................. 2

Definition............................................................................................................... 2

History of Cryopreservation ................................................................................... 2

Introduction ........................................................................................................... 3

Natural cryopreservation ........................................................................................ 3

Conditions Required for Cryopreservation ............................................................ 4

Process of cryopreservation ................................................................................... 7

Applications of Cryopreservation .........................................................................11

1) Embryos ...................................................................................................11

2) Ovarian tissue ...........................................................................................11

3) Oocytes.....................................................................................................12

4) Semen .......................................................................................................12

5) Testicular tissue ........................................................................................13

Advantages of Cryopreservation ...........................................................................15

Challenges of Cryopreservation ............................................................................15

Conclusion ............................................................................................................17

References ............................................................................................................18

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 Biological Techniques 2

Cryo-Preservation

Definition
Cryo is Greek word Kayos meaning frost. It literally means preservation in “frozen or cold state”.
Cryo-preservation or cryo-conservation is a process in which organelles, cells, tissues,
extracellular matrix, organs, or any other biological constructs susceptible to damage are preserved
by cooling to very low temperatures (typically −80 °C using solid carbon dioxide or −196 °C using
liquid nitrogen). At low enough temperatures any enzymatic or chemical activity which might
cause damage to the biological material is effectively stopped.

Fig: Liquid Nitrogen Tanks

History of Cryopreservation

• In 1949 Ernest John Christopher Polge: He was the first person to solve the problem of
how to preserve living cells and tissues at very low temperatures. He accidentally discovered
the cryoprotective properties of glycerol on fowl sperm. Polge reported high pregnancy rates
in cattle using sperm that had been frozen for periods in excess of a year. His work had far-
reaching consequences for the future of artificial insemination and genetic improvement in
livestock.
• In 1953 Jerome K. Sherman: He was a doctoral candidate at the University of Iowa. His
research led him to successfully freezing and thawing human sperm. In 1953, he founded the
world’s first sperm bank, and it was from that pioneering bank that the first human birth from
cryopreserved sperm was recorded.

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• In 1964 cryobiology invention: In 1964 the term cryobiology was invented. It can be literally
translated as cryo mean cold, bios mean life, and logos mean science. Cryobiology is the
science of how biological activity and its architecture is affected at low sub-zero temperatures.
Storage by cryopreservation, will be in the – 80 °C to – 196 °C temperature range.
• In 1983 Alan Trounson: Alan Trounson is credited for successfully achieving a pregnancy
after freezing early human embryos one to three days after fertilization.
• In 1986 Christopher Chen: 1986 Christopher Chen, another Australian biologist, was the
first to successfully freeze and thaw human oocytes. A twin pregnancy was achieved after
insemination and replacement in utero.
• In 1988 Yves Menezo and Edouard Servy M.D.: Obtained in Augusta Georgia the first
viable pregnancies following blastocyst culture with fresh and frozen transfers in the USA.

Introduction
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. Numerous research papers on cryopreservation have studied the underlying
physical and biological factors affecting the survival of cells at low temperatures during the
cooling and warming processes. Cryopreserved cells or tissues possess some advantages for basic
research and current and future clinical applications. With the constant availability of
cryopreserved cells and tissues, extensive quality testing can be performed to determine the
suitability of the cells or tissue for transplantation without the need to obtain fresh samples. The
successful cryopreservation of cells and tissues has been gradually increasing in recent years with
the use of CPAs and temperature control equipment.

Natural cryopreservation
Water-bears, microscopic multicellular organisms, can survive freezing by replacing most of their
internal water with the sugar trehalose, preventing it from crystallization that otherwise damages
cell membranes. Mixtures of solutes can achieve similar effects. Some solutes, including salts,
have the disadvantage that they may be toxic at intense concentrations. In addition to the water-
bear, wood frogs can tolerate the freezing of their blood and other tissues. Urea is accumulated in

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tissues in preparation for overwintering, and liver glycogen is converted in large quantities to
glucose in response to internal ice formation. Both urea and glucose act as "cryoprotectants" to
limit the amount of ice that forms and to reduce osmotic shrinkage of cells. Frogs can survive
many freeze/thaw events during winter if no more than about 65% of the total body water freezes.

