Advances in Pluripotent Stem Cell Expansion

We are only just beginning to realize the potential of pluripotent stem cells (PSCs), both in research and therapeutic applications—despite the success of autologous stem cell therapies in treating some blood cancers and disorders. Such treatments use cells harvested from each individual patient. However, the future use of allogeneic treatments—using stem cells previously donated from healthy volunteers, expanded, and prepared for therapy—could revolutionize immunotherapy.

Easily accessible, off-the-shelf immunotherapy products will require large numbers of PSCs, which necessitates maximizing human PSC (hPSC) expansion. “Human PSCs are unique in their requirements as we maintain them in their pluripotent state and prevent them from differentiating to other cell types,” says Matthew Hildebrandt, Product Manager for Pluripotent Stem Cell Biology at STEMCELL Technologies. “It’s critical to balance their metabolic needs, reduce buildup of waste products, and reduce cell stress.” This article will review some areas of innovation today in PSC expansion, as well as challenges that remain in improving efficiency and scalability.

Surfaces and media

The tried-and-true method of cell production, 2D cell cultures grown on coated plates or wells, is still going strong. Corning has long offered the widely used extracellular matrix Matrigel^® for coating surfaces to which stem cells adhere and grow. Matrigel matrix continues to be in widespread use within research and other non-clinical environments, but the rise of stem cell therapies has caused a shift to using more defined components in the production of stem cell therapies. For applications such as PSC expansion, Corning’s Synthemax^® II-SC substrate is a synthetic, xeno-free attachment surface compatible with PSCs destined for human use. “Synthemax substrate is a vitronectin mimetic, specifically designed to be a consistent attachment surface for stem cells, including pluripotent stem cells,” says Tom Bongiorno, Senior Support Scientist at Corning Life Sciences.

Similarly, standardized, animal-free media can also improve reproducibility and consistency. According to Yelena Bronevetsky, Director of Product Management at Xcellbio, today’s cell culture media are tailored to support current cell production needs. “These formulations ensure PSCs receive optimal concentrations of growth factors, amino acids, and vitamins, maximizing growth and maintaining pluripotency even at high cell densities,” she says. “Additionally, media designed for low-oxygen conditions can further enhance proliferation by optimizing energy sources for hypoxic metabolism or incorporating hypoxia-specific growth factors.” Media additives such as antioxidants can also help to reduce stress-related apoptosis during PSC expansion.

Media can meet other stem cell needs beyond nutritional requirements. STEMCELL Technologies recently released eTeSR™, an enhanced medium for culture and expansion of single human PSC cells. Optimized for single-cell passaging, it improves genetic stability and reduces the need for daily media exchanges. “Identifying more supportive supplements to further augment early stages of culture has greatly improved expansion at the clonal scale,” says Hildebrandt. “Obtaining more clones, and sooner, increases the throughput of those working to develop unique gene-edited lines for their downstream applications.” For example, STEMCELL’s seeding supplement CloneR™2 results in 20 to 40 times more clones that expand faster than when using a standard media supplement, according to Hildebrandt, which enabled them to also pick clones 2 days earlier.

Tools for scaling up

When growing adherent cells on surfaces, increasing the surface area is key for scaling up growth, and Corning offers many such tools. For example, their CellSTACK^® culture chambers support expansion with stacked surfaces, up to 40 layers. The Corning HYPERStack^® vessel offers a similar stacked-vessel format with specialized features that maximize the surface area per volumetric footprint. “HYPERStack is similar to a traditional T-flask, but with a lot more surface area in a single vessel, so you can have one operator working with one HYPERStack instead of 100 T-flasks,” says Bongiorno. A scaled-down version, the HYPERFlask^® vessel, with the footprint of a T175 flask, contains 10 different layers to increase surface area.

Bongiorno notes a trend toward greater demand for closed system process tools. Corning offers accessories to convert CellSTACK culture chambers to closed systems, and the HYPERStack vessel has closed system tubing built in. Corning’s Ascent^® fixed bed bioreactor (FBR) and CellCube^® cell culture system illustrate another recent trend toward bioreactors and automation in cell culture. “The CellCube system contains stacked layers of cell attachment surface connected to a bioreactor for process monitoring and automation, offering greater consistency from batch to batch,” says Bongiorno. “The Ascent FBR offers 1–5 m² surface area, while the CellCube system is smaller but modular, so you can attach them together to get the surface area you need.”

3D suspension cultures

When it comes to cell production, 3D suspension cultures grown in bioreactors offer a number of advantages over traditional 2D cultures. In addition to higher cell yields, bioreactors allow for greater control of environmental factors such as levels of O₂, CO₂, pH, and nutrients. Bioreactors often automate some processes, which generally increases throughput and reduces costs and human error. “Transitioning to 3D suspension culture using bioreactors significantly impacts PSC expansion,” says Bronevetsky. “The AVATAR Foundry [bioreactor] from Xcell Biosciences facilitates hypoxic culture environments that mimic the native stem cell niche, promoting PSC proliferation while reducing oxidative stress and maintaining pluripotency.”

Though 3D suspension cultures in bioreactors typically offer greater yields than planar cultures, scaling up this method remains challenging. “hPSCs are more sensitive to shear stress compared to other cells that have been previously optimized for suspension culture,” says Hildebrandt. To remedy this, he suggests technologies such as lower shear vertical wheel bioreactors, or protective encapsulation methods, but more development is needed in this space.

Suspension cultures may also benefit from alternative feeding methods. STEMCELL Technologies developed several TeSR™ media specifically for 3D suspension cultures that include an additional concentrated supplement that is spiked into low-density cultures as opposed to large volume perfusion. “This method reduces required media volumes, processing times, and interruptions in cell mixing [resulting in less cell aggregation],” notes Hildebrandt. Such creative methods of enhancing PSC expansion may help to bring accessible and affordable allogeneic stem cell therapies into reality sooner rather than later.