Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The overall goal of this procedure is to adapt and maintain human induced pluripotent stem cells to feeder free culture using complete knockout serum replacement, feeder free medium. This is accomplished by first preparing gelt, TRX coated, or cell start coated plates. The second step of the procedure is to passage your IPS from MEFs to feeder free medium using disc space.

The final step of the procedure is to continually maintain your IPS cells feeder free. Ultimately, results can be obtained that show similar cell growth and stemness found with meth cultures through immuno staining with pluripotent markers. Hi, I'm Kate Wagner at the GIB co site at Life Technologies.

The main advantage of this technique over existing techniques is that you can save time and increase consistency in your cell culture when you use knockout serum replacement throughout your entire workflow. Today the family of knockout products includes a full list of media and reagents for feeder based, feeder free and xeno free conditions for human embryonic stem cells, mouse embryonic stem cells, and induced pluripotent stem cells. KSR can be used for derivation growth, expansion, cryopreservation, and the initial steps of differentiation.

This following video will show you adapting and packaging your cells using KSR in feeder free conditions. Gel TRX coated culture dishes are required for adapting and packaging human-induced pluripotent stem cells in feeder free conditions one day prior to the preparation of these coated dishes, thaw one tube of gelt TRX slowly by leaving it in the refrigerator overnight on the following day, dilute the tube of thaw gelt TRX one to 100 in basal medium by transferring one milliliter of gel Rex to a bottle containing 99 milliliters of basal medium. Mix the solution gently.

We recommend using knockout D-M-E-M-F 12 as basal medium, but please see the protocol Text for other options. Cover the whole surface of each culture dish with gel Rex solution. For each 60 millimeter dish, add 1.5 milliliters of gel trek solution.

Incubate the dishes for one hour at 37 degrees Celsius. Additional plates may be wrapped in parfum and stored in the fridge for up to four weeks. Additional diluted gelt TRX may be allotted and stored at minus 20 to minus 80 degrees Celsius, but multiple freeze thaw cycles should be avoided prior to using.

Transfer the gelt TRX coated dishes to a lemon or flow hood and allow them to equilibrate to room temperature. In addition, prepare all other required reagents and ensure that all media are equilibrated to 37 degrees Celsius and appropriately gassed. To adapt human IPCs to feeder free medium First culture.

The IPSC is on meth feeder cells until they are 70 to 80%confluent. Aspirate the medium from each 60 millimeter culture dish and add an appropriate amount of dispa solution, which is one milliliter. In this case, incubate the dishes at 37 degrees Celsius for three to five minutes.

Aspirate the dispa solution from each culture dish and wash off the meth feeder cells gently with DPBS two to three times. Next, add an appropriate amount of complete serum replacement medium to each culture dish. Using a cell scraper, gently scrape the cells off the surface of the dish.

Collect the cell suspension from each dish into separate 15 milliliter conical tubes. Rinse each culture dish with an appropriate amount of complete serum replacement medium and add the rinse medium to the 15 milliliter conical tubes containing the cell suspension centrifuge. The 15 milliliter conical tubes at 200 G for five minutes at room temperature to pellet the cells.

After centrifugation, carefully aspirate and discard the supernat without disturbing the cell pellet. Gently flick the tube to fully dislodge the cell pellet from the tube bottom. Add an appropriate amount of complete serum replacement, feeder free medium according to the desired split ratio.

And gently resuspend the pellet. Do not break the cell clumps to a smaller size because the smaller clumps do not attach well to the surface. Aspirate any residual gel TRE solution from the previously prepared gel Rex coated culture dishes and slowly add an appropriate amount of cell suspension to each culture dish.

Move the culture dish back and forth and side to side several times to disperse the cells across the surface of the dish. Gently place the culture dishes in a 37 degree Celsius incubator with a humidified atmosphere of four to 6%CO2 in air error. Replace the spent medium daily thereafter until the cells become approximately 70 to 80%confluent.

When the human-induced pluripotent stem cells growing a complete serum replacement, feeder free medium are 70 to 80%confluent, they're ready to be subculture. If there are any differentiated cells, remove them prior to packaging. Aspirate the spent medium from the 60 millimeter culture dish using a pipette and rinse the cells twice.

With DPBS gently add one milliliter of prewarm disc based solution to the culture dish. Swirl the culture dish to coat the entire cell surface. Incubate the culture at 37 degrees Celsius for three to five minutes.

Next, aspirate the dis space solution and gently wash the cells twice with DPBS, add complete serum replacement, feeder free medium, and gently scrape the cells off the surface of the culture dish. Using a cell scraper, transfer the cells to a sterile 15 milliliter centrifuge tube. Rinse the culture dish twice with complete serum replacement.

Feeder free medium gently spraying off any cells that have not detached. Pull the medium from both rinses with the cells in the 15 milliliter tube. Centrifuge the tube at 200 G for five minutes at room temperature to pellet the cells without disturbing the cell pellet.

Carefully aspirate and discard the supernatant. Gently flick the tube to fully dislodge the cell pellet from the tube bottom. Gently resuspend the cells in complete serum replacement.

Feeder free medium using a five milliliter serological pipette. Do not tri rate. Transfer the cells to a fresh 60 millimeter gelt TRX coated dish at the desired split ratio.

Move the culture dish back and forth and side to side several times to disperse the cells across its surface. Place the culture dish in a 37 degree Celsius incubator with a humidified atmosphere of four to 6%carbon dioxide in air on the following day. Gently replace the spent medium with complete serum replacement.

Feed of free medium to remove cell debris. Replace the spent medium every day thereafter. This phase 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 immunos staining shows that IPC's cultured with knockout serum replacement medium plus the growth factor cocktail, expressed the pluripotent markers, OCT four and SSEA four. After watching this video, you should have a good understanding of how to adapt and maintain your IPCs and knockout serum replacement, feeder free conditions. So that's it.

Thanks for watching and good luck with your experiments.