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 (RUO) StemFlex Medium (Figures 2C and 2D).​

Figure 2. CTS StemFlex 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. (C) Cells also maintained high expression of pluripotency as assessed by flow cytometric analysis of OCT4 and NANOG markers. (D) RUO StemFlex and CTS StemFlex media achieve comparable editing efficiencies via next generation sequencing (NGS) at the CITTA target.

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).

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

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.

Figure 5. CTS StemFlex Medium maintains normal morphology, pluripotency and karyotype in long-term culture. (A) PSCs cultured in CTS StemFlex Medium demonstrate typical PSC morphology. Pluripotency is maintained over long-term culture in CTS StemFlex Medium. PSC lines grown for >10 passages exhibited high expression of pluripotent markers by (B) flow cytometry and (C) by immunocytochemistry. (D) CTS StemFlex supports genomic stability in long-term culture as confirmed by the KaryoStat Assay.

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. Trilineage differentiation potential. ESC (A) and iPSC (B) were spontaneously differentiated as embryoid bodies using CTS Essential 6 Medium. Gene expression was analyzed using the TaqMan® hPSC Scorecard Assay and showed upregulation of ectoderm, mesoderm and endoderm markers.

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 (RUO) StemFlex Medium 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 (Figure 7B) and is formulated for clinical PSC expansion and to support 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 (Figure 8A) that maintain expansion potential (Figure 8B) and high levels of pluripotency (Figure 8C).

Figure 8. Transitioning PSCs adapted to CTS StemFlex Medium to suspension culture. Adherent PSCs adapted to CTS StemFlex Medium were dissociated with CTS TrypLE Select and seeded for 3D spheroid initiation in CTS StemScale PSC Suspension Medium. This resulted in high-quality PSC spheroids with rounded morphology (A and B) consistent expansion (C) and maintenance of pluripotency (D).

*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.