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As cell and gene therapies, drug discovery, and fundamental stem cell research continue to advance, the need for scalable, reproducible, and efficient differentiation of human pluripotent stem cells (hPSCs) is more pressing than ever. STEMCELL Technologies recently hosted a webinar exploring the transition from 2D adherent culture to 3D suspension culture, the optimization of differentiation workflows, and the scale-up process. This webinar—available to watch on demand—featured insights from STEMCELL Technologies bioengineers Tia Sojonky and Sarah McManus, who provided detailed guidance on maintaining high-quality hPSC aggregates, transitioning differentiation protocols to suspension culture, and troubleshooting common challenges.

This blog post provides a high-level summary of the presentation, including a Questions & Answers section addressing attendee questions. Due to time limitations of the webinar, some of these answers are being shared here for the first time to provide additional insights for researchers working with 3D suspension culture.

Understanding the Transition from 2D to 3D Culture

Why Move to 3D Suspension Culture?

The shift from traditional 2D adherent culture to 3D suspension culture is driven by the need for scalability and efficiency when expanding large numbers of hPSCs. In 2D culture, cells grow as a monolayer on a matrix-coated surface, experiencing uniform nutrient exposure in a relatively static environment. In contrast, cells in 3D culture grow as aggregates in three dimensions without a matrix or attachment surface, experiencing constant movement and more cell-cell interactions.

Advantages of 3D Culture

1. Enhanced Scalability – 3D culture enables large-scale production of hPSCs and their derivatives, making it ideal for therapeutic applications.
2. Elimination of Matrix Dependence – Unlike 2D cultures, 3D systems do not require an attachment surface, reducing reliance on extracellular matrices.
3. Efficient Media Use – A fed-batch approach in 3D cultures minimizes labor and media costs while preventing aggregate loss.
4. Environmental Control – Some 3D bioreactor systems enable continuous monitoring of environmental factors such as temperature, pH, and oxygen in a controlled setting.

STEMCELL Technologies has developed specialized TeSR™ 3D media to support hPSC expansion in 3D suspension culture, including:

• mTeSR™ 3D – The first media that enabled fed-batch workflows. mTeSR™ 3D saves both time and media with daily feeds replenishing nutrients and eliminating the need for medium exchanges on non-passaging days.
• TeSR™-AOF 3D – The only animal-origin free (AOF) media for fed-batch workflows, enabling more straightforward traceability and enhanced viral safety. Additionally, TeSR™-AOF 3D provides the most consistent expansion across passages.

If you’re interested in adopting 3D suspension culture workflows, consider taking advantage of STEMCELL’s free, on-demand course, Expansion of hPSCs in 3D Suspension Culture.

Optimizing hPSC Differentiation in 3D Suspension Culture

To successfully differentiate hPSCs in suspension culture, researchers must carefully adapt protocols originally designed for 2D adherent culture. Here is a structured workflow for transitioning differentiation protocols to 3D:

Step 1: Confirm High-Quality hPSCs Before Differentiation

• Expand hPSCs in TeSR™-AOF 3D for at least two passages to confirm viability, expansion rates, and pluripotency.
• Assess key quality metrics, including aggregate morphology, marker expression (OCT4, TRA-1-60), and genetic stability.

Step 2: Validate the Standard 2D Differentiation Protocol

• Use STEMCELL's interactive product finder to choose from 40+ STEMdiff™ kits currently available.
• Follow the protocol in the technical manual for the relevant STEMdiff™ kit to confirm differentiation efficiency in 2D culture before attempting 3D differentiation.
• If the protocol does not work in 2D, it is unlikely to succeed in 3D.

Step 3: Develop Reproducible 3D hPSC Culture Techniques

• Master aggregate formation, media change techniques, and passaging before differentiation.
• Resources such as STEMCELL’s 3D hPSC On-Demand Course and technical manuals provide valuable guidance.

Step 4: Optimize Differentiation at Small Scale

• Begin with 6-well plates on an orbital shaker before scaling up.
• Optimize key parameters including media change strategy, differentiation timing, and seeding density.

