Biotechnology Journal

Automated platforms such as Chi.Bio enable simultaneous monitoring of optical density and fluorescent reporter expression in 20 ml reactor cultures with controllable pump systems. As such, they provide an appealing option for contemporary gene expression quantification, quantitative physiology, and laboratory evolution and ecology experiments. While optical density calibration for this device is well established, no equivalent calibration framework exists for fluorescence, making quantitative comparison with reference instruments unreliable. Here, we characterize Chi.Bio fluorescence capabilities using fluorescent calibration microspheres and fixed GFP-expressing S. cerevisiae and E. coli cells, compared with orthogonal plate-reader measurements. We show that microsphere fluorescence is detectable and scales linearly with concentration, whereas the GFP signal from both species falls below the device detection limit. Comparison of background-correction strategies indicates that direct subtraction of a non-fluorescent control measured within the same device yields more reliable fluorescence estimates than the commonly used on-line normalization method. Knowledge of these sensitivity boundaries of the device provides practical guidelines for experimental design of future studies.

The suspension culture of pluripotent stem (PS) cells in stirred bioreactors poses a delicate balance between maintaining homogeneous cell dispersion and avoiding excessive shear stress that can compromise cell viability and pluripotency. In this study, we used computational fluid dynamics (CFD) coupled with a discrete particle method (DPM) to simulate iPS cell behavior in a 5 mL delta-impeller stirred tank. Our analysis revealed that upward flow at the tank bottom and downward flow at the top are critical for maintaining a stable suspension. To optimize the stirring protocol, we applied Bayesian optimization to identify a time-dependent stirring schedule that begins with a high-speed phase for resuspension, followed by a low-speed phase for sustained suspension with minimal hydrodynamic stress. The optimized schedule demonstrated improved suspension ratio and reduced slip velocity, indicating lower mechanical stress on cells. These findings provide engineering insights into scalable bioreactor operation, contributing to the design of robust iPS cell manufacturing systems.

Extremophilic red microalgae are promising platforms for sustainable biotechnology, combining robust growth under selective thermoacidophilic conditions with production of thermostable phycobiliproteins and carbon-rich biomass. However, reactor-dependent effects on growth, product formation and biomass composition remain insufficiently resolved. Here, we systematically evaluated the extremophilic red microalga Cyanidioschyzon merolae across cultivation scales and reactor formats and benchmarked its performance against the well-established Galdieria javensis and Limnospira platensis. In small-scale multi-cultivator photobioreactors and microfluidic growth chambers, C. merolae showed superior growth, reaching a maximum growth rate of 0.034 {+/-} 0.001 h-1 and 8.3 {+/-} 0.3 g l-1 cell dry weight. Microfluidic cultivation enabled growth analysis at single-cell resolution and matched growth rates obtained in photobioreactors. To identify scalable production strategies, C. merolae was further cultivated in a flat-panel photobioreactor and a custom-designed internally illuminated photobioreactor. The custom-designed photobioreactor delivered the highest biomass concentration and productivity, yielding 11.5 {+/-} 0.6 g l-1 cell dry weight and 1.07 {+/-} 0.06 g l-1 d-1, and comparable yields with regard to R-phycocyanin and R-allophycocyanin. Biomass analysis revealed substantial carbon and nitrogen contents, starch accumulation up to > 20 % of cell dry weight, and fatty acids dominated by palmitic, linoleic and oleic acids. Despite its reduced cell wall fraction, C. merolae contained structurally diverse, cultivation-dependent polysaccharides. These results establish C. merolae as a versatile chassis for thermostable pigment production and renewable feedstock generation, highlighting photobioreactor design as a key determinant of productivity and biomass quality.

Methanogenic archaea, such as Methanothermobacter marburgensis, represent a powerful biological platform for carbon capture and valorization, directly converting carbon dioxide (CO2) and molecular hydrogen (H2) into proteinogenic amino acids (AAs). In this study, we present a controlled and scalable strategy for tailoring AA production (biosynthesis and secretion) in continuous gas fermentation. By applying various Design of Experiments (DOE) techniques, we systematically identified and optimized key process parameters governing AA biosynthesis and shaping a targeted AA secretion profile. A hybrid modeling framework combining experimental data with scale-independent parameters derived from computational fluid dynamics (CFD) enabled robust performance prediction across bioreactor scales. This model-driven approach successfully translated the process from 120 mL glass bottles via 2 L to 150 L reactors, corresponding to a reaction-volume scale-up factor of 2000. These findings set the foundation for a robust and predictive platform for sustainable AA production, positioning archaea as a high-potential alternative in industrial biotechnology.

