Process development for the production of mesenchymal stromal cell-derived extracellular vesicles in conventional 2D systems.

BACKGROUND: In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. METHODS: We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. RESULTS: We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm-2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. CONCLUSIONS: Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases.

Large-Scale Expansion of Human Liver Stem Cells Using Two Different Bioreactor Systems.

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm-2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h-1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm-2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm-2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.

Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system

BACKGROUND The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis. RESULTS We produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41C. CONCLUSIONS Although, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore rovides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets