Human Mesenchymal Stromal Cells (hMSCs) are a safe option for allogeneic cell therapy across various diseases, but their manufacturing process requires improvement to broaden accessibility. In this study, a state-of-the-art planar multi-vessel process was transferred to a stirred tank bioreactor using microcarriers to support the growth of adherent cells. The frequent medium exchange strategy from planar culture guided the design of the bioreactor process. However, complete medium changes in the bioreactor resulted in limited cell expansion and higher glucose consumption compared to planar culture. To enhance expansion, a 0.1 L perfusion bioreactor was tested, enabling continuous medium exchange. Three perfusion approaches were evaluated: (1) maintaining a target cell-specific glucose consumption rate, (2) varying the perfusion rate, and (3) applying a cell-specific perfusion rate. Implementing targeted glucose feeding (TAFE) reduced lactate production, while increasing perfusion rates improved cell density. The highest expansion was achieved using a cell-specific perfusion rate of 5 nL cell- 1 day- 1 combined with a target glucose consumption rate (qglc) of 15 pmol cell- 1 day- 1, resulting in a 5.4-fold higher expansion factor than daily medium changes in stirred tank bioreactors. This optimized process represents a key advancement toward producing clinically relevant quantities of hMSCs.

Background & Aim

Cell therapies hold the promise to fundamentally change health care by curing previously uncurable diseases. Both autologous and allogeneic cell therapies rely on donor cells for expansion. The variety of disease, cell type, and inherent growth kinetic donor variability puts a high demand on the manufacturing process. This production process has to be robust, follow cGMP guidelines, and be scalable in order to reduce the cost for patient and society. To this end, we developed a perfusion process for the expansion of allogeneic human Mesenchymal Stromal cells (hMSCs) in controlled bioreactors.

Methodology

A perfusion system, where medium is continuously replenished inside the bioreactor, was developed for the expansion of one donor. We then cultured cells from 2 additional donors on microcarriers using the same perfusion process in 100 mL stirred-tank bioreactors. The process was then scaled 20x to a 2L process.

Results

After initial development, the process achieved an expansion factor of 14 within 8 days of culture with one donor. After further optimizations, three tested donors were cultured. They exhibited different growth characteristics, resulting in some variation in lag phase after inoculation. A similar growth rate was observed in two out of the three donors in the exponential phase, while the third was slower. All donors achieved an expansion factor of 12 or higher (20 highest). Following this confirmation of growth, the process was scaled 20x to 2 L. In the 2 L bioreactor process, an expansion factor of 19.2 was achieved, displaying good comparability to the downscaled model. The final process achieved a 4x higher volumetric productivity than planar culture at equivalent passage.

Conclusion

The perfusion process was successfully applied to three donors, despite differences in growth characteristics and could be scaled to 2L with great results. The reported results are an important step toward achieving an economically feasible scalable production process for hMSCs.

BackgroundClostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures.ResultsTo separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (P-clo1313_2638::ldh* adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 degrees C for ethanol concentrations >= 10 g L-1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600 and relative growth rates were higher at 45 and 50 degrees C, compared to 55 degrees C, for ethanol concentrations >= 15 g L-1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L-1 at 45 and 50 degrees C compared to 55 degrees C.ConclusionPositive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.