Impact of microcarrier concentration on mesenchymal stem cell growth and death: Experiments and modeling.

Mesenchymal stem cell (MSC) products are promising therapeutic candidates to treat a wide range of pathologies. The successful commercialization of these cell therapies will, however, depend on the development of reproducible cell production processes. For this, using microcarriers as growth supports within controlled conditions may be a viable process option. Although increasing microcarrier concentration may be associated with greater productivity due to the increased available culture surface, additional friction or shocks between microcarriers are likely to lead to undesired cell death. However, data detailing the impact of microcarrier collisions on MSC growth remains scarce. The following work demonstrates that MSC growth on microcarriers is greatly influenced by particle concentration even when little impact is observed on the apparent growth rate. It is suggested that the apparent growth rate may result in an equilibrium between growth and death kinetics which are independently affected by particle concentration and that certain MSC quality attributes may be progressively degraded in parallel. In addition, the theoretical reduction of the MSC growth rate was modeled according to the ratio between the average interparticle distance and the Kolmogorov scale. This study is an original contribution toward understanding the hydrodynamic effects in microcarrier-based stem cell cultures.

Pristinamycin production using Streptomyces pristinaespiralis and date sirup as substrate-process modeling, optimization, and scale-up.

Pristinamycin biosynthesis using Streptomyces pristinaespiralis and date sirup (DS) as substrates was optimized before scale-up. DS was filter sterilized as heat sterilization primes Maillard reactions having negative effects on antibiotic production. Multilinear regression modeling (MLR) predicted optimum medium composition, specifying components with positive and negative effects on production. The MLR showed that to maximize bacterial growth, DS, arginine, CaCl2, and KH2PO4 must be fixed at the highest concentration, but to maximize antibiotic production, these factors have to be fixed at a low level. A noticeable difference in productivity was observed in a shake flask experiments with 50.4 and 43.1 mg/L pristinamycin final concentration for the DS and the glucose substrates, respectively. In the 2 L bioreactor, the DS medium resulted in a 66.6 mg/L antibiotic, while the scale-up in the 100 L resulted in 39.0 mg/L. The low yield in the 100 L bioreactor could be attributed to the relatively high stirring rate applied which was the minimum possible in the bioreactor used. This high stirring rate prevented pellet formation by the cells, which is described as necessary for antibiotic formation by the bacterium. Hence, a successful scale-up to pilot-scale should consider the effect of stirring rate.

A new perfusion mode of culture for WJ-MSCs expansion in a stirred and online monitored bioreactor.

As a clinical dose requires a minimum of 106 cells per kilogram of patients, it is, therefore, crucial to develop a scalable method of production of Wharton Jelly mesenchymal stem cells (WJ-MSCs) with maintained inner characteristics. Scalable expansion of WJ-MSCs on microcarriers usually found in cell culture, involves specific cell detachment using trypsin and could have harmful effects on cells. In this study, the performance of batch, fed-batch, and perfused-continuous mode of culture were compared. The batch and fed-batch modes resulted in expansion factors of 5 and 43, respectively. The perfused-continuous mode strategy consisted of the implementation of a settling tube inside the bioreactor. The diameter of the tube was calculated to maintain microcarriers colonized by cells in the bioreactor whereas empty microcarriers (responsible for potentially damaging collisions) were removed, using a continuous flow rate based on MSCs physiological requirements. Thanks to this strategy, a maximal number of 800 million cells was obtained in a 1.5 L bioreactor in 10 days. Lastly, online dielectric spectroscopy was implemented in the bioreactor and indicated that cell growth could be monitored during the culture.

Quality by design to define critical process parameters for mesenchymal stem cell expansion.

Stem cell-based therapeutic products could be the key to treat the deadliest current pathologies, ranging from neuro-degenerative to respiratory diseases. However, in order to bring these innovative therapeutics to a commercialization stage, reproducible manufacturing of high quality cell products is required. Although advances in cell culture techniques have led to more robust production processes and dramatically accelerated the development of early-phase clinical studies, challenges remain before regulatory approval, particularly to define and implement science-based quality standards (essential pre-requisites for national health agencies). In this regard, using new methodologies, such as Quality By Design (QBD), to build the production process around drug quality, could significantly reduce the chance of product rejection. This review-based work aims to perform a QBD approach to Mesenchymal Stem Cell (MSC) manufacturing in standard two-dimensional flasks, using published studies which have determined the impact of individual process parameters on defined Critical Quality Attributes (CQA). Along with this bibliographic analysis, parameter criticality was determined during the two main manufacturing stages (cell extraction and cell amplification) along with an overall classification in view of identifying the Critical Process Parameters (CPP). The analysis was performed in view of an improved standardization between research teams, and should contribute to reduce the gap towards compliant Good Manufacturing Practice (cGMP) manufacturing.

Corynebacterium glutamicum, a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro-aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild-type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L-1) corresponded to the lower kLa value of 5 h-1. Above 5 h-1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L-1. Taking into account this threshold, a fed-batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild-type strain was able to produce 93.6 g L-1 of succinate, which is one of the highest concentration reported in the literature.

Impact of the type of microcarrier and agitation modes on the expansion performances of mesenchymal stem cells derived from umbilical cord.

