10 Cryopreservation of primary culture and cell line-Ashwini Emmanuel M-6285.pptx

cryopreservation of primary culture

1. Cryopreservation of primary culture and cell line Animal Cell Culture- Principle And Applications BTY 604 Department of Veterinary Biotechnology ICAR- Indian Veterinary Research Institute, Izatnagar Submitted by DR. ASHWINI EMMANUEL ROLL NO: M-6285 MVSc , VMC DISCIPLINE Cryopreservation of primary culture and cell line 1

2. INTRODUCTION Primary Culture S tage of the culture, after the cells are isolated from the tissue and proliferated under the appropriate conditions, till it reach confluence. Cell Line After the first subculture, the primary culture becomes the cell line or subclone. Cell lines derived from primary cultures have a limited life span. Cryopreservation is a method of choice for preservation of important and rare cell stocks Cryopreservation of primary culture and cell line 2

3. WHY CELL LINE PRESERVATION? Minimizes • Genotypic drift • Senescence (biological aging, gradual deterioration of functional characteristics) • Contamination by microorganisms • Cross-contamination by other cell lines • Discontinue regular sub-culturing • Saving time and money • Provides a back-up Cryopreservation of primary culture and cell line 3

4. WHEN TO PRESERVE? Cryopreservation of primary culture and cell line 4

5. METHODS OF PRESERVATION Two popular preservation methods are- Lyophilization Cryopreservation Preservation of animal cell culture Preservation of cells in Liquid Nitrogen at - 196˚C (for longer period) or at -80 to -85˚C (for up to 4 months) Cryopreservation of primary culture and cell line 5

6. PRINCIPLE OF CRYOPRESERVATION Cell freezing Cell concentration Freezing medium Optimal freezing & maximum viability depends on minimizing intracellular ice crystal formation. Achieved by- Freezing slowly to allow water to leave the cell Using a cryoprotectant to sequester water Storing the cells at the lowest possible temperature to minimize the effects of high salt concentrations Cryopreservation of primary culture and cell line 6

7. Cont.. Freezing medium Recovery™ Cell Culture Freezing Medium F or mammalian cell cultures Dulbecco’s Modified Eagle Medium; optimized levels of fetal bovine serum, bovine serum and DMSO(10%) Synth-a-Freeze® Cryopreservation Medium P rotein free, sterile cryopreservation medium containing 10% DMSO S tem and primary cell types with the exception of melanocytes. Note: Always use the recommended freezing medium for cryopreserving different cell line. Cryopreservation of primary culture and cell line 7

8. CRYOPROTECTANTS Commonly used cryoprotectants are Dimethyl sulfoxide (DMSO) and Glycerol (Lovelock & Bishop, 1959) DMSO appears to be the more effective, penetrates the cell better than glycerol Other cryoprotectants are polyvinylpyrrolidone (PVP) (Suzuki et al., 1995) , polyethylene glycol (PEG) ( Monroy et al., 1997) and hydroxyethyl starch (HES) ( Pasch et al., 2000). Cryopreservation of primary culture and cell line 8

9. CRYOPROTECTANTS : DMSO Cryopreservation of primary culture and cell line 9 It is a commonly employed cryoprotectant. It has a low molecular weight and easily permeates the cells. The functions are: • To protect the cells from excessive dehydration during freezing process • To inhibit intracellular ice formation (Lovelock and Bishop, 1959). The mixture of 10% DMSO (ranging from 5 to 20%) and 10–20% FCS in the cryopreservation solution can be regarded as optimal concentrations for these reagents in the freezing of most mammalian cells ( Katen et al. 2007). FCS protects the cells from injury induced by the generation of free oxygen radicals during the freeze cycle ( Gutteridge and Quinlan 1993).

10. GUIDELINES Freeze your cultured cells at a high concentration and at as low a passage number Freeze the cells slowly by reducing the temperature at approximately 1°C per minute Store the frozen cells below –70°C; frozen cells begin to deteriorate above -5 0°C. S terile cryovials for storing frozen cells. W ear personal protective equipment. S olutions and equipment that come in contact with the cells must be sterile. Standard operative technique and work in a laminar flow hood Cryopreservation of primary culture and cell line 10

11. PROCEDURE Prepare freezing medium and store at 2° to 8°C until use. For adherent cells, gently detach cells from the tissue culture vessel Determine the total number of cells and percent viability Centrifuge the cell suspension at approximately 100–200 × g for 5 to 10 minutes Resuspend the cell pellet in cold freezing medium Dispense aliquots of the cell suspension into cryogenic storage vials. Freeze the cells in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute. Or Place the cryovials containing the cells in an isopropanol chamber and store them at - 80°C overnight. Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen. Cryopreservation of primary culture and cell line 11 Mr. Frosty Containers

12. IMPORTANT For adherent cell lines: Pre-centrifugation step to remove cryoprotectant is not normally necessary as the first media change will remove residual. For suspension cell lines: A pre-centrifugation step to remove cryoprotectant is recommended. Cryopreservation of primary culture and cell line 12

13. Cont.. • Make aliquots of 1ml in freezing vial Two types of Freezing Vials generally used Borosilicate Glass Ampoules Plastic Screw capped Vials Caution: If the Cryovial leaks it may accumulate liq.N2 and explode when thawing Cryopreservation of primary culture and cell line 13

14. Cont.. Label the cryovial properly Name of the cell Passage number Date of preservation Cryopreservation of primary culture and cell line 14 Common liquid nitrogen storage tanks used for cell cryopreservation

15. Cont.. Cool and freeze the vial in the following manner Cryopreservation of primary culture and cell line 15 Long-term storage

16. Cooling rate Cooling @ at 1◦C/min Slow cooling encourages the extracellular migration of water Cryopreservation of primary culture and cell line 16

17. REFERENCES American Type Culture Collection. 1992. Quality Control Methods for Cell Lines, 2nd Ed. Coriell , L.L. 1979. Preservation, storage, and shipment.In:Methods in Enzymology, vol. 58:29-36. Morris, C.B. 1995 Cryopreservation of animal and human cell lines. In: Methods in Molecular Biology, Vol. 38:179-187. Freezing cells. Invitrogen & Gibco. Part 4. Cell Culture Basics. Pp:37-39. R. Ian Freshney.2010. Culture of Animal Cells-A Manual of Basic Technique and Specialized Applications. Wiley-Blackwell Publication.6 th Ed. Chapter 19. Cryopreservation. Pp:317-334. Cryopreservation of primary culture and cell line 17

18. THANK YOU Cryopreservation of primary culture and cell line 18