Human Pluripotent Stem Cells


Induction - generating iPSCs

At what stage of reprogramming the generated iPSCs should be transferred to NutriStem® hPSC XF?

When the first PSCs colonies arise the cells may be shifted to NutriStem hPSC XF.

What is the preferred method for generating iPSCs?

There are many methods for generating iPSCs including, but not limited to, viral vectors (DNA, RNA, dsRNA etc.), small molecules, plasmid transfection and more. All methods involve the reprogramming of cells on the genetic level and return them to their naïve state. The recommended method depends on the origin of donor cells and your downstream applications. BI products are compatible for growth, expansion, maintenance and cryopreservation of hiPSC generated by all methods.

Which culture medium should be used for the cells of origin in the induction stage?

The preferable method (including all the media and supplements) should be stated in the induction protocol and followed to the latter.

Culturing PSCs

What are the common methods for culturing pluripotent stem cells?

The cells need to grow in multi-well plates, flasks or dishes coated with a layer to support the pluripotent cells. There are two categories of coating, the first is protein based, usually ECM proteins; the second is feeder cells based, murine or human fibroblasts usually.

Do I need to add supplements to Nutristem® hPSC XF before use?

NutriStem® hPSC XF is a complete medium. This medium is low protein content, and specifically low on growth factors to prevent cellular differentiation and keeping the pluripotent state.

What are the advantages of Serum-free and Xeno-free medium?

The medium should be serum-free for 4 reasons:
• To eliminate influence of the unknown ingredients within the serum
• Support direct or indirect medical applications which require known ingredients .
• Elimination of contagious health risks (prions, bacterial and viral).
• Reproducibility from lot to lot

Should I add TGF beta and bFGF to NutriStem® hPSC XF Medium before using it?

No. NutriStem® hPSC XF Medium is ready-to-use medium which contains TGF beta and bFGF.

What is the purity grade of the proteins within the Nutristem® hPSC XF medium?

Nutristem® contains transferrin, and human serum albumin (HSA) which is approved for medical uses.

Is it necessary to add bactericides and/or fungicides to the media?

It is not advisable if sterile work and culturing environments are kept.

How long can the cells be kept in an undifferentiated state?

The cells can be kept in an undifferentiated state for more than 50 passages, depending on the culture conditions and origin of cells.

What are the plating conditions after the initial reprogramming of the pluripotent cells?

The cells must be plated as per the induction protocol

Subculturing pluripotent cells

How long can a culture keep without subculturing?

Even if the culture doesn't show more than 15% differentiation, or the colonies don't grow and touch each other, a Feeder-dependent culture should be re-plated on new coated plate after a week from previous subculturing, since the feeder layer cells grow old and less supportive of the pluripotent colonies

When should the medium be changed after subculturing?

Medium should not be changed for the 48 hours after subculturing

Differentiating culture

Why should differentiating cells be removed from the culture?

Pluripotent cells culture should have an undifferentiated appearance. When more than 15% differentiation, the culture is not homogenous, when there are colonies that show differentiating cells, a manual removal of colonies should be performed.

Does "cleaning" culture procedure damage the culture integrity?

The removal of colonies (either differentiated or undifferentiated) should not damage the entire culture, as long as proper laboratory practices are maintained.

Why is there differentiation in a pluripotent cell culture?

There are several physical and biological reasons for differentiation appearing in a pluripotent culture, among them are the culture environment (changes in temperature/CO2/pH), the age of the culture (passage) and changes of media. Because the reasons are physical and biological, plates from the same cell line, under the same culturing conditions may exhibit different ratios of differentiation.

Cryopreservation

What freezing medium should be used for cryopreservation of pluripotent cells? (As clumps or single cells)

The recommended medium is CryoStem™ Freezing Medium. This medium was developed for the cryopreservation of hESCs and hiPSCs. This is an animal component free (ACF) medium, containing DMSO and methylcellulose. Pluripotent cells preserved in this medium show a very good recovery rate after thawing.

What is the recommended ratio for cryopreservation?

One ready for passage well of 6-wells plate per 1 ml cryopreservation vial.

For what period of time can pluripotent cells be preserved?

Indefinitely, as long as the freezing conditions are maintained - liquid nitrogen in vapor or liquid phase.

Should serum be added to the freezing medium?

Serum should not be added, CryoStem™ Freezing Medium is ACF and ready-to-use.

NutriStem® hPSC XF medium stability and storage conditions

How should NutriStem® hPSC XF medium be kept prior to use?

The NutriStem® hPSC XF medium is stored frozen (-20°C). The medium should be thawed in 2-8°C or at room temperature and aliquoted to appropriate work volumes which should be re-frozen. As long as the aliquoting is performed in sterile conditions the medium's expiry date is as noted on the original bottle.
Thawed medium can be stored up to two weeks in 2-8°C, in dark conditions.

How many freeze-thaw cycles can be performed to NutriStem® hPSC XF medium?

The medium can be thawed twice - the first thaw when aliquoting the original bottle and the second thaw when working with the medium.

What is to be done if sediments appear after thawing?

NutriStem® hPSC XF medium does not contain ingredients that cause sedimentation and therefore the medium should not be used.

What should I do if NutriStem® hPSC XF medium was partially thawed?

It is not recommended to re-freeze partially thawed medium. Partially thawed medium must be fully thawed before re-freezing, gently mixed and frozen again.

Do cells need adaptation when transferred to NutriStem® hPSC XF medium?

Most lines will not require adaptation, simply seed cells directly into NutriStem® hPSC XF medium. Some lines will require gradual adaptation or elevated bFGF concentration for the first few passages.

Why does NutriStem® hPSC XF medium change color when used?

NutriStem® hPSC XF medium contains Phenol-Red which is a pH indicator. In case of acidifying (due to prolonged exposure to CO2, from dry ice for instance) the medium changes color to yellow; In exposure to alkali substances the medium changes color to purple. In either case the medium should not be used.