Figure 1. CTS StemScale medium offers higher-fold expansion compared to other PSC suspension media.(A) Spheroids grown in CTS StemScale medium are larger and exhibit a more consistent spheroid morphology compared to Medium "M". (B) CTS StemScale medium exhibits greater expansion potential than other commercially available suspension culture media (Media A, C, and M) in a 3-passage study. (C) CTS StemScale medium outperforms Medium "M".
A new dimension to PSC culture for cell therapy
Gibco CTS StemScale PSC Suspension Medium is designed to support the expansion of pluripotent stem cells (PSCs) at scale in suspension culture. It’s scalable, easy-to-use workflow and xeno-free formulation make it excellent for translational and clinical applications.
- Maximal expansion capability—achieve up to 30% higher expansion than other PSC suspension media, helping reduce manufacturing time and cost
- Scalable expansion for clinical applications—grow PSCs across multiple culture vessel formats and sizes, from small to large scale
- No cell strainer required—remove scale limitation, reduce contamination risk and hands-on time, and improve cell viability and yield
- Intended for cell therapy manufacturing†—streamline regulatory filings with GMP manufacturing, extensive safety testing, traceability of raw materials, and regulatory documentation
Maximal expansion capability
PSCs cultured in CTS StemScale PSC Suspension Medium can achieve 5–10-fold expansion per passage and up to 30% higher expansion versus other PSC suspension media (Figure 1). Long-term expansion of PSCs in CTS StemScale PSC Suspension Medium (>30 passages) can be accomplished without the loss of expansion potential, pluripotency, or genomic stability (Figure 2).
Figure 2. CTS StemScale medium maintains pluripotency and genomic stability during long-term expansion of spheroids. (A) Both feed schedules support growth of spheroids with consistent, rounded morphology over the culture duration. (B) Cells grown as spheroids in CTS StemScale medium exhibit consistent cell growth over time, as evidenced by the 30-passage study. Cultures fed daily exhibited consistent growth. Cultures fed every other day began to show reduced expansion after 7 passages. Daily feeding of cultures growing in CTS StemScale medium is recommended to maximize expansion potential and cell viability. Cells grown 30 passages exhibited high expression of pluripotent markers, assessed through the PluriTest Assay for specific gene expression. (C) Cells grown for 30 consecutive passages exhibited high expression of pluripotency markers, as analyzed through the PluriTest Assay for specific gene expression. (D) Cells also maintained pluripotency as assessed by flow cytometric analysis of OCT4 and NANOG markers. Additionally, cell lines were confirmed to remain genomically stable (data not shown).
Scalable expansion for clinical applications
CTS StemScale PSC Suspension Medium promotes consistent growth of PSCs in plates, flasks, and bioreactors. PSCs can be efficiently expanded in CTS StemScale PSC Suspension Medium across vessel types using the same culture parameters at both small and large scale (Figures 3 and 4).
Figure 3. CTS StemScale medium supports efficient growth across multiple culture vessel sizes. (A) Regardless of scale, cultures grown in CTS StemScale medium achieve a consistently high fold expansion using the same seeding density of 200,000 cells/mL. (B) Spheroids remain pluripotent in all different sizes of suspension culture vessels, as assessed through flow cytometric analysis of OCT4 and NANOG markers.
Figure 4. CTS StemScale medium promotes spheroid growth in liter-scale bioreactors. (A) PSCs can aggregate into spheroids and be expanded when cultured in bioreactors with either horizontal blade or vertical wheel impellers. (B) Spheroids grown in CTS StemScale medium continue to show high expansion potential even when grown in closed-system 3L bioreactors. The yields from the 3L bioreactors are similar to cultures grown in CTS StemScale medium in small-scale vessels. (C) Spheroids remain pluripotent in the 3L bioreactors, as assessed through flow cytometric analysis of OCT4 and NANOG markers.
Simplified workflow
PSCs will readily self-aggregate into spheroids in CTS StemScale PSC Suspension Medium and are fed with a 50% medium replacement strategy. This method reduces metabolic waste build-up while maintaining consistent media volumes in the culture, a critical factor in the maintenance of spheroid sizes. Cell strainers are not required during passaging when dissociating the PSC spheroids, thereby helping minimize cell loss, improving cell viability, reducing contamination risk, and simplifying scale-up to large vessels with high culture volumes (Figure 5).
Figure 5. Simplified workflow for initiation of small-scale suspension cultures that can be scaled up. Cultures grown in CTS StemScale are initiated by seeding single cells in the presence of a ROCK inhibitor and DNase I. (A) Cultures are fed daily by replacing 50% of the medium. Once spheroids reach 300–400 µm in diameter, they can be passaged via enzymatic dissociation without the need for a cell strainer. (B) The resulting single-cell suspension can then be scaled up into larger vessels or utilized in downstream applications.
Transition from research-use media
CTS StemScale PSC Suspension Medium has a formulation similar to that of Research Use Only (RUO) StemScale PSC Suspension Medium with specific modifications in line with regulatory guidance. With a few minor protocol differences (Table 1), CTS StemScale PSC Suspension Medium enables comparable performance, including cell yield, morphology, and pluripotency (Figure 6).
Figure 6. CTS StemScale medium offers similar performance to RUO StemScale medium. (A) Spheroids grown in RUO StemScale medium typically reach an average 400 μm diameter in 5 days, while spheroids grown in CTS StemScale medium require an additional day to reach a similar diameter. (B) RUO StemScale and CTS StemScale media achieve similar total cell yields, reported as fold expansion, when harvested once spheroids reach a similar diameter (Day 5 and Day 6, respectively). (C) The spheroid diameter of spheroids grown in RUO StemScale medium and in CTS StemScale medium are similar on the respective days of harvest, with both close to the upper recommendation of 400 μm in diameter.