O_LIThe effect of some bioactive compounds on living organisms is dependent on their concentration and gradients, as is the case of hormones and signalling peptides, determining cell identity, activity and organism development. C_LIO_LIThere are a handful of methods that allow to produce spatially confined peaks of concentration local application of biochemicals on plants, such as agar blocks and microinjection, but they lack in precision, throughput and/or simplicity. C_LIO_LIWe developed the MicroTron, a microfluidics-based method specifically for filamentous organisms or life cycle stages, like the moss plant Physcomitrium patens protonemata, that serves as a platform for the application of chemicals on single cells and study the cell response. C_LIO_LIWe show how chemical applications could be performed on cells, either on the side or apically with dyes and hormones, targeting the cell wall, cell membrane, cytosol and nucleus. C_LIO_LITreatments could be applied on single filaments and with a precision of up to single cells in optimal conditions. C_LIO_LIThis method could be used to study live responses to chemicals with high spatiotemporal resolution. C_LI

A subset of extracellular vehicles (EVs) with particle diameters >200 nm, large vesicles (lEVs) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (SC) is the most reported protocol to separate lEVs from the smaller EVs (<200 nm)/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (BECs) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (DGC) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/732469v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@12d5e90org.highwire.dtl.DTLVardef@19b44a5org.highwire.dtl.DTLVardef@b7ad75org.highwire.dtl.DTLVardef@dd1a3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tumor models that recapitulate 3D architecture are essential for understanding how cellular organization and microenvironmental interactions govern therapeutic response in human cancers. Here, we developed a microfluidic microphysiological system that enables controlled and scalable culture and drug testing of non-small cell lung cancer spheroids and patient-derived organoids. The platform integrated U-shaped microwells with dual-channel loading to support de novo spheroid formation, efficient trapping of pre-formed spheroids, and loading of intact organoids with reduced size heterogeneity. Tumor spheroids and organoids maintained high viability and structural integrity during long-term on-chip culture, and constrained microscale confinement produced ellipsoidal geometries that deviate from idealized spherical assumptions. Baseline genotype-dependent responses to KRAS G12C and EGFR inhibitors were preserved across agarose and microfluidic formats, establishing a validated reference state. Building on this baseline, fibroblast- and endothelial-derived cues consistently attenuated responses to targeted therapies across conditioned media, mixed co-culture, and spatially organized configurations. Resistance phenotypes converged on a dominant role for paracrine signaling, while increasing architectural complexity primarily enhanced morphological fidelity rather than altering therapeutic response. These findings establish a microphysiological framework that decouples tumor-intrinsic drug sensitivity from microenvironment-mediated modulation, enabling the systematic evaluation of paracrine resistance mechanisms in NSCLC.

The growing demand for sustainable aquafeeds has intensified interest in alternative protein ingredients capable of reducing reliance on fishmeal without compromising fish performance. Here, we evaluated microbial community-based single-cell protein (SCP) as a fishmeal substitute in juvenile Asian seabass (Lates calcarifer) diets in two independent feeding trials of juvenile fish conducted over 49 and 56 days, respectively and compared them to a previous study that lasted 24 days. SCP was produced from nutrient-rich soybean-processing side streams by microbial communities in fermenters and incorporated into experimental diets at inclusion levels ranging from 10% to 100% fishmeal replacement. In the 24-day trial, a diet containing 50% fishmeal replacement with lab-scale produced SCP achieved 100% survival and a feed conversion ratio (FCR), specific growth rate (SGR), and weight gain comparable to the fishmeal control diet. In the 49-day trial using pilot-scale produced SCP, a 50% fishmeal replacement also maintained an FCR and feed intake comparable to the control, whereas complete replacement reduced feed intake and growth performance. In a 56-day pilot-scale trial that used 500-L fish tanks, diets containing up to 50% fishmeal replacement maintained comparable survival, weight gain, and SGR, although moderately higher FCR values were observed at higher SCP inclusion levels. Proximate composition and essential amino acid profiles of fish fed control or SCP-containing diets were comparable. Genome-resolved metagenomic analyses revealed diverse microbial taxa associated with the SCP. Collectively, these findings support microbial community-based SCP as a scalable and reproducible alternative protein platform for aquaculture feeds across independent trials and production scales.

