Biotechnology Journal

Our prior works have demonstrated the therapeutic potential extracellular vesicles (EVs) derived from batch cultures of brain endothelial cells (BECs) owing to their ability to transfer mitochondria to injured recipient cells. However, conventional batch/flask cultures limit scalable and reproducible production of EVs. Here, we evaluated a CELLine AD1000 bioreactor as a platform for sustained production of EVs from mouse b.End3 BECs and compared bioreactor-derived (BR) EVs with EVs obtained conventional batch cultures (BT). Large EVs (lEVs) and small (sEVs) were isolated using a sequential centrifugation protocol and were characterized using dynamic light scattering, nanoparticle tracking analysis, microBCA assay, western blotting, proteomics analysis, and their ability to rescue bioenergetics in oxygen-glucose deprived (OGD) BECs. Bioreactor cultures maintained high cell viabilities (~90%) for up to eight weeks and produced approximately three- to four-fold greater EV protein yields than batch cultures. BR EVs showed reproducible particle characteristics consistent with EV subtypes derived from BT cultures. CD63, CD9 and Arf6 were detected in EV fractions, whereas the mitochondrial outer-membrane protein TOMM20 was enriched in lEVs. Proteomic analyses revealed that bioreactor-derived lEVs retained the mitochondrial cargo signature observed in batch-derived lEVs. Both bioreactor and batch EVs restored relative ATP levels in ODG-exposed BECs. These findings suggest that bioreactor BEC cultures are a scalable approach for generating functionally active BEC-EVs for downstream characterization and therapeutic development.

Influenza A virus (IAV) defective interfering particles (DIPs) are regarded as promising broad-spectrum antivirals due to their high efficacy demonstrated in animal trials. Previously, an OP7 chimera DIP was generated that showed markedly enhanced antiviral efficacy compared to conventional IAV DIPs. In addition, a high-yield cell culture-based production process for OP7 chimera DIPs was established in perfusion mode. However, due to the technical complexity of this technology, we aimed to combine the advantages of perfusion in terms of yield with that of a simple and less complex batch process. In the present work, we established a fed-batch production process for OP7 chimera DIPs that allows a more straightforward implementation into existing production facilities. Using a feeding regime that was able to overcome the cell density effect, we achieved productive virus infection in stirred tank reactors until a cell density of 10E+6 cells/mL. Furthermore, we obtained a high virus titer of 3.8 log10(HAU/100 uL), corresponding to a 3.4-fold increase compared to a reference batch production. Moreover, the produced material demonstrated a significantly increased interfering bioactivity shown by an in vitro potency assay. Interestingly, this efficacy was similarly high as compared to OP7 chimera DIP material previously produced in perfusion mode, despite twofold lower virus titers. Finally, we demonstrate robustness of OP7 chimera DIP seed virus trains, which is significant step towards later pharmaceutical production. Further, corresponding GMP MDCK-PB2(sus) cell and OP7 chimera virus banks are now available for later biomanufacturing further use. In conclusion, the established fed-batch process offers a valid n additional option for pharmaceutical grade OP7 chimera DIP GMP manufacturing production under GMP conditions required for subsequent clinical testing and for later large-scale manufacturing.

The unprecedented rise in greenhouse gases such as carbon dioxide (CO2), and their detrimental effects on the atmosphere have intensified the current climate emergency. This challenge has inspired the development of novel carbon capture and utilisation (CCU) technologies, specifically microbial electrosynthesis (MES), for the bioelectrochemical fixation of CO2 into commodity chemical compounds (CCCs) using microbial biocatalysts. In this study, we systematically enriched and maintained robust mixed microbial communities of electroactive bacteria (EAB) from wastewater treatment sludge to serve as biocatalysts for MES. The MES performance of these biocatalysts under different operational conditions in bioelectrochemical systems (BES) reactors were investigated. BES reactors with enriched biocatalysts incubated and maintained under controlled conditions produced higher CCCs yields than those developed at ambient temperatures, while strict anaerobic operations further improved the MES efficiency compared to aerobic conditions. At an applied potential of -1000 mV vs Ag/AgCl, CO2 conversion reached up to 88.11%, significantly outperforming the lower potential (-600 mV) operation. These findings underscore the importance of systematic development of robust and stable biocatalysts, as well as maintaining strict anaerobic conditions, for achieving efficient MES performance, thereby demonstrating the potential of our approach for scalable industrial CCU applications.

