Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products

Mesenchymal stem cells (MSCs) are advanced therapy medicinal products used in cell therapy applications. Several MSC products have already advanced to phase III clinical testing and market approval. The manufacturing of MSCs must comply with good manufacturing practice (GMP) from phase I in Europe and phase II in the US, but there are several unique challenges when cells are the therapeutic product. Any GMP-compliant process for the production of MSCs must include the expansion of cells in vitro to achieve a sufficient therapeutic quantity while maintaining high cell quality and potency. The process must also allow the efficient harvest of anchorage-dependent cells and account for the influence of shear stress and other factors, especially during scale-up. Bioreactors are necessary to produce clinical batches of MSCs, and bioprocess development must therefore consider this specialized environment. For the last 10 years, we have investigated bioprocess development as a means to produce high-quality MSCs. More recently, we have also used bioreactors for the cocultivation of stem cells with other adult cells and for the production of MSC-derived extracellular vesicles. This review discusses the state of the art in bioprocess development for the GMP-compliant manufacture of human MSCs as products for stem cell therapy

Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use

Citation: Florian Petry, J. Robert Smith, Jasmin Leber, Denise Salzig, Peter Czermak, and Mark L. Weiss, “Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use,” Stem Cells International, vol. 2016, Article ID 4834616, 12 pages, 2016. doi:10.1155/2016/4834616The great properties of human mesenchymal stromal cells (hMSCs) make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, we performed a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cells growth in 12-well plate and spinner flask. The goal to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor was achieved

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method

Citation: Smith, J. R., Pfeifer, K., Petry, F., Powell, N., Delzeit, J., & Weiss, M. L. (2016). Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method. Stem Cells International, 14. doi:10.1155/2016/6810980Umbilical cord derived mesenchymal stromal cells (UC-MSCs) are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP) for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed-vessel dissociation following enzymatic digestion which reduces contamination risk and manipulation time. The new method produced >10 times more cells per cm of UC than our previous method. When biographical variables were compared, more UC-MSCs per gram were isolated after vaginal birth compared to Caesarian-section births, an unexpected result. UC-MSCs were expanded in medium enriched with 2%, 5%, or 10% pooled human platelet lysate (HPL) eliminating the xenogeneic serum components. When the HPL concentrations were compared, media supplemented with 10% HPL had the highest growth rate, smallest cells, and the most viable cells at passage. UC-MSCs grown in 10% HPL had surface marker expression typical of MSCs, high colony forming efficiency, and could undergo trilineage differentiation. The new protocol standardizes manufacturing of UC-MSCs and enables clinical translation

Policy and prevention efforts for gaming should consider a broad perspective : Commentary on: Policy responses to problematic video game use: A systematic review of current measures and future possibilities (Király et al., 2018)

Internet gaming disorder is gaining attention around the world. Some efforts have been directed toward preventing gaming problems from developing or persisting, but few approaches have been empirically evaluated. No known effective prevention intervention exists. Reviewing the broader field of prevention research should help research and best practices move forward in abating problems that arise from excessive gaming

Predisposing and Precipitating Risk Factors for Delirium in Elderly Patients Admitted to a Cardiology Ward: An Observational Cohort Study in 1,042 Patients.

Aim: Although the risk factors for delirium in general medicine are well-established, their significance in cardiac diseases remains to be determined. Therefore, we evaluated the predisposing and precipitating risk factors in patients hospitalized with acute and chronic heart disease. Methods and Results: In this observational cohort study, 1,042 elderly patients (≥65 years) admitted to cardiology wards, 167 with and 875 without delirium, were included. The relevant sociodemographic and cardiac- and medical-related clusters were assessed by simple and multiple regression analyses and prediction models evaluating their association with delirium. The prevalence of delirium was 16.0%. The delirious patients were older (mean 80 vs. 76 years; p < 0.001) and more often institutionalized prior to admission (3.6 vs. 1.4%, p = 0.05), hospitalized twice as long (12 ± 10 days vs. 7 ± 7 days; p < 0.001), and discharged more often to nursing homes (4.8 vs. 0.6%, p < 0.001) or deceased (OR, 2.99; 95% CI, 1.53-5.85; p = 0.003). The most relevant risk factor was dementia (OR, 18.11; 95% CI, 5.77-56.83; p < 0.001), followed by history of stroke (OR, 6.61; 95% CI 1.35-32.44; p = 0.020), and pressure ulcers (OR, 3.62; 95% CI, 1.06-12.35; p = 0.040). The predicted probability for developing delirium was highest in patients with reduced mobility and institutionalization prior to admission (PP = 31.2%, p = 0.001). Of the cardiac diseases, only valvular heart disease (OR, 1.57; 95% CI, 1.01-2.44; p = 0.044) significantly predicted delirium. The patients undergoing cardiac interventions did not have higher rates of delirium (OR, 1.39; 95% CI 0.91-2.12; p = 0.124). Conclusion: In patients admitted to a cardiology ward, age-related functional and cognitive impairment, history of stroke, and pressure ulcers were the most relevant risk factors for delirium. With regards to specific cardiological factors, only valvular heart disease was associated with risk for delirium. Knowing these factors can help cardiologists to facilitate the early detection and management of delirium

