CRYOPRESERVATION

Bundelkhand University, Jhansi

CONTENTS

Introduction

Historical overview

Cryopreservation methods

Summary

References

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HISTORICAL OVERVIEW
The initiation of cryopreservation in 1948 by C. Polge et al. marked a significant historical milestone, with the accidental discovery of successfully

freezing fowl spermatozoa

1972: Mazur et al. proposed the "Two-factor Hypothesis" linking cooling rates and survival.

Mid-1980s: Fahy et al. and Rall and Fahy introduced the vitrification strategy for cell preservation.

1998: Studies revealed the impact of cryopreservation on the cell's proteome and genome.

In 1949, Polge, Parks, and Smith discovered glycerol's cryoprotective function while preserving avian spermatozoa.

Smith extended the observation by successfully cryopreserving human red blood cells in glycerol in 1950.

Lovelock and Bishop introduced dimethyl sulfoxide (DMSO) as a cryoprotective agent in 1959.

"The Origins of Cryopreservation" delves into the storing of biological material at low temperatures. It draws connections to ancient practices,

such as icehouses in Mesopotamia around 2000 BC and experiments conducted by Boyle in the 17th century. The preservative effect observed in

these instances is linked to slowing cellular processes through low temperatures.

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 INTRODUCTION
Cryopreservation is the process of preserving organelles, cells, tissues,
or biological constructs by cooling the samples to very low
temperatures. Cryopreservation holds paramount importance in
maintaining the fine structure of cells and tissues for long-term
storage. The primary challenge in this process lies in avoiding cell
death, which can be caused by ice crystal formation, osmotic shock,
and membrane damage during freezing and thawing.

The definition of cryopreservation involves the storage of material at

ultralow temperatures, specifically around -196°C. Its significance
extends to tissue and organ banking, tissue-engineered products,
clinical applications, and regenerative medicine.

Example: Embryo cryopreservation, also known as embryo freezing,

is a defined process aimed at freezing and storing fertilized eggs
(embryos) for future use. This method plays a crucial role in fertility
treatments, providing options for delayed pregnancies and fertility
preservation.

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 MATERIAL SELECTION
• Material chosen for cryopreservation should be as far as possible to meristematic state.

• For selecting a material, several facts are taken into account.

1. The nature of cells.

2. The density of cells in the vials to be preserved.

Cell cultures are generally preserved in lag or early exponential phase of growth. Young, highly
cytoplasmic & and small cells that are non-vacuolated & and in small aggregates are good materials to be
selected for cryopreservation. In some species, it may be important to use highly embryonic cell cultures
since non-embryonic or poorly embryogenic cultures show poor or no regrowth after thawing. The

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20XX Presentation title 6
 CRYOPRESERVATION OF PLANT STOCK CELL
Due to the gradual disappearance of economic and rare species, the necessity for storage of genetic resources increases.

The conventional methods of storage fail to prevent losses caused by

1. Attack of pathogens & pest.

2. Climatic disorders.

3. Natural disorders.

4. Political & and economic causes.

The materials to be preserved are stored at low temperatures due to which the growth rate of cells retards; consequently, biological
activities are conserved for a long time.

3 principal methods are:

1. Alteration of the physiological condition of culture i.e. temperature of gas composition in the vessels.

2. Changing the composition of the basal medium. EX: Using sub or supra optimal concentration of nutrients.

3. Supplementing the nutrients with growth retarders or osmoregulatory compounds.

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20XX Presentation title 8
 CRYOPRESERVATION BY NATURE
Nature’s One potential application in cryopreservation is Marine antifreeze proteins that have two main functions firstly thermal hysteresis

(TH) and ice recrystallization inhibition (IRI). TH is the ability of the protein to lower the freezing point of a solution, while IRI is the ability

of the protein to prevent the growth of ice crystals. These functions make marine antifreeze proteins potential cryoprotectants, which are

substances that protect cells from damage during freezing.

The are some antifreeze several proteins, including AFPs, AFGPs, and IBPs.

Type I AFPs, such as HPLC6 from winter flounder and ss3 from shorthorn sculpin

Type II AFPs, such as the calcium-dependent AFP

Type III AFPs, such as HPLC12

Hyperactive AFPs, such as Maxi from winter flounder and FfIBP from Flavobacterium frigorific

Antifreeze glycoproteins (AFGPs) are a type of AFP found in some fish.

ice-binding proteins (IBPs) which are a broader category of proteins that include AFPs.
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 ADDITION OF CRYOPROTECTANTS

Cryoprotectants are chemicals that decrease cryodestruction.

“CRYODESTRUCTION” refers to the formation of large ice crystals inside the cells that rupture the
cells themselves & and cell organelles.
It is of 2 types:
INTRA CELLULAR CRYOPROTECTANTS.
Example: Glycerol and dimethyl sulfoxide.
EXTRA CELLULAR CRYOPROTECTANTS. Example : Polyvinyl pyrolidone.
5-10% protectants is added.

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 FREEZING.
After the addition of cryoprotectants, freezing is done in such a way that it does not cause intracellular freezing & and crystal
formation.

The following types of freezing is done in the process of cryopreservation:

RAPID FREEZING :

This method is simple and easy..

Freezing is done quickly so that there should be the least change or development of intracellular crystals.

SLOW FREEZING:

In this method, the rate of freezing is slow ( 0.1 - 10 0 C min). It is commonly used for animal germplasm.

In this due to cooling below freezing point, extracellular crystals are formed not intracellular.

STEPWISE FREEZING:

In this temperature gets lowered by - 20 0 C to – 40 0 C and allows protective freezing of the cells.

