Cryopreservation

1. CRYOPRESERVATION

2. CONTENTS

3. INTRODUCTION
→ Cryo (Greek) = krayos. Meaning- frost. Literally cryopreservation is preservation in “frozen state”.
→ Cryo-preservation or cryo-conservation is a process where organelles, cells, tissues, extracellular
matrix, organs, or any other biological constructs susceptible to damage caused by unregulated chemical
kinetics are preserved by cooling to very low temperatures.
→ The frozen cells are stored at a temperature of -196°C in liquid nitrogen. At low temperatures, any
enzymatic or chemical activities which might cause damage to the biological material in question is
effectively stopped.
→ The most common procedures are cryopreservation of embryos, spermatozoa and some somatic cells
and tissue.

4. HISTORY

5. PRINCIPLE OF CRYOPRESERVATION
• In cryopreservation, very low temperatures is used to preserve living cells and tissues and maintain their
viability. Unprotected freezing is normally lethal.
• Cryopreservation is based on the conversion of water present in the cells from a liquid to a solid state.
• When cooling below 0°C, the biological effects are dominated by the freezing of water, which typically
constitutes at least 80% of the tissue mass.
• The cell water requires much lower temperature to freeze (even up to -68°C) due to the presence of salts
and organic molecules in the cells, in comparison to the freezing point of pure water (around 0°C).
• The metabolic processes and biological divisions in the cells/tissues are almost stopped when stored at
low temperature.

6. REQUIREMENTS FOR CRYOPRESERVATION

7. REQUIREMENTS FOR CRYOPRESERVATION

8. SPERM
CRYOPRESERVATI
ON
o It is commonly known as sperm banking Sperm.
o Sperm banking is a procedure to preserve sperm cells for future
use.
o The first successful cryopreservation of spermatozoa was
initiated over 50 years ago.
o For human sperm, 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 for men
undergoing a vasectomy to still have the option to have children.
o It is also useful for men suffering from azoospermia & Gonadial
cancer. These frozen sperms can be used in association with
one of the Assisted Reproductive Techniques to induce
pregnancy.
scientistlive.co
m

9. PROCESS OF
SPERM
CRYOPRESERVATI
ON
Obtaining semen sample:
Semen samples are collected in sterile container. The semen so
collected is suspended in 10-20% glycerol in egg yolk buffer.
Semen analysis:
Semen samples are analyzed for volume, viscosity and pH levels,
and microscopically evaluated to determine motility, sperm count
and morphology.
Freezing:
Freezing of semen is done either in straw or ampoules.
Freezing can be achieved in three ways:
iStoc

10. PROCESS OF SPERM CRYOPRESERVATION
→ Slow freezing:
 Liquid nitrogen is poured into the tank and cooling rate is obtained from 20°C to −80°C at rate of 1.5°C/min and
then at 6°C/min; at completion of the freezing the straws are removed and stored into liquid nitrogen at −196°C.
This takes about 40 min.
→ Rapid freezing:
 It requires direct contact between the straws and the nitrogen vapors for 8–10 min and immersion in liquid
nitrogen at −196°C. The sample is initially mixed in dropwise manner with equal volume of cold cryoprotectant;
the mixture is loaded into the straws and left to incubate at 4°C for 10 minutes. The straws are then placed at a
distance of 15–20 cm above the level of liquid nitrogen (−80°C) for 15 min; after this stage, the straws are
immersed in liquid nitrogen.

11. PROCESS OF
SPERM
CRYOPRESERVATI
ON
→ Thawing:
→ It is done by putting ampoule containing the sample in a warm
water bath (35 to 40°c).
→ Frozen tips of the sample in tubes or ampoules are plunged into the
warm water with a vigorous swirling action just to the point of ice
disappearance.
→ Just a point of thawing quickly transfer the tubes to a water bath
maintained at room temperature and continue the swirling action for
15 sec to cool the warm walls of the tube.
onlinelibrary.wiley.com

12. EMBRYO
CRYOPRESERVATI
ON
→ Embryo cryopreservation or embryo freezing is a method used to
Embryo preserve embryos, generally at embryogenesis stage by
cooling and storing them at low temperatures.
→ Started in 1984.
→ It is one of the most common and well established fertility
preservation treatments, with proven successful pregnancy rates.
→ In addition, the duration of storage had no significant effect on
clinical pregnancy, miscarriage, implant, or live birth rate.
→ Although women are most fertile from their teens until age 35, that
time frame is not always ideal for a woman to start a family.
Freezing eggs allows women to harvest their eggs when they are
most viable and healthy.
newlifefertilityclinic.com

13. PROCESS OF
EMBRYO
CRYOPRESERVETI
ON
Embryo collection:
•Egg retrieval is done under ultrasound guidance and
subsequent fertilization and embryo culture is carried
out. If there is surplus of embryos, then embryos of
sufficient quality are collected for cryostorage.
Embryo selection:
While the embryos can be frozen at any pre
implantation stage between one cell to the blastocyte
stage, generally embryos at blastocyte stage are
chosen to cryopreserve. In cases where embryo needs
to be frozen without a fresh transfer, freeze all the
embryos the day after egg collection at one cell-stage.
fertilityclinic.com

14. PROCESS OF EMBRYO CRYOPRESERVATION

15. PROCESS OF
EMBRYO
CRYOPRESERVATI
ON
Thawed human embryos in which two or three blastomeres have undergone
fusion.
researchgate.ne

16. APPLICATIONS OF CRYOPRESERVATION
→ This method of preservation is widely used in different sectors including cryosurgery, molecular biology, ecology, food science,
plant physiology, and in many medical applications. Other applications of cryopreservation process are:- Seed Bank, Gene
Bank,
o Blood transfusion.
o In vitro fertilization.
o Organ transplantation.
o Artificial insemination.
o Storage of rare germplasm.
o Freezing of cell cultures.
o Conservation of endangered plant species.
o Biodiversity conservation.

17. BENEFITS OF CRYOPRESERVATION
→ There are many benefits of cryopreservation technique. These include:
• Fertility treatments.
• Minimal space and labour required.
• Safety from genetic contamination.
• Safeguards genetic integrity of valuable stains.
• Safeguards the germplasm of endangered species.
• Biological samples can be preserved for a longer period of time.
• Protects the samples from disease and microbial contamination.
• Prevents genetic drift by cryopreservation of gametes, embryos, etc.

18. CONCLUSION
→ Different types of cells have different requirements for optimum preservation and recovery. For example,
extracellular ice formation can be avoided by vitrification and controlled rate freezing, storage
temperatures need to be selected based on the anticipated storage period, and crystallization may be
avoided during recovery by rapid thawing. Selecting the best cryopreservation equipment for your lab,
based on the cell type, budget, and the required features, takes some research of the modern equipment
and methods available today.

19. REFERENCES
→ researchgate.net
→ fertilityclinic.com
→ newlifefertilityclinic.com
→ scientistlive.com
→ iasoivf.com
→ immunoprecise.com