Human mesenchymal stem/stromal cells (MSC) from juvenile donors (juvMSC) are crucial for studying bone development and for modeling pediatric skeletal diseases. However, the limited availability of these cells and the lack of physiologically relevant in vitro models hinder preclinical research. To address these issues, we established and characterized a new human bone marrow-derived MSC line under advanced culture conditions. Primary MSC from a 12-year-old donor in good health were immortalized via lentiviral transduction using a library of expansion genes. The resulting clone, C15 juvMSC, retained key features of MSC, including typical morphology, high proliferation rate, expression of stemness surface markers, and trilineage differentiation in a 3D format. Spectral karyotyping confirmed genomic stability without chromosomal aberrations. In 3D spheroid cultures, the C15 juvMSC demonstrated strong osteogenic potential, as evidenced by mineralization and alkaline phosphatase (ALP) activity. However, they exhibited a distinct differentiation pattern compared to primary cells. Overall, the C15 juvMSC line exhibits stable, scalable, and physiologically relevant characteristics, making it a valuable model for studying osteogenesis and for in vitro research on pediatric bone disorders.

Mesenchymal stem cells (MSCs) hold potential for several applications, but inefficient nonphysiological culturing methods constantly prevent clinical translation. Automated cell encapsulation in small hydrogels (microgels) facilitates physiologically relevant MSC expansion in bioreactors. Unfortunately, encapsulation processes are poorly characterized, biological variability is seldomly considered, and dynamic culturing is marginally explored. Here, a high-throughput millifluidic encapsulation process is introduced and standardized. This platform enables highly viable MSC networks within gelatin methacryloyl microgels. The impact of biological variability and crosslinking variations under strong dynamic culturing conditions is closely monitored through cell proliferation, microgel shrinkage, and metabolic activity. The effect of carboxymethyl cellulose on microgel's architecture is observed with cryogenic scanning electron microscopy. Increased crosslinking controls the formation of an outer layer on the microgels, which improves the microgel's resistance to shrinking, prevents cell proliferation on the material's surface and increases overall MSC expansion. Cell proliferation, microgel shrinkage, glucose uptake, and cell metabolism show interdependencies observable thanks to the high encapsulation output. Cell proliferation and metabolic activity depend strongly on donor-to-donor variability and change during culture. However, metabolic readouts reliably follow cell expansion, which makes this simple and mechanically robust platform promising for large-scale bioreactor applications.

Mesenchymal stem/stromal cells (MSCs) are a valuable source of paracrine factors, as they have a remarkable secretory capacity, and there is a sizeable knowledge base to develop industrial and clinical production protocols. Promising cell-free approaches for tissue regeneration and immunomodulation are driving research towards secretome applications, among which extracellular vesicles (EVs) are steadily gaining attention. However, the manufacturing and application of EVs is limited by insufficient yields, knowledge gaps, and low standardization. Facing these limitations, hydrogels represent a versatile three-dimensional (3D) culture platform that can incorporate extracellular matrix (ECM) components to mimic the natural stem cell environment in vitro; via these niche-mimicking properties, hydrogels can regulate MSCs' morphology, adhesion, proliferation, differentiation and secretion capacities. However, the impact of the hydrogel's architectural, biochemical and biomechanical properties on the production of EVs remains poorly understood, as the field is still in its infancy and the interdependency of culture parameters compromises the comparability of the studies. Therefore, this review summarizes and discusses the reported effects of hydrogel encapsulation and culture on the secretion of MSC-EVs. Considering the effects of cell-material interactions on the overall paracrine activity of MSCs, we identify persistent challenges from low standardization and process control, and outline future paths of research, such as the synergic use of hydrogels and bioreactors to enhance MSC-EV generation.

The Special Issue “Advanced 3D Cell Culture Technologies and Formats” presents a collection of original research and review articles that explore recent innovations in three-dimensional (3D) cell culture systems aimed at enhancing the physiological relevance and therapeutic utility of cells and cell-derived products and assays [...]

Purpose 3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized. Methods Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers. Results Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 10 9 ± 1.5 × 10 9 and 9.7 × 10 9 ± 3.1 × 10 9 particles/mL) compared to static 2D culture (4.2 × 10 9 ± 7.5 × 10 8 and 3.9 × 10 9 ± 3.0 × 10 8 particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 10 7 ± 1.1 × 10 7 particles/µg protein in 2D to 1.6 × 10 8 ± 8.3 × 10 6 particles/µg protein in 3D. Total MSC-EVs as well as CD73 − CD90 + MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions. Conclusion The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models.