Sustainable hydrogen production from microalgae remains limited by intrinsic physiological constraints and the need to preserve biomass value for food and feed applications. Transgenic approaches to overcome these limitations were proven successful, yet result in genetically modified (GMO) strains that face major regulatory and deployment barriers. Here, we present a non-GMO experimental platform that enables systematic isolation of hydrogen-producing phenotypes through high-throughput UV mutagenesis pipline coupled with targeted physiological screening. Applying this approach across phylogenetically distinct algal species, including the industrial strain Chlorella vulgaris and the extremophile Chlorella ohadii, we achieve high discovery efficiency, recovering 0.4-0.6% validated hydrogen-producing mutants and achieving 6.7-25% validation rates among screen-positive candidates, indicating strong enrichment at the primary screening stage. We show that sustained hydrogen production represents a physiologically accessible state emerging across diverse genetic backgrounds. This state is consistently associated with reorganization of photosynthetic electron partitioning, yet arises through multiple distinct configurations that differentially balance hydrogen production, oxygen metabolism, and carbon fixation. This framework provides a scalable route to identify hydrogen-producing strains in industrially relevant algae without introducing foreign DNA and expands the accessible design space for photobiological hydrogen production.

- Thalassiosira rotula produces extracellular chitin fibers of interest for material science. Tailored monomeric iminosugars, designed as substrate analogues for carbohydrate-active enzymes, unexpectedly elongate these fibers in vivo, yet their impact on chitin metabolism remains unclear. - T.rotula was exposed to three L-isoleucine-derived iminosugar analogues immediately before cell division, when chitin fibers are produced. RNA-sequencing, combined with differential expression and pathway enrichment analyses, as well as transcriptome mining for chitin-related genes was performed. - Gene mining identified 84 chitin-associated genes (including 42 chitin synthases). Two iminosugars globally repressed carbohydrate- and energy-related pathways including photosynthesis, glycolysis/gluconeogenesis, and Calvin cycle while simultaneously inducing ribosome biogenesis. ImOH specifically downregulated 29 chitin-related genes, including two strongly repressed chitinases and a {beta}-N-acetylhexosaminidase. - Tailored monomeric chitin-modulating iminosugars not only alter chitin fiber length but also trigger a broad metabolic shift from carbohydrate synthesis toward ribosome biogenesis, indicative of a cellular stress response to non-metabolizable iminosugars.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Chimeric antigen receptor (CAR)-T cell therapy is an important therapy involving provirus insertions in the genome. Characterizing these insertions is important for understanding the safety and efficacy of cell products, but the sequence of these insertions is not fully characterized. We generate clonal CD19 CAR-T cell lines with one to five copies of the lentiviral provirus insertions. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR), which revealed that most of the elements (LTR, Psi, RRE, CD19, and WPRE) were 1 to 5 or 6 copies per cell. DdPCR data also revealed that there was an additional copy of eGFP gene in VCN4 and VCN5 cell lines. To fully characterize the sequences and locations of these insertions, we use short- and long-read whole genome sequencing as well as digital PCR and flow cytometry. Long-reads enable full resolution of each insertion, and we find that of 10 insertion events, 3 have the expected insertion sequence, 2 differ from the expected only in small variants, 3 have structural abnormalities, and 2 are small partial insertions missed by most other approaches. One particularly important structural abnormality resolved only by long-reads is a 724 bp deletion of the EF1 promoter disrupting expression of the CD19 CAR. Standard short-read and ddPCR approaches miss this deletion due to this commonly used promoter being in the unengineered human genome. These results demonstrated that these cell lines are suitable VCN reference standards for 1 to 5 or 6 copies and highlight the utility of long-read sequencing in characterizing both quantity and quality of insertions in lentiviral-engineered cells.

Single-cell CRISPR screens have enabled systematic investigation of gene function, but studies have largely focused on gene-level effects, overlooking transcriptional complexity and isoform usage. Methods capable of capturing splicing and isoform usage have emerged, including long-read sequencing and alternative library preparation strategies, but their suitability for large-scale perturbation screens remains unevaluated. We compare two library preparation methods (10x Genomics and Parse Biosciences) across Illumina short-read, Oxford Nanopore, and PacBio long-read sequencing, applying CRISPRi to silence three genes with distinct regulatory roles (DDX6, GEMIN5, GFI1B) in K562 cells. While short-read methods detected some splicing events, only long-read sequencing consistently captured isoform-level changes. Although Parse provided even transcript coverage, we observed strong intronic read enrichment, limiting its utility for splicing analysis. The primary constraint of long-read approaches was sequencing depth: ~21 million reads are needed for 80% saturation of splicing events in a single perturbation. Notably, GEMIN5 knockdown produced only modest differential expression but the most extensive splicing changes, an effect invisible to gene-level analysis, underscoring the value of isoform-level screens. We provide a practical framework for isoform-level analysis in single-cell CRISPR screens, identifying current capabilities and limitations. As perturbation studies scale, long-read sequencing will be essential for comprehensive functional interpretation, capturing biology missed by gene-level analysis.

Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we implement precision molecular medicine and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high genomic selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive malignancy affecting children and adolescents. We describe the development of a non-viral and programmable polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in patient-derived xenografts (PDXs) of EWS. Moreover, silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. To our knowledge, we describe the first non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which can be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

Intestinal organoids are powerful in vitro models, but their use in large-scale analyses remains constrained by the low throughput, labor-intensive handling, and high reagent consumption of conventional Matrigel dome culture. Here, we present Organoid-in-Bead (OrB), a vortex-based compartmentalization workflow that partitions organoid fragments into thousands of discrete Matrigel microbeads, enabling scalable, high-density culture from a single batch preparation. OrB maintains dome-comparable organoid growth and epithelial polarity, supports passaging-based culture expansion, yields more than 5,000 organoids in the final 10 cm dish format, and reduces Matrigel and medium consumption by approximately 70% on a per-organoid basis. OrB therefore provides a practical and scalable upstream workflow for generating screening-scale intestinal organoids. HighlightsO_LIOrB generates Matrigel microcompartments by vortexing without microfluidics C_LIO_LIOrB enables scalable, high-density intestinal organoid culture in one batch C_LIO_LIOrB maintains dome-comparable growth and epithelial polarity and supports passaging C_LIO_LIOrB yields >5,000 organoids per batch with [~]70% less Matrigel/medium per organoid C_LI

The development of cost-effective, serum-free media is critical for scalable cultivated meat production. This study used high-throughput screening through a Design of Experiments (DoE) approach to develop an animal-free, serum-free medium (MMM1) specifically for the C2C12 murine myoblasts model cell line with applicability in cultivated meat research including for pet food. Low cost, food-grade inputs such as methylcellulose and spirulina extract resulted in significant cell growth improvements. The optimised MMM1 formulation containing low cost, food-grade inputs, achieved cumulative population doublings comparable to 10% (v/v) fetal bovine serum over four consecutive passages. Furthermore, MMM1 supported scalable cell expansion on commercially available dextran-based microcarriers (Cytodex-3) in both static and agitated conditions in spinner flasks, matching growth rates of serum-based controls. Finally, transitioning to a food-grade DMEM/F12 basal medium maintained cell proliferation equivalent to the pharmaceutical-grade DMEM/F12, but at a significantly lower cost, thus offering a viable strategy to substantially reduce biomanufacturing costs which is a critical challenge in cultivated meat production.

Viroids are plant infectious agents that threaten agricultural production. Current viroid detection methods rely on RT-PCR-based assays, which require specialized laboratory equipment and can sometimes produce false-negative results or non-specific amplification due to the high sequence conservation among closely related viroid species. CRISPR-based diagnostics, particularly Cas12-based systems for DNA detection (DETECTR) and Cas13a-based systems (SHERLOCK) for RNA detection, have emerged as powerful tools for nucleic acid diagnostics. However, most existing workflows still rely on target amplification and, in the case of Cas13a systems, require additional in vitro transcription steps, limiting their simplicity and direct applicability for plant diagnostics. Here, we developed a direct amplification-free Cas13a-based detection platform for viroids using potato spindle tuber viroid (PSTVd) as a model. We optimized CRISPR RNA (crRNA) design, identified inhibitory effects of plant total RNA on readout signal, and employed simplified viroid RNA enrichment workflows enabling robust detection in plant samples. The system further supported both PSTVd-specific and broad-spectrum pospiviroid (genus Pospiviroid) detection and was successfully extended to avocado sunblotch viroid (family Avsunviroidae), demonstrating its adaptability across distinct viroid families. Together, these results establish a practical and modular Cas13a-based platform, not only for viroid diagnostics, but also for broader applications in RNA-derived plant pathogen detection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/736049v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1d04170org.highwire.dtl.DTLVardef@1783aa3org.highwire.dtl.DTLVardef@51baa7org.highwire.dtl.DTLVardef@1b542b9_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementA simplified RNA enrichment workflow combined with CRISPR-Cas13a enables direct, amplification-free detection of plant viroids. The assay supports early and reliable diagnosis across different tomato varieties and provides a practical strategy for improving molecular detection of plant pathogens.

