Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors.

BACKGROUND: While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified. METHODS: This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture. RESULTS: The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology. CONCLUSIONS: This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.

Multifunctional fully biobased aerogels for water remediation: Applications for dye and heavy metal adsorption and oil/water separation.

Water ecosystem contamination from industrial pollutants is an emerging threat to both humans and native species, making it a point of global concern. In this work, fully biobased aerogels (FBAs) were developed by using low-cost cellulose filament (CF), chitosan (CS), citric acid (CA), and a simple and scalable approach, for water remediation applications. The FBAs displayed superior mechanical properties (up to ∼65 kPa m3 kg-1 specific Young's modulus and ∼111 kJ/m3 energy absorption) due to CA acting as a covalent crosslinker in addition to the natural hydrogen bonding and electrostatic interactions between CF and CS. The addition of CS and CA increased the variety of functional groups (carboxylic acid, hydroxyl and amines) on the materials' surface, resulting in super-high dye and heavy metal adsorption capacities (619 mg/g and 206 mg/g for methylene blue and copper, respectively). Further modification of FBAs with a simple approach using methyltrimethoxysilane endowed aerogel oleophilic and hydrophobic properties. The developed FBAs showed a fast performance in water and oil/organic solvents separation with more than 96% efficiency. Besides, the FBA sorbents could be regenerated and reused for multiple cycles without any significant impact on their performance. Moreover, thanks to the presence of amine groups by addition of CS, FBAs also displayed antibacterial properties by preventing the growth of Escherichia coli on their surface. This work demonstrates the preparation of FBAs from abundant, sustainable, and inexpensive natural resources for applications in wastewater purification.

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.

Visible light laser direct-writing of high-resolution, biocompatible, super-multifunctional and tough hydrogels without photoinitiators in 30 s.

Currently, the lack of bioinks and long printing time limits the further development of biofabrication. Here we report a novel biocompatible, multi-functional and tough 3D printable hydrogel via visible light photocrosslinking of polyvinyl alcohol bearing styrylpyridinium group (PVA-SbQ). The high-resolution PVA-SbQ hydrogels with different designed shapes can be generated via laser direct-writing in 30 s without extra toxic crosslinkers or photoinitiators, and demonstrates excellent biocompatibility. The rapid laser direct-writing technology also results in a super-strong, tough hydrogel with excellent adhesive, swelling, self-healing, and photo-tunable properties due to the photodimerization of styrylpyridinium (SbQ) groups and the left-over massive amount of free hydroxyl groups in the hydrogel. For example, the maximum tensile strength, elongation, compressive strength adhesive strength of printed PVA-SbQ hydrogels can reach 1.0 MPa, 810 %, 33 MPa, 31 kPa, and 25,000 % respectively. And these properties can be adjusted by controlling the parameters for laser direct-writing. In addition, the introduced nitrogen cations by SbQ groups further endow hydrogels with the potential to develop novel functionality, which is demonstrated by integrating negatively charged nanocelluloses in the PVA-SbQ system to develop underwater adhesives, anti-freezing (-24.9 °C), and anti-bacterial hydrogels. This discovery opens multiple doors for developing PVA-SbQ based multi-functional hydrogel for various applications including biofabrication and tissue engineering.

Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier-based stirred suspension bioreactors.

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.

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation.

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 (109 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.

In Situ Alignment of Bacterial Cellulose Using Wrinkling.

A scalable method for the assembly of oriented bacterial cellulose (BC) films is presented based on using wrinkled thin silicone substrates of meter-square size as templates during biotechnological syntheses of BC. Control samples, including flat templated and template-free bacterial cellulose, along with the oriented BC, are morphologically characterized using scanning electron microscopy (SEM). Multiple functional properties including wettability, birefringence, mechanical strength, crystallinity, water retention, thermal stability, etc., are being characterized for the BC samples, where the wrinkling-induced in situ BC alignment not only significantly improved material mechanical properties (both strength and toughness) but also endowed unique material surface characteristics such as wettability, crystallinity, and thermal stability. Owing to the enhanced properties observed, potential applications of wrinkle templated BC in printing and cell culture are being demonstrated as a proof of concept, which renders their approach promising for various biomedical and packaging applications.