Abstract Ewing sarcoma (ES) is an aggressive pediatric bone tumor in adolescents primarily driven by the expression of the fusion oncogene EWS::FLI1. Although the etiology of ES remains controversial, human mesenchymal stem cells (hMSCs) are considered to be the likely cell of origin. This is not only due to their ability to tolerate ectopic EWS::FLI1 expression, but also because the transcriptome of EWS::FLI1 expressing hMSCs is highly similar to that of primary Ewing tumors. However, to date a cellular differentiation-based model of ES does not exist. With the intent to understand ES histogenesis, this study aims to establish the first comprehensive single-cell differentiation atlas (both transcriptome and epigenome) of juvenile bone marrow-derived hMSCs with and without EWS::FLI1 expression. We sought to study the consequences of inducing the EWS::FLI1 oncogene expression at various time points of hMSC differentiation into adipo-, chondro-, osteo- and neuro-genic lineages. Our workflow utilizes physiology-mimicking conditions including serum-free differentiation, hypoxia, and growth/differentiation as spheroids. We pooled bone marrow-derived hMSCs isolated from eleven healthy juvenile donors, engineered them to conditionally express EWS::FLI1, and carried out multi-lineage differentiation for 10 days where EWS::FLI1 was induced daily for 24h or 72h. This temporally resolved atlas of 105 samples representing normal and perturbed differentiation allowed us to map ES patient tumor data to multiple lineages and time-points across stages of lineage commitment. Single-cell RNA-seq analysis of ~271,000 transcriptomes revealed an apparent and dynamic dedifferentiation in the presence of EWS::FLI1 across all four lineages. Preliminary analysis of genes detected in the EWS::FLI1 high cluster uncovers a specific lineage preference and time window that shares the transcriptomic signature with tumor cells, indicating the potential cell/stage of tumor-origin. Our results provide new insight into the enigmatic cell of origin of Ewing sarcoma, laying the foundation for the development of much-needed pre-clinical models for tailoring therapies. This study is supported by a generous grant (#20) of the Alex’s Lemonade Stand Foundation (ALSF) Crazy8 Program. Citation Format: Utkarsh Kapoor, Christoph Hafemeister, Lisa Shaw, Karin Muelbacher, Julia Moldaschl, Dominik Egger, Stefan Toegel, Cornelia Kasper, Matthias Farlik, Florian Halbritter, Heinrich Kovar. Dedifferentiation unveils origin of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3950.

Animal models lack physiologic relevance to the human system which results in low clinical translation of results derived from animal testing. Besides spheroids or organoids, hydrogel-based 3D in vitro models are used to mimic the in vivo situation increasing the relevance while reducing animal testing. However, to establish hydrogel-based 3D models in applications such as drug development or personalized medicine, high-throughput culture systems are required. Furthermore, the integration of oxygen-reduced (hypoxic) conditions has become increasingly important to establish more physiologic culture models. Therefore, we developed a platform technology for the high-throughput generation of miniaturized hydrogels for 3D cell culture. The Oli-Up system is based on the shape of a well-plate and allows for the parallel culture of 48 hydrogel samples, each with a volume of 15 µl. As a proof-of-concept, we established a 3D culture of gelatin-methacryloyl (GelMA)-encapsulated mesenchymal stem/stromal cells (MSCs). We used a hypoxia reporter cell line to establish a defined oxygen-reduced environment to precisely trigger cellular responses characteristic of hypoxia in MSCs. In detail, the expression of hypoxia response element (HRE) increased dependent on the oxygen concentration and cell density. Furthermore, MSCs displayed an altered glucose metabolism and increased VEGF secretion upon oxygen-reduction. In conclusion, the Oli-Up system is a platform technology for the high-throughput culture of hydrogel-based 3D models in a defined oxygen environment. As it is amenable for automation, it holds the potential for high-throughput screening applications such as drug development and testing in more physiologic 3D in vitro tissue models.

