CTS StemFlex Medium

Gene editing technologies offer the potential to advance PSC-based therapies by enabling the creation of hypoimmune cells that evade immune rejection or by correcting genetic defects for therapeutic purposes. The workflow for gene editing in PSCs is shown in Figure 1. PSC recovery after electroporation and single cell clonal expansion can be challenging and require a medium that can maintain viability and pluripotency throughout this process. CTS StemFlex Medium supports the maintenance of high quality PSCs through stressful applications.

The optimized formulation of CTS StemFlex Medium supports recovery of PSCs after the Cas9 complex is introduced via electroporation (Figures 2A and 2B ). CTS StemFlex Medium also maintains high levels of pluripotency and delivers editing efficiencies similar to research use only StemFlex Medium (RUO) (Figures 2C and 2D).​

Figure 2. CTS StemFlex Medium enables electroporation-based delivery of the CRISPR Cas9-gRNA complex targeting the CIITA gene. CTS StemFlex Medium supports fast recovery following gene editing as shown by (A) confluency and (B) fold change in the post-electroporation passage. PSCs cultured in CTS StemFlex Medium or RUO StemFlex Medium exhibit similar recovery after electroporation. PSCs in CTS StemFlex or RUO StemFlex Medium also (C) maintain high expression of OCT4 and NANOG pluripotency markers as assessed by flow cytometry and (D) achieve comparable editing efficiencies at the CIITA target as determined by next-generation sequencing (NGS).

Following electroporation, edited PSC clones can be efficiently expanded in CTS StemFlex Medium (Figures 3A and 3B) without loss of pluripotency (Figure 3C) or genomic stability (Figure 3D).

Figure 3. CTS StemFlex Medium supports expansion of high-quality PSC clones. PSCs edited at the CIITA target and cultured in CTS StemFlex Medium or RUO StemFlex Medium were seeded by limited dilution at a clonal seeding density of 1 cell/well. (A) CTS StemFlex Medium supported expansion of PSC clones from 3 PSC lines edited at the CIITA locus, with efficiencies similar to RUO StemFlex Medium. (B) Editing efficiency at the CIITA target was determined by next-generation sequencing (NGS) for PSC clones expanded in CTS StemFlex Medium. These PSC clones also (C) exhibited high expression of pluripotency markers, as assessed using the PluriTest Assay and (D) maintained genomic stability as assessed using the KaryoStat assay. A representative KaryoStat plot is shown for a TFSi001-A iPSC clone.

PSCs cultured in CTS StemFlex Medium exhibit high viability and cumulative fold expansion similar to cells grown in research use only StemFlex Medium (RUO) (Figure 4A). In addition, PSCs expanded in CTS StemFlex Medium maintained pluripotency and genomic stability after consecutive single cell passages (Figures 4B and 4C).

Figure 4. CTS StemFlex provides consistent expansion and maintains pluripotency and genomic stability during consecutive single cell passaging.(A) PSCs in CTS StemFlex Medium exhibited consistent cumulative fold expansion over 10 single cell passages with growth similar to RUO StemFlex Medium. These PSCs also (B) maintained high expression of OCT4 and NANOG pluripotency markers as assessed by flow cytometry, and (C) maintained genomic stability as assessed by the KaryoStat assay after 10 single cell passages.

CTS StemFlex Medium promotes consistent growth in long-term culture while also maintaining normal morphology (Figure 5A), pluripotency (Figures 5B and 5C), and genomic stability (Figure 5D) across multiple PSC lines.

Long-term expansion of PSCs can be achieved in CTS StemFlex Medium without the loss of expansion potential, pluripotency, or genomic stability (Figure 5). In addition, PSCs maintain the ability to differentiate to all three germ layers (Figure 6).

Figure 6. PSCs expanded in CTS StemFlex Medium maintain the ability to differentiate into all three germ lineages. An iPSC and an ESC line cultured in CTS StemFlex Medium were spontaneously differentiated as embryoid bodies followed by assessment of trilineage potential. Gene expression analysis using the TaqMan® hPSC Scorecard Panel demonstrated differentiation potential to ectoderm, mesoderm and endoderm.

Gibco Cell Therapy Systems (CTS) products have been developed to help facilitate the transition from discovery to clinical and commercial manufacturing by providing high-quality, GMP-manufactured ancillary materials, helping ensure commercial-scale production. These materials are extensively qualified, traceable, and accompanied by regulatory documentation.

CTS StemFlex Medium has a formulation similar to that of research use only StemFlex Medium (RUO) with specific modifications in line with regulatory guidance.Using the same protocol, CTS StemFlex Medium delivers comparable performance to RUO StemFlex Medium, including cell yield (Figure 7A) and expression of pluripotency markers (Figures 7B). CTS StemFlex Medium is formulated to support expansion of PSCs in the development of PSC-derived therapies.

For large-scale expansion of PSCs, three-dimensional or suspension culture may be preferred. Cells cultured in CTS StemFlex Medium can be transitioned to suspension culture in CTS StemScale PSC Suspension Medium. After adapting to CTS StemScale medium, PSCs readily aggregate into high-quality spheroids (Figures 8A and 8B) that maintain expansion potential (Figure 8C) and high levels of pluripotency (Figure 8D).

Figure 8. PSCs in CTS StemFlex Medium can be transitioned to 3D culture. Adherent PSCs adapted to CTS StemFlex Medium were dissociated with CTS TrypLE Select reagent. Single cells were then seeded in CTS StemScale PSC Suspension Medium to initiate nucleation of cells into 3D spheroids. PSCs transitioned from CTS StemFlex Medium readily aggregated into (A) high-quality spheroids that exhibit (B) consistent cumulative fold expansion and (C) high expression of OCT4 and NANOG pluripotency markers.

*Gibco CTS products are manufactured at a site that uses methods and controls that conform with cGMP for medical devices, 21 CFR Part 820. Our FDA-registered manufacturing site is ISO 13485-certified. Specific intended-use statements, full documentation traceability, and convenient access to our Drug Master File (DMF) are available for CTS StemFlex Medium.