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Background Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. Methods Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone. Results Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. Conclusions Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.

Processes : open access journal 9(4), 575 (2021). doi:10.3390/pr9040575 special issue: "Special Issue "Cell, Gene and Regenerative Therapy Processes" / Special Issue Editors: Dr. Qasim Rafiq, Guest Editor; Dr. Ioannis Papantoniou, Guest Editor; Ms. Jelena Ochs, Guest Editor", Published by MDPI, Basel

Frontiers in Bioengineering and Biotechnology 8, 580352 (2020). doi:10.3389/fbioe.2020.580352, Published by Frontiers Media, Lausanne

Human cardiomyocytes (CMs) have potential for use in therapeutic cell therapy and high-throughput drug screening. Because of the inability to expand adult CMs, their large-scale production from human pluripotent stem cells (hPSC) has been suggested. Significant improvements have been made in understanding directed differentiation processes of CMs from hPSCs and their suspension culture-based production at chemically defined conditions. However, optimization experiments are costly, time-consuming, and highly variable, leading to challenges in developing reliable and consistent protocols for the generation of large CM numbers at high purity. This study examined the ability of data-driven modeling with machine learning for identifying key experimental conditions and predicting final CM content using data collected during hPSC-cardiac differentiation in advanced stirred tank bioreactors (STBRs). Through feature selection, we identified process conditions, features, and patterns that are the most influential on and predictive of the CM content at the process endpoint, on differentiation day 10 (dd10). Process-related features were extracted from experimental data collected from 58 differentiation experiments by feature engineering. These features included data continuously collected online by the bioreactor system, such as dissolved oxygen concentration and pH patterns, as well as offline determined data, including the cell density, cell aggregate size, and nutrient concentrations. The selected features were used as inputs to construct models to classify the resulting CM content as being “sufficient” or “insufficient” regarding pre-defined thresholds. The models built using random forests and Gaussian process modeling predicted insufficient CM content for a differentiation process with 90% accuracy and precision on dd7 of the protocol and with 85% accuracy and 82% precision at a substantially earlier stage: dd5. These models provide insight into potential key factors affecting hPSC cardiac differentiation to aid in selecting future experimental conditions and can predict the final CM content at earlier process timepoints, providing cost and time savings. This study suggests that data-driven models and machine learning techniques can be employed using existing data for understanding and improving production of a specific cell type, which is potentially applicable to other lineages and critical for realization of their therapeutic applications.

Reactive oxygen species (ROS) can act as signaling molecules involved in the acclimation of plants to various abiotic and biotic stresses. However, it is not clear how the generalized increases in ROS and downstream signaling events that occur in response to stressful conditions are coordinated to modify plant growth and development. Previous studies of maize (Zea mays L.) primary root growth under water deficit stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex, and that the rate of cell production is also decreased. It was observed that apoplastic ROS, particularly hydrogen peroxide (H2O2), increased specifically in the apical region of the growth zone under water stress, resulting at least partly from increased oxalate oxidase activity in this region. To assess the function of the increase in apoplastic H2O2 in root growth regulation, transgenic maize lines constitutively expressing a wheat oxalate oxidase were utilized in combination with kinematic growth analysis to examine effects of increased apoplastic H2O2 on the spatial pattern of cell elongation and on cell production in well-watered and water-stressed roots. Effects of H2O2 removal (via scavenger pretreatment) specifically from the apical region of the growth zone were also assessed. The results show that apoplastic H2O2 positively modulates cell production and root elongation under well-watered conditions, whereas the normal increase in apoplastic H2O2 in water-stressed roots is causally related to down-regulation of cell production and root growth inhibition. The effects on cell production were accompanied by changes in spatial profiles of cell elongation and in the length of the growth zone. However, effects on overall cell elongation, as reflected in final cell lengths, were minor. These results reveal a fundamental role of apoplastic H2O2 in regulating cell production and root elongation in both well-watered and water-stressed conditions.

BACKGROUND AIMS: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial).METHODS: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14(+) cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product.RESULTS: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest.CONCLUSIONS: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.

Sandy surface sediments of tidal flats exhibit high microbial activity due to the fast and deep-reaching transport of oxygen and nutrients by porewater advection. On the other hand during low tide, limited transport results in nutrient and oxygen depletion concomitant to the accumulation of microbial metabolites. This study represents the first attempt to use flow-through reactors to investigate virus production, virus transport and the impact of tides and season in permeable sediments. The reactors were filled with intertidal sands of two sites (North beach site and backbarrier sand flat of Spiekeroog island in the German Wadden Sea) to best simulate advective porewater transport through the sediments. Virus and cell release along with oxygen consumption were measured in the effluents of reactors during continuous flow of water through the sediments as well as in tidal simulation experiments where alternating cycles with and without water flow (each for 6 h) were operated. The results showed net rates of virus production (0.3–13.2 × 106 viruses cm−3 h−1) and prokaryotic cell production (0.3–10.0 × 105 cells cm−3 h−1) as well as oxygen consumption rates (56–737 μmol l−1 h−1) to be linearly correlated reflecting differences in activity, season and location of the sediments. Calculations show that total virus turnover was fast with 2 to 4 days, whereas virus-mediated cell turnover was calculated to range between 5–13 or 33–91 days depending on the assumed burst sizes (number of viruses released upon cell lysis) of 14 or 100 viruses, respectively. During the experiments, the homogenized sediments in the reactors became vertically structured with decreasing microbial activities and increasing impact of viruses on prokaryotic mortality with depth. Tidal simulation clearly showed a strong accumulation of viruses and cells in the top sections of the reactors when the flow was halted indicating a consistently high virus production during low tide. In conclusion, cell lysis products due to virus production may fuel microbial communities in the absence of advection-driven nutrient input, but are eventually washed off the surface sediment during high tide and being transported into deeper sediment layers or into the water column together with the produced viruses.

The purpose of the present study was to characterize the progression of red blood cell volume (RBCV) expansion and potential volumetric and endocrine regulators of erythropoiesis during endurance training (ET). Nine healthy, untrained volunteers (age = 27 +/- 4 yr) underwent supervised ET consisting of 3-4 x 60 min cycle ergometry sessions per week for 8 wk. Plasma volume (PV), RBCV, and overnight fasting hematological markers were determined before and at weeks 2, 4, and 8 of ET. In addition, plasma erythropoietin (EPO), cortisol, copeptin, and proatrial natriuretic peptide concentrations were measured during a 3-h morning period at baseline and postexercise at weeks 1 and 8. PV increased from baseline (2,405 +/- 335 ml) at weeks 2, 4, and 8 (+/- 374 +/- 194, +505 +/- 156, and + 341 +/- 160 ml, respectively, P