Mesenchymal stem cells (MSCs) hold immense therapeutic potential due to their regenerative and immunomodulatory properties. However, to utilize this potential, it is crucial to optimize their in vitro cultivation conditions. Three-dimensional (3D) culture methods using cell-laden hydrogels aim to mimic the physiological microenvironment in vitro, thus preserving MSC biological functionalities. Cellulosic hydrogels are particularly promising due to their biocompatibility, sustainability, and tunability in terms of chemical, morphological, and mechanical properties. This study investigated the impact of (1) two physical crosslinking scenarios for hydrogels derived from anionic cellulose nanofibers (to-CNF) used to encapsulate adipose-derived MSCs (adMSCs) and (2) physiological culture conditions on the in vitro proliferation, differentiation, and extracellular vesicle (EV) production of these adMSCs. The results revealed that additional Ca2+-mediated crosslinking, intended to complement the self-assembly and gelation of aqueous to-CNF in the adMSC cultivation medium, adversely affected both the mechanical properties of the hydrogel spheres and the growth of the encapsulated cells. However, cultivation under dynamic and hypoxic conditions significantly improved the proliferation and differentiation of the encapsulated adMSCs. Furthermore, it was demonstrated that the adMSCs in the CNF hydrogel spheres exhibited potential for scalable EV production with potent immunosuppressive capacities in a bioreactor system. These findings underscore the importance of physiological culture conditions and the suitability of cellulosic materials for enhancing the therapeutic potential of MSCs. Overall, this study provides valuable insights for optimizing the in vitro cultivation of MSCs for various applications, including tissue engineering, drug testing, and EV-based therapies.

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Mesenchymal stem cells (MSCs) are the most prominent type of adult stem cells for clinical applications. Three-dimensional (3D) cultivation of MSCs in biomimetic hydrogels provides a more physiologically relevant cultivation microenvironment for in vitro testing and modeling, thus overcoming the limitations of traditional planar cultivation methods. Cellulose nanofibers are an excellent candidate biomaterial for synthesis of hydrogels for this application, due to their biocompatibility, tunable properties, availability, and low cost. Herein, we demonstrate the capacity of hydrogels prepared from 2,2,6,6-tetramethylpiperidine-1-oxyl -oxidized and subsequently individualized cellulose-nanofibrils to support physiologically relevant 3D in vitro cultivation of human MSCs at low solid contents (0.2-0.5 wt %). Our results show that MSCs can spread, proliferate, and migrate inside the cellulose hydrogels, while the metabolic activity and proliferative capacity of the cells as well as their morphological characteristics benefit more in the lower bulk cellulose concentration hydrogels.

This chapter gives a general overview on the basic requirements for designing bioreactors for tissue engineering. It comprises the general components of a bioreactor and discusses the choice of suitable culture parameters for a tissue engineering process. Furthermore, the necessity to mimic physiologic conditions with the help of bioreactor systems in order to achieve sufficient tissue maturation is explained. As cell expansion is a prerequisite for tissue engineering processes, this chapter covers also bioreactors for expansion of cells and the issue of scale-up versus scale-out. Finally, commercially available bioreactor systems for the application of defined mechanical forces for tissue engineering processes are presented and discussed.

Understanding the growth and functionality variation of lymphocytes during expansion is fundamental for process control and endpoint determination. However, monitoring CQAs along lymphocyte expansion requires offline cell characterization, hindering timely identification of process deviations and transitions. We developed a culturing protocol that allowed to investigate day-to-day variation of NK-92 cells’ cytotoxicity against K-562 cells. Replacing 20% of the culture with fresh medium every day while disrupting the formed lymphocyte aggregates, morphological and functionality changes of the NK-92 cells were induced. Our experiments revealed that the adaptation to the pseudo-static protocol divided the culture to two distinctive phases with exponential growth in each. In between, there was an intermittent drop in viability. Significant functionality fluctuations were observed, despite the regular stimulation strategy. Scale-independent descriptors, such as growth rate, were calculated for each phase and the whole culture and correlated with cell morphology parameters and functionality. A novel correlation was found between, the cell size-derived, biomass volume and cytotoxicity that was stronger during the first phase, but it was apparent throughout the entire culture. This was possible to capture, only because of the day-to-day monitoring of cytotoxicity. By utilizing the pseudo-static culturing protocol and the methodology, variables, such as biomass volume, were discovered and by this, we are getting closer to find performance indicators suitable to monitor CQAs in a straightforward, non-invasive manner.

