Human induced pluripotent stem cell (hiPSC)-derived neurons offer a promising, physiologically relevant alternative to animal-based neurotoxicity models. However, the influence of the surface coating, a basic but important variable, has often been neglected. Here, the impact of commonly used surface coatings including polymers (polyethylenimine, poly-L-ornithine, poly-D-lysine, poly-L-lysine) as well as extracellular matrix proteins (laminin, fibronectin, Matrigel) on NGN2 neurons, primary human astrocytes, and neuron/astrocyte co-cultures was systematically investigated. Neurons cultured on polymer-only coatings exhibited a less mature neuronal network. Interestingly, neurons on polymers with laminin, laminin only, fibronectin and Matrigel exhibited no differences in protein and gene expression but displayed different electrophysiological profiles in co-culture with human primary astrocytes. The peak in number of network bursts was reached at different time points, with fibronectin and Matrigel as early as week 2. Co-cultures on fibronectin exhibited stable cell-electrode coupling and robust activity patterns up to week 6, despite being rarely used in MEA. In a proof-of-principle experiment, laminin, fibronectin and Matrigel were further evaluated in a donor-matched hiPSC-derived co-culture system, supporting the observed effects. This study highlights the importance of the coating selection for establishing neural cultures, ultimately improving the maturation and robustness of hiPSC-derived neuronal models for drug development.

Protein-coated ultra-high viscosity (UHV)-alginate hydrogels are essential to mimic the physiological in vivo environment of humans in several in vitro applications. This work presents an optimized bioreactor-integrated freeze-drying process for MatrigelTM-coated UHV-alginate microcarriers in the context of human induced pluripotent stem cell (hiPSC) expansion. The impact of freeze-drying on the UHV-alginate microcarriers using trehalose 100 mg/mL in 0.9% NaCl as a lyoprotective agent, as well as the stem cell response using hiPSCs, was analyzed using microscopy-based screenings. First observations of the process showed that the integrity of the cake was preserved in the samples with a maximum vapor exchanging rate. Following rehydration, the UHV-alginate microcarriers retained their original morphology. Upon the addition of Poloxamer 188, stickiness and bubble formation were reduced. The expansion of hiPSCs in a suspension bioreactor resulted in a 5-7-fold increase in total cell count, yielding at least 1.3 × 107 cells with viability exceeding 80% after seven days of cultivation. In flow cytometry analysis, the pluripotency factors OCT3/4 and SSEA4 resulted in positive signals in over 98% of cells, while the differentiation factor SSEA1 was positive in fewer than 10% of cells. Supported by preceding in silico predictions of drying time, this study presents, for the first time, basic steps toward a "ready-to-use" bioreactor-integrated freeze-drying process for UHV-alginate microcarriers in the iPSC context.

Alginate hydrogels are integral to many cell-based models in tissue engineering and regenerative medicine. As a natural biomaterial, the properties of alginates can vary and be widely adjusted through the gelation process, making them versatile additives or bulk materials for scaffolds, microcarriers or encapsulation matrices in tissue engineering and regenerative medicine. The requirements for alginates used in biomedical applications differ significantly from those for technical applications. Particularly, the generation of novel niches for stem cells requires reliable and predictable properties of the resulting hydrogel. Ultra-high viscosity (UHV) alginates possess alginates with special physicochemical properties, and thus far, numerical simulations for the gelation process are currently lacking but highly relevant for future designs of stem cell niches and cell-based models. In this article, the gelation of UHV alginates is studied using a microscopic approach for disc- and sphere-shaped hydrogels. Based on the collected data, a multiphase continuum model was implemented to describe the cross-linking process of UHV alginate polysaccharides. The model utilizes four coupled kinetic equations based on mixture theory, which are solved using finite element software. A good agreement between simulation results and experimental data was found, establishing a foundation for future refinements in the development of an interactive tool for cell biologists and material scientists.

