Hi, I am Leah Kent from STEM Gen. Today we're gonna show you how to thaw and freeze human embryonic stem cells. So let's get started.
To thaw human embryonic stem cells, a cryo vial is removed from liquid nitrogen and quickly thawed. In a 37 degrees Celsius water bath, the cell suspension is transferred from the cryo vial to a conical tube diluted with human ES cell culture medium and centrifuged. To form a small cell pellet, the supernatant is then removed and the cell pellet is resuspended with fresh human ES cell culture medium in the tube before plating on one well of a six well cell culture plate previously plated with an irradiated meth feeder layer.
One day prior to thawing human ES cells plate a feeder layer of irradiated mouse, embryonic, fibroblasts, or mes in meth culture. Medium in one well of a gelatin coated six well tissue culture plate. Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide.
To ensure an efficient and successful thaw, be sure to have all necessary equipment and reagents ready. Before removing the human ESL from liquid nitrogen. Using metal forceps, remove a cryogenic vial of human E ESL from the liquid nitrogen storage tank.
Roll the vial between gloved hands for three to five seconds. To remove the frost record the information on the label of the vial using metal forceps. Immerse the vial into a 37 degree Celsius water bath.
Swirl the vial gently and observe the progress of the thaw often but quickly by holding the vial up to the light. To see the size of the ice crystal. Do not submerge the cap of the vial in the water bath as this could contaminate the cells.
When only a small ice crystal remains submerge the vial in ethanol to sterilize it in a sterile biological safety cabinet, transfer the contents of the cryogenic vial directly to the bottom of a 15 milliliter conical tube. Slowly add four milliliters of human ES cell culture. Medium to the tube.
Gently rock the tube to continually mix the cells as the new medium is added to the tube. Centrifuge the human E es cells for five minutes at 200 Gs while the cells are in the centrifuge. Retrieve the previously prepared meth feeder plate from the 37 degree Celsius incubator and label it with the appropriate human ESL information such as cell line, passage number, and thaw.Date.
Aspirate the MEF culture medium and add one milliliter of PBS to the well. Bring the pelleted human E ESL back to the biological safety cabinet and carefully aspirate the supernatant. Be careful not to aspirate the cell pellet, but remove as much supernatant as possible as this solution contains DMSO from the freezing medium.Resus.
Suspend the pellet very gently by adding 2.5 milliliters of human ESL culture medium. Using a five milliliter glass pipette and pipetting three to four times aspirate the PBS from the MEF feeder. Well and slowly add all 2.5 milliliters of the human ES cell suspension to the prepared well of the six well plate.
Place the plate into the 37 degrees Celsius incubator and carefully slide the plate forward to back and side to side to evenly distribute the cells throughout the well. To avoid concentrating the colonies in the center. Do not swirl the plate in a circular pattern.
Allow the cells to attach at 37 degrees Celsius and 5%carbon dioxide overnight on the day following the thaw. Remove the medium with any floating cells and add 2.5 milliliters of fresh human ES cell culture. Medium to the well.
Incubate the plates at 37 degrees Celsius and 5%carbon dioxide overnight and continue to culture until the colonies are ready to be passaged. The human E es cell freezing protocol begins with healthy medium sized human ES cell colonies cultured on a six well plate. After incubating the culture with collagenase four enzyme, the colonies are scraped with a five milliliter glass pipette collected in a conical tube and centrifuge to form a loose cell pellet.
After aspirating the supernatant, the cell pellet is resuspended. First with human ES cell culture medium and then diluted with two x cryo-preservation.Medium. The cells are evenly distributed in one milliliter, aliquots, and frozen in cryogenic vials.
While the cells are in the centrifuge, label the cryogenic vials inside the safety cabinet with the cell line passage number and freezing date. The typical density for freezing human E es cells is one well of cells from a six well plate per cryogenic vial. When the human E ESL have been centrifuged, bring the tube back to the biological safety cabinet.
Aspirate the supernatant from the tube. Being careful not to disturb the loosely packed cell pellet Resus. Suspend the cell pellet with the appropriate amount of human ESL culture.
Medium 0.5 milliliters of human ESL culture. Medium per cryogenic vial. Be very gentle when pipetting, as the human E es cells recover better when frozen in relatively large colony pieces, slowly while gently shaking the tube, add the appropriate amount of two x human E es cell freezing medium to the cells.
Once the freezing medium is added mixed by pipetting the solution extremely gently, one to two times quickly, but gently add one milliliter of the cell suspension to each cryogenic file. Transfer the vials into an isopropanol freezing container and place in a minus 80 degrees Celsius freezer overnight. The cells will freeze at one degree Celsius per minute in the isopropanol freezing container On the following day.
Quickly transfer the frozen vials to a liquid nitrogen storage tank using metal forceps. The first day after human ES cells are t thought the small colonies may appear transparent and can be difficult to see under the microscope. Since newly thought, human e ESL tend to proliferate rather slowly.
They may take a few days in culture to appear as established colonies. We've just shown you how to thaw and freeze human embryonic stem cell cultures. So that's it.
Thanks for watching and good luck with your experiments.