Step 5: Scale Up in Nalgene Storage Bottles and PBS-MINI Bioreactors

• Once a small-scale protocol is established, move cultures to Nalgene Storage Bottles (15-60 mL) and then to PBS-MINI Bioreactor Vessels (100-500 mL). 
• Monitor differentiation efficiency through marker expression and yield.
• Optimize agitation rates and media exchange protocols to ensure consistent aggregate size and differentiation outcomes.
• Consider using real-time monitoring for environmental parameters such as pH, oxygen levels, and metabolite concentrations to maintain optimal culture conditions.
• Implement sampling strategies at different time points to track differentiation progress and make data-driven adjustments to feeding schedules or aggregate density.
• If transitioning to bioreactors, validate scalability by comparing key differentiation markers between small-scale and large-scale cultures.
• Work with STEMCELL Technologies' Product and Scientific Support team to troubleshoot and refine differentiation conditions for specific cell types and applications.

Questions & Answers

Numerous questions were submitted during the webinar and not all questions could be addressed live due to time constraints. Below, we provide answers to all the submitted questions.

1.     What are the key advantages of 3D culture over 2D culture in your system?

The choice between 2D and 3D culture depends on experimental goals, including required cell yields and cell type. Advantages of 3D culture include scalability, the ability to generate large cell numbers in a homogeneous environment, and the elimination of extracellular matrix requirements. Additionally, 3D bioreactor systems enable continuous monitoring of environmental factors such as temperature, pH, and oxygen in a controlled setting.

2.     What is the expected adaptation time when transitioning hPSCs to 3D suspension culture? What indicators should be monitored during the transition from 2D to 3D?

The adaptation time depends on the cell line, culture medium, and culture conditions (e.g. vessel type and mixing dynamics). With TeSR™-AOF 3D medium and recommended culture vessels, we have not observed an adaptation phase. In mTeSR™ 3D, some cell lines experience lower expansion during the first one or two passages when transitioning from 2D to 3D, but by passage three, hPSCs should be fully adapted to suspension culture. During scale-up, key quality attributes—including aggregate morphology, viability, and expansion—should be monitored at the end of each passage to ensure optimal culture conditions. More details on these metrics can be found in the original webinar presentation.

3.     Can hPSCs be expanded in 3D culture without first undergoing 2D expansion?

Yes, cryopreserved hPSCs can be thawed directly into 3D suspension culture, bypassing 2D expansion. For optimal recovery, we recommend freezing clumps generated from 3D hPSC cultures using CryoStor® CS10. Using a 70-micron reversible strainer instead of a 37-micron strainer helps maintain slightly larger clumps, improving post-thaw viability. If a detailed protocol is needed, please contact your STEMCELL sales representative.

4.     Which bioreactors and culture vessels have been effective for hPSC expansion?

hPSCs are highly shear-sensitive, and we have tested multiple bioreactor configurations. The most reproducible results have been observed using Nalgene Storage Bottles and PBS-MINI Bioreactors, both of which support consistent expansion across different hPSC lines without requiring additional media additives. For cultures larger than 500 mL, further optimization is ongoing.

5.     Does the dissociation method for 3D aggregates differ from that used for 2D cultures?

3D aggregates can be dissociated into single cells similarly to 2D cultures but typically require a longer incubation time with the dissociation reagent. Protocols for enzymatic dissociation are outlined in Appendix 5 (Section 14.0) of the TeSR™-AOF 3D Technical Manual. Alternatively, aggregates can be dissociated using Gentle Cell Dissociation Reagent (GCDR) with a 10-15 minute incubation at 37°C and trituration to achieve a single-cell suspension.

6.     What markers or methods are used to assess spontaneous differentiation in 3D aggregates? What indicators signal whether a culture is successful?

We observe minimal spontaneous differentiation in 3D aggregates. However, we monitor several cell quality metrics regularly. At each passage, we assess expansion, as expected daily fold expansion ranges from 1.4 to 2. Deviations from this range may indicate suboptimal culture conditions. Viability and expansion are key quality checks, and automated cell counting is highly useful for this.