For the preclinical phase of the drug development, the ICH guideline S7B describes assays to assess cardiotoxicity focusing on potentially life threatening, acute effects causing delayed ventricular repolarization and an increase in proarrhythmic risk. However, there exist no guidelines to assess long-term cardiotoxicity although a variety of treatments e.g. with anti-cancer drugs like anthracyclines cause life threatening conditions including cardiomyopathy. HESI's Cardiac Safety Committee Stem Cell Work Group organized and executed a multi-site study with 12 blinded compounds (doxorubicin, erlotinib, sunitinib, pentamidine, arsenic trioxide, BMS-986094, milrinone, nilotinib, endothelin-1, vinblastine, vincristine, vinorelbine) of different known mechanism causing long-term cardiotoxicity in humans. We participated in HESI's multi-site study with the objective to reveal whether microelectrode array (MEA) recordings from hiPSC-derived cardiomyocytes are suitable for assessing drug-induced long-term cardiotoxicity. Cardiomyocytes were cultured on microelectrode arrays. On day of experiment, baseline recordings were taken before compounds were added as single dose in 4 concentrations and 5 replicates each. Recordings were taken at 1 h, 24 h, 48 h, 72 h, and 96 h post compound addition. On each MEA, doxorubicin was added at 300 nM (n = 4) and dofetilide at 3 nM (n = 4) as positive controls for long-term or acute cardiotoxic response, respectively. The MEA parameters FPD, FPDc, beat period, field potential amplitude and beating arrest were analyzed. After 1 h treatment, only Nilotinib prolonged FPD and FPDc and endothelin-1 decreased beat period and amplitude. Long-term dose and time dependent FPD/FPDc prolongations were observed with sunitinib, pentamidine, nilotinib, and endothelin-1. Vincristine and vinblastine slightly prolonged FPD/FPDc. Vinorelbine decreased the FP amplitude. Sunitib, pentamidine, vinorelbine, BMS-986094 and doxorubicin arrested beating at higher doses. As expected, the non-cardiotoxic drug erlotinib did not reveal any effect. As exception for the cardiotoxic drugs, only arsenic trioxide and milrinone did not show any effect. Overall, we assessed 9 of 11 compounds correctly with clear long-term cardiotoxic effects on hiPSC-CMs from 8 compounds and erlotinib with no effects as expected. Two compounds appeared to be false negative, namely milrinone and arsenic trioxide. Thus, MEA recordings from hiPSC-CM are a powerful tool to assess long-term cardiotoxicity.

The penetration kinetics of small-molecule compounds like nutrients, drugs, and cryoprotective agents into artificial cell aggregates are of pivotal relevance in many applications, from stem cell differentiation and drug screening through to cryopreservation. Depending on compound and tissue properties as well as aggregate size and shape, the penetration behavior can differ vastly. Here, we introduce bioorthogonal Raman microspectroscopy as a contactless technique to investigate the penetration of various compounds into spheroids, organoids, and other tissue models in terms of diffusion coef fi- cients and perfusion times. We showcase the potential of the method by applying it to the radial perfusion of neural stem cell spheroids with the prevalent cryopreservation additive dimethyl sulfoxide. Employing a diffusion model for spherical bodies, the spectroscopic data were quantitatively analyzed. Perfusion times were obtained for spheroids in the sub-mm region, and interesting findings about the spheroid-size dependence of the diffusion coef ficient are reported.