Introduction: Tumors are known to develop genomic heterogeneity over time, particularly in metastatic disease, raising concerns that a single-site biopsy may be insufficient to accurately predict response to medications. Methods: We used our 3D microtumor functional profiling platform that preserves key features of the native tumor microenvironment. We tested (i) six split samples from primary tumors and (ii) seven samples from primary tumors (ovarian, colon, and breast cancer) matched with their locoregional or distant metastases. Fresh tissue specimens were processed within 24 hours after surgery and treated with NCCN guideline-recommended chemo or targeted therapies at concentrations over four orders of magnitude. Cytotoxicity was evaluated after three days to generate dose response curves. The response correlations between the split sample or between the two matched sites were quantified using the concordance correlation coefficient ({rho}c) for the drug efficacy metrics. Results: We compared a total of 275 drug responses. The six split samples resulted in a strong correlation between the two tumor areas ({rho}c=0.95, p<0.001), supporting high assay reproducibility across distinct regions of the same tumor. Locoregional metastases were highly correlated with their primaries (ovarian to omentum {rho}c=0.97, p<0.001; ovarian to liver capsule {rho}c=0.93, p<0.001; colon to peritoneum {rho}c=0.91, p<0.001). A lesser correlation was observed with lymphogenic metastases (breast to axilla {rho}c=0.87, p<0.001), and the correlations of hematogenic metastases were even lower (colon to lung {rho}c=0.69 p<0.001; ovarian to soft tissue/colon {rho}c=0.58, p<0.001). No correlation was observed among specimens that were resistant to all tested medications. Conclusion: Tumor drug responses were highly preserved in locoregional metastases, whereas somewhat greater divergence was observed in lymphogenic or hematogenic metastases. Single-site functional profiling may remain informative for identifying treatment resistance and might reduce the risk of ineffective therapy selection in metastatic disease.

An increasing number of researchers, educators, and at-home growers are turning to hydroponics as an alternative method of food production, diversifying and strengthening the food supply chain. This has led to a substantial increase in the amount of academic work involving hydroponic production of plants for a variety of purposes, including but not limited to a more controlled and customizable environment than conventional production methods, utilizing hydroponics as a method of wastewater treatment, and investigating the vertical production of plants on a much smaller footprint than traditional commercial-scale production. Because so many unique stakeholders are interested in hydroponics for a variety of reasons, academic studies use a wide variety of hydroponic systems with varying levels of technology and automation, making comparisons across studies and systems challenging. A few institutions have sophisticated plant growth and research facilities, like those used by commercial companies, but these systems are often prohibitively expensive, costing upwards of $10,000 USD for a very limited amount of growing space for experimental trials. To enable more researchers, educators, and citizen scientists to conduct experiments and easily compare their results, this study details three pilot-scale hydroponic production systems (Kratky, NFT, and DWC), utilizing entirely off-the-shelf components, for under $5,000 USD each, that are capable of maintaining the desired consistent temperature and relative humidity levels, and have space for 216, 108 (6 irrigation loops), and 64 (16 irrigation bins) plants, respectively. The material lists and prices are provided along with assembly instructions and general guidelines for each hydroponic system. The testing and optimization process of environmental conditions within each growth system is also detailed, with suggested customizations for potential experimental setups. The authors developed these systems during their Masters and Doctoral degree programs, enabling improved hydroponic research at the University of Illinois Urbana-Champaign. They hope that other aspiring hydroponic researchers can benefit from this work in the development and operation of their hydroponic systems, increasing research efficiency and collaboration opportunities in this field of study.

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

By decoupling biological function from cellular viability, fixed abiotic cellular materials have emerged as a promising platform for next-generation immunotherapies, preserving essential immunomodulatory activities while enabling unprecedented control over manufacturing, storage, and standardization. Nevertheless, the coupled effects of fabrication history and post-fabrication environmental cues on their biological functionality remain elusive. Here, we show that both preparation route and storage time critically affect the bioactivity of mesenchymal stromal cell (MSC)-derived microtissues. By exploring different devitalization agents, we demonstrate that an ethanol-based strategy provides a green fixation option with comparable preservation of volumetric and fixed cell architecture, as well as immunomodulatory function. Processing protocols did not merely substitute conventional fixation but tailored specific immune-related bioactivity. An unexpected processing protocol and time-dependent enhancement of immunomodulatory performance was observed, with ethanol-fixed microtissues maintaining a stable baseline bioactive profile while increasing functional potency over 8 weeks of storage. We establish a framework for the design of immunomodulatory abiotic cell-derived materials with potential for the treatment of immune-mediated diseases and regenerative therapies, and we hope that the principles uncovered here may contribute to a broader understanding of how devitalized tissues can be engineered, preserved, and functionally tuned, whether lab-made or derived from natural biological systems.

