A Multi-Stage Bioprocess for the Expansion of Rodent Skin-Derived Schwann Cells in Computer-Controlled Bioreactors.

Regenerative therapies for the treatment of peripheral nerve and spinal cord injuries can require hundreds of millions of autologous cells. Current treatments involve the harvest of Schwann cells (SCs) from nerves; however, this is an invasive procedure. Therefore, a promising alternative is using skin-derived Schwann cells (Sk-SCs), in which between 3-5 million cells can be harvested from a standard skin biopsy. However, traditional static planar culture is still inefficient at expanding cells to clinically relevant numbers. As a result, bioreactors can be used to develop reproducible bioprocesses for the large-scale expansion of therapeutic cells. Here, we present a proof-of-concept SC manufacturing bioprocess using rat Sk-SCs. With this integrated process, we were able to simulate a feasible bioprocess, taking into consideration the harvest and shipment of cells to a production facility, the generation of the final cell product, and the cryopreservation and shipment of cells back to the clinic and patient. This process started with 3 million cells and inoculated and expanded them to over 200 million cells in 6 days. Following the harvest and post-harvest cryopreservation and thaw, we were able to maintain 150 million viable cells that exhibited a characteristic Schwann cell phenotype throughout each step of the process. This process led to a 50-fold expansion, producing a clinically relevant number of cells in a 500 mL bioreactor in just 1 week, which is a dramatic improvement over current methods of expansion.

Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. METHODS: The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. RESULTS: CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. CONCLUSIONS: Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.

Long-Term Stability and Differentiation Potential of Cryopreserved cGMP-Compliant Human Induced Pluripotent Stem Cells.

The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products.

Computational fluid dynamics modeling, a novel, and effective approach for developing scalable cell therapy manufacturing processes.

Induced pluripotent stem cells (iPSCs) hold great potential to generate novel, curative cell therapy products. However, current methods to generate these novel therapies lack scalability, are labor-intensive, require a large footprint, and are not suited to meet clinical and commercial demands. Therefore, it is necessary to develop scalable manufacturing processes to accommodate the generation of high-quality iPSC derivatives under controlled conditions. The current scale-up methods used in cell therapy processes are based on empirical, geometry-dependent methods that do not accurately represent the hydrodynamics of 3D bioreactors. These methods require multiple iterations of scale-up studies, resulting in increased development cost and time. Here we show a novel approach using computational fluid dynamics modeling to effectively scale-up cell therapy manufacturing processes in 3D bioreactors. Using a GMP-compatible iPSC line, we translated and scaled-up a small-scale cardiomyocyte differentiation process to a 3-L computer-controlled bioreactor in an efficient manner, showing comparability in both systems.

Improved expansion of equine cord blood derived mesenchymal stromal cells by using microcarriers in stirred suspension bioreactors.

Equine mesenchymal stromal cells (MSCs) are increasingly investigated for their clinical therapeutic utility. Such cell-based treatments can require cell numbers in the millions or billions, with conventional expansion methods using static T-flasks typically inefficient in achieving these cell numbers. Equine cord blood-derived MSCs (eCB-MSCs), are promising cell candidates owing to their capacity for chondrogenic differentiation and immunomodulation. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to generate clinically relevant numbers of cells while decreasing cost, time and labour requirements and increasing reproducibility and yield when compared to static expansion. As eCB-MSCs have not yet been expanded in stirred suspension bioreactors, a robust protocol was required to expand these cells using this method. This study outlines the development of an expansion bioprocess, detailing the inoculation phase, expansion phase, and harvesting phase, followed by phenotypic and trilineage differentiation characterization of two eCB-MSC donors. The process achieved maximum cell densities up to 75,000 cells/cm2 corresponding to 40 million cells in a 100 mL bioreactor, with a harvesting efficiency of up to 80%, corresponding to a yield of 32 million cells from a 100 mL bioreactor. When compared to cells grown in static T-flasks, bioreactor-expanded eCB-MSC cultures did not change in surface marker expression or trilineage differentiation capacity. This indicates that the bioreactor expansion process yields large quantities of eCB-MSCs with similar characteristics to conventionally grown eCB-MSCs.

An Integrated Approach toward the Biomanufacturing of Engineered Cell Therapy Products in a Stirred-Suspension Bioreactor.

Recent advances in stem cell biology have accelerated the pre-clinical development of cell-based therapies for degenerative and chronic diseases. The success of this growing area hinges upon the concomitant development of scalable manufacturing platforms that can produce clinically relevant quantities of cells for thousands of patients. Current biomanufacturing practices for cell therapy products are built on a model previously optimized for biologics, wherein stable cell lines are established first, followed by large-scale production in the bioreactor. This "two-step" approach can be costly, labor-intensive, and time-consuming, particularly for cell therapy products that must be individually sourced from patients or compatible donors. In this report, we describe a "one-step" integrated approach toward the biomanufacturing of engineered cell therapy products by direct transfection of primary human fibroblast in a continuous stirred-suspension bioreactor. We optimized the transfection efficiency by testing rate-limiting factors, including cell seeding density, agitation rate, oxygen saturation, microcarrier type, and serum concentration. By combining the genetic modification step with the large-scale expansion step, this not only removes the need for manual handing of cells in planar culture dishes, but also enables the biomanufacturing process to be streamlined and automated in one fully enclosed bioreactor.

Non-Newtonian rheology in suspension cell cultures significantly impacts bioreactor shear stress quantification.

The fields of regenerative medicine and tissue engineering require large-scale manufacturing of stem cells for both therapy and recombinant protein production, which is often achieved by culturing cells in stirred suspension bioreactors. The rheology of cell suspensions cultured in stirred suspension bioreactors is critical to cell growth and protein production, as elevated exposure to shear stress has been linked to changes in growth kinetics and genetic expression for many common cell types. Currently, little is understood on the rheology of cell suspensions cultured in stirred suspension bioreactors. In this study, we present the impact of three common cell culture parameters, serum content, cell presence, and culture age, on the rheology of a model cell line cultured in stirred suspension bioreactors. The results reveal that cultures containing cells, serum, or combinations thereof are highly shear thinning, whereas conditioned and unconditioned culture medium without serum are both Newtonian. Non-Newtonian viscosity was modeled using a Sisko model, which provided insight on structural mechanisms driving the rheological behavior of these cell suspensions. A comparison of shear stress estimated by using Newtonian and Sisko relationships demonstrated that assuming Newtonian viscosity underpredicts both mean and maximum shear stress in stirred suspension bioreactors. Non-Newtonian viscosity models reported maximum shear stresses exceeding those required to induce changes in genetic expression in common cell types, whereas Newtonian models did not. These findings indicate that traditional shear stress quantification of cell or serum suspensions is inadequate and that shear stress quantification methods based on non-Newtonian viscosity must be developed to accurately quantify shear stress.

Bioreactor Expansion of Skin-Derived Precursor Schwann Cells.

Scaling up the production of cells in a culture process is a critical step when trying to develop cell-based regenerative therapies. Static cultures often cannot be easily scaled up to clinically relevant cell numbers. Alternatively, bioreactors offer a highly valuable means to develop a clinical-ready process. To culture adherent cells in suspension, such as skin-derived precursor Schwann cells (SKP-SCs), microcarriers need to be used. Microcarriers are small spherical beads suspended within the vessel that allow for higher growth surface area to volume ratio. Here we describe the procedure of combining microcarriers with the controllability of bioreactors to generate higher cell densities in smaller reactor volumes leading to a more efficient and cost-effective cell production for applications in regenerative medicine.

Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.