Adherent cell systems are usually dissociated before being cryopreserved, as standard protocols are established for cells in suspension. The application of standard procedures to more complex systems, sensitive to dissociation, such as adherent monolayers, especially comprising mature cell types or tissues remains unsatisfactory. Uncontrolled cell detachment due to intracellular tensile stress, membrane ruptures and damages of adhesion proteins are common during freezing and thawing of cell monolayers. However, many therapeutically relevant cell systems grow adherently to develop their native morphology and functionality, but lose their integrity after dissociation. The hypothesis is that cells on stretchable substrates have a more adaptable cytoskeleton and membrane, reducing cryopreservation-induced stress. Our studies investigate the influence of stretchable surfaces on the cryopreservation of adherent cells to avoid harmful dissociation and expedite post-thawing cultivation of functional cells. A stretching apparatus for defined radial stretching, consisting of silicone vessels and films with specific surface textures for cell culture, was developed. Adherent human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultivated on a stretched silicone film within the vessel, forming a monolayer that was compressed by relaxation, while remaining attached to the relaxed film. Compressed hUC-MSCs, which were cryopreserved adherently showed higher viability and less detachment after thawing compared to control cells without compression. Within three to seven days post-thawing, the hUC-MSCs recovered, and the monolayer reformed. These experiments support the hypothesis that cryopreservation success of adherent cell systems is enhanced by improved adaptability of the cytoskeleton and cell membrane, opening up new approaches in cryobiotechnology.

For the investigation of diseases and other harmful environmental influences (e.g., chemicals) epidemiological studies rely on high quality human samples, among others. Collecting samples and data in the field can pose an enormous challenge to the study team with regard to health protection and occupational safety, especially in the context of a pandemic where there was great uncertainty about the biological risks associated with SARS-CoV-2. The German Environmental Specimen Bank (German ESB) is a key element of environmental and human biomonitoring in Germany with the aim to document and assess trends of human and environmental exposure to chemicals over time and to provide scientific data for policy decision makers. Starting with a pilot study in 1978 human samples are now collected at four sampling locations annually, while sampling is carried out with a highly standardized mobile laboratory since 2013. Due to the corona pandemic 3 of 4 ESB sampling campaigns had to be cancelled in 2020. However, a continuous sampling is crucial to generate current policy relevant data on chemical exposure. Hence, a protection and hygiene concept has been developed including COVID-19 testing with the goal to protect the health of participants and employees during sampling and to meet legal requirements, while sustaining the standardized procedures of sampling and sample preparation. The concept is based on a flexible approach to allow adjustments to changing government regulations and recommendations in the course of the pandemic. By implementing this concept, all samplings were successfully carried out in 2021 & 2022, with the pandemic still ongoing. This paper provides an example of good practice and valuable insights in how to collect human samples during a pandemic.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent a valuable tool for in vitro modeling of the cardiac niche and possess great potential in tissue engineering applications. However, conventional polystyrene-based cell culture substrates have adverse effects on cardiomyocytes in vitro due to the stress applied by a stiff substrate on contractile cells. Ultra-high viscosity alginates offer a unique versatility as tunable substrates for cardiac cell cultures due to their biocompatibility, flexible biofunctionalization, and stability. In this work, we analyzed the effect of alginate substrates on hPSC-CM maturity and functionality. Alginate substrates in high-throughput compatible culture formats fostered a more mature gene expression and enabled the simultaneous assessment of chronotropic and inotropic effects upon beta-adrenergic stimulation. Furthermore, we produced 3D-printed alginate scaffolds with differing mechanical properties and plated hPSC-CMs on the surface of these to create Heart Patches for tissue engineering applications. These exhibited synchronous macro-contractions in concert with more mature gene expression patterns and extensive intracellular alignment of sarcomeric structures. In conclusion, the combination of biofunctionalized alginates and human cardiomyocytes represents a valuable tool for both in vitro modeling and regenerative medicine, due to its beneficial effects on cardiomyocyte physiology, the possibility to analyze cardiac contractility, and its applicability as Heart Patches.

Philipp Kreisz was not included as an author in the original publication [...]

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.