Conditions Required for Cryopreservation

1. Freezing Medium: Always use the recommended freezing medium for cryopreserving your
cells. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol.
2. Temperature: Storage at very low temperatures is presumed to provide an indefinite longevity
to cells, although the actual effective life is rather difficult to prove. However, only few
biological materials can be frozen to (-196 degrees) without affecting the cell viability. The
principle is to bring plant cells or tissues to a zero metabolism and non dividing state by
reducing the temperature in the presence of cryoprotectant.

It can be done:

• Over solid carbon dioxide (at-79 degree)

• Low temperature deep freezer (at-80 degree)
• In vapour phase nitrogen (at-150 degree)
• In liquid nitrogen (at -196 degree)

While refrigerators, freezers and extra-cold freezers are used for many items, generally the ultra-
cold of liquid nitrogen is required for successful preservation of the more complex biological
structures to virtually stop all biological activity.

3. Cryoprotectants: Traditional cryopreservation has relied on coating the material to be frozen

with a class of molecules termed cryoprotectants.
4. Cryoprotective agents CPAs: The CPA, which is usually a fluid, reduces the freezing injury
from the cryopreservation process. Various CPAs have been developed and are used to reduce
the amount of ice formed at any given temperature, depending on the cell type, cooling rate,
and warming rate. In order to achieve the best survival rate of cells and tissues, the sample

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volume, cooling rate, warming rate, and CPA concentrations should be optimized depending
on the different cell types and context of tissues. CPAs can be divided into two categories:

1) Cell membrane-permeating cryoprotectants, such as dimethyl sulfoxide (DMSO),

glycerol, and 1,2-propanediol

•Glycerol: Sterile glycerol, is used at a final concentration of 10% in the medium. Glycerol
is a nonelectrolyte and thus may act by reducing the electrolyte concentration in the
residual unfrozen solution 4in and around a cell at any given temperature. It is widely used
in the storage of bacteria and animal sperm.

•DMSO: The primary choice is sterile dimethyl sulfoxide (DMSO) at a final concentration
of between 7% and 10%, with ten percent being optimum in most cases. It has been
commonly used for the cryopreservation of cultured mammalian cells because of its low
cost and relatively low level of cytotoxicity. Like glycerol, DMSO acts by reducing the
electrolyte concentration in the residual unfrozen solution in and around a cell at any given
temperature. However, a decline in the survival rate and the induction of cell differentiation
caused by DNA methylation and histone alteration have been reported. These negative
effects of DMSO in cryopreservation create some difficulties for its use in routine clinical
applications.

2) Nonmembrane-permeating cryoprotectants, such as 2-methyl-2,4-pentanediol and

polymers such as polyvinyl pyrrolidone, hydroxyethyl starch, and various sugars.

• Polymers: Vinyl-derived polymers, such as polyethylene glycol, polyvinyl alcohol, and

hydroxyethyl starch have a capacity to decrease the size of formed ice crystals.
• Proteins: Sericin is a water-soluble sticky protein (∼30 kDa) isolated from the silkworm
cocoon and has been developed as a fetal bovine serum- or DMSO-replacing CPA for human
adipose tissue-derived stem or progenitor cells, or hepatocytes. Small antifreeze proteins
derived from marine teleosts or fishes have also attracted attention as CPAs.
• Cell Banker series: A newly developed Cell Banker series allows for rapid cell
cryopreservation at −80 °C, and has been shown to achieve better survival rates following
freezing and thawing. The Cell Banker series of cryopreservation media contain 10%

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DMSO,glucose, a prescribed high polymer, and pH adjustors.33 Serum-containing Cell

Bankers 1 and 1+ can be used for the cryopreservation of almost all mammalian cells.
Advantages of Cryoprotectants

• Helps the material from rapid cooling

• Prevents from formation of ice in the intracellular region.
• When the cell undergoes high concentration of solute it helps to prevent from dehydration.
• Helps the cell to function even after the rewarming.

