Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system.

OBJECTIVE: To provide a standardized protocol for large-scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC). METHODS: The hPSC were expanded and differentiated into PTEC on matrix-coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin-treatment was assessed to check the potential use of PTEC to model drug-induced nephrotoxicity. RESULTS: hPSC expansion and PTEC differentiation could be performed directly on matrix-coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10-fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico-basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM-1 after Cisplatin treatment. CONCLUSION: We demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix-coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.

An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds.

The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract •The embryonic stem cell test to predict teratogenicity was made automation-compatible. •Several key improvements to the assay procedure have been introduced to increase performance. •The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.

Distributed automated manufacturing of pluripotent stem cell products.

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site-decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing 'live' corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation.

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture.

Tyramine-conjugated alginate hydrogels as a platform for bioactive scaffolds.

Alginate-based hydrogels represent promising microenvironments for cell culture and tissue engineering, as their mechanical and porous characteristics are adjustable toward in vivo conditions. However, alginate scaffolds are bioinert and thus inhibit cellular interactions. To overcome this disadvantage, bioactive alginate surfaces were produced by conjugating tyramine molecules to high-molecular-weight alginates using the carbodiimide chemistry. Structural elucidation using nuclear magnetic resonance spectroscopy and contact angle measurements revealed a surface chemistry and wettability of tyramine-alginate hydrogels similar to standard cell culture treated polystyrene. In contrast to stiff cell culture plastic, tyramine-alginate scaffolds were found to be soft (60-80 kPa), meeting the elastic moduli of human tissues such as liver and heart. We further demonstrated an enhanced protein adsorption with increasing tyramine conjugation, stable for several weeks. Cell culture studies with human mesenchymal stem cells and human pluripotent stem cell-derived cardiomyocytes qualified tyramine-alginate hydrogels as bioactive platforms enabling cell adhesion and contraction on (structured) 2-D layer and spherical matrices. Due to the alginate functionalization with tyramines, stable cell-matrix interactions were observed beneficial for an implementation in biology, biotechnology, and medicine toward efficient cell culture and tissue substitutes. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 114-121, 2019.

Poly(amidoamine)-alginate hydrogels: directing the behavior of mesenchymal stem cells with charged hydrogel surfaces.

The surface charge of a biomaterial represents a promising tool to direct cellular behavior, which is crucial for therapeutic approaches in regenerative medicine. To expand the understanding of how the material surface charge affects protein adsorption and mesenchymal stem cell behavior, differently charged surfaces with zeta potentials spanning from -25 mV to +15 mV were fabricated by the conjugation of poly(amidoamine) to alginate-based hydrogels. We showed that the increase of the biomaterials surface charge resulted in enhanced quantities of biologically available, surface-attached proteins. Since different surface charges were equalized after protein adsorption, mesenchymal stem cells interacted rather with diverse protein compositions instead of different surface features. Besides an enhanced cell attachment to increasingly positively charged surfaces, the cell spreading area and the expression of adhesion-related genes integrin α5 and tensin 1 were found to be increased after adhesion. Moreover, first results indicate a potential impact of the surface charge on mesenchymal stem cell differentiation towards bone and fat cells. The improved understanding of surface charge-related cell behavior has significant impact on the design of biomedical devices and artificial organs.