Purpose Human induced pluripotent stem cell (hiPSC)-derived lung cell types such as alveolar epithelial cells are promising for toxicological and pharmaceutical in vitro screenings. Reproducible differentiation processes are highly demanded, but protocols which are suitable for the high-throughput generation of lung cell types from hiPSCs are lacking. Methods In this study, a new approach for the hiPSC-differentiation in alveolar epithelial-like cells type 2 under dynamic 3D-conditions in a suspension bioreactor is presented. Gene and protein expression analyses of key markers during the embryonal lung development have been performed in comparison to cells differentiated under static 2D-conditions to evaluate the differentiation efficacy of the new bioreactor-based approach. Finally, the resulting cells were infected by SARS-CoV-2 pseudotypes to demonstrate their functionality and suitability for e.g. COVID-19 drug development. Results The dynamic bioreactor is suitable to differentiate hiPSCs in spheroids, which express relevant lung markers in each developmental stage on gene and protein level. The 3D method is able to significantly increase the expression of some markers in comparison to conventional 2D differentiation. 3D-differentiated alveolar epithelial-like cells express functional SARS-CoV-2 receptors and can display the viral infection. Conclusion The presented dynamic 3D-differentiation is a promising, new approach to generate alveolar epithelial-like cells from hiPSCs as cell source for in vitro lung models.

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.

Stem cell-based therapies are promising tools for regenerative medicine and require bulk numbers of high-quality cells. Currently, cells are produced on demand and have a limited shelf-life as conventional cryopreservation is primarily designed for stock keeping. We present a study on bulk cryopreservation of the human iPSC lines UKKi011-A and BIONi010-C-41. By increasing cell concentration and volume, compared to conventional cryopreservation routines in cryo vials, one billion cells were frozen in 50 mL cryo bags. Upon thawing, the cells were immediately seeded in scalable suspension-based bioreactors for expansion to assess the stemness maintenance and for neural differentiation to assess their differentiation potential on the gene and protein levels. Both the conventional and bulk cryo approach show comparative results regarding viability and aggregation upon thawing and bioreactor inoculation. Reduced performance compared to the non-frozen control was compensated within 3 days regarding biomass yield. Stemness was maintained upon thawing in expansion. In neural differentiation, a delay of the neural marker expression on day 4 was compensated at day 9. We conclude that cryopreservation in cryo bags, using high cell concentrations and volumes, does not alter the cells' fate and is a suitable technology to avoid pre-cultivation and enable time- and cost-efficient therapeutic approaches with bulk cell numbers.

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.

Preliminary studies of Vγ9Vδ2 T cells and zoledronate (ZOL) present promising reasons to exploit their immunotherapeutic potential for osteosarcoma treatment (OS). ZOL is a third-generation aminobisphosphonate (ABP) and is well established in the management of cancer-induced bone disease. However, ZOL is characterized by high tropism for bone matrix, and the efficacy of ZOL for sensitizing tumors remains to be optimized. Vγ9Vδ2 T cells are important effectors of antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we investigated whether Vγ9Vδ2 T cell-mediated killing of ZOL-pretreated OS cells could be increased by the anti-HER-2 monoclonal antibody trastuzumab (TTZ). The cytotoxic activity of Vγ9Vδ2 T cells against osteosarcoma was assessed by an MTS assay in the presence or absence of TTZ. A CD107a assay was used to measure degranulation in cytotoxic Vγ9Vδ2 T cells. Blocking studies were used to determine the effect of relative ligands on Vγ9Vδ2 T cell recognition. TTZ induced an ADCC response in the OS cell line, U2OS; however, it had no effect on another OS cell line HOS with low levels of surface HER2 expression. Although the OS cells pretreated with ZOL for clinically relevant time periods (2 hours) stimulated a suboptimal immune response of Vγ9Vδ2 T cells, TTZ could further enhance the cytotoxicity of Vγ9Vδ2 T cells. These results demonstrate that combining TTZ and ZOL significantly increases the cytotoxic potential of Vγ9Vδ2 T cells. This study raises the possibility of utilizing ZOL and TTZ in Vγ9Vδ2 T cell-based immunotherapy for OS.

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.