Why cells are Freezed?

• cells are stored for future study.

• Cell storage acts as an insurance policy in case of contamination, failure, or cell shortage.

• Freezing and storage of cells is a crucial step to ensure long-term cell use and reproducible results.

• Save time and money. Obtaining new cells is costly and time-consuming.

Fig: Cryogenically Preserved Samples

How much Does it Cost?

Cryopreservation process is expensive. Fees starts at $28,000 for body organs preservation to
$200,000 for whole body preservation.

Why do people go for it?

• Fear of Death

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• Love of life

• Hope for a cure of their disease

• Curiosity about their future

• Or even wanting to be immortal

Process of cryopreservation

Fig: Cryopreservation Process

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. The most
important stages of the survival of samples is the:

• Selection of material
• Addition of cryoprotectant
• Vitrification(freezing)
• Storage in liquid medium
• Thawing
• Washing and reculturing
• Measurement of viability
• Regeneration

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Selection of material: Selection of proper material or sample is important.it depends on two

factors:

• Nature
• Density

Density should be high. Any tissue can be selected e.g. embryo, ovules, seed etc.

Fig: Samples are grown, collected, vitrified, labeled and stored, retrieved, and analyzed

Addition of cryoprotectant: They are the chemicals which decrease cryodestruction.

cryodestruction refers to the formation of large ice crystals inside cells that rupture cells and cell
organelles.it is of two types:

Intracellular cryoprotectant

• Glycerol
• dimethyl sulfoxide (DMSO)

Extracellular cryoprotectant

• Polyvinyl pyrolidone
• 5-10% protectant

Cryopreservation is based on ability of small molecules to enter cells and prevent dehydration and
formation of intracellular ice crystals, which cause cell death and destruction of cell organelles
during freezing process. Glycerol is used for cryoprotection of red blood cells, and DMSO is used
for protection of most other cells and tissues.

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A sugar called trehalose, which occurs in organisms capable of surviving extreme dehydration, is
used for freeze-drying methods of cryopreservation.it stabilizes cell membranes and useful for the
preservation of sperm, stem cells, and blood cells.

Freezing: It is done in such a way to prevent intracellular freezing and crystal formation. As the
freeze point temperature is approached, ice crystals can form outside of the cell creating an osmotic
imbalance with the cell and it causes water to exit the cell resulting in cellular dehydration.
Excessive dehydration will create nonviable cells. types of freezing are:

• Rapid freezing
• Slow freezing
• Stepwise freezing

Rapid freezing: It is simple, done quickly and easy so that least change or development of crystals.
Slow freezing: The rate of freezing is slow ( 0.1-10oC ),mostly use in animal germplasm.Due to
cooling below freezing point,extra celluler crystals are formed not intrecelluler.
Stepwise freezing: In this, temperature get lowered by ( -20to -40oC) and allow protective
freezing of cells.freezing stop for 30 minutes, then rapidly freeze in liquid N,good results are obtain
by decline in temperature.

The detrimental effects can be minimized by doing the following:

• Controlling the cooling rate
• Using cryoprotective agents
• Maintaining appropriate storage temperatures
• Controlling the thawing rate
All of these events interact to influence the viability of thawed cells.
Freezing equipment:

Blast freezer and direct plunge into LN 2 for rapid freezing is used.
Laboratory freezer

LN 2 controlled rate freezer

Glacier G50
Controlled rate cooling can be accomplished with an ultra low temperature laboratory freezer with
an insulated accessory that holds the vials and is designed to limit the heat transfer . Or it can be
done using a programmed cooling rate that controls the flow of liquid nitrogen (LN2) around the
sample vessels. Another way is to use an ultra low temperature recirculating bath programmed
with a temperature ramp rate. These two later methods also have the advantage of an adjustable
cooling rate.