The success of Chimeric Antigen Receptor (CAR) T cell therapy is heavily dependent on the quality of the final cellular product. Current expansion protocols often rely on reagents that require removal from cell culture media, posing logistical challenges in manufacturing, and can also lead to terminal differentiation. Here, we evaluate the use of a soluble, bead-free T cell activator, T cell expansion protein (T-CEP), as a streamlined alternative for generating potent CAR-T cells. Human T cells were activated with T-CEP or known T cell activators (Dynabeads and TransAct) and transduced with either CD19 or interleukin-13 (IL-13) mutein (tetravariant-13; TV-13)-based CAR lentiviral vectors. Our results demonstrate that T-CEP supports robust CAR-T cell expansion and achieves transduction efficiencies comparable to commercial reagents for both types of CAR-T cells. Notably, T-CEP significantly favored the expansion of CD8+ T cells, yielding an enhanced CD27+ phenotype and a lower CD4:CD8 ratio compared to TransAct. Cytotoxicity assays confirmed that T-CEP-expanded CAR-T cells possess cytolytic function equivalent to commercial reagents for both CARs, while exhibiting lower levels of inflammatory cytokine secretion. In summary, T-CEP represents a competitive alternative to existing expansion agents, as it does not require its removal during CAR-T manufacturing and generates a CD8+ dominant, less-differentiated phenotype without compromising efficacy.

High-grade serous carcinoma (HGSOC) is the deadliest subtype of ovarian cancer, characterized by high metastatic rates. HGSOC is typically diagnosed at late stages, and treatment options are limited, resulting in a 60% recurrence rate. HGSOC cells exhibit metabolic plasticity, dynamically shifting between glycolysis and oxidative phosphorylation (OXPHOS) to meet energy demands for tumor progression. To evaluate therapeutic strategies that target metabolic vulnerabilities, we developed a microphysiological system (MPS) that recapitulates the heterogenous cell states and bioenergetic distribution of HGSOC solid tumors. Our platform utilized HGSOC spheroids embedded in a collagen hydrogel that mimics the extracellular matrix to capture tumor progression in the ovary. We used atovaquone (ATO), an FDA-approved OXPHOS inhibitor, to prototype the capabilities of our platform to investigate metabolic plasticity in HGSOC. Treatment with ATO decreased viability and invasion of HGSOC spheroids. Crucially, ATO exhibited no cytotoxicity toward biomimetic blood vessels, preserving their integrity and permeability. Metabolic imaging revealed that ATO induces an oxidative state in the outer region of the spheroids. At the invasive front, ATO disrupted mitochondrial organization, forcing collective cell migration and eventually inducing breakdown of mitochondrial networks. Furthermore, ATO decreased YAP/TAZ pathway activity in the outer region of the spheroid, providing a potential mechanism for hindered cell invasion. Collectively, our data demonstrates that a low-potency OXPHOS inhibitor like ATO can effectively target metabolic plasticity to suppress HGSOC spheroid progression. Overall, this platform successfully recapitulated metabolic heterogeneity and provided a workflow for safely testing other drugs that target cancer metabolism.

Mesenchymal stem/stromal cells (MSCs) are key players in regenerative medicine due to their immunomodulatory properties and ability to promote tissue repair. However, their therapeutic efficacy is often limited by rapid clearance following transplantation. MSC spheroids have shown enhanced functional properties, and we hypothesize that encapsulating them within hydrogel microbeads could offer additional protection and improve their viability. In this study, we developed a novel droplet-based microfluidic protocol for human MSCs derived from the apical papilla (SCAP) encapsulation and In-Bead spheroid formation within alginate microbeads. Optimization of the protocol allowed the formation of MSC spheroids in alginate droplets overnight (In-Bead), before alginate cross-linking and retrieval of alginate beads loaded with MSC spheroids. SCAP were successfully encapsulated within 275 {micro}m alginate microbeads, forming spheroids of approximately 80 {micro}m in diameter. Encapsulated SCAP spheroids retained their immunomodulatory properties. The process was further optimized by incorporating nanomedicines into the alginate solution before the formation of droplets and then spheroids, forming thus hybrid beads (Sph.Beads/NP). Nanomedicines were loaded with NecroX-5, a necrosis inhibitor, to improve SCAP viability further. Live/Dead assays indicated a protective effect of the nanomedicines, supporting the potential of this system for advanced cell delivery in regenerative applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/732109v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@278324org.highwire.dtl.DTLVardef@12bad2org.highwire.dtl.DTLVardef@1a7489forg.highwire.dtl.DTLVardef@190e793_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractA combination strategy enhancing MSC viability through spheroid formation, microencapsulation, and nanomedicine association achieved by microfluidic encapsulation with In-Bead spheroid formation. Created with BioRender