Extracellular vesicles (EVs) are nanosized, lipid bilayer-enclosed particles that mediate intercellular communication and hold considerable promise as biomarkers and therapeutic agents. However, their functional heterogeneity remains incompletely understood, largely due to the absence of reliable methods for isolating specific EV subpopulations. Here, we established a Nanoscale Flow sorting (NanoFACS) workflow and evaluated its potential for EV detection, sorting, and molecular characterization. We assessed critical parameters, including fluorescent staining and labeling approaches, background signal discrimination, dilution and flow-rate effects, and scatter triggering. We achieved high-purity sorting of CFSE-stained and antibody-labeled EVs, preserving vesicle integrity and sufficient RNA yields for small RNA sequencing of sorted populations. We identified EV subtype-specific small RNAs despite partial cargo overlap in single-marker sorted populations. Our results establish a reproducible antibody-based NanoFACS workflow that defines key operational parameters for reliable EV detection and sorting, with potential adaptability across conventional and spectral flow cytometry platforms. By reducing background and non-EV-associated events, the workflow enables the isolation of phenotypically defined EV subpopulations suitable for downstream molecular analysis, providing a practical approach to investigate EV heterogeneity.

Fermentation is a process through which microorganisms are used to transform basic molecules into high value substances in various applications such as biopharma, food production, and biofuel generation. In such processes, monitoring the health of microbial cells is essential, as it is a key factor in the final product yield. Traditional methods of assessing the health of fermenter populations have involved manually extracting samples from the fermenter for benchtop testing, which lacked the accuracy and efficacy of real-time continuous measurements. This has opened the door for inline sensors to be integrated into fermenters for continuous measurement; including microfluidic based Electrochemical Impedance Spectroscopy (EIS). EIS utilizes a range of frequencies to gain insight into the characteristics of microbial populations, such as cell size and membrane capacitance. EIS cytometry, in particular, has emerged as a relevant tool, as it enables single-cell level detection in real-time. However, current EIS cytometry devices cannot be deployed directly within fermenters due to the high cell density conditions. This has limited EIS analysis in fermenters to bulk measurements averaged across a population. Here, we present a microfluidic chip that uses passive cell-flux-partitioning geometry to direct cells from high-density culture through a single-cell-constrained flow path containing integrated impedance-sensing electrodes. The proposed chip combines geometric channel design and embedded electrodes to enable continuous impedance measurements from cell-containing flow while operating at input cell densities on the scale of 10^9 CFU/mL. This work demonstrates a first step toward integrating single-cell-constrained microfluidic handling with impedance sensing for inline monitoring in industrial biomanufacturing.

Oxygen (O2) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O2-permeable polydimethylsiloxane (PDMS), while retaining high O2 permeability. We measured O2 permeability in 50-m-thick PMP membranes and found approximately 200-fold greater O2 permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-m-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O2 from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O2-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O2-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.

Lack of robustness and high variability in cardiomyocyte (CM) differentiation from human induced pluripotent stem cells (hiPSCs) remain critical challenges for cardiac regenerative medicine and biological research. Guided by developmental principles of lineage specification, we investigated whether early WNT inhibition could expand the process window of CM differentiation and thereby improve robustness. To test this, we compared early WNT inhibition from days 1--5 with standard inhibition from days 3--5 following a 1-day CHIR99021 (CHIR) treatment for WNT activation. Focusing on CHIR concentration as a critical parameter, we found that early WNT inhibition substantially broadened the concentration window yielding [&ge;]80% differentiation efficiency across distinct hiPSC lines. Extending the analysis to a two-dimensional parameter space incorporating cell density revealed a markedly expanded [&ge;]80% process window. Temporal analysis of lineage-specific transcription factor expression showed that early WNT inhibition suppressed paraxial mesoderm (PM) specification, likely through MSGN1 suppression, in the range of high initial WNT activation that otherwise favored PM fate, thereby extending the process window. It also suppressed definitive endoderm (DE) specification within the expanded window, promoting a more favorable lateral plate mesoderm/cardiac fate. We further demonstrated substantially reduced variability under parameter perturbations and across wells and batches, accompanied by higher differentiation efficiency, without compromising CM phenotypes. These findings demonstrate early WNT inhibition as an effective strategy for expanding the process window of CM differentiation and improving robustness, while exemplifying the process engineering principle that a broader window can support more robust differentiation, with potential relevance to stem cell--based bioprocesses.