Aggregate morphology is also a key indicator. In smaller culture vessels, aggregates can be visually assessed more easily. Pockmarking is often correlated with undifferentiated hPSC marker expression. Every five passages, we dissociate aggregates for flow cytometry analysis of undifferentiated hPSC markers, and every 5-10 passages, we assess genetic stability. Anecdotally, spontaneous differentiation tends to favor the ectoderm lineage, so markers such as Nestin and Pax6 may also be useful to assess differentiation status. If all metrics are within expected ranges, the culture is considered high quality and suitable for differentiation.

7.     How do you achieve and count specific cell densities, such as 10,000 or 100,000 cells per mL, given that hPSC passaging is typically done in clumps rather than single cells? Additionally, does GCDR allow for single-cell generation?

For counting cells, we use the NucleoCounter NC-250, which supports multiple assays. The standard method is single-cell counting with AO/DAPI, but for clumpy suspensions, we lyse the clumps to obtain total and viable cell counts using DAPI staining. Some researchers prefer clump counting using a microscope and maintaining consistent clump sizes, though automated cell counting is generally more robust.

Regarding GCDR for single-cell passaging, we are actively developing protocols and have preliminary data available. The process is similar to clump-based GCDR dissociation but with a longer incubation time to yield a single-cell suspension. We have found that expansion following single-cell passaging with GCDR is superior to enzymatic dissociation methods. If you are interested in the protocol, please contact your STEMCELL sales representative.

8.     How do you ensure consistency and quality control in hPSC cultures at a larger scale? Are there specific markers or assays recommended for monitoring differentiation fidelity in 3D systems?

During scale-up, we assess key quality attributes such as aggregate morphology, viability, and expansion at the end of each passage to ensure optimal culture conditions. Additional quality control measures include undifferentiated hPSC marker expression, functional pluripotency testing with the STEMdiff™ Trilineage Differentiation Kit, and genetic stability analysis before differentiation. For larger culture vessels, a representative sample may be needed for assessment.

The specific markers and assays for differentiation fidelity depend on the target cell type. If you need guidance on cell quality assessments for a specific STEMdiff™ kit, please reach out to your STEMCELL sales representative or our Product and Scientific Support team for more details.

9.     How can necrotic core formation in 3D cultures be avoided? Do media volume, aggregate size, or other factors affect nutrient access to the center of the aggregates?

To prevent necrotic core formation, aggregates should remain below 400 microns in diameter. Using the recommended culture vessels, media volumes, and agitation rates helps maintain this size threshold by the end of each passage. Since cell lines exhibit different aggregation kinetics, agitation rates may need to be adjusted. During differentiation, aggregates may grow slightly larger than 400 microns, but we have not observed necrotic cores under our 3D differentiation protocols.

10.  Have you encountered difficulties staining 3D aggregates for immunofluorescence? Any tips for improving staining?

Whole aggregate immunocytochemistry (ICC) staining is possible, though antibody penetration is often a challenge. Typically, staining is clearest around the edges. For more quantitative analysis, dissociating aggregates into single cells and performing flow cytometry is recommended. If you are interested in a preliminary protocol, please contact your STEMCELL sales representative.

11.  How efficient is self-aggregation in 6-well plates? Are spheroids typically uniform in size?

Aggregation efficiency depends on the cell line, culture conditions, and passaging method. Our TeSR™-AOF 3D medium supports robust aggregation, with GCDR-based passaging improving aggregation efficiency over enzymatic dissociation. Aggregate size distribution is influenced by seeding density, and we have data and images available upon request.

12.  How should feeding be managed when thawing 3D-adapted cells for expansion in bioreactors? Should a full media change be done daily with ROCK inhibitor?

Regardless of whether cells originate from a 2D culture, 3D culture, or cryopreserved sample, we recommend the same feeding schedule with TeSR™ 3D media: a fed-batch feed with Feed Supplement on Days 1 and 2, followed by a 50% media exchange on Day 3 of a four-day passage. If the seeding density is low or proliferation is slower, an additional fed-batch feed on Day 3 may be beneficial. ROCK inhibitor remains in the culture throughout as minimal media is removed, and it is included in the initial seeding media.

