FreezingandThawingofMEFs

Introduction

During experimental needs the preservation of cell lines and their recovery after preservation are important steps in laboratory. Cryopreservation is a best procedure to preserve the cell lines, but accuracy during handling is an important factor which gives high yield of cells after storage. Rapid freezing and slow thawing gives low recovery of cells after cryopreservation, thereby slow freezing gives maximal survival of mammalian cells provided that a cryoprotective agent and rapid thawing are used. It could be supposed, therefore, that this type of freezing would cause minimal structural and functional damage to the cells.

Embryonic stem cells are usually grown on a layer of mitotically inactivated primary mouse embryonic fibroblasts (MEFs) to promote growth and prevent differentiation. These cells stop dividing after a couple of passages, so embryonic fibroblasts need to be isolated freshly from time to time. To avoid isolation at time to time we need to be preserve them safely.

Materials and reagents

• Cryovials: Marked cryovials

• Freezing media: 7.5% DMSO and 92.5% fetal bovine serum

• Minicooler: A minicooler unit which decreases the temperature at 1°C/ min

• Liquid nitrogen container

• Water bath: should be maintained at 37°C

• 50 mL tubes

• 10% FBS-DMEM

• Petriplates

Procedure

Freezing

1. Harvest MEF cells by typsinizing from petripaltes and centrifuging in 20 mL DMEM media

2. Dissolve cell pellet in freezing media
   -Note: Here cell numbers should be more the 106 cell/ mL

3. Transfer cell suspension in cryovials immediately and simultaneously keep vials in minicooler unit.

4. Finally transfer cryovials in an appropriate rack of liquid nitrogen and record the position of samples in your register or computer.

Thawing

1. Keep cryovials in 37°C waterbath to thaw the cells, until just a small ice-clump remains.
   - Note: Minimizing the time and process swiftly give high yield of viable cells.

2. Transfer the cell suspension in a labeled petriplates using a sterile pipette. Here cell suspension should be mixed well by sucking and dispensing by pipette to breakdown the clumps of the cells.

3. Dilute the cell suspension in a 20 mL of DMEM media.

4. Allow to grow them in CO₂ incubator.
   -Note: In cryobiology DMSO has been used as a cryoprotectant and is still an important constituent of cryoprotectant vitrification mixtures used to preserve organs, tissues, and cell suspensions. Without it, up to 90 percent of frozen cells will become inactive. It is particularly important in the freezing and long-term storage of embryonic stem cells and hematopoietic stem cells, which are often frozen in a mixture of 10% DMSO and 90% fetal calf serum.