Stem cell research has significantly increased over the past few years, as shown by activity in the lab and clinic and investments made across the industry.
In vitro stem cell research is fueling new precision approaches in tissue engineering, cancer immunotherapies, neurological and endocrine diseases, and many other fields, and is expected to continue to drive the biotech boom.
Image Credit: ShutterStock/pinkeyes
Despite the upsurge in activity and promise surrounding applications in mesenchymal stem cells, induced pluripotent stem cells, and emerging cellular therapies, such as extracellular vesicles, challenges in making these innovative therapies production-ready for clinical trials and entry into the market remain.
Obstacles ranging from optimizing cellular expansion and downstream processing to the ongoing requirement to prioritize cell quality in tandem with cell quantity have prevented many bench projects from reaching the milestones for commercial success.
One scientist told Nature in 2021 that it can seem as though stem cell researchers are around-the-clock babysitters with a full-time effort to maintain healthy cultures.1
For lab scientists trying to realize the potential of stem cell therapies while avoiding the many pitfalls, new technologies, culturing platforms, and materials have reduced costs, obligations, and pressures.
Here, experts from Corning Life Sciences examine how new technologies have ushered in a new era of more effective and efficient workflows and explain how the field of stem cell research and cell therapy is evolving.
Corning® CellSTACK® Culture Chambers are a scalable culture vessel system that may be used to culture larger quantities of adherent cells, with a growth surface area ranging from 636 cm2 to 25,440 cm2. Image Credit: Corning Life Sciences
Stem cell applications and therapies: An overview
Human tissue can regenerate in healthy bodies, but many injuries and diseases can prevent this. However, stem cell therapies can support the body’s fight against cancer and tissue repair and regeneration if this occurs. Since various cell types behave differently depending on the environment, scientists’ approaches to these therapies vary significantly.
That strategy is frequently tailored to fit the particulars of the cell type, such as pluripotent cells (which have a broad range of differentiation options) or multipotent or unipotent adult stem cells (which do not have the same differentiation potential as pluripotent stem cells but may be more widely accessible).
By reprogramming adult stem cells to behave similarly to embryonic stem cells, certain technologies, such as induced pluripotent stem cells, enable researchers to achieve the best of both worlds.
You have this continuum with pluripotent cells on one end and more differentiated cells on the other end that are already well defined and can turn into just a few cell types. There is a delicate balance where you want to match the right cell type to your therapeutic application, but you might not want fully differentiated cells because they won’t replicate as quickly.”
Tom Bongiorno, PhD, Field Application Scientist, Corning Life Sciences
While stem cells and their uses in contemporary medicine hold a lot of promise, this continuum also adds a great deal of complexity as they are living things. The cells are the therapy and billions may be needed for one dose.
High volumes call for a scaling-up strategy using either 2D adherent culturing or, more recently, 3D methods. To choose such a system, one must sift through a variety of options, including culture ware, substrates, and scale-up platforms designed specifically for the characteristics of the cells.
Operational considerations, such as labor or equipment costs and time to market, go along with these scale-up options.
However, labs are frequently left to make these choices on their own because there is little industry guidance on how to effectively culture these complex cells to obtain more of them without compromising quality. This section looks at things to consider and best practices.
Factors that influence cell quality
The key to successful stem cell therapy is cell quality. Users may have billions of these cells, but if the quality is compromised in the process, all of that effort, money, and time could be wasted.
In cell therapy, cells are part of the actual product, so the fact that cells are healthy and performing as they should is integral. If the cell isn’t behaving as it should, the therapy could be less effective, or maybe even dangerous if you have a situation where cells are differentiating into something they shouldn’t.”
Hilary Sherman, Senior Applications Scientist, Corning Life Sciences
The scale-up seed train includes a number of variables that can affect cell quality, including the substrate, where anchorage-dependent cells adhere and proliferate. After factoring in various considerations such as synthetic or biological materials, some substrates are superior for particular applications.
Teams will be best prepared for a successful and timely scale-up if they choose their surfaces carefully and cautiously.
Sherman stated, “Substrate choices can depend on your ultimate needs. You might not want the added variability of a biological component, or maybe you do because you are trying to replicate something that is happening in the body within an in vitro environment.”
Substrates can also impact cell behavior. For instance, one study discovered that mesenchymal stem cells differentiated into various cell types depending on the stiffness of the substrate when the cells were cultured on different substrates.2
Alejandro Montoya, senior product line manager at Corning Life Sciences, says it can come down to a cell’s sensitivities.
