Cryopreservation of gamtes by bhawan.pptx

Cryopreservation of gamaetes

1. CRYOPRESEVATION OF GAMETES Bbb Presented by: Bhawanpreet Kaur Ph.D Scholar Pres Presented to: Dr. Jaspreet Arora Assistant Scientist

2. AIM OF THIS PRESENTATION Cryopreservation Cryoprotective agents (CPAs) or Cryoprotectants and its types . Techniques for Cr yopreservation Cr yopreservation of gametes : Spermatozoa & oocyte preservation Cryodamage or Cryoinjury or Cryodestruction Advantages of Cryopreservation Applications Limitations

3. Cryopreservation comes from Greek word Cryos , which means “ cold ”. It is a process that maintains biological samples in a state of suspended animation at cryogenic temperature -196℃ using liquid nitrogen to preserve the fine structure of cells. At sub zero temperature, all biological activities of cells and tissues is ceased or paused. It is the use of very low temperatures to preserve structurally intact living cells and tissues for long period of time. Depending upon the cell types, there is great diversity in cryobiological response and cryosurvival . The Science pertaining this activity is known as “ CRYOBIOLOGY ”. Cryopreservation

4. Cryoprotective agents (CPAs) or Cryoprotectants CPAs which is an organic and inorganic additive reduces the freezing injuring from cryopreservation process. CPAs is biologically acceptable and have low toxicity. It reduces the amount of ice formed. low molecular weight. Non electrolyte. Miscible with water. E xample : G lycerol , DMSO, PEG, Propylene glycol etc. Characteristics

5. They aim to protect the cells from damage called cold shock . Cryoprotectants minimize the damage caused by ice formation, as they cause water to form a glass rather ice crystal. They reduce the toxic effects of high concentrations. Water permeability affects the amount of water in cell . Too much water— intracellular ice—physical damage.

6. Protective efficiency of CPAs vary from cell to cell types. Human embryos are best frozen by propanediol and while human blastocyst by glycerol.

7. Mechanism of Cryoprotectants. . Cryoprotectants act as salt buffering action (binds water and less crystal formed) and gives more time to cell for dehydrate and remains intact with the cell membrane to make it less brittle during freezing .

8. Cr yoprotectants , including alcohols, sugars, oils, and starches, and each type acts through different mechanisms . These cryoprotectants have similar properties: solubility in water at low temperatures, cell permeability, and relatively low toxicity. CPAs themselves can be damaging to cells, especially when used in high concentrations. For example: DMSO may alter chromosome stability, which can lead to a risk of tumor formation.  Apart from endogenous changes in cells, the possible infection or contamination with cells such as tumorous ones should be prevented .

9. Based on their ability to diffuse across the membrane . CPAs can be divided into two categories . Membrane permeable CPAs (penetrate the plasma membrane) It penetrates into the cell membrane and enter the cytosol. They are exclusively small molecules. Stabilize cell plasma membrane proteins and reduce concentration of electrolytes. They form hydrogen bond with water to prevent ice formation. It also prevents the excessive dehydration during preservation. Such as dimethylacetaldehyde , glycol, glycerol , ethylene glycol etc.

10. Membrane Nonpermeable CPAs (unable to penetrate cell membrane). It don't penetrate into cell membrane. These are usually large molecules. Minimize intracellular crystallization by increasing viscosity of sample. They inhibit ice formation by same mechanism but don't enter into the cell. It prevents the damage of cell by preventing solutes, protoplasmic elements escaping from the cell. Such as albumins, egg yolk, citrate, sucrose, glucose etc.

11. Nonpermeable CPAs are classified as: Low molecular weight : It changes the water balance of cell by shrinking the cells before freezing. For eg : Maltose, Sucrose, Sorbitol . High molecular weight : It repairs the cell during thawing. For eg : Dextran, Serum, BSA .

12. Commonly used cryoprotective agents : Polge discovered cryoprotective effect of glycerol in 1949 . It is non electrolyte . It reduces the electrolyte concentration in unfrozen solution and around cell at any temperature. It is used in the storage of animal semen , human blastocyst. : Alexander Zaytsev discovered in 1866. It has low level of cytotoxicity and its cost is less . It is widely used for mammalian cells. Glycerol : DMSO :

13. C ryoinjury / Cryodestruction /Cryodamage . The mechanism states the damage of cells associated with phase changes of water in both extra and intracellular environment at low temperature. Cryodamage can occurs during freezing process. Causes of Cryodamage : Intracellular ice formation. Extracellular ice formation. Solution effects. Dehydration.

14. Intracellular ice formation : When some tissues tolerate extracelluar ice but the formation of intracellular ice might be fatal for cell. Extracellular ice formation : When tissues are cooled slowly, water migrates out of the cell and ice formed in extracellular space. Too much ice formation cause mechanical damage to the cell membrane. Dehydration : Migration of water causing extracellular ice formation can also cause of cellular dehydration. This can lead to directly damage. Solution effects : As the ice crystals formed in freezing water, solutes excluded and causing them to concentrated in remaining liquid. High concentration of solutes cause the major damaging .

15. Major steps in Cryopreservation Mixing of CPAs with cells or tissues before cooling Cooling of the cells or tissues to a low temperature Warming of the cells or tissues Removal of CPAs from or cells or tissues after thawing

16. DIFFERENT METHODS OF CRYOPRESERVATION

17. Techniques for Cryopreservation

18. Storage in liquid nitrogen If cells are not stored at low temperature, an additional injury to the material be caused. The storage temperature such that it stops all the metabolic activities and also prevents biochemical activity. This can be achieved with the help of liquid Nitrogen at -196°C.

