Cryopreservation ( methods and application)

Cryopreservation is an method to store an cells and culture for long term. It's contain various procedure and practices .it's also contain an various application in several industries and specific branches.

1. CRYOPRESERVATION SUBMITTED BY: K. JAYALAKSHMI II-MSC., BIOTECHNOLOGY

2. INTRODUCTION; Long time storage of animal cells at super freeze temperature is called cryopreservation. Cell lines, hybridoma , ova and sperms have been frequently stored for long time to preserve the cells for future use. During cryopreservation animal cells are kept in solid carbon dioxide (-79ᵒ)or in deep freezer (-80ᵒc) or in nitrogen vapour (-150ᵒc) or in liqued nitrogen (-196ᵒc).

3. MECHANISIMS: While preserving cells at these super low temperature, the enzyme and metabolic activities become zero so that they remain inert but live for a long time. Since the animal cells, except zygote, have no totipotency , they could not regenerate into whole animals, but they could resume growth when suitable nutrient media and microenvironment are provided to them.

4. REASONS …. it is not possible to develop desired cell lines instantly when we are in need to use them in industries. It would be hardly possible to isolate the same cell line while repeating the cell culture for the second time. Equipment failure may lead to failure of certain phases of cell culture.

5. METHODS.. I) SELECTION OF CELL TYPES : Both finite cell line and continuous cell lines have been selected for long-time storage at - 196 ᵒc. The selected cell lines are subjected to various tests such as cytological tests , biochemical tests , physiological tests and immunological tests . Finite cells cannot grow indefinitely so that they are selected at early stage of culture { 5 th subculture).

6. Continuous cell lines are capable of growing continuously in artificial media so that they can be grown for a longtime to get bulks of cells. Prior to storage, the cell culture are kept at standard growth condition ; the cell suspension must contain 10 ^6-10^7 cells/ml , which is the best cell density for cryopreservation. II) Prerequisites for cryopreservation: Optimum medium is selected to grow cells and cell lines for storage. Serum free medium is likely for cryopreservation. In many cases, phenotypic expression of cell types is influenced by serum variation. Only a particular serum is used.

7. In order to overcome complication, a batch of a cell type has always to be maintained at each step. Standaridized substrates are selected for use. Cells are subjected to sterility tests to identify cultures free from microbial contaminations. III) Stages of cryopreservation in lab: Ampoules ( ampul ) : A small sealed vial which is used to contain and preserve a sample, usually a solid or liquied . Made up of glass

8. Cell culture Maintained by organiser 1-5 ampules “ T oken free” Maintained by organiser 12-15 ampoules “Seed stock” Maintained by organiser 50-100 ampoules “Distribution stock” Not send to users Not send to users send to users Maintained by user 1-3 ampoules “User stock”

9. IV)Protocol of cryopreservation: The cell suspension to be cryoprerserved is carried in a tube and centrifuged at 1000 rpm for 5 minutes; cells settled at bottom of tube. The supernatant thus obtained is discarded. The cells are suspended in a liquied medium containing 10% DMSO so that the cell density of 10^6-10^7 cells maintained. The vials are maintained at -80 ᵒ proper freezing effect in nitrogen liquied . V)Thawing( gentle warming of cell lines). VI) Removal of DMSO

10. EX.. Cell bank , ova bank , frozen semen, semen bank

11. F rozen semen…

12. Ova bank.. Q o P o T W o m e n G o n o d o t r o p h i n i n j e c tion C o l d t r e a t ment f r e e zing M u l t i ple o v u l a tion S u s p e n d i ng e g g s i n n u t r i e n t m e d i u m U l t r a a o n i c a t i o n I s o l ation o f e g g s using l a p r o s c p e T e m p e r ature r e d u c t ion t o 8 * c C r y o f r e e z e r , ( - 1 9 6 * c ) P o l y v i n y l 1 m l m e d i u m + 1 - 2 5 e g g s

13. Advantage: The method of keeping the live cells , tissues and other biological samples in a deep freeze at subzero temperature. (sperm, embryos, tissues, bone marrow, organs). Disadvantage: Which can cause damage to cells during cryopreservation mainly occur during the freezing stage, extracellular ice formation, dehydration, and intracellular ice formation. Cyroprotectant : A substance used to protect biological tissues from freezing damage. (ethylene glycol, dimethyl sulfoxide , glycerol)