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Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Mesenchymal stromal cells (MSC) are a promising platform for regenerative medicine applications because of their multilineage differentiation abilities and ease of collection, isolation, and growth ex vivo. To meet the demand for clinical applications, large-scale manufacturing will be required using three-dimensional culture platforms in vessels such as stirred suspension bioreactors. As MSCs are an adherent cell type, microcarriers are added to the culture to increase the available surface area for attachment and growth. Although extensive research has been performed on efficiently culturing MSCs using microcarriers, challenges persist in downstream processing, including harvesting, filtration, and volume reduction, which all play a critical role in the translation of cell therapies to the clinic. The objective of this review is to assess the current state of downstream technologies available for microcarrier-based MSC cultures. This includes a review of current research within the three stages: harvesting, filtration, and volume reduction. Using this information, a downstream process for MSCs is proposed, which can be applied to a wide range of applications., (© 2022 Wiley Periodicals LLC.)

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (10 9 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency., (© 2020 Wiley Periodicals, Inc.)