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Storage: Maintainance offrozen cellsor material at specific temperatureis important.the

temperature generally use is -70 to -196 oC. Prolong and indefinite storage is done at -130 to -
196o C temperature at liquid nitrogen.to prevent damage by continuous supply of N is done and
biological time has stop. the lower the storage temperature, the longer the viability storage period
can be.

Thawing: The warming rate of cryopreserved samples can also impact cell viability. the thawing
rate has considered less critical than the controlled rate of cooling. In general cells that were cooled
at a controlled rate, cryoprotective agent was used slow thawing better to allow time for the cell to
rehydrat, loss of cryoprotective agent. Cells that were frozen rapidly generally require rapid
thawing to prevent crystallization that would cause cell damage.

Washing: Washing of material is done to remove toxic cryoprotectant.when it is low toxic or

nontoxic cryoprotectant,culture should not wash but simply reculture. After washing Dilution is
done than after resuspension process centrifugation is done and cells are removed.

Measurement of viability: There is possibility of death of cells due to storage stress.thus viability
can be found at any time.
Formula is no. of cells growing divided by no. of cells thawed into loo.

Regeneration: Viable cells are cultured on non specific growth medium.suitable environmental
conditions are maintain.

Fig: Cryopreservation Process

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Applications of Cryopreservation
The applications of cryopreservation can be categorized into the following areas:
cryopreservation of cells or organs; b) cryosurgery; c) biochemistry and molecular biology; e)
food sciences; e) ecology and plant physiology; and f) many medical applications, such as blood
transfusion, bone marrow transplantation, artificial insemination, and in vitro fertilization
(IVF).Some suggested advantages of cryopreservation include the possible banking of cells
for human leukocyte antigen typing for organ transplantation; the allowance of sufficient
time for transport of cells and tissues among different medical centers; and the provision of
research sources for identifying unknown transmissible diseases or pathogens. Furthermore, the
long-term storage of stem cells is still the initial step toward tissue engineering, which holds
promise for regeneration of the soft tissue esthetic function and for the treatment of known diseases
that have currently no therapy option.

1) Embryos

Cryopreservation for embryos is used for embryo storage, e.g., when in vitro fertilization (IVF)
has resulted in more embryos than is currently needed. Pregnancies have been reported from
embryos stored for 16 years. Many studies have evaluated the children born from frozen embryos,
or “frosties”. The result has been uniformly positive with no increase in birth defects or
development abnormalities. A study of more than 11,000 cryopreserved human embryos showed
no significant effect of storage time on post-thaw survival for IVF or oocyte donation cycles, or
for embryos frozen at the pronuclear or cleavage stages. Additionally, the duration of storage did
not have any significant effect on clinical pregnancy, miscarriage, implantation, or live birth rate,
whether from IVF or oocyte donation cycles. Rather, oocyte age, survival proportion, and number
of transferred embryos are predictors of pregnancy outcome.

2) Ovarian tissue

Cryopreservation of ovarian tissue is of interest to women who want to preserve their reproductive
function beyond the natural limit, or whose reproductive potential is threatened by cancer
therapy, for example in hematologic malignancies or breast cancer. The procedure is to take a part
of the ovary and perform slow freezing before storing it in liquid nitrogen whilst therapy is
undertaken. Tissue can then be thawed and implanted near the fallopian, either orthotopic (on the
natural location) or heterotopic (on the abdominal wall), where it starts to produce new eggs,

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allowing normal conception to occur. The ovarian tissue may also be transplanted into mice that
are immunocompromised (SCID mice) to avoid graft rejection, and tissue can be harvested later
when mature follicles have developed.

Fig: Cryopreserved Tissues

3) Oocytes

Human oocyte cryopreservation is a new technology in which a woman’s eggs (oocytes) are
extracted, frozen and stored. Later, when she is ready to become pregnant, the eggs can be thawed,
fertilized, and transferred to the uterus as embryos. Since 1999, when the birth of the first baby
from an embryo derived from vitrified-warmed woman’s eggs was reported by Kuleshova and co-
workers in the journal of Human Reproduction, this concept has been recognized and widespread.
This break-through in achieving vitrification of woman’s oocytes made an important advance in
our knowledge and practice of the IVF process, as clinical pregnancy rate is four times higher after
oocyte vitrification than after slow freezing. Oocyte vitrification is vital for preservation fertility
in young oncology patients and for individuals undergoing IVF who object, either for religious or
ethical reasons, to the practice of freezing embryos.

