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In this video, we will demonstrate how to thaw the frozen aggregates of a human induced pluripotent stem cell line.

Proper thawing and handling are essential for optimal recovery of your frozen cells and to ensure your cell cultures are set up successfully. Refer to the Product Information Sheet specific to the cell line you're working with. The latest version of the Product Information Sheet can be found on the product page.

For this demonstration, we'll be using the SCTi003-A cell line. For detailed lot-specific information on quality testing results and recommended culture conditions, please refer to the lot-specific Certificate of Analysis.

In a biological safety cabinet, let an aliquot of mTeSR™ Plus media and coated cultureware, such as a Matrigel®-coated plate, warm up at room temperature before thawing the cells. Do not warm mTeSR™ Plus in a 37°C water bath. Begin by taking the vial from the cold storage unit and place it on dry ice when transporting it to the biological safety cabinet. We recommend thawing one vial of cells at a time. Wipe the outside of the vial with 70% ethanol or isopropanol. Twist the cap a quarter turn to relieve internal pressure, then retighten the cap.

The cell thawing process should be performed quickly to ensure optimal cell viability and recovery. For consistent thawing and reduced variability, we recommend using the automated Thawstar® CFT2, or you can thaw the cells in a 37°C water bath for 2 to 3 minutes by gently shaking the vial. Do not allow the vial to thaw completely. Instead, remove the vial when a small frozen cell pellet remains.

Wipe the outside of the vial again with 70% ethanol or isopropanol. Use a 2 mL serological pipette to transfer the contents of the cryovial to a 15 mL conical tube. It's advisable to use a 2 mL serological pipette instead of a 1 mL pipette to minimize breakage of cell aggregates. Next, add 5 to 7 mL of room temperature mTeSR™ Plus dropwise to the 15 mL tube, gently mixing the cells as the medium is added. Gently flick the tube to help with mixing the cell suspension.

Centrifuge the cell suspension at 300 g for five minutes at room temperature. Remove the tube from the centrifuge, then aspirate the supernatant carefully, leaving some media behind while keeping the cell pellet undisturbed. Add 1 mL of mTeSR™ Plus to the cell pellet. Then, resuspend the cell pellet by gently flicking the tube. Avoid pipetting up and down. Ensure the cells remain as aggregates for optimal attachment and recovery.

Next, take the Matrigel®-coated plate and aspirate the Matrigel® solution from each well. Then, add 2 mL of mTeSR™ Plus to each well of the six-well plate. Aliquot the cell suspension into the coated six-well plate containing mTeSR™ Plus at six different densities, as outlined in the cell line-specific Product Information Sheet. Gently flick the tube as many times as needed to ensure a uniform cell suspension prior to seeding the cells into the wells. Flick the tube again between seeding into each well, as the cell aggregates will sink to the bottom of the cell suspension. The repeated flicking allows for an equal distribution of the cell aggregates between the wells.

Place the plates in an incubator at 37°C and 5% CO₂. Move the plate in several quick, short back-and-forth and side-to-side motions to evenly distribute the cell aggregates within each well. Note that uneven distribution of cell aggregates may result in increased differentiation of human iPSCs, so it's important to move the plate sufficiently in quick motions. Do not disturb the plate for 24 hours.

Before performing the first medium change, it's good practice to check the plate using the microscope to confirm the cell aggregates have attached to the matrix. Perform medium changes as needed using mTeSR™ Plus. Refer to the mTeSR™ Plus technical manual for examples of a flexible feeding schedule.

Visually assess your cultures on a regular basis to monitor growth and morphology. This will help determine the optimal day to passage the cultures.

For additional information on passaging or managing spontaneous differentiation in your cultures, please refer to the Product Information Sheet relevant to the cell line you are using. For more information, visit our frequently asked questions page about iPSCs on the �����ƽ�� Technologies website.