Large-Scale Expansion of PSCs To Support Cell Therapy Manufacturing

Scalable and efficient expansion of pluripotent stem cells (PSCs) continues to present a challenge for PSC-derived allogeneic therapies that require large numbers of cells. Three-dimensional (3D) suspension culture offers a promising solution for large-scale production of high-quality PSCs required for cell therapy manufacturing applications. However, a suspension culture medium that supports the transition from bench to clinic has not been commercially available. Therefore, we developed Gibco™ CTS™ StemScale™ PSC Suspension Medium, which promotes the self-aggregation of single cells into 3D spheroids that can be effectively scaled to multi-liter culture volumes with consistent performance across PSC lines. This PSC culture medium delivers similar benefits and performance as our research use only (RUO) Gibco™ StemScale™ PSC Suspension Medium but in a xeno-free formulation.CTS StemScale medium overview CTS StemScale PSC Suspension Medium is a xeno-free medium that enables robust expansion of PSCs in suspension for translational and clinical research. The medium is scalable and easy to use (Figure 1), utilizing the self-assembly method of spheroids to promote PSC growth without the use of microcarriers. PSCs expanded in CTS StemScale medium maintain pluripotency and genomic stability across multiple passages and can be differentiated to the three germ layers. The benefits of CTS StemScale PSC Suspension Medium include: • Enhanced expansion capability versus other PSC suspension media, reducing manufacturing time and cost • Scalable expansion of PSCs with a simplified workflow that does not require a cell strainer • Consistent growth of multiple PSC lines • Supported regulatory filings with GMP manufacturing, traceability of raw materials, and regulatory documentation Seed single cells: 10 µM Y-27632 0.1 U/mL DNase I 200,000 cells/mL 3 L bioreactor Place on orbital shaker (recommended 70 RPM) Single cells can be passaged and reseeded for continued expansion or utilized for downstream applications Replace 50% medium daily Replace 50% medium daily (allow spheroids to settle first) Place on orbital shaker (recommended 70 RPM) Passage 300–400 µm diameter spheroids with 0.25X CTS TrypLE Select Enzyme Place single cells in larger vessel: 10 µM Y-27632 0.1 U/mL DNase I 200,000 cells/mL Figure 1. Simplified CTS StemScale PSC Suspension Medium workflow for initiation of small-scale cultures that can be scaled up. Cultures in CTS StemScale medium are initiated by seeding single cells in the presence of Y-27632 (to promote spheroid nucleation) and DNase I (to prevent undesirable cell aggregation from cell lysis during passaging). Cultures in CTS StemScale medium should be fed daily using 50% medium replacement. Once spheroids reach approximately 300–400 μm in diameter, they can be passaged using diluted (0.25X) Gibco™ CTS™ TrypLE™ Select Enzyme. The resulting single-cell suspension can then be scaled up into larger vessels or utilized in downstream applications. CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale 2RUO and CTS StemScale media comparison Achieving consistent cell yields is critical for cell therapy manufacturing and for developing PSC-based therapies. CTS StemScale medium facilitates the transition to the clinic as it supports robust expansion of PSCs and comparable cell yields as the RUO StemScale medium. The formulation of CTS StemScale medium is similar to RUO StemScale medium but with specific modifications in line with regulatory guidance. These formulation changes necessitate a few minor protocol differences between the RUO and CTS versions of StemScale PSC Suspension Medium to provide similar performance; these protocol differences affect seeding density, feeding strategy, and days of growth (Table 1). For example, a higher seeding density is recommended for cultures in CTS StemScale medium as compared to cultures in RUO StemScale medium. Furthermore, cultures in CTS StemScale medium will require an additional day of growth for spheroids to reach the recommended passaging metric of an average 300–400 μm spheroid diameter. To align the CTS StemScale protocol with cell therapy manufacturing applications and to obtain similar cell yields as with the RUO StemScale medium, the CTS StemScale protocol incorporates two additional slight modifications to the RUO StemScale protocol (Table 1). The CTS StemScale protocol utilizes CTS TrypLE Select Enzyme during passaging rather than the Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent. DNase I is also added while seeding new cultures in CTS StemScale medium. Table 1. Protocol differences between RUO StemScale and CTS StemScale media. Modifications to the RUO StemScale protocol allow for similar cell yields to be obtained with the CTS StemScale medium. StemScale PSC Suspension Medium Dissociation reagent Inclusion of DNase I Days of growth Feeding strategy Seeding density StemPro Accutase Cell Dissociation Reagent* DNase I not required CTS StemScale PSC Suspension Medium CTS TrypLE Select Enzyme (diluted**) DNase I should be added 4–5 days† Every other day 150,000 cells/mL * Contains an animal-origin component. ** CTS DPBS(–/–) can be used to dilute CTS TrypLE Select Enzyme to a lower concentration. † Estimated time to achieve an average spheroid diameter between 300–400 µm. 5–6 days† Daily 200,000 cells/mL CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale 3Figure 2 shows a comparison between spheroids grown in RUO StemScale medium and spheroids grown in CTS StemScale medium from the same initial cell bank. Spheroids grown in RUO StemScale medium and CTS StemScale medium typically reach the upper limit (400 µm) within 5 days and 6 days, respectively (Figures 2A and 2C). A 1,000 µm CTS StemScale medium (day 6) 1,000 µm B StemScale medium (RUO) (day 5) Fold expansion 14 12 10 8 6 4 2 0 RUO spheroids (day 5) CTS spheroids (day 6) Spheroid media condition The additional day of growth for cultures in CTS StemScale medium enables the spheroids grown in this medium to achieve the same fold expansion as the spheroids grown in RUO StemScale medium (Figure 2B). Daily feeding of cultures with CTS StemScale medium will help ensure that spheroids remain highly viable through this additional day of growth. Cultures in CTS StemScale medium can also be seeded at a higher density to promote spheroid nucleation and greater cell yields. C Spheroid diameter (µm) 500 400 300 200 100 0 Day 1 Day 2 Day 3 Day 4 Days in culture RUO spheroids Day 5 CTS spheroids Day 6 Red = Maximum recommended average diameter for passaging Green = Minimum recommended average diameter for passaging Figure 2. CTS StemScale medium provides similar performance to RUO StemScale medium. As indicated in Table 1, spheroids grown in CTS StemScale medium will require an additional day of growth as compared to spheroids grown in RUO StemScale medium to achieve similar cell yields. (A) Spheroid morphology on passage day. Spheroids grown in RUO StemScale medium will typically reach an average of 400 µm in diameter in 5 days, while spheroids grown in CTS StemScale medium will require an additional day to reach a similar diameter. (B) Cumulative cell expansion on passage day. By harvesting spheroids grown in RUO StemScale medium on day 5 and spheroids grown in CTS StemScale medium on day 6, it is possible to achieve similar total cell yields (reported as fold expansion). (C) Spheroid diameter comparison. The spheroid diameter of the spheroids grown in RUO StemScale medium and the spheroids grown in CTS StemScale medium are similar on the respective days of harvest, with both close to the upper recommendation of 400 µm in diameter. 4 thermofishe