Freezing stopped for 30 minutes and then rapidly froze in Liquid Nitrogen this results in a decline in temperature & and good results
are obtained.
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 VITRIFICATION.
Researchers Greg Fahy and William F. Randall helped to introduce
vitrification to reproductive cryopreservation in the mid-1980s.
It means the addition of cryoprotectants before cooling. They act as anti-
freeze i.e. they decrease the freezing temperature and the viscosity.
Researchers claim that vitrification provides the benefits of
cryopreservation without damage due to ice crystal formation.
Rather than a phase change from liquid to solid by the crystallization the
amorphous state is like a ‘solid-liquid’ and the transformation is over a
small temperature range described as "Glass Transition" temperature.

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 CRYOPRESERVATION
METHODS

Electric freezing

Vapor phase freezing

Liquid phase nitrogen

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 ELECTRIC FREEZING

Cryopreservation is a process of preserving living cells and tissues by freezing them at

very low temperatures. Electrical freezing, also known as electro freezing, is a relatively
new technique for cryopreservation that uses an electric field to control the formation of
ice crystals.
Disadvantages
Advantages

• Chance of electrical short-circuits or

• Low running cost
breakage.
• Easy in maintenance
• Storing temperature is higher then
• Uniform temperature maintenance liquid nitrogen

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 VAPOR PHASE NITROGEN
Vapour phase nitrogen cryopreservation is a method of preserving biological samples by
freezing them in the vapour phase of liquid nitrogen. This method is often used for long-
term storage of samples, as it is less expensive and more convenient than liquid nitrogen
storage.

Disadvantages
Advantages

• Simple and reliable method • High running cost

• Low temperature can be achieved • Vertical temperature gradient in the

Vapour phase
• No cross contamination due to liquid
• Requires regular supply of liquid
nitrogen
nitrogen

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 LIQUID PHASE NITROGEN
Liquid phase nitrogen cryopreservation is a method of preserving biological samples by
freezing them at very low temperatures (-196°C) in liquid nitrogen. This method is often
used for long-term storage of samples, as it can preserve samples for many years or even
decades.

Advantages Disadvantages

• Simple and low mechanical • It requires a regular nitrogen supply.

equipment.
• High Running Cost
• Continues maintenance of ultra load
• Cross contamination chances are
temperature.
high.

• contamination

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 APPLICATIONS OF CRYOPRESERVATION
Suitable combinations of cryoprotectants and regimes of cooling & and
rinsing allow successful cryopreservation of biological materials such as
semen, embryos, oocytes, ovarian tissues, testicular tissue, meristems etc.
Some examples are given below:
1. EMBRYO CRYOPRESERVATION: It is used for embryo storage
when in-vitro fertilization has resulted in more embryos than is
currently needed. Pregnancies have been reported from embryos
stored for 16 years. The result has been uniformly positive with no
increase of birth defects or developmental abnormalities.

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 OVARIAN CRYOPRESERVATION
Ovarian tissue cryopreservation is of interest to women who want to preserve their reproductive
function beyond the natural limit or whose reproductive potential is threatened by chemotherapy.

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
N 2.

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 OOCYTES CRYOPRESERVATION

Human oocyte cryopreservation is a new technology in which a woman’s eggs(oocytes) are

extracted, frozen & and stored.

Later, when a woman is ready to become pregnant, the eggs can be thawed, fertilized and transferred
to the uterus as embryos.

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20XX Presentation title 20
 REFERENCES
• Baust, J.G., Gao, D. and Baust, J.M. (2009). Cryopreservation. Organogenesis, [online] 5(3), pp.90– 96. Doi:
HTTPs://doi.org/10.4161/org.5.3.10021.
• Bojic, S., Murray, A., Bentley, B.L., Spindler, R., Pawlik, P., Cordeiro, J.L., Bauer, R. and de Magalhães, J.P. (2021).
Winter is coming: the future of cryopreservation. BMC Biology, 19(1). Doi https://doi.org/10.1186/s12915-021-00976-8.
• Jang, T.H., Park, S.C., Yang, J.H., Kim, J.Y., Seok, J.H., Park, U.S., Choi, C.W., Lee, S.R. and Han, J. (2017).
Cryopreservation and its clinical applications. Integrative Medicine Research, [online] 6(1), pp.12–18. Doi:
HTTPs://doi.org/10.1016/j.imr.2016.12.001.
• Moo-Young, M. (2019). Comprehensive Biotechnology | ScienceDirect. [online] www.sciencedirect.com. Available at:
https://www.sciencedirect.com/referencework/9780444640475/comprehensive-biotechnology.
• Murray, K.A. and Gibson, M.I. (2022). Chemical approaches to cryopreservation. Nature Reviews Chemistry, 6(8),
pp.579–593. doi:https://doi.org/10.1038/s41570-022-00407-4.
• Whaley, D., Damyar, K., Witek, R.P., Mendoza, A., Alexander, M. and Lakey, J.R. (2021). Cryopreservation: An Overview
of Principles and Cell-Specific Considerations. Cell Transplantation, 30, p.096368972199961. Doi:
HTTPs://doi.org/10.1177/0963689721999617.
• www.youtube.com. (n.d.). Cryopreservation | application and mechanism. [online] Available at:
https://www.youtube.com/watch?v=6jx8bD6wWTs&t=7s&pp=ygUQY3J5b3ByZXNlcnZhdGlvbg%3D%3D [Accessed 11
Dec. 2023].

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THANK YOU
Any Queries?!

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