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.

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

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.

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.

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.

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.

Biological ethanol production is important for the circular carbon economy and makes up 73% of the U.S. biological fuels market. Previously, we produced ethanol by reversing methanogenesis and capturing methane by cloning methyl-coenzyme M reductase (Mcr) from an unculturable population of anaerobic methanotrophic archaea; this process was predicated on the generation of the intermediate acetate and its conversion by the methanogenic host to ethanol. Moreover, methanogens are generally thought to be detrimental for converting acetate to ethanol and are usually intentionally inhibited. Here, we demonstrate that direct growth on acetate as the sole carbon and energy source by the methanogen Methanosarcina acetivorans C2A results in 40% of the metabolized acetate becoming ethanol and that there is 430% more ethanol produced, compared to growth on methane via Mcr. In addition, we found growth on methanol results primarily in methane generation and low levels of ethanol. Therefore, acetate may be readily converted by the methanogen M. acetivorans to ethanol at high yields.

In vitro regeneration of Citrus plants is a widely used method, however, induction of adventitious roots from regenerated shoots remains a major bottleneck, limiting the recovery of healthy plants for commercial production and genomic research for crop improvement. We established an in vitro regeneration system producing profuse, healthy roots for sweet orange (Citrus sinensis cv. Benyenda) by optimising combinations and concentrations of auxins. Prior to optimising the rooting media (RTMs), we obtained a shoot regeneration rate of 90.6% from sweet orange epicotyl explants using a cytokinin, 6-benzylaminopurine (BAP). Across twelve auxin-supplemented RTMs containing different concentrations of indole-3-butyric acid (IBA) and/or 1-naphthaleneacetic acid (NAA), rooting percentages ranged from 8 - 87.5%. The combination of IBA 1.0 mg L-1 and NAA 0.1 mg L-1 promoted the best overall performance, 75 {+/-} 7.2% rooting percentage with healthy, callus-free roots ([≥]5 cm in length), whereas other RTMs with other auxin combinations induced callus and limited root elongation. The best-performing SRM and RTM were subsequently used for selection and recovery of transgenic sweet orange lines carrying an empty CRISPR/Cas9 construct, resulting in an 4.8% transformation efficiency. Both transgenic and non-transgenic rooted plantlets were successfully acclimatised under glasshouse conditions with a survival rate of 90%. This enhanced regeneration system overcomes rooting bottleneck and improves plant survival,enabling faster recovery of transgenic citrus lines within four months. It supports accelerated development for commercial applications and advances in citrus genetic improvement.

Culture conditions were optimized for the production of melanin nanoparticle by the bacterial strain Pseudomonas stutzeri BTCZ 305. Response surface methodology was employed for determining the most significant fermentation conditions using variables including, pH, temperature and L-tyrosine concentration identified through one-factor-at-a time approach. Box-behnken design consisting of 17 different combinations of all these factors were performed. Using this methodology, a quadratic regression model was built and the optimal combinations of media constituents for maximum melanin production 1192.27 microg/mL were determined as temperature (32.5 degreeC), pH (8.5) and L-tyrosine concentration (7 g/L). Melanin production was obtained experimentally coincident with the predicted value and the model was proven to be adequate. The nanostructural distribution, its stability in colloidal suspension and particle size were also characterized with the help of TEM, particle size analysis and Zeta potential. The potent applicability of this molecule in anti-inflammation and wound healing was also elucidated.