We have seen that adult mesenchymal cells are comparatively easier to grow because they have less sensitivity to not having an extracellular matrix (ECM), while more complex cells, like induced pluripotent or embryonic stem cells, may need the signaling and structure of an ECM, such as Corning® Matrigel® matrix or a more defined biological relevant substrate such as Corning rLaminin-521. So much can be optimized according to these unique cell types.”
Alejandro Montoya, Senior Product Line Manager, Corning Life Sciences
The vessel itself should be considered as a factor. Most labs typically begin with a small-scale option, such as a T-flask, before progressing to options offering more surface area without consuming a lot of space, like multilayer flasks and stacked solutions.
The Corning® HYPERFlask® provides the same gas exchange, footprint, and operator responsibilities as a single flask while increasing the attachment area from a standard flask’s 175 cm2 to 1720 cm2.3
When more scale is required, closed-system tubing manifolds can be expanded to accommodate stacked systems such as Corning HYPERStack® or CellSTACK cell culture vessels.
Microcarrier options in high-volume bioreactors offer an adherent surface for maximum output. Each of these various systems highlights the potential for using more effective vessels to produce more cells while maintaining quality and affordability.
“You want to develop a very efficient seed train that can yield as many cells as you can while replicating them as few times as you can. That is because one of the big challenges with some stem cells, such as mesenchymal stem cells, is that after you isolate them, they can only replicate a limited number of times in vitro,” Bongiorno added.
Extracellular vesicles (EVs), spheroids, organoids, and advances in organ regeneration are all examples of new stem cell culture techniques. They highlight the need for more sophisticated microenvironments that offer the molecular signaling and scaffolding required to expand stem cells in 3D, more vivo-like environments.
Extracellular matrices (ECMs), hydrogels, and specialized surface treatments and coatings are important factors to take into account when creating the ideal microenvironment. These 3D structures, such as spheroids, are housed in vessels, microplates, and substrates.
Bongiorno pointed out, “Many hydrogels have some degree of lot-to-lot variability, so using specialized products that are screened and made for stem cells is critical. “It may be a minor change, but it can have a big impact on stem cell quality.”
Products that are exclusively optimized for organoid cultures or those that are hESC-qualified can sometimes fall under this category.
“Corning Matrigel matrix versions are optimized and qualified for these specific applications, minimizing the lot-to-lot variability. Matrigel matrix has been an integral part of feeder free stem cell culture and just as important if not more in the origin, pioneering and continuous development of organoid research,” Montoya stated.
Human iPSCs cultured on hESC qualified Corning® Matrigel® matrix. Image Credit: Corning Life Sciences
The right stimuli must be activated at the right time with the correct intensity in addition to the vessels, specialized media, and reagents. These stimuli can be produced with accurate geometry and fluid dynamics, whether in the form of static, dynamic, or perfusion media conditions.
With developing fields such as electric vehicle (EV) production and acellular therapies, in addition to the potential for producing EVs in MSCs and other stem cells4, it is crucial that what occurs in whole tissues and organs is accurately represented as researchers move from 2D cultures to 3D models.
Replicating nature is the ultimate goal of 3D models. To promote quality cultures for the production of desired acellular products, such as EVs, high-volume production without compromising quality will require the need to continue learning about and applying these crucial stimuli.
Simple principles such as geometry and surface chemistry can also govern microenvironments. Without scaffolding or hydrogels, 3D cell cultures can be created in microwells or cavities with hydrophilic, nonstick coatings. Adherent cells in a single-cell suspension will naturally aggregate and self-assemble into spheroids because they lack localized points of attachment.
The size and possibly the shape of the resulting spheroids will be determined by the geometry of the cell culture substrate or surface. Simplicity is crucial to creating 3D culture models that enable the best exchange of nutrients, gases, and other essential requirements without the scaffold or hydrogel imposing any impediments to the culture environment.
Image Credit: Corning Life Sciences
Breaking through expansion barriers with scale-up strategies
The need to simultaneously harvest billions of attachment-dependent cells while preventing problems with quality, senescence, or variability, calls for a scale-up strategy that addresses these cells’ diverse and complex needs. Here are a few things to consider:
Seed train compatibility
An effective scale-up strategy requires that the various components of the seed train remain compatible and consistent. For instance, it is ideal if the surface treatment used on a smaller vessel, such as a single-layer flask, remains the same as production increases. Through this upstream work, time and money can be saved downstream.
Bongiorno stated, “You do need to carefully consider how different cell types will respond to different variables, including surface treatment as you are moving from one platform to the other. Sometimes that can require a bit of testing at small scale to make sure that the cells are compatible with the surface you eventually want to use later on.”
