Cryopreserving en herstellen van de Mens iPS cellen met behulp van complete KnockOut Serum Vervangende Feeder-vrij medium

The overall goal of this procedure is to cryopreserve and recover IPS cells in feeder based or feeder free culture conditions. This is accomplished by first beginning packaging your IPCs as you normally would. The second step of the procedure is to transfer the cells into serum free freezing medium.

The third step of the procedure is to cryopreserve as you normally would. The final step of the procedure is to thaw the cells and begin maintenance. Ultimately, results can be obtained that show successful freezing and thawing of IPS cells in serum free freezing medium through healthy morphology and recovery post thaw.

Hi, I'm Kate Wagner at the GCO site at Life Technologies. Visual demonstration of this protocol is critical because IPCs can be very sensitive to freezing and T fine. What makes this protocol easier is that knockout serum replacement can be used throughout your entire workflow.

Today the KSR family of products includes a full list of media and reagents for feeder based feed free and xeno free culture of your embryonic stem cells and induced pluripotent stem cells. KSR can be used for derivation growth, cryo-preservation, expansion, and the first initial steps of differentiation. The following video will show you thaw and freezing yourselves using KSR in a Peter free machine.

To begin this procedure. First warm in an appropriate amount of complete feeder free medium to 37 degrees Celsius in a water bath aspirate spent medium from the culture vessel, which is a 60 millimeter dish. In this case, rinse the human induced pluripotent stem cells twice with DPBS.

Then add one milliliter of dispa to the dish. Incubate the dish at 37 degrees Celsius for three minutes. After that, aspirate the dispa solution and gently wash the cells with DPBS.

Next, add complete feeder free medium to cover the dish and use a cell scraper to gently scrape the cells transfer cells to a sterile 15 milliliter centrifuge tube. Rinse the dish with complete feed of free medium, gently spraying off any cells that have not detached. Transfer the rinse solution to the same 15 milliliter tube containing the cells.

Rinse the dish a second time with medium and pull that rinse solution with the cells pellet cells by centrifugation at 1000 RPM for three minutes at room temperature. After centrifugation, carefully aspirate and discard the supernatant without disturbing the cell pellet. Gently flick the tube to fully dislodge the cell pellet from the bottom of the tube.

Then add one milliliter of freezing medium A, using a five milliliter serological, pipette, and resuspend the cells by gently pipetting up and down. Following uniform suspension of clumps, add one milliliter of freezing medium B to the tube in a dropwise manner while gently swirling the cell suspension to mix. Allocate one milliliter of cell suspension into each cryo vile freeze cells at minus 80 degrees Celsius overnight in an isopropanol chamber.

On the following day, transfer the vials of cells into a liquid nitrogen tank for long-term storage. Rapidly thaw the frozen vial of cells by placing it in a 37 degree Celsius water bath for about three to four minutes until just a small frozen chunk remains in the vial. After removing the vial from the water bath spray with 70%isopropanol to decontaminate it, then use a five milliliter pipette to aseptically transfer the entire contents of the vial into a 15 milliliter conical tube.

The next step may be the most difficult aspect of the procedure. The feeder free medium must be added slowly and carefully to the cells. To reduce osmotic shock Slowly add four milliliters of complete feeder free medium dropwise to the cells in the 15 milliliter conical tube.

While adding the medium gently move the tube back and forth to mix the cells. This will reduce osmotic shock to the cells. Complete feeder free medium may be replaced with knockout serum replacement containing meth conditioned medium or knockout serum replacement medium for use with feeders, refer to the protocol text for media preparation instructions.

Once all four milliliters of medium have been added, centrifuge the cells at 1000 RPM for five minutes at room temperature. After centrifugation, carefully aspirate and discard the sane without disturbing the cell pellet. Gently resuspend the cell pellet in four milliliters of complete feeder free medium.

Using a five milliliter pipette, gently add the cell suspension dropwise to a gelt trucks coated 60 millimeter culture dish. Place the culture dish in a 36 to 38 degrees Celsius incubator with a humidified atmosphere of four to 6%carbon dioxide in air. Carefully swirl the dish in a north to south, east to west pattern to evenly distribute the cells.

24 hours post thaw. Replace the medium in the dish with fresh medium. Replace the spent medium daily thereafter until cells become approximately 70 to 80%confluent.

This contrast image shows human induced pluripotent stem cells grown on gelt TRX coated culture dishes using complete serum replacement, feeder free medium. The IPC's exhibit morphology similar to human embryonic stem cells characterized by large nuclei and scant cytoplasm. An example of cells post thaw and knockout serum replacement.

Feeder free medium on gelt TrackX is shown here. When following this procedure, it is important to remember to keep all your cell colonies the same size. When cryo preserving this improves cell recovery.

So that's it. Thanks for watching and good luck with your experiments.