Cryopreservation

Cell Culture Technique

1. CRYOPRESERVATION: Presented By: Naveen K L 1 st (Sem) M pharma Dept of Pharmacology Srinivas college of pharmacy Mangalore

2. Introduction: Cryo is Greek word. (krayos- frost) It literally means preservation in “ frozen state.” It’s a process where tissues, organelles, cells, extracellular matrix, organs or any biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temp 196˚ using liquid nitrogen . Cryopreservation is long term storage technique with very low temperature to preserve the structurally intact living cells & tissues for extended period of time at a relatively low cost. The science pertaining this activity known as “Cryobiology”

3. Cryopreservation: Cryopreservation is a non-lethal storage of biological material at ultra-low temperature. At the temperature of liquid nitrogen (196˚) almost all metabolic activities of cells are ceased and the sample can be preserved in such state for extended periods. however only few biological material can be frozen without affecting the cell viability.

4. Why Preservation is important ? Until two decade ago the genetic resource were getting depleted owing to the continuous depredation by man. It Was imperative therefore that many of the elite, economically important and endangered species are preserved to make them available when needed. The conventional method of storage failed to prevent losses caused due to various reason . A new methodology had to be devised for long term preservation of material.

5. history: Ernest John Christopher Polge, an English biologist, was the first person to solve the mystery of how to preserve living cells at very low temperature in 1949. he accidently discovered the cryoprotective properties of glycerol on fowl sperm. During 1950s James E. Lovelock suggested that increasing salt concentration in a cell as it dehydrated due to lose of water results in cell damage and he also proposed that the mechanism of action of cryoprotectant. In1953, research work of Jerome K. Sherman led him to successfully freeze and thaw human sperm. In1983, Alan Trounson, was credited for Sucessfully achieving a pregnancy after freezing early human embryos one to three days after fertilization.

6. Storage methods: Cryopreservation - generally in liquid nitrogen. Cold storage – in low and non freezing temperature Low pressure – reducing the atmospheric pressure partially. Low oxygen storage – involve reducing the oxygen level but maintaining the pressure.

7. PRINCIPLE: To bring plant cells or tissues to a zero metabolism and non - dividing state by reducing the temperature in the presence of cryoprotectant. Done by : Over solid CO2 (at 79˚) Low temperature deep freezer (at 80˚) In vapor phase N2 (AT 150˚) In liquid N2 (at 196˚)

8. Mechanism of Cryopreservation: Selection of plant material. Pregrowth Addition of cryoprotectant . Freezing. Storage in liquid nitrogen . Thawing . Washing and Reculturing . Measurement of viability. Regeneration of plant.

9. selection of plant material: Morphological and Physiological conditions of plant material influences the ability of explant to survive during cryopreservation. Cells or tissues from the late lag phase or log phase. Different types of tissues can be used as follows: Ovules Anther or pollen Embryos Endosperm Protoplast

10. Factors to be consider in selection of plant material……. Tissue must be selected from healthy plants. Small. Young. Rich in cytoplasm. Meristic cells can survive better than the larger. Highly vacuolated cells. Water content of cell or tissue used for cryopreservation should be low freezable water, tissue can withstand extremely low temperature.

11. Pregrowth: Pregrowth treatment protect the plant tissue against exposure to liquid nitrogen. Pregrowth involves the application of additives known to enhance plant stress tolerance. E.g. Abscisic acid Praline Trehalose

12. Addition of cryoprotectant: A cryoprotectant is a substance that is used to protect biological tissues from freezing damage (due to ice formation) or prevent cryodestruction. they acts like antifreezing. They lower freezing temperature. Increase viscosity and prevents damage to the cells. E.g. Sucrose, Alcohols, Glycols, A mino acid (Proline), DMSO (dimethyl sulfoxide) * Generally two cryoprotectant should be used together instead of single.

