Cryopreservation of cells is an important aspect of tissue culture for a variety of reasons. This lab allowed me to learn the cryopreservation technique and put viable cells to -196°C and bring them back up and culture them.

Materials

Prelab Preparation

Please refer to lab 1: Making MEM media for Biosafety hood and water bath setup

Please refer to lab 2: Trypsinization and Photography for cell splitting protocol.

The freshly made 1:16 split was then placed into the incubator for future use. The cap of the tissue culture flask was only tightened around 3/4 and was made sure to to be loose. This is done to allow oxygen to reach the cells that are growing. 1:16 split process depicted in pictures above.

Shown above is the centrifugation of the cells a second time and the removal of the media using a pasture pipette. Since the cell count fell between 1x10⁶ and 1x10⁷, at 9,772,000 only 1 ml of the freeze down media was necessary. The 1ml of freeze down media was then added to the cell pellet in the 15ml tube, resuspending the cells.

The 1ml of resuspended cells were then transferred to a cryopreservation Nuc tube. The Nuc tube was then labeled with the date, the type of cells as well as the my initials. After completion, the tube was placed in the freeze down box located in a -20°C freezer. This box was then transferred to the -80°C freezer by the TA, Ennid Dulaney.

After the cells spent an adequate time in the -80°C freezer, it was time to submerge the cells into liquid nitrogen. This process required the supervision of the TA, or Dr. Rohde, as it is a dangerous procedure due to the extremely low temperature of the liquid nitrogen. The cells in our case were stored in rack 5, so that rack was carefully brought up.

When bring up the sample rack it was placed at an angle at the top of the cryosafe to allow any excess LN2 to drip back into the storage container. Avoiding any spillage. Sample box A was then pulled from the rack.

The cells were then quickly transferred from the -80°C freezer into the storage box A, noting the position in which the cell were placed in. The position was then cataloged in a binder containing documentation regarding the cryosafe. In my case, I placed the cells into the 90th position.

When submerging the cells, the storage box was placed back into the rack and it was fastened with the locking rod. The rack was then slowly submerged into the LN2 avoiding overboiling of the nitrogen. The cryosafe lid was then placed over top, completing this process.

My cells were left in cryopreservation for 168 hours before being brought up from the liquid nitrogen.

When bring up my cells from LN2, the reverse of the freeze down procedure was done. The rack was allowed to completely drain of LN2 before working with the storage boxes.

When finished working with the cryosafe the cells were then thawed in the 37°C water bath. This was done by carefully shaking the NUC tube in the water without letting the water level to reach the cap, as shown above. The NUC tube was then sprayed with EtOH, and placed in the hood to adhere to the sterile technique.

The 1ml from the NUC tube was then transferred to a 15ml tube, where it was resuspended in 10ml of MEM media. The cells were then centrifuged to allow for the removal of the DMSO freeze down media, which is cytotoxic to the cells. This process is shown in the pictures above.

As stated above, after the cells were pelleted the 11ml media and DMSO freeze down media were removed using a pasture pipette. Then 10ml of MEM media were added to the cell pellet to resuspend.

A 75cm² TC flask was then labeled and the entirety of the 10ml from the 15ml tube were transferred to the flask. The flask was then placed in the incubator with the cap tightened 3/4 of the way. I was made use that the cap was loose.

After 24 hours the media in the flask was changed in order to remove any excess freeze down media. The cells were then placed back into the incubator and the confluence was noted.

The image above is a 10x magnification of the cells after cryopreservation and media replacement. It can be seen that the cells are starting to adhere and grow. This image does not represent confluency as it was around 50%.