4) Semen

Semen can be used successfully almost indefinitely after cryopreservation. The longest reported
successful storage is 22 years. It can be used for sperm donation where the recipient wants the
treatment in a different time or place, or as a means of preserving fertility for men
undergoing vasectomy or treatments that may compromise their fertility, such
as chemotherapy, radiation therapy or surgery.

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Fig: Semen Cryopreservation

5) Testicular tissue

Cryopreservation of immature testicular tissue is a developing method to avail reproduction to

young boys who need to have gonadotoxic therapy. Animal data are promising, since healthy
offsprings have been obtained after transplantation of frozen testicular cell suspensions or tissue
pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension
transplantation, tissue grafting and in vitro maturation (IVM) has proved efficient and safe in
humans as yet.

6) Stem cells

Adult stem cells are capable of differentiating into multiple types of specific cells and can be
obtained from various locations other than bone marrow, including fat tissue, the periosteum,
amniotic fluid, and umbilical cord blood. Stem cells can be subdivided into embryonic stem cells,
mesenchymal stromal cells, and hematopoietic stem cells, all of which are considered as goldmines
for potential application in regenerative medicine. Clearly, the fields of tissue engineering, gene
therapy, regenerative medicine, and cell transplantation are largely dependent on the ability to
preserve, store, and transport these stem cells without modification of their genetic and/or cellular
contents.

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Fig: Haematopoietic Stem cells Preservation

7) Hepatocytes

Primarily isolated hepatocytes have found important applications in science and medicine over the
past 40 years in a wide range of areas, including physiological studies, investigations on liver
metabolism, organ preservation and drug detoxification, and experimental and clinical
transplantation.In addition, there is currently increasing interest in the applications of liver
progenitor cells across a range of scientific areas, including both regenerative medicine and
biotechnology, which raises a need for cryobanking.

8) Others

Although primary neuronal cells and cardiomyocytes are routinely used for neuroscience and
cardiology research, a gold standard protocol for the preservation of these cells has not yet been
developed. With the discovery of glucocorticoid-free immunosuppressive regimens,51 pancreatic
islet transplantation may be considered as an alternative for the treatment of type 1 diabetes. For
this reason, the development of islet cryopreservation methods has been ongoing, but results are
still suboptimal, with a survival rate of less than 50%.

Is it Possible to Preserve Human Body?

The low temperatures are needed to allow the cells to survive dehydration after death - but
uncontrolled dehydration and freezing is also lethal to living cells, so it has to be done carefully.
The eventual aim is that one day they will be rewarmed and revived, but there is no evidence or
guarantee that they can be.

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Fig: Human Body Preservation

Advantages of Cryopreservation
There are many advantages to cell freezing. Proper cryopreservation of cells is the best way to
save money, improve reproducibility of experiments, and protect important stocks of cells that
may be difficult to replace.

• Cryopreservation helps in the preservation of biological Specimens.

• Sperm, Gametes, Embryo, Tissues, bone marrow and Organs can be preserved.
• It helps to study the adapting nature of plants and animals under the low temperature.
• Used to preserve Genetic material of plants which are on the verge of its extinction.
• Reduced risk of microbial contamination.
• Reduced risk of Genetic Drift and Morphological changes.
• Reduced risk of cross contamination with other cell lines.
• Cryopreservation technology is important in breeding programs to preserve desired genes,
but also provides an opportunity to save endangered species.

Challenges of Cryopreservation
Although numerous usages of the cryopreservation technique exist, both in basic and clinical
research, some limitations still exist. The challenge of cryopreservation is to help cells to survive
both cooling to extreme temperatures and thawing back to physiological conditions.