13.  How is single-cell sequencing performed on 3D cultures?

Similar to 2D cultures, 3D hPSC aggregates can be dissociated into single cells for sequencing. However, dissociation may require an extended incubation period with the dissociation reagent. A protocol for enzymatic dissociation is provided in Appendix 5 (Section 14.0) of the TeSR™-AOF 3D Technical Manual. Alternatively, GCDR can be used by incubating for 10-15 minutes at 37°C with trituration to achieve a single-cell suspension suitable for sequencing.

14.  How long do hPSCs need to be cultured in 3D prior to differentiation?

For differentiation experiments, we maintain consistency by performing either three four-day or four four-day passages in 3D suspension culture before initiating differentiation. However, we have found that a single passage can also be sufficient. If using a less robust medium, at least two passages may be required before differentiation.

15.  Are there quick ways to assess whether differentiation is progressing successfully?

Each STEMdiff™ differentiation kit produces distinct 3D aggregate morphologies. Over multiple differentiations, tracking changes in morphology using microscope images can help identify successful differentiation. A reduction in pockmarking and a shift in cell morphology are positive signs. Additionally, healthy cultures have few single cells, while excessive single cells or debris may indicate issues. If hPSC clumps fail to aggregate within 24 hours, the culture should be restarted using TeSR™ media before transitioning to differentiation media.

16.  Can cells be cryopreserved post-differentiation and what is the recovery like?

For hPSCs, freezing in clumps yields the best post-thaw recovery. We recommend using a 70-micron reversible strainer rather than a 37-micron strainer to maintain larger clumps. Differentiated cells can also be cryopreserved. When using STEMdiff™ kits, we typically dissociate differentiated aggregates into single cells before freezing, using STEMCELL’s cryopreservation media.

17.  What are the best passaging reagents to use during differentiation?

The optimal dissociation reagent depends on the cell type. When passaging hPSCs in 3D suspension culture—whether for maintenance or differentiation—we recommend using GCDR for non-enzymatic clump or single-cell dissociation. For differentiated cells, the appropriate dissociation reagent is specified in the Product Information Sheet (PIS) or Technical Manual for the corresponding STEMdiff™ kit.

18.  What automated systems are commonly used for hPSC workflows?

Many automated systems are available for hPSC culture. In-house, we have experience using Hamilton liquid handling robots, but numerous other options are available on the market.

19.  How can hPSCs be differentiated into brain endothelial cells? Do different cell lines show variation in differentiation efficiency?

STEMCELL offers differentiation kits designed to generate specific cell types from hPSCs. While these kits are optimized for robustness across multiple cell lines, there is inherent variability among cell lines, which may require optimization for your specific application. For more details, please contact your STEMCELL sales representative.

20.  What is the best scale-up platform for pancreatic cell differentiation?

For pancreatic differentiation, we recommend Nalgene Storage Bottles (15-60 mL culture volumes) or PBS-MINI Bioreactors (100-500 mL culture volumes), as discussed in the webinar. We do not currently have a protocol for using the STEMdiff™ Pancreatic Progenitor Kit in 3D suspension culture, but general optimization guidelines were covered in the webinar.

21.  What is the best protocol for 3D intestinal-like cultures?

The IntestiCult™ Suspension Culture Protocol describes passaging intestinal organoids into suspension culture. For additional guidance, please contact your STEMCELL sales representative or our Product and Scientific Support team.

22.  Does 3D culture support hematopoietic differentiation?

Yes. We have established a preliminary protocol using the STEMdiff™ Hematopoietic Kit in 3D suspension culture, yielding comparable or improved marker expression and cell yields relative to standard 2D protocols. These findings were discussed in the webinar—please review the recording for details. If you are interested in using STEMdiff™ Hematopoietic - EB Reagents, contact your STEMCELL sales representative for more information.

23.  What are the optimal conditions for hPSC culture and mesoderm differentiation in PBS-MINI Bioreactors?

Culture volume and impeller speed recommendations for PBS-MINI Bioreactors (100 and 500 mL) are summarized in Table 1 of the TeSR™-AOF 3D Technical Manual. For transitioning mesoderm differentiation to 3D culture, general guidelines were provided in the webinar. If you need additional recommendations for using STEMdiff™ Mesoderm Induction Medium in 3D suspension culture, please reach out to your STEMCELL sales representative.