Gangliosides are an indispensable glycolipid class concen-trated on cell surfaces with a critical role in stem cell differentiation. Nonetheless, owing to the lack of suitable methods for scalable analysis covering the full scope of ganglioside molecular diversity, their mechanistic properties in signaling and differentiation remain undiscovered to a large extent. This work introduces a sensitive and comprehensive ganglioside assay based on liquid chromatography, high-resolution mass spectrometry, and multistage fragmentation. Complemented by an open-source data evaluation workflow, we provide automated in-depth lipid species-level and molecular species-level annotation based on decision rule sets for all major ganglioside classes. Compared to conventional state-of-the-art methods, the presented ganglioside assay offers (1) increased sensitivity, (2) superior structural elucidation, and (3) the possibility to detect novel ganglioside species. A major reason for the highly improved sensitivity is the optimized spectral readout based on the unique capability of two parallelizable mass analyzers for multistage fragmentation. We demonstrated the high-throughput universal capability of our novel analytical strategy by identifying 254 ganglioside species. As a proof of concept, 137 unique gangliosides were annotated in native and differentiated human mesenchymal stem cells including 78 potential cell-state-specific markers and 38 previously unreported gangliosides. A general increase of the ganglioside numbers upon differentiation was observed as well as cell-state-specific clustering based on the ganglioside species patterns. The combination of the developed glycolipidomics assay with the extended automated annotation tool enables comprehensive in-depth ganglioside characterization as shown on biological samples of interest. Our results suggest ganglioside patterns as a promising quality control tool for stem cells and their differentiation products. Additionally, we believe that our analytical workflow paves the way for probing glycolipid-based biochemical processes shedding light on the enigmatic processes of gangliosides and glycolipids in general.

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-β1 (TGFβ1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGFβ1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.

Extracellular vesicles (EVs) are cell-derived membrane structures exerting major effects in physiological as well as pathological processes by functioning as vehicles for the delivery of biomolecules to their target cells. An increasing number of effects previously attributed to cell-based therapies have been recognized to be actually mediated by EVs derived from the respective cells, suggesting the administration of purified EVs instead of living cells for cell-based therapies. In this review, we focus on the heterogeneity of EVs derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions. Hereby, we discuss the effects of the cell source, medium composition, 3D culture, bioreactor culture and hypoxia. Furthermore, aspects of the isolation and storage strategies influences EVs are described. Conclusively, optimization of upstream process parameters should focus on controlling MSC-derived EV heterogeneity for specific therapeutic applications.

Mesenchymal stem cells (MSCs) are primary candidates in tissue engineering and stem cell therapies due to their intriguing regenerative and immunomodulatory potential. Their ability to self-assemble into three-dimensional (3D) aggregates further improves some of their therapeutic properties, e.g., differentiation potential, secretion of cytokines, and homing capacity after administration. However, high hydrodynamic shear forces and the resulting mechanical stresses within commercially available dynamic cultivation systems can decrease their regenerative properties. Cells embedded within a polymer matrix, however, lack cell-to-cell interactions found in their physiological environment. Here, we present a “semi scaffold-free” approach to protect the cells from high shear forces by a physical barrier, but still allow formation of a 3D structure with in vivo-like cell-to-cell contacts. We highlight a relatively simple method to create core–shell capsules by inverse gelation. The capsules consist of an outer barrier made from sodium alginate, which allows for nutrient and waste diffusion and an inner compartment for direct cell-cell interactions. Next to capsule characterization, a harvesting procedure was established and viability and proliferation of human adipose-derived MSCs were investigated. In the future, this encapsulation and cultivation technique might be used for MSC-expansion in scalable dynamic bioreactor systems, facilitating downstream procedures, such as cell harvest and differentiation into mature tissue grafts.

Approved cells and cellular products (i.e. extracellular vesicles) for cell-based therapy applications 20 carry a huge promise for the treatment of a broad variety of diseases and several (stem) cell therapies. 21 However, the therapeutic potential of cells is not fully exploited at present. On the one hand, outdated 22 culture conditions are still used during in vitro cultivation. On the other hand, technological hurdles 23 block the way to efficient and safe cell-based therapy products. Traditional cell culture conditions do 24 not represent the physiological conditions of the cellular native environment (Yamada et al., 2022 In summary, this article collection provides a comprehensive overview of the state of the art in 85 advanced cell culture technologies to boost cell-based therapies, which we think will be of significant 86 interest to the journal readership. 87The authors declare that the research was conducted in the absence of any commercial or financial 89 relationships that could be construed as a potential conflict of interest. 90Author Contributions 91 DK and DE wrote the editorial which was proof-read, revised and approved by all other authors. 92Please add any funding here. 94 4

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants.