Ask the Expert: Streamlining Mesenchymal Stem Cell Production — From Expansion Through Removal of Cryoprotectant

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The number of cell therapy product candidates based on mesenchymal stem cells (MSCs) has grown steadily since their clinical debut in 1995. As of June 2020, clinical investigators were evaluating more than 1,100 such therapies. Scaling up MSC production remains challenging, however. On 31 May 2021, Hilary Sherman (senior scientist at Corning Life Sciences) presented an “Ask the Expert” webinar describing her company’s efforts to facilitate MSC workflows.

Sherman’s Presentation
Easing Expansion: MSCs have strong differentiation capability and can be isolated easily from several parts of the body — often from bone marrow, but also from umbilical cord blood, adipose tissue, and other sources. However, MSCs can be difficult to expand. A therapeutic dose can require 100 million to 1.2 billion cells, yet MSC densities upon harvest can vary from 20,000 cells/cm2 to 100,000 cells/cm2 depending on source, culture medium, and application.

Sherman demonstrated use of Corning HYPERStack culture vessels to streamline MSC expansion. They leverage gas-permeable layers to enable gas exchange beneath each culture chamber. That eliminates the need for headspace above the culture medium and reduces the requisite footprint for each culture. HYPERStack systems come in 12- and 36-layer formats, respectively providing 6,000 cm2 and 18,000 cm2 of surface area for cell growth.

In three independent studies, using bone-marrow–derived MSCs, Corning seeded 36-layer HYPERStack vessels at densities of 3,000 MSCs/cm2. After five days of culture, viable cell densities ranged from 40,000 cells/cm2 to 50,000 cells/cm2. Cell viability exceeded 90% in all three studies. The MSCs also maintained their differentiation capacity. Subsequent analysis showed that >99% of harvested cells bore markers of multipotency (e.g., CD90, CD105, and CD73), and <0.5% of cells expressed markers that indicate differentiation.

Simplifying Storage: MSCs are cryopreserved to increase their range of administration and make time for quality testing. Selecting appropriate containers for frozen storage can help streamline MSC production workflows. Made from a proprietary film blend that remains flexible in ultracold conditions, Corning cryopreservation bags are designed for aseptic transfer and storage of cells in large volumes. The bags come in four sizes, ranging in volume from 10 mL to 190 mL. They can be tube-welded, and they come with thin-walled membrane ports or Leur connections.

Washing Cells: Dimethyl sulfoxide (DMSO) often is added to cell-therapy solutions before cryopreservation to prevent ice-crystal formation. Because the compound can be cytotoxic, it must be removed from a therapy before administration. But many available methods for DMSO removal diminish cell viability or require open process steps.

Although the Corning X-WASH system was developed for whole-blood products, it also can be used to remove DMSO from cryopreserved MSC products. The system comprises a disposable, multichamber cartridge and a mutliuse control module. Thawed cells and buffer are added to the cartridge’s primary chamber. Next, the cartridge and control module are added to a centrifuge to pellet the cells. Buffer is removed, then cells are resuspended and placed back in the centrifuge for additional pelleting. At that point, users can perform an additional buffer exchange or proceed to harvest. Infrared sensors in the control module measure cell–media interactions to help coordinate cell transfer to a harvesting chamber.

Unlike other cell-wash units, the X-WASH system enables closed, semiautomated processing. It also reduces cryoprotectant from MSC products without compromising cell recovery and quality. Corning recently performed three studies of MSC density and viability after X-WASH system processing. Sherman’s team preserved MSCs in a 10-mL solution of serum (90%) and DMSO (10%), then thawed the material and processed it in an X-WASH unit. In all three studies, MSC recoveries exceeded 70%, and cell viability remained >90%. Cells showed high expression of multipotency markers and low expression of differentiation markers. And DSMO concentrations had been reduced to less than 200 parts per million. Sherman added that other washes could be performed to minimize cryoprotectant levels further.

Questions and Answers
How much material can an X-WASH system process? Initially, users can add 240 mL of cells and buffer. That can be pelleted, aspirated, and supplemented with another 240 mL of buffer. Corning recommends performing no more than two buffer exchanges, after which cells should be centrifuged and harvested.

How can users visualize cells that are cultured in a HYPERStack vessel? The 12-layer option can be placed under a microscope. Handheld microscopes are a good option for the 36-layer format.

How much reagent is needed to harvest cells from a HYPERStack system? The 12- and 36-layer formats need ~200 mL and ~600 mL of reagent, respectively.

Watch the complete presentation to learn more about novel tools for MSC production.