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Gibco StemFlex Medium supports the robust expansion of feeder-free pluripotent stem cells (PSCs) and is optimized to support novel applications, including single-cell passaging, gene editing, and reprogramming.

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Robust support of PSC cultures during expansion and maintenance

The newest stem cell medium from the Gibco brand, StemFlex Medium, is the only medium designed to deliver superior performance in the innovative applications and technologies used in today’s stem cell research. StemFlex Medium is uniquely formulated to maximize performance in today’s more challenging applications that have traditionally stressed pluripotent stem cell cultures, such as reprogramming, single-cell passaging, and gene editing. In addition to core performance enhancements, it also delivers the modern conveniences of a flexible feeding schedule (including weekend-free options) and the ability to switch between the matrix and passaging reagents best suited for a chosen application. StemFlex Medium is provided in a convenient two-component kit (450 mL basal medium & 50 mL supplement), and when used with Geltrex Matrix, provides a cost-effective, robust system for superior feeder-free culture of human PSCs. StemFlex Medium enables long-term feeder-free culture of PSCs without karyotypic abnormalities and supports the ability to differentiate into all three germ layers (Figures 1 and 2).

Figure 1. StemFlex Medium provides long-term maintenance of normal PSC properties. Culture displays normal morphology (left); a normal karyotype is observed after 20 passages on a weekend-free flexible feeding schedule (right).

Figure 2. Confirmation of tri-lineage differentiation potential. Following 22 passages on the weekend-free feeding schedule, iPSCs expanded in StemFlex Medium maintain the ability to differentiate into (A) mesoderm, as shown by expression of TNNT2 following differentiation using the PSC Cardiomyocyte Differentiation Kit, (B) endoderm, as shown by the CXCR4+/PDGFRalpha phenotype following differentiation using the PSC Definitive Endoderm Induction Kit, and (C) ectoderm, as shown by expression of Sox1 and nestin following differentiation using the PSC Neural Induction Medium.

Superior performance in today's modern applications

Faster recovery following gene editing

Figure 3. StemFlex Medium supports up to 2-fold faster recovery following gene editing. PSCs expanded in various media formulations were singularized using Gibco TrypLE Select reagent and subjected to delivery of a Cas9 protein/HPRT guide RNA complex via electroporation. Upon seeding at 100,000 viable cells/well in the absence of ROCK inhibitor it was shown that StemFlex Medium supported optimal recovery from this stressful event.

Superior clonal expansion following single-cell passaging

Figure 4. StemFlex Medium supports PSCs in recovery from flow sorting, demonstrating as much as 5-fold improvement in clonal expansion following single-cell passaging in the absence of ROCK inhibitor. PSCs expanded in StemFlex Medium on the rhLaminin-521 substrate for >3 passages were singularized using TrypLE Select reagent. They were then subjected to flow cytometric sorting to isolate live pluripotent stem cells (Tra1-60+/propidium iodide (PI)-negative) and were seeded at 1, 3, or 5 cells/well in a 96-well plate. Following plating, cells were fed with fresh medium every 3 days, and the percentage of wells achieving >5% confluency by day 14 was assessed via whole-well imaging on the IncuCyte ZOOM system. These data indicate that StemFlex Medium provides superior performance in the cells’ recovery from this stressful event.

Improved workflow and robust colony formation

Figure 5. StemFlex Medium provides an improved workflow and robust colony formation for easy clone selection following reprogramming of somatic cells. Human dermal fibroblasts were reprogrammed using the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit. Beginning on day 8 (upon introduction of the stem cell medium per the standard workflow), cells were fed either daily with mTeSR1 Medium or every other day using the StemFlex Medium system. (A) Robust colony formation was observed for the StemFlex Medium system. (B) Improved reprogramming efficiency was also observed for all three donors fed every other day using StemFlex Medium, relative to mTeSR1 cultures on a daily feed schedule.

Superior results with improved stem cell workflows

Traditionally, pluripotent stem cell culture has required daily feeding schedules to maintain pluripotency. This frequent handling of cells has created additional challenges for researchers, including the increased potential for errors and contamination, and increased variation and contamination should multiple users handle a single culture. As with the applications mentioned above, protocol recommendations have been developed to ease some of these challenges with traditional culture systems. However, these recommendations are not well suited for continued, long-term use as they may have an impact on downstream pluripotency. StemFlex Medium (along with Gibco Essential 8 Flex medium) enables a truly weekend-free feeding schedule by consistently maintaining FGF2 activity—a key factor for pluripotency (Figure 6–8).

Figure 6. StemFlex Medium more consistently maintains pluripotency. StemFlex Medium provides prolonged stability of FGF2 when incubated at 37°C, 5% CO2, allowing for flexible feeding schedules, including the weekend-free option, and eliminating daily feeding requirements.

Figure 7. Long-term maintenance of pluripotency in weekend-free feeding. PSCs maintain expression of self-renewal factors using StemFlex Medium for 21 passages in wells coated with GelTrex matrix. The cells were stained for pluripotency markers using the Invitrogen PSC 4-Marker Immunocytochemistry Kit.

Figure 8. Recommended weekend-free feeding schedule. Unlike traditional PSC media, StemFlex Medium eliminates the need to manage cultures daily, enabling a truly weekend-free schedule for expansion and maintenance of PSCs.

Recommended reagents

It is not uncommon for complementary reagents used within a PSC culture system (like matrices and passaging reagents) to have an impact on performance. Traditional PSC media are typically only compatible with one matrix and passaging method, which can require significant protocol adjustments or less-than-desirable results. StemFlex Medium offers three-way usage flexibility, not only allowing you to decide what matrix and passaging reagent will best fit your needs but also giving you flexibility in your feeding schedule.

Table 1. Flexibility to choose the best matrix and passaging reagent.

Component Product Best for
Matrix Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix Replacement for Matrigel matrix

An ideal economical choice for expansion and maintenance of PSCs
Vitronectin Recombinant Human Protein, Truncated (VTN-N) Applications requiring a more lean, defined matrix
Recombinant Human Laminin-521 (rhLaminin-521) Recommended for highest performance in stressful applications including gene editing and single-cell passaging
Passaging reagent Versene Solution Clumped-cell passaging
StemPro Accutase Cell Dissociation Reagent 2- to 3-cell clusters
TrypLE Select Enzyme, with or without RevitaCell Supplement Single-cell passaging