Enzymatic Detachment of Therapeutic Mesenchymal Stromal Cells Grown on Glass Carriers in a Bioreactor

Abstract

Cell therapies require the in vitro expansion of adherent cells such as mesenchymal stromal cells (hMSCs) in bioreactor systems or other culture environments, followed by cell harvest. As hMSCs are strictly adherent cells, cell harvest requires cell detachment. The use of hMSCs for cell therapy requires GMP production in accordance with the guidelines for advanced therapeutic medical products. Therefore, several GMP-conform available proteolytic enzymes were investigated for their ability to promote hMSC detachment. An allogeneic hMSC cell line (hMSC-TERT) that is used in clinical trials in the form of alginate cell capsules was chosen as a model. This study investigated the influence of several factors on the outcome of proteolytic hMSC-TERT detachment. Therefore, hMSC-TERT detachment was analyzed in different cultivation systems (static, dynamic) and in combination with further cell processing including encapsulation. Only two of the commercially available enzymes (AccutaseTM, TrypZeanTM) that fulfill all process requirements (commercial availability, cost, GMP conditions during manufacturing and non-animal origin) are found to be generally suitable for detaching hMSC-TERT. Combining cell detachment with encapsulation demonstrated a high impact of the experimental set up on cell damage. It was preferable to reduce the temperature during detachment and limit the detachment time to a maximum of 20 minutes. Cell detachment in static systems was not comparable with detachment in dynamic systems. Detachment yields in dynamic systems were lower and cell damage was higher for the same experimental conditions. Finally, only TrypZeanTM seemed to be suitable for the detachment of hMSC-TERT from dynamic reactor systems.

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

“…Microcarriers 2-5 mm in diameter are often used for FBR processes, whereas those used in fluidized bed reactors are typically 1 mm in diameter and those used in STRs are generally 100-300 µm in diameter [80]. Glass has been used as a cell culture growth surface for decades [81], and low-density microcarriers with a copolymer plastic core and a high-silica glass coating were used for the expansion of hMSC-TERT in serum-containing medium [51,82]. These results are based on a microcarrier choice for the hMSC-TERT cell line resulting in strong proliferation and a good yield of detached cells for glass and polystyrene microcarriers [36].…”

“…Researchers often use recombinant trypsin for process development, but there is an intensive ongoing search for alternatives, mostly studied using static cultures [82]. Here, we discuss the harvesting procedure for the dynamic systems described above and then consider alternatives based on studies using static cultures.…”

“…Studies reported by Weber [89] and Salzig and colleagues [82] have shown how cells can be harvested from a FBR system. Success depends on the combination of enzyme choice, incubation time and temperature, and the effects of downstream processing and formulation [82].…”

“…Success depends on the combination of enzyme choice, incubation time and temperature, and the effects of downstream processing and formulation [82]. After flushing the reactor twice with phosphate buffered saline (PBS), harvesting was carried out by incubating the cells with TrypZean for 15 min at 21°C and cells were flushed out of the reactor with media (37°C) at a superficial velocity of 19 cm/s.…”

“…Viability testing and cell counting are achieved using methods such as flow cytometry, including dye exclusion. Different dyes make it possible to detect viable and dead cells even in encapsulated formulations [82].…”