CRYOPRESERVATION

Background/History:
Cryopreservation also known as cryoconservation is a process wherein cells, tissues,
organs and many more that relates to other biological constructs that are susceptible to damage
unregulated reaction kinetics are being preserved at a low enough temperature that is suitable for
the biological material not to cause further damage. The intention of cryopreservation is to
preserve structurally intact living cells and tissues for a long period of time to prevent the loss of
genetic diversity.
In 1949, an English biologist namely Ernest John Christopher Polge, was the first to solve
the mystery of how to preserve living cells and tissues at very low temperatures. One of the most
important early theoreticians that uses cryopreservation was James Lovelock in 1953. In the mid-
1950’s he experimented the rodents using cryopreservation, discovering that hamsters could be
frozen by 60% of the water in the brain crystallized into ice with no adverse effects on the other
hand, other organs were unfortunately susceptible to damage.
Cryopreservation was applied to humans beginning in 1954 with three pregnancies
resulting from the insemination of previously frozen sperm. Fowl sperm was cryopreserved in
1957 by a team of scientists in the UK directed by Christopher Polge.

Cryopreservaion Freezing Methods:

Many methods have been developed for various types of cells, tissues and organs due to
an unanticipated discovery by Polge and co-workers of the cryoprotective effect of glycerol. The
progression in the field has come from empirical work as well as from fundamental cryobiology
and due to more in-depth understanding of the causes of cryo-injury the cryopreservation
methods continuously improved over time. The most commonly used methods that would
prevent cryopreservation damages are a well established combination of controlled rate and slow
freezing and a newer flash-freezing process known as vitrification. These are quite different
methods, but relate to the same physico-chemical relationships. The difference between the two
main methods will be explained further:

 Slow Freezing
Cells in slow-freezing are to be cooled below freezing point. At some stage, ice
masses containing pure crystalline water will form and what remains between the growing ice
masses is the so-called unfrozen fraction. Slow cooling is needed in order to allow sufficient
efflux of water to minimize the chance of intracellular ice formation. As cooling continues, the
viscosity of the unfrozen fraction ultimately becomes too high for any further crystallization. The
remaining unfrozen fraction turns into an amorphous solid that contains no ice crystals.
  Virtrification
Virtrification also known as flash-freezing process is the transformation of a
substance into a glass, that is to say a non-crystalline amorphous solid. According to this
definition,cells that are properly slow frozen become “vitrified”. Vitrification methods involve
the use of a medium that has a very high solute concentration to begin with. Thus, ice cannot
form in any part of the sample. As no ice forms, cooling does not have to be slow. In fact, it may
be beneficial to cool very rapidly. The vitrified state and the associated physico-chemical
conditions obtained using vitrification methods, are to some extent similar to those obtained by
slow cooling, but the way of reaching this point is quite different.

Application fields of Cryopreservation:

According to Yusuf Bozkurt (2018), In human medicine, cryopreservation has gained its
importance when its usage in infertility treatment was realized. Since then, gamete
cryopreservation has been developed to solve fertility problems in this field . In the field of
cryopreservation, sperm was the first reproductive cell to be successfully frozen and still remains
the easiest to freeze because of containing low amounts of cytoplasm and consequently low
quantity of water. Sperm and semen can be used almost indefinitely after proper
cryopreservation. Overall, cryopreservation can be used as a first-line means of preserving
fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as
chemotherapy, radiotherapy, or surgery.
The first case of embryo cryopreservation for fertility preservation took place in 1996,
with the application of a natural In Vitro Fertilization (IVF) cycle prior to chemotherapy in a
woman diagnosed with breast cancer. Cryopreservation of mature oocytes is a proven technique
for preserving the reproductive capacity. Results from a retrospective study of 11,768
cryopreserved human embryos that underwent at least one thaw cycle from 1986 to 2007 showed
that there was no significant impact of the duration of storage on clinical pregnancy, miscarriage,
implantation, or live birth rate, whether from IVF or oocyte donation cycles. Since oocytes are
highly prone to chilling injury; cryopreservation of immature oocytes and ovarian tissue is a
promising approach with reports of live births-but the need for investigational improvements
remain.
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. 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.
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. There is a current increase of interest in the applications of liver progenitor cells
across a range of scientific areas, including both regenerative medicine and biotechnology, which
raises the need for cryobanking.
References:
 "The Cryobiological Case for Cryonics" (PDF). Cryonics. Vol. 9(3) no. 92. Alcor Life
Extension Foundation. March 1988. p. 27.
 Lovelock JE (March 1953). "The haemolysis of human red blood-cells by freezing and
thawing". Biochimica et Biophysica Acta. 10 (3): 414–26. doi:10.1016/0006-
3002(53)90273-X. PMID 13058999.
 Yusuf Bozkurt (2018,November 15th). Introductory Chapter: Application Fields of
Cryopreservation Biotechnology, Cryopreservation Biotechnology in Biomedical and
Biological Sciences, Yusuf Bozkurt, IntechOpen, DOI: 10.5772/intechopen.82229.
Retrieved from: https://www.intechopen.com/books/cryopreservation-biotechnology-in-
biomedical-and-biological-sciences/introductory-chapter-application-fields-of-
cryopreservation-biotechnology