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3D cell culture has emerged as a relevant and promising alternative model for improving the preclinical phase of pharmaceutical development. It mimics cell-cell and cell-matrix interactions, as well as drug penetration and toxicity responses. However, there are currently no standardized methods that could lead to highly predictive treatment responses. In this context, the focus of this study was the adaptation of a liquid overlay technique, to generate a large and reproducible panel of six spheroid models, including melanoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, non-tumorigenic breast tissue and healthy kidney tissue. In this way, four cell concentrations (200 to 10,000 cells per well) with four matrix percentages (0 to 3%) were tested to determine the optimal combination based on their compaction, proliferation and viability. The surface characterization of each model was then assessed using scanning electron microscopy. Afterwards, the cytostatic and cytotoxic responses of these models to three targeted anti-PARP therapies, Olaparib, Rucaparib and Niraparib, were analyzed, revealing their sensitivity. These results demonstrated that our liquid overlay-based technique provides both a large cell culture panel, whatever the tissue type or pathological level, and an automated drug screening process that could lead to highly predictive efficacy results.

In plant cells, cortical microtubules (CMTs) generally control morphogenesis by guiding cellulose synthesis. CMT alignment has been proposed to depend on geometrical cues, with microtubules aligning with the cell long axis in silico and in vitro. Yet, CMTs are usually transverse in vivo, i.e., along predicted maximal tension, which is transverse for cylindrical pressurized vessels. Here, we adapted a microwell setup to test these predictions in a single-cell system. We confined protoplasts laterally to impose a curvature ratio and modulated pressurization through osmotic changes. We find that CMTs can be longitudinal or transverse in wallless protoplasts and that the switch in CMT orientation depends on pressurization. In particular, longitudinal CMTs become transverse when cortical tension increases. This explains the dual behavior of CMTs in planta: CMTs become longitudinal when stress levels become low, while stable transverse CMT alignments in tissues result from their autonomous response to tensile stress fluctuations.

Dental pulp stem cells (DPSCs) maintained high viability and secreted extracellular vesicles (EVs) in hollow-fiber bioreactors (HFBs) for up to 29 days.Tuning the pore size yielded more uniform DPSC-EV populations, supporting product standardization.HFB cultivation demonstrated a tunable upstream strategy to balance yield and standardization, advancing scalable DPSC-EV manufacturing for clinical translation.HFB upscaling produced DPSC-EVs with preserved angiogenic and improved immunomodulatory potency compared with T-flask cultivation.Qualitative proteomic analysis showed condition-dependent differences in EV composition, reinforcing the role of manufacturing in the final product. Dental pulp stem cells (DPSCs) reduce acute inflammation and induce angiogenesis, primarily through paracrine factors, in which extracellular vesicles (EVs) are pivotal. However, low production yields and poor standardization have hindered clinical translation. Here, we investigated hollow-fiber bioreactor (HFB) cultivation of DPSCs to improve the scalability and standardization of DPSC-EV production. We approached this problem by investigating bioreactors with membranes of two molecular weight cut-off (MWCO) pore sizes (5 kDa and 20 kDa) and evaluating DPSC-EV production over 26 days. The quantity of EVs decreased gradually with every harvest. The 5-kDa MWCO cartridge produced a more uniform EV size distribution and marker profile compared with the 20-kDa cartridge, whereas DPSC-EVs derived from both HFBs exhibited comparable angiogenic and anti-inflammatory properties. These findings demonstrate that HFB-based upscaling is a viable route for standardized production of functional DPSC-EVs upon determination of the optimal reactor parameters, thereby holding promise for advancing their clinical potential. [Display omitted] An hollow-fiber bioreactor (HFB) represents a promising, innovative technology for large-scale, standardized extracellular vesicle (EV) manufacturing for clinical applications. According to the Technology Readiness Level (TRL) framework, this technology is currently at TRL 3, with initial potency validation completed but requiring further method optimization and standardization, and in vivo preclinical and clinical testing. Our findings demonstrate that pore size tuning in HFB-based EVs can standardize size distribution without compromising biological activity, a critical step toward regulatory acceptance. However, full-scale clinical translation requires larger donor cohorts, more in-depth characterization of EV cargo, and rigorous validation in preclinical models to ensure functional reliability. While lab-scale upscaling improves yield, reproducibility, and standardization, key challenges remain, including Good Manufacturing Practice (GMP)-compliant manufacturing, long-term stability, and regulatory approval. Collaboration with researchers to refine mechanistic understanding, regulatory bodies to define potency assays and quality benchmarks, and industry to advance downstream processing and automation for GMP-compatible purification will be crucial for developing scalable and standardized EV manufacturing pipelines, ultimately enabling EV-based therapies to progress to higher TRL levels. Hollow-fiber bioreactor cultivation enables scalable and standardized production of extracellular vesicles from dental pulp stem cells (DPSC-EVs). The system preserves their angiogenic and anti-inflammatory functions while enhancing manufacturing consistency. This strategy enhances the translational potential of DPSC-EVs, supporting their consistent and scalable use in regenerative therapies.

We introduce a novel fabrication method for developing a 3D-printed perfusion bioreactor (3D-PBR) to facilitate the in situ growth and differentiation of human bone marrow (BM)-derived mesenchymal stem cells (MSCs) while enabling coculture with vascular cells. To recapitulate human physiology, in vitro platforms must incorporate several key features of their native target organ. This often entails a supportive 3D architecture for growing and differentiating multiple human cell types in situ under perfusion. Other essential characteristics include reproducibility, ease of customization, and biocompatibility. Our 3D-PBR combines these features and was fabricated using a biocompatible resin-based polymer, which was 3D-printed, followed by the addition of a permeable membrane to create a coculture microenvironment. MSCs were encapsulated in a collagen-fibrin gel alongside human endothelium within the 3D-PBR. The physical cues that our 3D-PBR provided facilitated the differentiation of MSCs into specific lineages, such as adipocytes and osteoblasts. Immunohistochemistry images demonstrated that cells grown in the 3D-PBR exhibited more physiologically relevant BM perivascular niche markers compared to static culture models. Our method utilizes emerging 3D printing techniques and alternative materials, departing from traditional PDMS-based soft lithography. These advancements in fabrication further enhance in vitro platforms for diverse cell culture models and vascular permeability assays.

Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

Mechanical loading on articular cartilage induces various mechanical stresses and strains. In vitro hydrodynamic forces such as compression, shear and tension impact various cellular properties including chondrogenic differentiation, leading us to hypothesize that shaking culture might affect the chondrogenic induction of induced pluripotent stem cell (iPSC) constructs. Three-dimensional mouse iPSC constructs were fabricated in a day using U-bottom 96-well plates, and were subjected to preliminary chondrogenic induction for 3 days in static condition, followed by chondrogenic induction culture using a see-saw shaker for 17 days. After 21 days, chondrogenically induced iPSC (CI-iPSC) constructs contained chondrocyte-like cells with abundant ECM components. Shaking culture significantly promoted cell aggregation, and induced significantly higher expression of chondrogenic-related marker genes than static culture at day 21. Immunohistochemical analysis also revealed higher chondrogenic protein expression. Furthemore, in the shaking groups, CI-iPSCs showed upregulation of TGF-β and Wnt signaling-related genes, which are known to play an important role in regulating cartilage development. These results suggest that shaking culture activates TGF-β expression and Wnt signaling to promote chondrogenic differentiation in mouse iPSCs in vitro. Shaking culture, a simple and convenient approach, could provide a promising strategy for iPSC-based cartilage bioengineering for study of disease mechanisms and new therapies.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.