24.  How can islet-like clusters maintain their shape after transitioning from AggreWell™ to 3D suspension culture?

We do not yet have a definitive solution, but some reports suggest that static culture may be better for islet maturation. Islets may be kept in AggreWell™ plates post-aggregation or maintained at a low density to prevent clustering. Alternatively, suspension culture on an orbital shaker at the appropriate speed (e.g. 70 RPM with a 1-inch orbital throw in a 6-well plate) may help prevent merging.

25.  How can hPSC-derived islets be dissociated without excessive endocrine cell death?

GCDR is not optimal for dissociating islets due to their strong cell-cell contacts. Accutase™ may also be too mild. TrypLE™ incubation at 37°C on a shaker for 8-10 minutes, followed by gentle trituration, should effectively dissociate islets while minimizing cell death.

26.  How can EC coupling in cardiomyocytes be maintained after differentiation, and how is it tested?

Using the STEMdiff™ Ventricular Cardiomyocyte Kit, EC coupling remains intact. We confirm this through physical contractions (beating), electrophysiological recordings using multielectrode arrays (MEAs), and intracellular calcium signal measurements via fluorimetry.

27.  Do you have any additional comments on cardiomyocyte differentiation?

We have recently optimized the STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit for 3D suspension culture at small scales and are currently conducting proof-of-concept studies at larger scales, including Nalgene Storage Bottles and PBS-MINI Bioreactors. If you are interested in preliminary protocol details, please reach out to your STEMCELL sales representative who can arrange a virtual meeting to discuss further.

28.  Can you provide an estimated cost for 500 mL differentiation cultures using the PBS-MINI Bioreactor?

The cost of a 500 mL ventricular cardiomyocyte differentiation in the PBS-MINI Bioreactor depends on several factors, including media usage. Based on our optimized protocol, differentiation at this scale would require approximately five full kits, though some components may not be fully consumed. For a more detailed cost estimate, we recommend scheduling a virtual meeting with your STEMCELL sales representative.

29.  What biosafety level (BSL) is recommended for hPSC culture?

We follow Biosafety Level 2 (BSL-2) practices for handling all hPSCs and hPSC-derived cells, as they originate from human tissue. Institutional requirements may vary, so please ensure compliance with local biosafety regulations.

30.  Can eTeSR™ be used for 3D hPSC culture?

eTeSR™ was designed for 2D hPSC monolayer culture to improve genetic stability during single-cell passaging. For 3D culture, we recommend using TeSR™ 3D media, which employ a fed-batch approach to minimize labor, reduce cell loss, and prevent aggregate merging. We do not have protocols for using eTeSR™ in 3D culture, but it could be tested.

31.  How can reprogramming efficiency be improved when using fibroblasts from donors over 80 years old, given the effects of cellular senescence?

While we do not have specific recommendations, choosing an optimal cell source is critical. Many researchers prefer blood-derived cells due to the ease of reprogramming. Using low-passage or fresh fibroblast samples may also improve efficiency. Even with low reprogramming efficiency, only a few high-quality clones are typically needed to establish a robust hPSC line with normal karyotype.

32.  Can RT-qPCR with TaqMan be used to compare 2D adherent and 3D suspension hPSC cultures?

While we have not specifically compared 2D and 3D cultures using RT-qPCR with TaqMan assays, we have used RNA sequencing and other gene expression analyses to characterize differences between the two culture methods.

About STEMCELL Technologies

Driven by science and a passion for quality, STEMCELL Technologies offers over 2500 tools and services to support discoveries in fields such as regenerative medicine, immunotherapy, and disease research. Whether you're culturing and editing hematopoietic stem and progenitor cells, differentiating pluripotent stem cells, or activating and expanding immune cells, we have the specialized cell isolation products, high-performance cell culture media, and accessory tools for your research. By increasing the accessibility of innovative techniques like gene editing and organoid cultures, we’re helping scientists accelerate the pace of discovery—so they can get therapies to patients faster. Researchers working towards clinical trials can also obtain guidance and customized support through our Services for Cell Therapy Program on qualifying our products for use as raw/ancillary materials. To learn more, visit www.STEMCELL.com.