BMEM Human Primed Pluripotent Kit

Instructions for Use

Use sterile technique to prepare complete BMEM Human Primed Pluripotent (basal medium + supplement). Complete BMEM can be used straight from the refrigerator (4 °C) and should not be warmed to RT or 37 °C prior to use.

Directions for Use

Incubator atmosphere: Humidified atmosphere of 5% CO2. Hypoxia (5% O2) can be used but is not essential.

Ensure that proper gas exchange is achieved in culture vessels.

BMEM can be used like any other traditional hiPSC medium. It is recommended to passage cells with BMEM Dissociation when they reach 70-80% confluence, split ratios should be modified to achieve this level of confluence every 4 days, selecting the highest split ratio possible. Cells should be passaged every 5 days irrelevant of confluence, adjusting the split ratio to compensate. Once adapted to BMEM, which may take 5-10 passages, cells will grow faster than in legacy media resulting in a requirement for higher split ratios. Consistency is beneficial for subsequent differentiation. Do not allow cells to become 100% confluent as it will result in slow growth and unreliable passaging.

ROCK Inhibitor

Addition of a ROCK inhibitor, such as 2 µM thiazovivin or 10 µM Y27632 for 24 hours after passage is recommended to provide more reproducible passaging, reduce selective pressure, and allow higher split ratios.

Feeding Schedule

Daily media changes provide optimal cell growth and should be used when adapting cells to BMEM. Once adapted, BMEM is compatible with day-skipping, weekend-free, and no-change protocols – all feeding schedules will maintain pluripotency as long as cells are split 70-80% confluence.

Adaptation from Legacy Media

BMEM has been specifically modified to ease the transition from legacy media. Spontaneous differentiation during transition should not be seen.

The morphology of cells in chemically defined media such as BMEM differs from that seen in mTeSR1™, mTeSR™ Plus, and StemFlex™. Cells 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 overgrow. It is common for cells transitioning from the tight mTeSR1™ morphology to the loose chemically defined morphology to be mistaken for spontaneous differentiation. This is not the case, spontaneous differentiation will not be seen in BMEM cultured cells and no ‘picking’ should be expected.

To Adapt Cells from Legacy Media 

NOTE: The aim is to eliminate the mTeSR1-type dense colony growth. Switching media mid-passage or using 50:50 mixes of media results in slower adaptation. 

Product Use

For Research Use Only. 

Not for diagnostic procedures. 

Reference 

1.         Lyra-Leite, D.M., Copley, R.R., Freeman, P.P., Pongpamorn, P., Shah, D., McKenna, D.E., Lenny, B., Pinheiro, E.A., Weddle, C.J., Gharib, M., et al. (2023). Nutritional requirements of human induced pluripotent stem cells. Stem Cell Reports 18, 1371-1387. 10.1016/j.stemcr.2023.05.004.

Troubleshooting

Slow growth and low confluency by day 4

Cause: Split ratio was too high.

Solution: Obtain new cells and split more gently at a variety of ratios.

Cause: Media is too old.

Solution: Complete media should be used within 30 days.

Cause: Cells were either overconfluent (>90%) or underconfluent (<60%).

Solution: Adjust split ratio of hiPSCs to achieve 70-80% confluency after 4 days of growth.

hiPSC did not attach

Cause: No extracellular matrix on the plates or matrix may be too old and have dried out.

Solution: Discard old plates and replace.

Cause: Cells were overconfluent before passaging and became contact inhibited.

Solution: Adjust split ratio of pluripotent cells to achieve 70-80% confluency after 4 days of growth.

Cause: Cells were kept in dissociation solution for too long.

Solution: Follow the specific dissociation times for each dissociation reagent.