Cryopreservation of Cells and TIssues

Cryopreservation is the process of preserving cells, tissues or other substances by freezing them to very low temperatures with the help of cryogenic fluids such as Liquid Nitrogen or methanol, etc which has a lower boiling points of -196 and -96 degree Celsius.when cells and tissues are subjected to low temperature freezing the viability of cells goes which makes impossible to grow those cells after cryo-preservation to avoid this cryoprotective agents(Cryoprotectants) are used, there by increasing the survivability of the preserved samples.some of the most common cryoprotectants are DMSO (dimethyl sulfoxide), Ethylene glycol, Glycerol, 2-Methyl-2,4-pentanediol, Propylene glycol, Sucrose.

Reasons for freezing cells:

1. Provides a continuous source of cells; unique cell lines might be impossible to replace.
2. Genotypic drift due to genetic instability.
3. Senescence
4. Contamination by microbes; cross contamination with/or by other cell lines.
5. Incubator failure
6. Saves time and material
7. Distribution to users

Cryoprotective Agent (CPA) / Cryoprotectant : a substance used to protect cells/tissues from damage during freezing. e.g. glycerol, DMSO

Equilibrium time: the period of time between mixing the cryoprotectants to the cell suspension in the beginning of the cooling process. (between 15-60 mins)

Living cells can be successfully preserved by freezing and can be stored for long time without much change in its viability, here cryopreservation of BHK-21 cell line is described.

Cryopreservation BHK (Baby Hamster Kidney) Cells.

AIM:

To prepare the cryoprotective medium and to subsequently cryopreserve the BHK – 21 suspension culture.

PRINCIPLE:

Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or −196 °C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. However, when vitrification solutions are not used, the cells being preserved are often damaged due to freezing during the approach to low temperatures.

PROCEDURE:

1. Prepared 2ml of the cryo protective medium (CPM) ( 7.5% DMSO, 10% serum, rest of volume made with GMEM media of 1X concentration).
2. Added 2.5ml of the seed and 2.5ml of CPM into a centrifuge tube and 1ml was transferred into 4 different cryovials.
3. The remaining 1ml was used to determine cell count. The cell concentration of the seed was found to be 1.24×10⁶ cells/ml.
4. After 15 minutes, the cells were then subjected to gradual cooling from room temperature to 4⁰C and 4⁰C to -20 ⁰C (30 min), -20⁰C to -80⁰C (1 hour), in different deep freezers maintained at corresponding temperatures.
5. The vials were further transferred to the canister which was sealed with aluminium foil and eventually stored in liquid nitrogen container to cool to temperature of -176°C in vapors of nitrogen and then to liquid nitrogen at -196°C gradually.
   
   
   

REVIVAL OF CULTURE AFTER CRYOPERSERVATION

1. The vials were then taken and rapidly thawed at 37⁰C.
2. The culture in the four vials was pooled into a centrifuge tube aseptically and add 16ml of media into it(4ml of media for 1ml of suspension).
3. Centrifugation was done at 1200rpm for 5minutes.
4. The supernatant was discarded and pellet was dissolved in fresh media .
5. The cell concentration of the cryopreserved cells was found to be 1× 10⁶cells/ml.
6. % of Survival growth was then calculated using the formula:

Survival growth = (Post freeze count / Pre freeze count) × 100

= (1.0*10^6 / 1.24*10^6) * 100

= 80.06%

RESULT: The BHK – 21 cells were successfully cryopreserved and revived and the survival growth was found out to be 80.6 %.

This Protocol was followed during my training session on Animal Tissue Culture (BHK 21 Cell Line)  and its Preservation.