Innovative fixed bed bioreactor platform: Enabling linearly scalable adherent cell biomanufacturing with real-time biomass prediction from nutrient consumption.

Scalable single-use adherent cell-based biomanufacturing platforms are essential for unlocking the full potential of cell and gene therapies. The primary objective of this study is to design and develop a novel fixed bed bioreactor platform tailored specifically for scaling up adherent cell culture. The bioreactor comprises a packed bed of vertically stacked woven polyethylene terephthalate mesh discs, sandwiched between two-fluid guide plates. Leveraging computational fluid dynamics modeling, we optimized bioreactor design to achieve uniform flow with minimal shear stress. Residence time distribution measurements demonstrated excellent flow uniformity with plug flow characteristics. Periodic media sampling coupled with offline analysis revealed minimal gradients of crucial metabolites (glucose, glutamine, lactate, and ammonia) across the bioreactor during cell growth. Furthermore, the bioreactor platform demonstrated high performance in automated cell harvesting, with ≈96% efficiency and ≈98% viability. It also exhibited linear scalability in both operational parameters and performance for cell culture and adeno-associated virus vector production. We developed mathematical models based on oxygen uptake rates to accurately predict cell growth curves and estimate biomass in real-time. This study demonstrates the effectiveness of the developed fixed-bed bioreactor platform in enabling scalable adherent cell-based biomanufacturing with high productivity and process control.

Long term expansion of bone marrow-derived hMSCs on novel synthetic microcarriers in xeno-free, defined conditions.

Human mesenchymal stem cells (hMSCs) present an attractive target for cell therapy given their wide availability, immunomodulatory properties, and multipotent nature for differentiation into chondrocytes, osteocytes, and adipocytes. With the progression of hMSC clinical studies, there is an increasing demand for development of technologies that enable efficient cell scale-up into clinically relevant quantities. Commercial scale manufacturing of hMSCs will require a large surface area which is not cost effective with available two-dimensional culture vessels. Recent studies showed that microcarriers provide a three-dimensional culture environment suitable for hMSC expansion. Traditionally, biological coatings and/or serum-containing medium are required to facilitate hMSC attachment and expansion in dynamic conditions. These limitations may hinder the use of microcarriers as a scale-up technology for hMSC therapeutics, where cell products, and therefore patient safety, are more controlled with the use of xeno-free, defined culture conditions. Here we report the long term culture of hMSCs on novel synthetic Synthemax II microcarriers in two different xeno-free media. Cells were maintained over 40 days on sterile, ready-to-use microcarriers in spinner flasks with programmed agitation. hMSC expansion was obtained by addition of fresh beads without the need for enzymatic dissociation. We achieved a cumulative cell expansion of >10,000 fold, and cells retained normal hMSC phenotype, karyotype, and tri-lineage differentiation potential. To our knowledge, this report is the first example of long term culture of hMSCs on synthetic microcarriers in xeno-free, defined conditions.

A synthetic, xeno-free peptide surface for expansion and directed differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax(®) Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs' lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α(v)ß(5) integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.

Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost, robust, scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined, xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally, PAS were scaled up to large culture-vessel formats. Synthetic, xeno-free, scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies.