Biotechnology Journal

Natural killer (NK) cells are increasingly recognized as a versatile therapeutic platform, yet their translation is hindered by limited ex vivo proliferation. Feeder cells serve as robust stimulatory component supplying activating signals required to initiate large-scale NK cell expansion. Here, using bench-scale cultures, we evaluated how distinct engineered K562-based feeder cells influence NK cell proliferation, phenotype maintenance, potential for activation, and post-cryopreservation function. Across conditions, feeder-based systems consistently enabled superior, up to 500-fold higher NK cell yield compared to feeder-free system. Variants incorporating membrane-bound costimulatory and cytokine cues yielded the most favorable balance between expansion and functional preservation. Simple adjustments to cryopreservation, including high-density-freezing and centrifuge-free-thawing, further supported NK cell recovery. Together, these findings highlight feeder cells as essential upstream reagents for effective NK cell bioproduction and provide foundational biological insights to guide the rational design and validation of future scalable NK cell manufacturing platforms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/718880v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1e8e8baorg.highwire.dtl.DTLVardef@720d4org.highwire.dtl.DTLVardef@1fc7ce2org.highwire.dtl.DTLVardef@16b0fca_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Human pluripotent stem cells (hPSCs) hold significant promise for regenerative medicine, yet optimizing their expansion in three-dimensional bioreactor systems remains challenging due to complex interactions between mechanical forces, metabolic constraints, and aggregate formation dynamics. This study developed and validated a mechanistic mathematical model to predict hPSC proliferation dynamics in vertical-wheel bioreactor (VWBR) systems, incorporating the effects of shear stress and energy dissipation rate (EDR) on cell growth and aggregate dynamics. Seven model variants employing different kinetic formulations for shear stress and energy dissipation rate effects were systematically evaluated through model selection, identifiability analyses, and experimental validation. Experimental data from six bioreactor conditions varying in initial cell density (2 x 104-15 x 104 cells/mL), agitation rate (30-60 RPM), and working volume (100-500 mL) were used for model calibration and selection. Bayesian Information Criterion analysis identified a model combining Michaelis-Menten kinetics for shear stress inhibition with a EDR-mediated aggregate detachment formulation as the best-performing variant, achieving a Mean Relative Prediction Error of 13.97%, comparable to the experimental variability of 16.29%. Independent validation experiments using leave-out data gathered under different media exchange schedules confirmed model accuracy with prediction errors below 14%, consistent with observed experimental variability around 12%. The validated model was used to optimize the media exchange protocol, leading to a 37.5% reduction in media consumption with only a 13.5% reduction in final cell yield, demonstrating its utility for prospective, quantitative bioprocess design in VWBR systems.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated transformative efficacy in hematologic malignancies, but its broader use remains constrained by complex ex vivo manufacturing, prolonged production timelines, high cost, and dependence on lymphodepleting chemotherapy. Emerging in vivo CAR-T generation strategies aim to address these limitations, but they introduce additional safety concerns associated with systemic delivery of gene-modifying vectors, including off-target transduction and insertional mutagenesis. This paper describes a novel antigen-driven CAR T-cell expansion platform (adCAR-T) based on co-culture of CAR T cells with engineered target cells expressing defined antigen density and lacking the inhibitory checkpoint ligand PD-L1. This system induces immediate activation, rapid proliferation, and sustained cytotoxic differentiation of CAR T cells without reliance on artificial CD3/CD28 bead stimulation or exogenous cytokine-driven expansion. In contrast to conventional methods, the platform eliminates the lag phase of CAR T-cell expansion and enables rapid scaling to clinically relevant doses (108-109 cells) within several days, depending on the initial cell input. Mechanistically, antigen-driven CAR engagement and target-cell lysis trigger cytokine release and amplification of CAR T cells in a physiologically relevant manner. This process promotes coordinated expansion of both directly antigen-engaged and non-engaged CAR T cells. The platform preserves "functional fitness", minimizes exhaustion, and avoids systemic exposure to gene-delivery vectors. Taken together, this strategy defines a hybrid manufacturing paradigm that bridges the control of ex vivo production with the physiological logic of in vivo activation. Proposed method has a potential to reduce manufacturing complexity, improve safety, and possibly decrease or eliminate the need for lymphodepleting conditioning. This work presents a potential alternative to both standard ex vivo manufacturing and emerging in vivo CAR-T generation approaches, with important implications for improving the accessibility, safety, and cost-effectiveness of CAR T-cell therapies.

The rapid advancement of mRNA therapeutics has imposed stringent requirements on both the quality and scalability of in vitro transcription (IVT) products. However, the accumulation of double-stranded RNA (dsRNA) byproducts and 3-terminal heterogeneity during T7 RNA polymerase (T7 RNAP)-mediated transcription can robustly trigger deleterious innate immune responses and compromise translation efficiency. Existing enzyme engineering strategies frequently struggle to reconcile the trade-offs between salt tolerance, volumetric productivity, and product purity. Here, we report a novel engineering strategy for halotolerant T7 RNAP by fusing optimized mutant polymerases with diverse DNA-binding domains (e.g., Sso7d, MC1). This approach orchestrated the development of a series of chimeric T7 RNAP mutants designed to bolster catalytic activity and template selectivity under high-salt conditions while concurrently suppressing RNA-dependent RNA polymerase (RdRP) activity. Our lead chimeric mutants exhibited exceptional salt tolerance and processivity in the presence of up to 270 mM NaCl. Notably, these mutants significantly diminished dsRNA formation to less than 0.001%, while markedly improving transcript integrity and 3 homogeneity, thereby facilitating superior translation efficiency for both linear mRNA and circular RNA (circRNA). Crucially, this heightened salt tolerance does not necessitate a trade-off in RNA yield, affording broader flexibility for downstream process optimization. In an enzymatic circRNA synthesis system, these mutants enabled a non-fed-batch configuration with high initial rNTP concentrations (15 mM each), resulting in a 50% increase in yield and achieving an unprecedented titer of 15 mg/mL. This research provides a robust enzymological solution that harmonizes quality and productivity for the industrial-scale manufacturing of high-concentration, low-immunogenicity RNA.

Biolistic transformation is a versatile tool in plant science, yet high equipment costs and tissue damage from high-pressure gas remain significant barriers. Building on our previously developed "TSGMAC", a low-cost, helium-free biolistic system, we report three major advancements to enhance its throughput, delivery quality, and quantitative capability. First, a "guide barrel" assembled from commercial DIY fittings was developed; it effectively eliminates physical tissue damage and ensures uniform particle distribution, even in soft tissues like bok choy (Brassica rapa subsp. chinensis). Second, a rapid gene expression platform using PCR products was characterized. Results demonstrate that linear DNA constructs are efficiently circularized via non-homologous end joining (NHEJ) in plant cells, and protein expression is robust regardless of the relative positions of the promoter, coding sequence, and terminator. This system bypasses time-consuming cloning. Third, a cost-effective, highly sensitive dual-luciferase assay system utilizing teal Luc (teLuc) and inexpensive firefly luciferase (FLuc) inhibitors was established. This integrated workflow enables rapid, quantitative molecular biology using supermarket-obtained materials and standard PCR reagents. Our findings provide a practical foundation for plant scientists, synergistically accelerating gene functional analysis and genetic tool development.

Advances in transcriptome profiling have revealed transcriptomic differences across different cellular states. However, functional interpretation requires precise perturbation tools and experimental frameworks. This study benchmarked two widely used modalities: CRISPR interference (CRISPRi) and Cas13d/CasRx. A standardized workflow was established to generate human pluripotent stem cells (PSCs) with inducible ZIM3-dCas9 or CasRx expression. The cell lines were subjected to flow cytometry, copy number, and immunocytochemical analyses. The knockdown performance was validated via robust OCT4 suppression and the expected downstream effects on pluripotency genes. Time-course measurements indicated that CRISPRi produced faster and stronger repression but slower recovery after inducer withdrawal. In contrast, CasRx yielded slower and typically weaker knockdown with rapid reversibility. Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions. In contrast, CasRx avoids these limitations and supports isoform-resolved targeting of circular and alternatively spliced transcripts, albeit with variable efficiency. These results provide practical guidance for selecting complementary knockdown tools to improve the interpretability of transcriptomic function studies. MOTIVATIONAdvances in transcriptome profiling have enabled the detection of subtle cell type-specific differences. However, mechanistic interpretation still depends on perturbation tools that can modulate transcripts with high precision and efficiency. Recent CRISPR-based modalities, CRISPRi and Cas13/CasRx, function as robust and orthogonal methods to achieve the knockdown of specific gene targets. However, a standardized approach for cell line preparation and comparative studies on their relative performances and limitations remains unclear. Consequently, this study presents a standardized workflow for generating cell lines that support high-efficiency knockdown using CRISPRi and CasRx. Moreover, it compares the trade-offs in potency, reversibility, and isoform resolution, along with a practical overview of method-specific pitfalls to guide tool selection and data interpretation in future studies. HIGHLIGHTSO_LIDoxycycline-inducible AAVS1 knock-in human PSC platforms for CRISPRi (ZIM3-dCas9) and CasRx (RfxCas13d) were generated to enable standardized RNA perturbation experiments. C_LIO_LIThe prepared cell lines demonstrated strong OCT4 knockdown, with expected downstream effects on the expression of another pluripotency gene, NANOG. C_LIO_LIA comparison of knockdown characteristics and their reversibility revealed rapid and sustained repression with CRISPRi, whereas slow but rapid recovery was observed with CasRx. C_LIO_LIA CRISPRi-specific off-target effect arising from TSS proximity/overlap (ATF5-NUP62) was identified, whereas CasRx achieved ATF5 knockdown without collateral repression of the neighboring NUP62 gene. C_LIO_LICasRx enables isoform-resolved knockdown of structural isoforms (circHIPK3 vs. linear HIPK3 mRNA) and splice isoforms (RAB6A-iso1 vs. RAB6A-iso2). C_LI

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.

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

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.

The development of clinically effective CAR-T cell therapies for solid tumors requires careful optimization of receptor design, functional fitness, and manufacturability. While advancing low-affinity HER2-targeting CAR-T cells toward clinical application, we found that the candidate with the strongest in vivo antitumor activity--comprising a CD8 hinge and transmembrane region and a 4-1BB co-stimulatory domain--exhibited measurable tonic signaling. This basal antigen-independent signaling, likely driven by high CAR surface expression, was associated with increased apoptosis and reduced ex vivo expansion under research-grade manufacturing conditions. Modification of the transmembrane domain reduced CAR surface expression but did not alleviate tonic signaling and instead impaired antitumor activity. By contrast, transient pharmacologic inhibition of CAR signaling with dasatinib rescued expansion and reduced apoptosis in small-scale research cultures. Notably, these tonic-signaling-associated defects were largely absent during large-scale, GMP-compliant manufacturing, which enabled robust CAR-T cell expansion without additional benefit from dasatinib supplementation. Together, these findings show that tonic signaling is not inherently detrimental to CAR-T cell performance and that its functional consequences are highly dependent on manufacturing context. Our study underscores the importance of evaluating CAR candidates within clinically relevant production platforms and supports the advancement of this 4-1BB-based HER2-specific CAR-T cell product toward clinical testing.

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.

In biotherapeutic manufacturing, product quality such as glycosylation profile is typically assessed only after harvest, limiting opportunities for corrective action during cell culture operation. Intracellular nucleotide sugar donors (NSD) directly determine glycosylation outcomes but are rarely measured, even offline, due to analytical complexity and process disruption. As a result, quality-related decisions remain constrained to fixed operating strategies. This work introduces a model-based soft sensing framework to infer NSD concentrations from readily available extracellular measurements. A Bayesian state estimation approach based on the Ensemble Kalman Filter (EnKF) is developed to reconstruct unmeasured intracellular states during CHO cell culture. An imperfect kinetic process model is combined with noisy extracellular measurements, explicitly accounting for process variability and measurement uncertainty through ensemble-based propagation and updates. The framework is validated using four independent experiments with distinct feeding perturbations that are not used for model calibration. Although the open-loop model exhibited substantial mismatch for both extracellular metabolites and intracellular NSDs, EnKF assimilation of extracellular measurements corrected key metabolic profiles. Building on these corrected extracellular dynamics, the EnKF demonstrated robust estimation of a growth-determining amino acid, asparagine, from correlated extracellular states. Based on the improved extracellular and amino acid estimates, the framework further enabled reliable inference of intracellular NSDs across all experiments.

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

Human induced pluripotent stem cells (hiPSCs) are the most accessible source material for derivation of stem-cell-based therapies at scale. However, a disconnect exists between quality characteristics of phenotype in the pluripotent state, and downstream metrics for efficacy and safety. Bridging this gap is a major challenge. Given hiPSC plasticity, environmental conditioning plays a crucial role in guiding phenotype. This work presents a parallelizable scale-down approach, acquiring real-time data to inform hiPSC phenotype throughout biomanufacturing. We developed an optoelectronic instrumentation suite capable of measuring pH, dissolved oxygen, and cell density as important surrogates for phenotype in a scale-down expansion bioprocess. We were successful in obtaining continuous, integrated parametric data throughout cultivation and estimating metabolic characteristics of hiPSC phenotype. This system functions as a proof-of-concept tool for development of predictive models and monitoring strategies around the elucidation of phenotypic dynamics within hiPSC biomanufacturing. We have demonstrated a feasible open-source multivariate continuous monitoring approach at research scale that combines common process parameters with a scattering measurement against aggregate density. The combination of these parameters enables surrogate measurement of a metric for metabolic phenotype. This contribution emphasizes monitoring how the bioprocess influences variables important in the context of cell state, in broader pursuit of better understanding the link to downstream functionality and global optima in hiPSC biomanufacturing for regenerative medicine.

The isolation of cellulose nanofibrils (CNFs), a promising precursor for sustainable and high-performance materials, has relied on chemically intensive, energy-demanding processes. As these processes were originally designed for the isolation of CNFs from wood, we herein show that the intrinsic ultrastructure of non-structural plant cells provides unique opportunities, namely direct access to loosely organized cellulose nanonetworks. We demonstrate that this loose nanofibrillar tissue can be transformed into CNFs with sizes down to elementary nanofibrils ([~]4 nm) at high yields (reaching [~]32%) under exceptionally mild hydrothermal conditions. Three distinct plants were evaluated and the physicochemical properties of the obtained nanonetworks and corresponding CNFs were thoroughly studied, including the hydrodynamics of the resulting gels. Films prepared from the obtained CNFs showed similar performance to those obtained from conventionally isolated wood-based CNFs. Overall, this study demonstrates that CNFs can be obtained through low-intensity, hazard-free, processes from widely available biomass. Thus, this approach offers a unique shift in the range of opportunities to produce CNFs facilitating the integration of their use into the food supply chain, biomedical applications, and other regulatory-constrained applications.

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biorefinery applications, yet efficient enzymatic saccharification remains challenging due to the recalcitrant structure of plant cell walls. This study presents a comprehensive comparative analysis of enzymatic activities, saccharification performance, and secretome composition of six fungal species cultivated under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using untreated flax shives as substrate. While SmF yielded approximately 4-fold higher total protein concentrations (0.38 {+/-} 0.13 g.L-1 vs. 0.08 {+/-} 0.02 g.L-1), SSF-derived enzymes demonstrated superior specific enzymatic activities, particularly for endo-xylanase and endo-cellulase, resulting in more efficient biomass saccharification. Proteomics analysis revealed distinct secretome profiles between fermentation modes, with SSF showing higher proportions of polysaccharide metabolism proteins (71.0%) compared to SmF (49.3%), while SmF exhibited greater enzyme diversity including more lytic polysaccharide monooxygenases (LPMOs) and auxiliary activity enzymes. Trichoderma species consistently demonstrated the highest saccharification efficiency, with glucose yields reaching 2.37 mM under SSF conditions. A Scheffe simplex-lattice mixture design comprising 65 enzyme cocktail combinations revealed significant synergistic interactions between several cocktails, with the binary mixture of Trichoderma 2SA21 and P. chrysogenum achieving 54% synergy - in terms of higher sugar release above expectations - and the highest total monosaccharide release (1.80 mM). These findings provide practical guidance for developing cost-effective enzyme cocktails for lignocellulosic biorefinery applications, emphasizing the importance of fermentation mode selection and strategic strain combination over enzyme supplementation complexity. The methodology established here, combining systematic screening, comparative proteomics, and statistical mixture design, offers a robust framework for optimizing fungal enzyme systems across diverse biomass substrates. BULLET POINTSSuperior enzymatic activity (xylanase, cellulase) and saccharification in solid-state fermentation Superior total protein content and diversity in submerged fermentation Specific enzyme cocktails combination can lead to synergistic effects, justifying a combinatorial approach GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/729743v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1f3704dorg.highwire.dtl.DTLVardef@151e4bforg.highwire.dtl.DTLVardef@180ced7org.highwire.dtl.DTLVardef@18bb42f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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