The present study proposed to compare the impact of agitation mode (static, orbital, and mechanical) on the culture of mesenchymal stem cells extracted from the Wharton's jelly of umbilical cords (WJ-MSC), in a clinical grade culture medium, using human platelet lysate and different xeno-free microcarriers. Attachment, expansion, and detachment performances were characterized by a new dedicated tool of microscopic image posttreatment, allowing an in situ cell counting without detachment step. Results showed that performances in static mode were not necessarily representative of those obtained in dynamic mode. Moreover, impacts on nutrient consumptions and metabolite productions were identified, such as a higher glutamine consumption when Cytodex-1 microcarriers were used. The detachment strategy used was relatively efficient for Star-Plus, Plastic-Plus, and Hillex II, but not sufficient for Cytodex-1. Despite Cytodex-1 presented promising attachment and expansion performances, Star-Plus and Plastic-Plus showed a better compromise, respectively, for the orbital and the mechanical agitation modes.

Pilot-scale biomethanation of cattle manure using dense membranes.

This study aimed at studying the biomethanation process using a 100 L pilot-scale digester equipped with a dense membrane for hydrogen injection. Hydrogen mass transfer was characterized and the impact of hydrogen flowrate, agitation rate and of the co-injection of CO2, on biogas production and composition, was precisely studied. A linear relationship between H2 flowrate and the CO2 and CH4 rates in biogas was found but no impact on biogas flowrate was shown. It was also noticed that, without exogenous CO2 injection, and for high H2 injection flowrates, residual H2 could be found at the digester outlet due to local CO2 limitation. Thus, this study suggested that biogas production in biomethanation process at the pilot scale was probably rather limited by the dissolved CO2 transport within the liquid phase than by the hydrogen mass transfer itself.

Extracellular hemoglobin combined with an O2 -generating material overcomes O2 limitation in the bioartificial pancreas.

The bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O2 supply after transplantation remains a major challenge. In this study, we address the O2 limitation by combining silicone-encapsulated CaO2 (silicone-CaO2 ) to generate O2 with an extracellular hemoglobin O2 -carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O2 -diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O2 species produced by silicone-CaO2 . While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone-CaO2 alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone-CaO2 alone on murine pseudo-islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O2 provider with hemoglobin as an effective strategy to overcome O2 limitations in tissue engineering.

Impact of shear stress and impeller design on the production of biogas in anaerobic digesters.

Today, intensification of anaerobic digestion is still a scientific and technical challenge. The present study proposed combined experimental and computational fluid dynamics simulations to characterize the impact of shear stress and impeller design on the biogas production after sequential additions of substrate. Liquid phase (cattle manure digestate) rheological law was experimentally determined and input in numerical simulations. The results showed that the original use of a double helical ribbon in digester allowed a significantly faster dispersion of fresh substrate than the use of a classical Rushton turbine, leading to a 50% higher methane production rate. However, with both impellers, too high agitation rates entailed a clear slow-down of production rate and a decrease in CH4 content. To avoid this loss of productivity, it was shown that the maximal value of shear stress, determined by numerical simulations, was a consistent parameter to set the upper agitation conditions in digesters.

Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates.

Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process.

The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.8 g/L in fed-batch culture, but the specific production rate in the fed-batch culture was four times lower than that in the batch culture. Compared to other investigated fermentation processes, our strategy resulted in the highest yield of lactic acid from biomass. Lactate production by C. glutamicum 2262 thus revealed the capability of the strain to produce various fermentation products from pyruvate.

Investigation of growth conditions for the expansion of porcine mesenchymal stem cells on microcarriers in stirred cultures.

The extensive use of mesenchymal stem cells (MCS) in tissue engineering and cell therapy increases the necessity to improve their expansion. Among these, porcine MCS are valuable models for tissue engineering and are classically expanded in static T-flasks. In this work, different processes of stirred cultures were evaluated and compared. First, the effect of glucose, glutamine, antioxidant, and growth factors concentrations on porcine MSC expansion were analyzed in a suitable medium by performing kinetic studies. Results showed that a lower glucose concentration (5.5 mM) enabled to increase maximal cell concentration by 40 % compared with a higher one (25 mM), while addition of 2 to 6 mM of glutamine increased maximal cell concentration by more than 25 % compared with no glutamine supplementation. Moreover, supplementation with 1 µM thioctic acid increased maximal cell concentration by 40 % compared with no supplementation. Using this adapted medium, microcarriers cultures were performed and compared with T-flasks expansion. Porcine MSC were shown to be able to proliferate on the five types of microcarriers tested. Moreover, cultures on Cytodex 1, Cytopore 2, and Cultispher G exhibited a MSC growth rate more than 40 % higher compared with expansion in T-flasks, while MSC metabolism was similar.

Combination of yeast hydrolysates to improve CHO cell growth and IgG production.

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L(-1) increased the maximal cell density by 70 %, while a mixture of YE (1 g L(-1)) and YP.A (4 g L(-1)) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.

Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers.

Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T-flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex-1®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50×10(6) cell g(-1) Cytodex-1®). Then, the MSC aggregates were put into contact with additional available surfaces (T-flask, fresh and used Cytodex-1®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T-flasks or fresh Cytodex-1® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex-1® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex-1® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number.