Table 1. Key protocol differences between RUO StemScale and CTS StemScale media.
| RUO StemScale medium | CTS StemScale medium | |
| Feeding strategy | Every other day | Daily |
|---|---|---|
| Days of growth | 4–5 days* | 5–6 days* |
| Seeding density | 150,000 cells/mL | 200,000 cells/mL |
| Dissociation reagent | StemPro Accutase Cell Dissociation Reagent** | CTS TrypLE Select Enzyme (diluted***) |
| Addition of DNase I | DNase I not required | DNase I should be added |
*Estimated time to achieve an average spheroid diameter between 300–400 μm.
**Contains an animal-origin component.
***CTS DPBS (-/-) can be used to dilute CTS TrypLE Select to a lower concentration.
Differentiation of PSCs to NK cells
PSCs grown in CTS StemScale PSC Suspension Medium can be differentiated to clinically relevant immune cell types, such as natural killer (NK) cells. Differentiation can be initiated directly from PSC spheroids and can be scaled to larger volumes.
Figure 7. Analysis of iNK phenotype and expansion over a 21-day period.(A) Flow cytometry analysis of changes in iNK cell phenotype over time. The depletion of CD3 indicates the iNK population is becoming pure over time. (B) Total CD56+ cells increase over time, showing the expandability of iNKs while viability remains stable.
Resources: CTS StemScale PSC Suspension Medium
Browse resources needed to support your PSC suspension culture using CTS StemScale PSC Suspension Medium.
How-to videos: 3 steps to culturing PSCs in suspension
Watch instructional videos to learn how to culture human pluripotent stem cells using CTS StemScale PSC Suspension Medium.
How should I store complete CTS StemScale PSC Suspension Medium?
Following reconstitution, the complete medium can be stored at 2–8 °C for up to 2 weeks.
Can I aliquot and freeze CTS StemScale PSC Suspension Supplement?
Yes. The CTS StemScale supplement may be aliquoted into smaller volumes upon receipt and stored at –5 °C to –20 °C for up to 6 months. Avoid repeat freeze-thaw cycles.
Is the formulation of CTS StemScale PSC Suspension Medium the same as RUO StemScale PSC Suspension Medium?
The formulation of CTS StemScale medium is similar to RUO StemScale medium but with specific modifications to adhere to regulatory guidance. These changes necessitate a few minor protocol differences between the RUO and CTS versions of StemScale medium to provide similar cell yields.
Why does the CTS StemScale PSC Suspension Medium protocol recommend diluting CTS TrypLE Select Enzyme using Gibco CTS DPBS (–/–)?
Cells can be very sensitive to dissociation with CTS TrypLE Select Enzyme, leading to some cells becoming stressed or lysing during dissociation. To minimize the sensitivity of these cells to the dissociation enzyme, we recommend diluting CTS TrypLE Select Enzyme to a lower concentration with CTS DPBS (–/–).
Dilution of CTS TrypLE Select Enzyme will help maintain highly viable suspension cultures post-dissociation. For orbital shaker cultures, we recommend diluting CTS TrypLE Select Enzyme to a final concentration of 0.25X CTS TrypLE Select Enzyme. This can be achieved by adding 3 mL of CTS DPBS (–/–) for every 1 mL of CTS TrypLE Select Enzyme.
For larger culture volumes (i.e., ≥100 mL), we recommend a range of 0.5–0.75X CTS TrypLE Select Enzyme. This higher concentration accounts for the increased cell mass during dissociation.
If I am using diluted CTS TrypLE Select Enzyme, do I still need to include DNase I with my suspension cultures?
Yes, it is important to always add DNase I when working with CTS TrypLE Select Enzyme. Even when the CTS TrypLE Select Enzyme is diluted, lysed cells may release genomic DNA and cause spheroid aggregation issues. We recommend adding DNase I to suspension cultures at a concentration of 0.1 U/mL. Higher concentrations (such as 1 U/mL) may also be used in large-scale vessels.
What is the recommended passaging schedule when using CTS StemScale PSC Suspension Medium?
The CTS StemScale PSC Suspension Medium protocol recommends passaging every 5–6 days, depending on spheroid size. It is recommended to passage suspension cultures when the average spheroid diameter is 300–400 μm.
Passaging cultures earlier is also an option but may result in a lower cell yield. Early passaging is an option to avoid passaging on a weekend.
Do I need to feed my cultures daily with CTS StemScale PSC Suspension Medium?
Yes. The CTS StemScale medium feeding schedule recommends daily feeding to maximize cell health. It is possible to skip one day on the weekend if desired, but otherwise CTS StemScale medium cultures should always be fed daily.
Can I thaw frozen cells directly into CTS StemScale PSC Suspension Medium?
Yes. Cells that have been cryopreserved can be thawed directly into CTS StemScale medium. The same protocol for thawing cells for adherent cultures can be utilized for suspension cultures. Once cell counts have been obtained, the thawed cells can then be seeded into suspension cultures using the recommended seeding conditions for StemScale medium.
Users should expect cells to require one passage in suspension before recovering from cryopreservation. After this recovery passage, suspension cultures will perform similarly to those seeded from adherent cultures.
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†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 StemScale PSC Suspension Medium.
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