Most proteins targeted to the organelles of endosymbiotic origin are encoded in the nuclear genome, placing them under the regulatory dominance of the nucleus. For photosynthetic eukaryotes, nuclear-encoded chloroplast proteins arise via two routes: First, genes of cyanobacterial origin were relocated to the nucleus through endosymbiotic gene transfer (EGT). Second, proteins of eukaryotic origin emerged to support chloroplast function and structure. These proteins are reimported into the chloroplast via an import machinery. Reversing the transfer of such genes from the nucleus to the chloroplast genome may offer insights into chloroplast regulation and evolution. In this study, we established a highly efficient and accessible electroporation protocol for chloroplast transformation in the green alga Chlamydomonas reinhardtii, and used it to reverse-transfer two nuclear-encoded genes encoding proteins arising via the two routes described above: the cyanobacteria-derived chloroplast division protein FtsZ1 and the Rubisco-linker EPYC1 of eukaryotic origin. Regardless of origin, both chloroplast-encoded FtsZ1 and EPYC1 showed proper localization and functionality comparable to their nuclear-encoded counterparts. Together, our study provides a robust protocol for chloroplast transformation, a platform for investigating the evolutionary drivers of EGT, and a foundation for advancing chloroplast bioengineering. SIGNIFICANCE STATEMENTO_LIEndosymbiotic gene transfer has resulted in the mass migration of genes from the chloroplast genome to the nuclear genome. Reversing the gene transfer could reveal the evolutionary significance of genome partitioning. C_LIO_LIUsing the green alga Chlamydomonas reinhardtii, this study developed an efficient, electroporation-based protocol for chloroplast transformation. Relocating the genes encoding two chloroplast-targeted proteins, FTSZ1 and EPYC1, to the chloroplast genome showed that the proteins maintained normal localization and function. C_LIO_LIThe established transformation protocol facilitates systematic testing of reverse gene transfer to elucidate the potential evolutionary advantages of genome partitioning and opens new avenues for chloroplast bioengineering. C_LI

Microbial fuel cells (MFCs) technology offers sustainable electricity production. Current research largely focuses on few select model organisms, therefore the true prevalence of exoelectrogenesis amongst bacteria remaining largely unknown. We present a broad-scale survey of monomicrobial electricity production among environmental bacterial isolates inoculated in MFCs, using model organism Shewanella oneidensis MR-1 as a benchmark. Of the assessed taxa, 11-22% displayed exoelectrogenic activity, exceeding current predictions and identifying a further three novel exoelectrogenic species. Phylogenetic analysis based on the 16S sequences enabled the evolutionary relationship between isolates to be visualised, revealing that exoelectrogenesis is non-randomly distributed and phylogenetically conserved. Polarisation studies were implemented, revealing that numerous electron transfer mechanism were being utilised to perform exoelectrogenesis. The results of this study imply that bacterial electricity production is more widespread amongst culturable bacteria than previously estimated, with implications for bioprospecting novel exoelectrogens and predicting electrogenic activity in diverse microbial communities.

Melanins are biological macromolecule with immense functionality synthesised by a wide spectrum of living organism. It is mainly synthesised by the oxidative polymerization of indolic and phenolic compounds through several enzymatic process. It has wide spread application in agriculture, cosmetic and therapeutic industry due to its various properties including antioxidation ability, UV protection efficiency and anticancer activity. Because of this wide range of application in different sectors, large scale production and commercialization attains enormous consideration. The present study deals with the effect of 12 different process parameters on melanin production viz., production media, incubation time, inoculum concentration, pH, temperature, agitation, carbon source, phosphate and magnesium source, CuSO4.5H2O, sodium chloride and L-tyrosine on melanin production by Pseudomonas stutzeri strain BTCZ 109 obtained from Arabian sea sediments was evaluated. After optimizing the important process parameters, the bacteria showed about ~4.65 fold increase in melanin production compared to unoptimized cultural conditions. The melanin optimized through this method was found to be nano sized. The Nano sized DOPA melanin in treating Skin cancer cell line SK ML28 which showed a dose-dependent activity with an IC50 value of 164 g/mL. All these results highlight the therapeutic efficiency of DOPA melanin Nano particle as promising bioactive molecule.

Pancreatic cancer remains a highly lethal malignancy with limited therapeutic options. Chimeric antigen receptor (CAR) therapy has revolutionized the treatment of hematological cancers but still faces major limitations in solid tumors, particularly due to the scarcity of tumor-specific targets. Cutaneous lymphocyte antigen (CLA) recently emerged as a promising PDAC target due to its high tumor expression and limited presence in healthy tissues. However, previously reported CLA-directed CAR constructs lacked antitumor functionality. Here, we investigated multiple strategies to generate functional CLA-targeting CAR approaches. We first hypothesized that impaired activity resulted from fratricide caused by CLA expression on activated T cells. CLA knockout was successfully achieved through deletion of fucosyltransferase-7, but not by knockout of the major CLA carrier backbones CD162, CD44 or CD43, suggesting additional CLA carriers or compensatory regulation. As CLA knockout alone did not restore CAR-mediated killing, we explored whether insufficient binding affinity limited CAR activity. Affinity maturation was performed in silico and in vitro using yeast surface display, identifying 39 candidate mutations, although none restored cytotoxicity. We finally switched to an AdCAR strategy using anti-biotin CAR T cells combined with biotinylated anti-CLA scFv-Fc adapters. This approach enabled efficient, concentration-dependent cytotoxicity with both CLA-targeting binders. Additionally, we identified a dynamic, cell density-dependent regulation of CLA expression. Finally, glycan profiling of CLA binders further revealed broader-than-expected glycan interactions, suggesting a potentially wider definition of the CLA family. Overall, our findings establish CLA as a functional PDAC immunotherapy target while revealing unexpected complexity in its regulation and molecular presentation.

Here we introduce OpenEvo, a fully open-source, low-cost turbidostat platform for automated continuous culture and directed evolution experiments. Existing tools are expensive, complex, or lack open-source hardware; OpenEvo addresses this gap. OpenEvo is a complete, fully automated evolution platform with detailed, illustrated construction instructions for beginners, open-source software and firmware, and a single device priced around $300. An optional PC-based version offers enhanced functionality, including remote access, programmable evolution cycles, programmable LED stimulation, and a data visualization tool. OpenEvo can cycle through three types of media for positive, negative, and neutral selection conditions, supporting a wide range of experimental designs. We validate the use of OpenEvo by evolving H. volcanii to grow from 15% to 12% salt over ~150 cycles, ~1,000 hours. Evolved cells grew 36% faster than wild-type at 12% salt. Whole-genome sequencing of adapted cells found SNPs and large deletions. We also demonstrate positive and negative selection using the OpenEvo LEDs to drive optogenetics via a Phytochrome B-based optogenetic tool, with light as the selection stimulus during over 4000 hours of growth. OpenEvo lowers the technical and cost barriers for continuous evolution experiments, serves as a teaching tool, and is designed to grow an open community of users who share modifications.

Glioblastoma (GBM) is a highly aggressive brain tumor with a five-year survival rate of less than 5%. The current standard of care established 20 years ago includes maximal surgical resection and administration of alkylating agent temozolomide (TMZ). GBM is highly invasive, and GBM cells that evade surgical resection can become resistant to TMZ and develop new aggressive secondary tumors. Post-relapse there are few treatment options available to patients. Tissue engineering approaches suggest the opportunity to develop in vitro models of the GBM tumor microenvironment that may accelerate the discovery of novel therapies for GBM. Here, we report the adaptation of hydrogel microdroplets (microgels) to encapsulate GBM cells in a tailorable 3D matrix to assess patterns of growth and to screen TMZ drug response using patient-derived xenograft (PDX) specimens. We exploit a unique aspect of the microgel system to account for the cellular heterogeneity within the tumor microenvironment (TME). We combine cell-laden microgels generated from TMZ-resistant and TMZ responsive variants of the same PDX specimens to create heterogeneous populations with varying levels of drug sensitivity. We demonstrate a range of drug resistance phenotypes as a function of the ratio of TMZ-responsive to resistance cells and identify the population required for TMZ-resistance to overtake take the response. We then investigate the influence of tumor mimetic shifts in hyaluronic acid bioavailability and hypoxia on patterns of TMZ resistance. We show exposure to matrix-bound hyaluronan increases TMZ resistance and the glioma stem cell population in both cell variants. Lastly, we report an increase in TMZ sensitivity but divergent changes in the GSC subfraction for TMZ resistant vs responsive GBM in the presence of hypoxia. Together, we demonstrate the versatility of cell-laden microgel approach to replicate heterogenous tumor populations, model shifts in the tumor microenvironment, and rapidly screen therapeutic response.