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Stemcell Technologies
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Human pluripotent stem cells (hPSCs) can be a powerful tool in your cell and gene therapy development toolkit. However, most clinical applications require many more hPSCs than can be generated in a typical 6-well plate. If your workflows require the generation of large numbers of cells (i.e., scale-up), you might have encountered one of several roadblocks: cost, time, expertise, or process development uncertainties. In this post, we offer an overview of the challenges and considerations related to scaling up hPSC cultures, some practical recommendations, and resources to help you get started on the right foot.
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1. Start with fully characterized, high-quality hPSCs
While some variation between cell lines is to be expected, proper reporting and standardized quality control measures can help limit variability and ensure that relevant, reproducible findings are shared. If your starting population of hPSCs is of insufficient quality, you are more likely to encounter poor survival and expansion rates, failed downstream differentiation, and the development of genetic abnormalities in your expanded population. Left unchecked, these issues could prevent you from publishing, proceeding to clinical trials, manufacturing a product, or achieving other desired endpoints. Starting with a fully characterized, high-quality cell bank will allow you to restart experiments with minimal delay in the case of genetic abnormalities or other issues experienced during maintenance and expansion. To learn more about cell quality attributes, including how to assess and maintain high-quality cells, explore our hPSC Quality Learning Center.
If you’re looking for a reliable starting cell source, our SCTi003-A cells have been fully characterized and adhere to the International Society for Stem Cell Research’s newly-released Standards for Human Stem Cell Use in Research.
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2. Understand the differences between scaling up in 2D vs. 3D
Scaling up in 3D refers to suspension culture whereby cells are expanded in a volume of media, often in flasks or bioreactors, and is what most people imagine when thinking about hPSC scale-up. In contrast, scaling up in 2D refers to expanding cells as an adherent, monolayer culture in vessels with large surface areas, such as cell stacks.
Suspension culture enables scale-up of hPSC manufacturing for applications requiring large numbers of cells, such as high throughput screening, generating master or working cell banks, and for use in cell and gene therapy research. However, media optimized for 2D adherent culture can result in lower volumetric productivity and laborious workflows when applied to 3D culture systems. STEMCELL Technologies has developed a portfolio of TeSR™ 3D-based media products for robust and scalable suspension culture of hPSCs as aggregates. These media can reduce time and labor with a fed-batch feeding strategy, using daily feed supplements to replenish nutrients and eliminating the need for medium exchanges on non-passaging days. Learn how to achieve robust scale-up of hPSC aggregates in 3D suspension culture by following this protocol.
Whereas 3D suspension culture requires additional training, reagents, and equipment, 2D monolayer culture enables you to expand your cells in an environment more similar to the one hPSCs are exposed to during routine maintenance. By gradually increasing the surface area available for cell growth and expansion from 6-well plates into flasks, and then to cell stacks, you can quickly and easily generate large numbers of hPSCs. Learn how to rapidly expand hPSCs as single cells in a 10-chamber cell stack by following this protocol.
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3. Consider both your current and desired workflows.
To gain expertise in maintaining hPSCs, a workflow that incorporates 3D suspension culture can be helpful. While somewhat more challenging than scaling up in 2D, 3D systems offer advantages in terms of avoiding the need for a matrix, and the additional costs or variability that can come with sourcing additional reagents. Additionally, if your downstream workflows call for 3D differentiation, 3D scale-up will give you a population of hPSCs ready to use in the next stage of your research or cell manufacturing.
Compared to 3D suspension culture, expanding cells in 2D monolayer culture using cell stacks is typically faster, less expensive, and more straightforward. If you’re new to hPSC culture and don’t plan to devote many resources to learning how to maintain this cell type, then 2D scale-up is likely best for you. Even if you already have lots of experience with hPSCs, considering aspects of your downstream workflow (e.g., 2D vs. 3D differentiation) and the characteristics of your cells (are you working with lines that are especially shear sensitive?) could guide you in making a decision about whether to scale up in 2D or 3D.
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4. Select a scale-up method that generates an appropriate number of cells for your workflow
The number of hPSCs you need to be able to generate to support your desired output is perhaps the most important consideration in selecting a scale-up method. If your long-term goals require tens or hundreds of billions of cells, why invest in developing processes that won’t be able to get you there? Likewise, if you need smaller numbers of cells, why commit precious time and resources to methods that generate orders of magnitude more than you actually need?
While your output will vary based on your specific culture conditions, 2D scale-up protocols using 1- to 10-chamber cell stacks can generate anywhere from 10 million to 3 billion cells, whereas 3D suspension culture protocols can generate even larger numbers of cells depending on the size of the vessel you use at the end of your scale-up protocol. Smaller bioreactors (0.1 L to 0.5 L), like the PBS-MINI Bioreactor, can generate anywhere from 80 million to 1 billion cells and can be very useful for developing your processes prior to transitioning to larger vessels. Larger bioreactors (3 L to 80 L) can generate anywhere from 6 to 160 billion cells.
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5. Reach out for support early and often.
To get you started, we have developed free, on-demand courses for both routine hPSC maintenance and the expansion of hPSCs in 3D suspension culture. Our scientists also offer real-time, instructor-led training courses to support your ongoing training needs. And if you have additional questions or need to troubleshoot issues in your experiments, our Product and Scientific Support team is only a call or email away.
Once you’re ready to take your hPSC production to the clinic, we can help you with that, too! Our Regulatory Support hub has lots of information to help you make sense of the ancillary material qualification process, and our dedicated Services for Cell Therapy team can provide tailored advice for your specific situation.
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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.
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