Adaptation from legacy media

BMEM has been specifically modified to ease the transition from legacy media and remove the risk of spontaneous differentiation during adaptation. It is important to note that the morphology of cells in chemically defined media such as BMEM differs from that seen in mTeSR1™, mTeSR™ Plus, and StemFlex™. hiPSCs in BMEM exhibit a looser morphology and do not grow as colonies but as looser non-colony ‘continents’ which will become a monolayer if allowed to grow as shown in the figure below.

Fig: Representative phase-contrast images of the same hiPSC line either kept in mTeSR for 36 passages (left), and 7 passages into BMEM culture (right). Zoomed in versions make it easier to see the significant differences in morphology between the two media.

Spontaneous differentiation should not be seen in BMEM cultured cells and no ‘picking’ should be expected.

Here, we present protocols with the goal of adapting the hiPSCs derived and cultured in legacy media to BMEM/Max as fast as possible. It is important to note that the faster the cells are growing before the adaptation (including pre-freezing), the easier and more reproducible the process will be.

CRITICAL: Before adapting your cells to BMEM, make sure they are a pure population of hiPSCs, presenting high expression of pluripotency markers. BMEM can help with selecting a population of more proliferative cells but will not make non stem cells become stem cells.

Adaptation of frozen cells

Timing: 20 to 40 days.

1. When thawing a new vial of cells, split the vial into two. Seed half of the cells into their original media (two wells, at 1:3 and 1:6), and the other half into BMEM-Ri (also at 1:3 and 1:6).

a. The cells kept in the original media can be used as a control for the adaptation, or a new source of cells if needed.

2. After 24h, check plate on a microscope to evaluate the level of attachment of the cells to the wells, but do not change the media.

a. Keeping the cells in BMEM-Ri for the first 48 h increases survival post-thawing.

3. After 24h (48h post thawing), change media to BMEM.

CRITICAL: Do not use weekend-free feeding schedule during adaptation.

4. After 4 days (or 5 days if confluence is very low), choose the well with the highest split ratio that achieved 70-80% confluence. Passage these cells using the same split ratio and two higher ones.

a. For example, if the 1:3 well achieved 70% confluency in 4 days, passage this well at 1:3, 1:6, and 1:12.

5. Keep repeating this ratio testing process until you settle on a split ratio that achieves 70-80% confluence after 4 days (typically 5-10 passages) increasing the ratios assessed (i.e. 1:15, 1:20, 1:25) as cells adapt.

Note: After a minimum of 4 passages post thawing, the ratio testing should be performed until only the lowest split ratio well is the only one to consistently achieve 70-80% confluence within the desired interval between passages. At that point, this split ratio should be used for further passages.

Adaptation of cells already in culture in legacy media

Timing: 20 to 40 days.

Note: Switching media mid-passage or using 50:50 mixes of media results in slower adaptation. Starting from the second passage in BMEM, cells should present a different morphology compared to those not cultured in chemically defined conditions.

1. If cells are already in culture in legacy media, on the day of their next passage, seed them straight into BMEM-Ri using the same ratio used for the legacy media, and two higher ratios to perform a split ratio test.

a. For example, if cells in legacy media have been split at 1:3 ratio, passage the into BMEM at 1:3, 1:6, and 1:12.

2. After 24h, and every day following, change media with BMEM.

3. After 4 days (or 5 days if confluence is very low), choose the well with the highest split ratio that achieved 70-80% confluence. Passage these cells using the same split ratio and two higher ones.

a. For example, if the 1:3 well achieved 70% confluency in 4 days, passage this well at 1:3, 1:6, and 1:12.

4. Keep repeating this ratio testing process until you settle on a split ratio that achieves 70-80% confluence after 4 days (typically 5-10 passages) increasing the ratios assessed (i.e. 1:15, 1:20, 1:25) as cells adapt.

Note: The faster the cells are already growing in culture, the faster the adaptation process will take. For example, mTeSR cells growing at 1:6 in a 7-day schedule took between 7-10 passages to completely change their morphology and become BMEM-adapted, achieving split ratios between 1:12 and 1:15 in 4 days. On the other hand, mTeSR and StemFlex cells that were growing at 1:10 in a 4-day schedule took 3 passages to completely change morphology and 5 to adapt to higher split ratios, in both cases to 1:20 in 4 days.