Genetic predisposition to mosaic Y chromosome loss in blood.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism1-5, yet our knowledge of the causes and consequences of this is limited. Here, using a computational approach, we estimate that 20% of the male population represented in the UK Biobank study (n = 205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes that are involved in cell-cycle regulation and cancer susceptibility, as well as somatic drivers of tumour growth and targets of cancer therapy. We demonstrate that genetic susceptibility to LOY is associated with non-haematological effects on health in both men and women, which supports the hypothesis that clonal haematopoiesis is a biomarker of genomic instability in other tissues. Single-cell RNA sequencing identifies dysregulated expression of autosomal genes in leukocytes with LOY and provides insights into why clonal expansion of these cells may occur. Collectively, these data highlight the value of studying clonal mosaicism to uncover fundamental mechanisms that underlie cancer and other ageing-related diseases.This research has been conducted using the UK Biobank Resource under application 9905 and 19808. This work was supported by the Medical Research Council [Unit Programme number MC_UU_12015/2]. Full study-specific and individual acknowledgements can be found in the supplementary information

Three-Dimensional Bioreactor Technologies for the Cocultivation of Human Mesenchymal Stem/Stromal Cells and Beta Cells

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess engineering of the cocultivation processes is necessary to achieve successful beta cell therapy

Synthesis and Characterization of A[W2O2F9] (A = Li – Cs)

Herein, we report upon the in situ formation of WOF4 and its subsequent reaction with AF (A = Li – Cs) in aHF to produce A[W2O2F9] (A = Li – Cs). Structural analysis of the obtained compounds was performed using single-crystal X-ray diffraction. A comparison of the single-crystal structures obtained here to the previously reported A[Mo2O2F9] (A = Li – Cs) and H3O[W2O2F9] salts is given. Additionally, IR and Raman spectroscopy were used to characterize each salt. These spectra were compared to calculated ones for the solid-state structure of each salt using the DFT-PBE0 density functional method. The calculated spectra were used to assign bands in the experimental spectra. Both MoOF4 and WOF4 gave similar reaction products under similar reaction conditions. It was concluded that the oxyfluorides show similar Lewis acid-base reactions with the alkali metal fluorides in aHF solutions.Peer reviewe

MoF5 revisited. A comprehensive study of MoF5

While the properties of molybdenum pentafluoride, MoF5, have been investigated in the past, there exists no comprehensive study of the compound. Additionally, many of these studies appear incoherent and offer contradictory explanations of some of the observed properties of MoF5. Consequently, a comprehensive examination of MoF5 is presented here, including a redetermination of the crystal structure of MoF5 using single crystal and powder X-ray diffraction, the reevaluation of its IR, Raman and UV–Vis spectrum, and a study of its density (3.50(2) g/cm3 @ 25 °C) and magnetic properties. Additionally, density functional theory (DFT) calculations were performed on the gas phase molecule Mo4F20 to provide a discussion of properties realized during investigation. Single crystal X-ray diffraction showed MoF5 to crystalize in the monoclinic, C2/m space group, as isolated tetramers having the formula Mo4F20. Magnetic measurements showed that when “MoF5” is cooled from the melt fast enough, paramagnetic species with S = ½ are present together with S = 0 species. These species may be described using the formula (MoF5)n (n = odd) and (MoF5)n (n = even, presumably n = 4). From the measurements, the content of the S = ½ species is estimated to be 6%. The preferred species of MoF5 under ambient conditions is Mo4F20.Peer reviewe