19. Vitrification Greg fahly and Willam F.R helped to introduce vitrification to reproductive cryopreservation in 1980s. Addition of cryoprotectants prior to cooling. They act as antifreeze. Vitrification provides benefits of cryopreservation without damage due to ice crystal formation.

20. Cryobank Several cell banks have been established to secure storage and distribution of validated cell line. Cryobanks plays vital role in genetic management and conservation of cultured stocks.

21. Cryopreservation of sperm It is referred to as Sperm banking to preserve sperm for future use . It is useful for the men suffering from Gonadial cancer and azoospermia. The longest reported for successful storage of human sperm is 22 years. Frozen sperms can be used in association with Assisted Reproductive Techniques (ART) to induce pregnancy.

22. Process involved for Sperm Cryopreservation Collecting semen sample . Semen samples are collected in sterile container. It is suspended in 10-20% glycerol in egg yolk buffer. Semen analysis . Semen samples are analysed for volume, viscosity, pH levels and microscopically evaluate sperm count, morphology, motility. Freezing Slow freezing Rapid freezing Thawing

23. Slow freezing : Liquid nitrogen is poured into tank and cooling rate is obtained from 20° to -80°C at rate of 1.5°C/min and 6°C/ min. At completion of freezing straws are removed at stored into liquid nitrogen at -196°C for 40 mins.

24. 2. Rapid freezing : It requires the direct contact of straws and nitrogen vapours for 8-10 mins and immersion in liquid nitrogen at -196°C. The sample is initially mixed in dropwise manner with equal volume of cryoprotectant, and the mixture is loaded into straws and incubate at 4°C for 10 mins. Then straws are placed at distance of 15 to 20 cm above the level of liquid nitrogen at 80°C for 15 mins. After this the straws are immersed in liquid nitrogen.

25. 3. Thawing : It is done by putting ampoule containing sample in warm water bath 35 to 40°C. Frozen tips of sample in tubes or ampoules are plunged in warm water with vigorously swirling action just to disappearance of ice. Just a point of thawing , quickly transfer the tubes to water bath maintained at room temperature. Continue the swirling action for 15 sec to cool the warm walls of tube.

26. Cryopreservation of Embryo It was started in 1984. It is a method to preserve embryos, at embryogenesis stage by cooling and storing at low temperatures. I t is called as e mbryo cryopreservation or embryo freezing. It is one of the most common and well established fertility preservation treatment. High efficiency rates with successful pregnancy rates. Duration of storage had no effect on miscarriage, clinical pregnancy.

27. Process of embryo cryopreservation 1 . Embryo collection 2. Embryo Selection 3. Embryo Freezing 4. Thawing

28. Embryo collection: The embryo cultured is carried out. If there is surplus embryo, then the embryos of sufficient quality are collected for cryostorage . 2. Embryo selection : While the embryos can be frozen at any pre-implantation between one cell to blastocyst stage. Generally, the blastocyst stage are chosen to cryopreserve.

29. 3. Embryo Freezing : Selected embryos are transferred to Freezing medium and kept for 20 mins at 20°C for equilibrium. Slowly cool the embryos in cryoprotectant fliud from -196°C at rate of 0-4°C/min. The embryos are contained within special labelled embryos straws or vials that are prior to freezing. Embryos are placed inside the labelled tubes inside the aluminum canes and stored in liquid nitrogen.

30. Thawing : It is the reverse of freezing process which involves warming the embryos. Embryo thawing takes 2 hours. During the process, embryo is placed in water bath. When embryos returned to room temperature, the embryos are passed through a series of solution to remove cryoprotectants. Then thaw embryos are kept in incubator until embryo transfer.

31. 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. For eg : In breast cancer . Oocytes cryopreservation: Human oocytes cryopreservation is a new technology in which a women’s eggs (oocytes) are extracted , frozen and stored. Later, when a women is ready to become pregnant , the eggs can be thawed, fertilized and transferred to uterus as embryos.

32. Benefits of Cryopreservation Sperm, oocyte, embryos, organ can be preserved for long time. First line means of preserving fertility for men undergoing vasectomy. Efficient method to conserve germplasm of endangered species. Larger range of stocks available. Easily disease free exchange of stocks nationally and internatinally as well. Fertility preservation. Stocks remain viable indefinately. Methods to reduce multiple pregnancies.

33. ApplicationS of Cryopreservation of gametes. In vitro fertilization (IFV) Embryo cryopreservation. Ovarian cryopreservation. Artificial insemination . Semen cryopreservation. Testicular cryopreservation

34. Limitations of Cryopreservation CPAs themselves can be damaging to cells, especially when used in high concentrations. Ice formation can lead to wide damage to cells. DMSO may alter chromosome stability, which can lead to a the tumor formation. The possible infection or contamination should be prevented .

35. REFERENCES Jang TH, Park SC, Yang HJ, et al. Cryopreservation and its clinical applications. Jour Integr med res. 2017;12-18. Michal J, Jana Z, Pavel V, Kempisty B, Igor C. Cryopreservation of human gametes and embryos: current state and future perspectives. Cryopreservation in eukaryotes. 2016. Barrett SL and Woodruff TK. Gamete preservation. Cancer Treat Res. 2010;25-39. Hafez ESE. Reproduction in Farm Animals, 6th edition. Philadelphia: Lea & Febiger , 1993.