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1.Ice Formation in and out the Cell

Intracellular ice formation in particular is a critical issue that has to be controlled to keep the cell
membrane intact and the cells alive. If optimal conditions are not provided during cell freezing
then damage and cellular apoptosis or necrosis may occur post-thaw. During the freezing process
ice forms in and out the cell and this is very much dependent on the cooling rate. If ice forms
externally to the cell, water migrates out of the cell causing dehydration, shrinkage and finally cell
death. If too much water remains inside the cell during the freezing process, intracellular ice
crystals form that damage cellular organelles and pierce the cell membranes during the thawing
process. The crucial elements to prevent this are the freezing rate (degrees per minute) and the
composition of the freezing medium used. The freezing medium generally consists of a diluter,
(sometimes) a protein source, as well as a cryoprotectant compound. The choice of most suitable
cryoprotectant will influence the preservation result and will be different between different cells
and different species.

2. Osmotic pressure imbalance

Osmotic pressure imbalance is the main concern during cryopreservation as it may cause
dehydration, deformation, and injury by effecting the water movement in or out the cell. When
freezing cells at too low cooling rate, water may leave the cell through the cell membrane to join
the ice on the exterior. Since solutes are excluded from ice crystals, the solute concentration
increases and water moves from low solute concentration to high solute concentration to achieve
osmotic balance.

To mitigate osmotic imbalance, freezing solutions with specific cryoprotectants are used to prevent
dehydration by replacing water and stabilizing the cell. These cryoprotectants are added to
cryopreservation media that may be individually formulated or bought commercially. Rapid
cooling may be used for cryopreservation by the vitrification technique. Vitrification is a fast
cryopreservation method that avoid ice crystals formation within the cell. Vitrification method is
usually successful when cryopreservation solutions contain high concentration of cryoprotective
agents and it is used routinely for the cryopreservation of gametes and embryos.

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Conclusion
Improvements in the freezing and thawing rates, osmotic conditions, choice and concentration of
cryoprotective agents (CPAs), and equilibration times in the CPAs might result in better survival
and functionality of human tissue and cell samples, permitting their successful future clinical
applications. New CPAs are constantly being investigated owing to the inherent toxicity of many
known agents. A better understanding of the chemistry and biology behind freezing and thawing
will be necessary for future development of this process and for finding the safest and most
effective cryopreservation method. The successful cryopreservation of biological samples may
play a pivotal role in research connected with clinical utility for all kinds of human trials.
Collectively, the most prominent future goals of cryopreservation should focus on the development
of procedures that minimally affect the intactness of cryopreserved cells or tissues, followed by
the standardization and optimization of the technique for routine use.

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References
• Scott D. Pratt. Oct 06, 2017.Cryopreservation Freezing Methods and Equipment.
• David H. Yawn.Cryopreservation
• Cryogenetics AS, Storhamargata 44, N-2317 Hamar, Norway Phone: +47 909 20 600
E-mail: post@cryogenetics.com VAT Reg. 985283885
• www.slideshare.net/richatiwari54/cryopreservation-88695759
• Tony V. L. Bui,Ian L. Ross,Gisela Jakob,Ben Hankamer Published: November 11, 2013
/journal.pone.0078668 Impact of Procedural Steps and Cryopreservation Agents in the
Cryopreservation of Chlorophyte Microalgae
• Tae Hoon Janga, Sung Choel Parka, Ji Hyun Yanga, Jung Yoon Kima, Jae Hong Seoka, Ui
Seo Parka, Chang Won Choia, Sung Ryul Leeb, Jin Han Cryopreservation and its clinical
applications College of Medicine, Inje University, Busan, Korea
• DAVID.E. PEGG Long-term preservation of cells and tissues .Journal of clinical
.Pathology.,1976,29,271-285
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395684/
• https://www.cryogenetics.com/products-and-services/cryopreservation/
• https://www.thermofisher.com/pk/en/home/references/gibco-cell-culture-basics/cell-culture-
protocols/freezing-cells.html

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