J. Murrell

Media and microcarrier surface must be optimized when transitioning mesenchymal stem/stromal cell expansion to stirred tank bioreactors

Background The long-term outlook for regenerative medicine predicts an increased need for high quality materials that are compatible with the limited number of downstream processing steps required for cell-based therapies. Large scale manufacturing of adherent-dependent cell types necessitates movement away from planar culture and toward technologies such as stirred tank bioreactors where suspension culture using microcarriers is enabled [1]. Microcarriers are available in a variety of base materials including glass, polystyrene or dextran, and have been coated or derivatized to carry charge, peptides or extracellular matrix proteins such as collagen that may be animal-derived. Cell culture medium may also contain animal-derived components. Fetal bovine serum (FBS) in particular is associated with regulatory, supply, and consistency challenges [2]. Eliminating this commonly-used reagent will require thorough evaluation of animal originfree materials for compatibility with cell therapy applications. Here, we evaluated growth of human mesenchymal stem/stromal cells (MSCs) with a variety of microcarriers and cell culture media formulations. Not only was a wide range of performance observed between the microcarriers and media screened, but positive performance in static culture was not necessarily predictive of that under agitated conditions.

Clinical-scale expansion of Adipose derived Stromal Cells starting from Stromal Vascular Fraction in a single-use bioreactor: Proof of concept for autologous applications

Adipose‐derived stromal cells (ASCs) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASCs are currently selected from stromal vascular fractions (SVFs) of adipose tissue and expanded in 2D flasks following good manufacturing practices. This process is limited in surface area, labour‐intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost‐effective production of ASCs. This study investigated a single‐use stirred‐tank bioreactor that can expand ASCs from SVFs on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASCs from the SVF (n  =  2). The process was then adjusted by two means: (1) decreasing the platelet lysate in the medium for enhancing cell adherence; and (2) adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVFs was not significantly different between the bioreactor and the 2D controls (n  =  3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASCs from SVFs can be performed in a stirred‐tank bioreactor combined with microcarriers. Copyright

466. Manufacturing Solutions for Robust Cell Therapy Expansion and Harvest

The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that ease scalability, utilize animal origin-free materials and are compatible with limited downstream processing steps. As more cell therapeutics progress through clinical testing, current in vitro culture methods are proving cumbersome to scale and lack robustness. Moreover, high quality animal origin-free reagents and downstream processing devices support the future implementation of large scale manufacturing solutions that will be required following clinical success. Here, we describe the implementation of single use bioreactors and high quality media for expansion of cell therapies. We include examples from allogeneic mesenchymal stem cells and autologous T cells. The presentation will review solutions addressing animal origin-free expansion of cells within the context of different upstream process development steps as well as scaling and downstream processing with good cell quality, high recovery, high viability and good activity. Start to finish solutions for expansion and harvest, including high quality reagents, are key enabling technologies for success in commercializing cell therapies.