Mitigating contamination and toxicity risk
Contamination and toxicity risks are increased as yield rises throughout the seed train. These risks can come from internal substances, such as cryoprotectants, and external contaminants.
Scientists are increasingly using closed-system platforms, such as Corning® HYPERStack® vessels, with tubing and connectors pre-attached to ensure a sterile fluid path. This reduces the risks mentioned above and their associated effects, including cost and time.
Such systems prevent opportunities for contamination by limiting how frequently or how long platforms are exposed to the environment.
Advanced closed system washing platforms, such as the Corning® X-WASH® system, are an essential component of the effective scale-up workflow for cryoprotectants like DMSO.5
Automation can significantly speed up processes as scale increases, enabling labs and smaller startups to accomplish more with fewer resources. Among those choices are automated manipulators with stack compatibility and adherent-supportive platforms, such as the Corning® Ascent™ FBR system.6
“When you have a lot of vessels, you need a lot of different operators to work with them. And that brings in some challenges of time, but also operator-to-operator variability. Bringing more automated solutions into your workflow allows you to accelerate the scale-up strategy and reduce labor costs and variability, while also getting much better repeatability,” added Bongiorno.
Work with a trusted scientific partner
Having an experienced partner to lead uncharted production territory is essential to success in the complex world of cell and gene therapy. Users can receive assistance from technical support staff from scientific vendors such as Corning Life Sciences. Corning Life Sciences can:
- Increase production output by manifolding multiple stack vessels in compact styles
- Streamline liquid manipulations with tailored closed systems
- Increase stem cell yields for clinical applications by optimizing culture conditions
Accelerate Adherent Scale up with the Corning® Ascent™ Fixed Bed Bioreactor System
Learn more about how to accelerate adherent scale-up to produce high cell yields more efficiently. Video Credit: Corning Life Sciences
Making the most of stem cell innovations
Even though stem cells hold great promise, researchers have just begun exploring their full potential. The need to grow research cultures to a production-ready scale—cost-effectively and quickly—is an ongoing challenge for life science companies with applications across therapeutic approaches and disease types.
Scale-up strategies need to consider the complexity of these cell types as the team investigates the route from the research bench to upstream bioproduction for the program. Even seemingly insignificant decisions, such as the substrate or vessel, can have a big effect.
Teams should search for opportunities to reduce the risk of contamination and automate where necessary. Generally, they should look for consistency and compatibility in the scale-up approach.
Image Credit: Corning Life Sciences
- Bender, E. Stem-cell start-ups seek to crack the mass-production problem. Nature. 2021;597(7878): S20-S21. doi:10.1038/d41586-021-02627-y
- Engler, A.J.; Sen, S.; Sweeney, H.L.; and Discher, D.E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell. 2006;126(4):677-689. doi:10.1016/j.cell.2006.06.044
- A Flask for Any Task. Accessed Jan. 6, 2022. https://www.corning.com/catalog/cls/documents/infographics/CLS-CC-114.pdf
- Teng, F., and Fussenegger, M. Shedding Light on Extracellular Vesicle Biogenesis and Bioengineering. Advanced Science. 2020;8(1):2003505. doi:10.1002/advs.202003505
- X-WASH System. Corning.com. Published 2022. Accessed Jan. 6, 2022. https://www.corning.com/worldwide/en/products/life-sciences/products/bioprocess/x-series-cell-separation-platform/x-wash.html
- AscentTM FBR System. Corning.com. Published 2022. Accessed Jan. 6, 2022. https://www.corning.com/worldwide/en/products/lifesciences/products/bioprocess/ascent-fbr-system.html
About Corning Life Sciences
A division of Corning Incorporated, Corning Life Sciences is a leading global manufacturer of cell culture products and solutions that enable academic, biotech and biopharma scientists to harness the power of cells to create life-changing innovations. Corning supports a range of application areas including core cell culture, 3D cell culture, bioprocess, cancer research, primary and stem cell research, drug screening, cell and gene therapy, disease modeling, lab automation and more.
Whether your goal is stem cell expansion or viral vector production, Corning Life Sciences platforms, including HYPERStack® vessels that maximize cell growth area in a small footprint, the high-yield Ascent® Fixed Bed Reactor platform, microcarriers, and closed system solutions can help get you there. Choose from hundreds of vessels, the widest selection of cell culture surfaces, and custom media in a variety of single-use technology configurations. Learn more at www.corning.com/lifesciences.