Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor. In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

Scalable expansion of iPSC and their derivatives across multiple lineages.

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

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.

Freeform direct laser writing of versatile topological 3D scaffolds enabled by intrinsic support hydrogel.

In this study, a novel approach to create arbitrarily shaped 3D hydrogel objects is presented, wherein freeform two-photon polymerization (2PP) is enabled by the combination of a photosensitive hydrogel and an intrinsic support matrix. This way, topologies without physical contact such as a highly porous 3D network of concatenated rings were realized, which are impossible to manufacture with most current 3D printing technologies. Micro-Raman and nanoindentation measurements show the possibility to control water uptake and hence tailor the Young's modulus of the structures via the light dosage, proving the versatility of the concept regarding many scaffold characteristics that makes it well suited for cell specific cell culture as demonstrated by cultivation of human induced pluripotent stem cell derived cardiomyocytes.

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.