StemFlex™ Medium - FAQs

The StemFlex Medium (Cat. No. A3349401) includes 3.151 g/L (~17.5 mM) of D-glucose (dextrose) in the Basal Medium. The StemFlex Medium Supplement does not include glucose.

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We recommend StemFlex Medium (Cat. No. A3349401) or Essential 8 (Cat. No. A1517001) for standard single-cell iPSC cloning in the presence of RevitaCell or ROCK inhibitor. StemScale PSC Suspension Medium (Cat. No. A4965001) is not recommended for iPSC cloning.

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StemFlex Medium does not contain pen/strep antibiotic.

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Essential 8 Flex Medium is a serum-free, xeno-free medium that contains proprietary material to extend the activity of the key heat-sensitive component, FGF2. Therefore, it supports the culture and expansion of feeder-free pluripotent stem cells (PSCs) without the need for daily or weekend feeding. Note: We also offer Essential 8 Medium that does not allow skipping of PSC feeding during the weekend.

StemFlex Medium also supports the expansion of feeder-free PSCs with a flexible feeding schedule (including weekend-free feeding) and also the ability to choose the matrix and passaging reagent that best suits specific applications.

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StemFlex Medium contains basic FGF which has been shown to result in differentiation of mouse embryonic stem cells (mESCs) to mouse-derived epiblast stem cells (mEpiSCs). Also, mESCs demonstrate dependence on leukemia inhibitor factor (LIF). Therefore, StemFlex Medium is not recommended for culture of mESCs.

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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of CD34+ blood cells. For CD34+ cells, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 39-44). On Day 3, you can utilize rhVTN-N (Cat. No. A14700), Geltrex (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, reprogrammed CD34+ cells should be fed every-other-day with StemFlex Medium.

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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of both neonatal and adult human dermal fibroblasts. For fibroblasts, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 16-20). On Day 7, you may use rhVTN-N (Cat. No. A14700), Geltrex matrix (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, we recommend that you feed reprogrammed fibroblasts every-other-day with StemFlex Medium.

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Yes. If improved reprogramming efficiency is required, then you may utilize Essential 7 (Essential 6 + bFGF) or Essential 8 Medium for reprogramming somatic cells and directly transition them into StemFlex Medium system upon colony selection.

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Yes. We have seen compatibility with the following differentiation kits provided by Thermo Fisher Scientific: PSC Cardiomyocyte Differentiation Kit (Cat. No. A2921201), PSC Definitive Endoderm Induction Kit (Cat. No. A3062601), PSC Neural Induction Medium (Cat. No. A1647801), and PSC Dopaminergic Neuron Differentiation Kit (Cat. No. A3147701).

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We have not yet evaluated the Geltrex matrix system for clonal expansion in the presence of StemFlex Medium. However, rhLaminin-521 does provide optimal survival of cells following single-cell passaging and thus this matrix is recommended for such critical applications.

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We recommend feeding of cells every three days, even in the presence of RevitaCell Supplement up to ˜14 days in culture.

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Yes. We use Antibiotic-Antimycotic (Cat. No. 15240062) for additional protection from contamination.

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We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/PG1584-PJT1313-COL31227-Demonstrated-Protocol-Performing-CRISPR-Cas9-FHR.pdf); see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.

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Yes. PSCs cryopreserved from cultures of other media systems may be thawed directly into the StemFlex Medium system. However, certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use Versene solution to passage the cells into the StemFlex Medium system.

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This transition is very straightforward. We recommend at least a 2-passage transition into the StemFlex Medium system. Briefly, if you have cryopreserved cells previously cultured in the mTeSR1/Matrigel system, we recommend thawing the cells back into mTeSR1/Matrigel until fully recovered. Upon reaching ~70-85% confluency, passage using Versene solution and seed directly into the StemFlex Medium system.

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Yes. The main reason that the workflow states this is to accommodate ROCK inhibitor or RevitaCell Supplement that have been added during passaging. In such circumstances where ROCK inhibitor or RevitaCell Supplement are added, you should feed your cells within 18-24 hours post-passaging. However, if ROCK inhibitor or RevitaCell Supplement are not included at the time of passaging, then you may skip the day post-passaging.

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In such circumstances, we recommend either full media change on Sunday or overlaying the culture with additional media on Sunday.

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StemFlex Medium is a more robust medium than TeSR-E8 but has fewer components than mTeSR. Performance of StemFlex Medium was shown to be superior to mTeSR1 in a number of applications including single-cell passaging, gene editing and reprogramming.

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The formulation of StemFlex medium does include BSA and thus is not considered xeno-free. It is more defined than mTeSR1 as it contains less material in the formulation. Essential 8 and Essential 8 Flex are recommended for the most defined and xeno-free PSC culture media.

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Yes. We recommend following the coating instructions for Vitronectin or rhLaminin-521 and using the culture and passaging recommendations in the StemFlex protocol.

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For optimal health of the cultures, cells should be passaged upon reaching ~85% confluency. We have observed improved cell health upon single-cell passaging for cells passaged between 40-85% confluency. If the cells are overly confluent, then inclusion of ROCK inhibitor/RevitaCell Supplement is recommended at the time of passaging. In general, we recommend carrying cells at a range of split ratios to prevent routine passaging at high confluencies as this can result in poor cell survival.

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StemFlex Medium will accommodate most feeding schedules including every day and weekend-free schedules. For a list of recommended feeding schedules, visit thermofisher.com/stemflex.

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The use of ROCK inhibitor is not required when culturing in StemFlex Medium on rhLaminin-521. However, supplementation with RevitaCell Supplement can provide additional support to PSCs during stressful transitions such as single-cell passaging.

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Versene passaging is recommended for routine culture of PSCs in StemFlex Medium.

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No. We recommend thawing the aliquot either at room temperature or overnight at 2 to 8 degrees C.

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Yes. Following reconstitution, complete StemFlex Medium can be aliquoted and stored at -5 to -20 degrees C for up to 6 months. Alternatively, aliquots of the supplement can be made and frozen at -5 to -20 degrees C for up to 6 months. Avoid multiple freeze-thaw cycles.

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