Cell cultures are utilized In a wide variety of research and diagnostic applications. It is sometimes desirable to store cell lines for future work to preserve cells, avoid senescence, reduce the risk of contamination, and minimize the effects of genetic drift. Cell lines may be frozen for long-term storage.
In this video, we show you how to freeze cells grown both in monolayer and in suspension, and how to thaw them for growth once again. Hi, I'm Ricky here at the Dr.LAN's laboratory at Molecular Pathology Laboratories Network in East Tennessee. Today I'm going to show you how to freeze cells for long-term storage and how to thaw them from liquid nitrogen.
To freeze human adherence Cells, you start by lifting them from the plate as you would if you were packaging them. Keep in mind that it's best to use cells that are in their log phase of growth for cryo-preservation. Once the cells have detached and you've stopped the tripsin reaction by adding serum or medium containing serum, transfer the cell suspension to a sterile centrifuge tube and add two mills.
Complete medium with serum cells from three or more dishes from the same subculture of the same source can be combined to one tube. Centrifuge is cells for five minutes at 300 to 350 Gs at room temperature. After the spin, remove the supernatant and resuspend the pellet in 10 mils of four degrees Celsius.
Complete medium, add an equal volume of four degrees freezing medium, mix the cells thoroughly, and then place the tube on ice count cells. Using a hemo cytometer dilute the suspension with more freezing medium in order to get a final cell concentration of 1 million or 10 million cells per milliliter to freeze cells from a nearly confluent 25 centimeter square flask. Resus suspend in approximately three mils of freezing medium pipette one mil aliquots of cell suspension into labeled two mil cryo vials.
Be sure to tighten the caps on the vials. Place vials one hour to overnight in a minus 80 freezer, then transfer them to a liquid nitrogen storage freezer. Freezing cells from suspension culture is similar in principle to freezing cells from a monolayer.
The major difference is that suspension cultures do not need to be tryps. Inized first, transfer the cell suspension to a centrifuge tube and spin for 10 minutes at 300 to 350 Gs at room temperature. Next, remove the supernatant and resuspend the pellet in four degrees, freezing medium at a density of 1 million to 10 million cells per mil.
Then transfer one mil aliquots of cell suspension into cryo vials and freeze. As done for the monolayer cultures, which was demonstrated in the previous Chapter to thaw and recover frozen vials. First, remove the vial of cells from the liquid nitrogen freezer and immediately place it into a 37 degree water bath.
Agitate the vial continuously until the medium is thawed, which generally takes less than 60 seconds. Once thawed wipe the top of the vial with 70%ethanol before opening. Then transfer the thaw cell suspension into a sterile centrifuge tube containing two mils of warm complete medium and 20%fetal bovine serum centrifuge.
This for 10 minutes at 150 to 200 Gs at room temperature. This step will remove any residual DMSO after centrifugation, discard the supernatant and gently resus suspend the cell pellet in a small amount around one mil of complete medium and 20%FBS transfer this to a properly labeled culture plate containing the appropriate amount of medium. Generally, one mil of cell suspension is needed in five to 20 mils of medium.
Now, you can put the cells in an incubator at 37 degrees Celsius to grow after 24 hours. You'll wanna make sure the cells have attached to the plate after five to seven days, or when the pH indicator in the medium changes color, you'll want to change the medium. You'll want to keep the cultures in a medium containing 20%FBS until the cell line is reestablished.
If the recovery rate is extremely low, only a subpopulation of the original culture may be growing. Be especially careful of this when working with cell lines known To be mosaic. If cultures need to be shipped, This can be done easily for both monolayer and suspension cultures.
In 25 centimeter tissue culture, flasks cells are grown to near co fluency in a monolayer or to desire density in suspension. For monolayer cultures, the medium is first removed and then the flask is filled with fresh medium. It is essential that the flak be completely filled with medium to prevent cells from drying out if the flasks are inverted during transport.
For suspension, cultures, fresh, medium is added to fill the flask. The cap is tightened and secured in place using parfum. The flask is sealed in a leakproof plastic bag or other leak proof container designed to prevent spillage.
In the event that the flask should become damaged, the primary container is then placed in a secondary insulated container to protect it from extreme temperatures during transport. A buyer hazard label is affixed to the outside of the package. Generally, cultures are transported by same night or overnight.Couriers.
We've just shown you how To freeze and thaw human cell cultures for both adherent and suspension cells. So that's it. Thanks for watching and good luck with all your experiments.