13. Freezing: Freezing is a phase transition where a liquid turns into a solid when its temperature is lowered below its freezing point. The sensitivity of cells to low temperature depends on the plant species. M ethods of freezing: Slow freezing Rapid freezing Step down freezing

14. SLOW FREEZING METHOD: * The tissue is slowly frozen with decrease in temp of -0.5 ˚C to -5˚C /min and transfer into liquid nitrogen . * Slow cooling permits flow of water from the cells to the outside , thereby extracellular ice formation instead of lethal intracellular freezing. * employed for cryopreservation of meristems of peas , potato , cassava. RAPID FREEZING METHOD: * The material is plugged into liquid nitrogen with decrease in temp from -300 ˚C to -1000 ˚C /min or more * This method is technically simple and easy to handle. * employed for cryopreservation of shots tips of potato , strawberry , brassica species . STEPWISE FREEZING METHOD: * in this method freezing is down to -20 ˚C to -400 ˚C , a stop for a period of approximately 30min and then additional rapid freezing to -196 ˚C is done by plugging in liquid nitrogen.

15. Storage: Storage of frozen material at correct temp is as important as freezing. The frozen cells / tissues are kept for storage at temp ranging from -70 to -196 ˚C. Temperature should be sufficiently low for long term storage of cells to stop all the metabolic activities are ceased and prevent the biochemical injury. Long term storage done at -196 ˚C…… & constant supply of liquid nitrogen essential. Temp above -130 ˚C ice crystal grow may occur inside the cells , which reduces the viability of cells , storage is ideally done at liquid nitrogen refrigerator at -150 ˚C In vapour phase or -196 ˚C in liquid phase.

16. Why liquid nitrogen ideal for cryopreservation ? Also called as cryogenic fluid. Chemically inert. Relatively low cost. Non toxic. Non inflammable. Readily available.

17. Thawing: Process in which its carried out by plugging the vials containing sample into warm water bath (35 to 40 ˚C) with vigorous swirling (15 sec). After thawing process vials are transfer into a another bath at 0 ˚C. Its imp for the survival of tissue that the tubes should not be left in the warm water bath after ice melts .

18. washing and reculturing: The preserved material is washed few minutes to remove the cryoprotectant. The material is then recultured in a fresh medium. When low toxic or non-toxic cryoprotectants are used , then the culture should not be washed but simply recultured.

19. Measurement of viability: Regrowth of the plants from stored tissues or cells is the only test of survival of plant materials. Various viability tests include Fluorescien diacetate staining (FDA), Growth measurement by cell number, dry and fresh weight. Imp staining methods are ….. * Triphenyl T etrazolium chloride. * Evan’s blue staining.

20. TRIPHENYL TETRAZOLIUM CHLORIDE ASSAY : Cell survival is measured by amount of red formazan product formed due to reduction of TTC assay which is measured by spectrometrically. Only the viable cells which contain the enzyme mitochondrial dehydrogenase which reduces TTC to red formazan will be stained and dead cells will not take up the dye.

21. Evan’s blue staining: One drop of 0.1% solution of Evan’s blue is added to cell suspension on a microscope slide and observed under light microscope. Only non viable cells (dead cells) stain with Evan’s blue. % of viable cells = Num of fluorescent cells ×100 total Num of cells (viable + non viable ) Individual cell viability assayed with Evan’s blue dye and fluorescein diacetate

22. Plant regeneration The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant. For appropriate plant growth and regeneration, the cryopreserved cells/tissues have to be carefully nursed, and grown. Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is often necessary for successful plant regeneration.

23. Application: Cryopreservation of Embryo, Ovarian tissue, Oocytes, Semen, Testicular tissue. Ideal method for long term conservation of material. Disease free plant can be conserved and propagated. Recalcitrant seeds can be maintained for long time. Endangered species can be maintained. Pollens can be maintained to increase longitivity .

24. Reference: Culture of animal cells, a manual of basic technique by R. Ian Freshney , 5 th edition Pg no 321-329 . www.biologydisscussion.com germiplasm conservation and cryopreservation article by Nandkishore Jha