Plasmid copy number (PCN) is a central determinant of recombinant gene expression levels and metabolic burden in bacterial cell factories. However, dynamic real-time control of PCN during microbial cultivation remains largely unexplored. In the ColE1/pUC19 replication system, PCN is governed by an antisense RNAI/RNAII circuit, but this native mechanism provides no external handle for process-level intervention. Here, we engineer and characterize a dual-layer control architecture that combines two orthogonal perturbations to direct plasmid replication in Escherichia coli populations i.e., temperature shifts, which modulate the secondary structure of RNAII and, consequently, its binding to RNAI and its inhibitory potency (ex vivo control), and arabinose-inducible overexpression of a supplementary RNAI cassette, integrated either on a secondary low-copy plasmid or directly on the high-copy target plasmid (in vivo control). Using a superfolder GFP validated fluorescent reporter and automated flow cytometry in continuous cultures, we track PCN dynamics at the population level in real time under chemostat conditions. Preliminary screening in microplates high-throughput cultivation device across three strains, two carbon sources, and three temperatures (30, 37, and 42C) reveals that the two control layers act synergistically. Neither temperature alone nor inducible RNAI alone achieves the dynamic range obtained by their combination. This synergistic effect is sustained during continuous cultivation at a fixed dilution rate of 0.1/h, with carbon source and temperature transitions producing predictable and reproducible shifts in population-level PCN. These results establish a scalable, dual-input framework for on-demand PCN tuning in continuous bioprocesses, with direct relevance to biopharmaceutical plasmid production and synthetic biology applications.

Microalgae are promising biological platforms for sustainable energy production because they can convert photosynthetic activity into electricity in biophotovoltaic (BPV) systems. Identifying strains that grow well at elevated temperatures and under reduced irradiance is an important step toward BPV applications in seasonally variable environments. Microalgae were isolated from Seondong Lake (Seoul, Republic of Korea) during three sampling periods and screened for growth at different temperatures. Among 45 isolates, KHUC-11 showed the highest weighted growth index based on growth rates at 25, 30, and 35 under dark conditions and was selected for further characterization. Phylogenetic reconstruction of the 45S rRNA gene cluster, internal transcribed spacer 2 (ITS2) secondary-structure comparison, and morphological characterization supported the description of KHUC-11 as Desmodesmus ecotolerans sp. nov. D. ecotolerans produced more biomass than Chlamydomonas reinhardtii CC-125 under both moderate- and low-light conditions. Under low light, its final dry biomass concentrations were 1.81 and 1.58 times those of CC-125 under mixotrophic and photoautotrophic conditions, respectively. Despite its smaller cell size, D. ecotolerans showed higher photosynthetic performance than CC-125, and screen-printed electrode (SPE)-based electrochemical assays demonstrated enhanced anodic current generation and CV-derived estimated power output. Taken together, these findings identify D. ecotolerans as a novel climate-resilient Desmodesmus species with bioelectrochemical properties that support further evaluation for BPV applications.

Cell fusion enables cell-based applications, yet systematic optimization remains constrained by low throughput (microfluidic devices) and manual operation (bulk protocols). Here, an automated microfluidic platform is presented for cell pairing and chemical fusion at a statistically relevant scale. High throughput is achieved by arranging ~350,000 deformability-based cell traps as concentric circular arrays in a radial chip. Using tumor and dendritic cells as a model for cancer vaccine, the device reproducibly co-occupies ~75% of traps (~250,000) of which ~140,000 hold exactly one cell of each type. This exceeds the yield of previously reported in hydrodynamic pairing arrays by more than an order of magnitude. A concentration step-up-and-down PEG profile yielded a fusion efficiency of ~20% (range 17.7-23.4%, n =3) at viability above 90% that results in producing at least ~50,000 hybrids per chip. This efficiency is comparable to that reported for PEG-based fusion in small-scale microfluidic devices but obtained at higher throughput and approximately four-fold higher than the conventional benchtop protocol. Fusion efficiencies were quantified by image analysis and validated by flow cytometry. By combining automation, large-area circular trap arrays and reagent control, the platform is useful for data-rich optimization of cell engineering processes previously constrained by limited microfluidic throughput.

Emerging efforts to engineer multicellular kidney tissues for drug discovery, disease modeling, and therapeutic repair require scalable generation of kidney organoids derived from human induced pluripotent stem cells (hiPSCs). Here, we report a method for generating kidney ureteric bud and collecting duct epithelial organoids in stirred tank bioreactors. We demonstrate that extracellular matrix microcarriers facilitate organoid expansion and are compatible with suspension bioreactor methods. The combination of cell-coated matrix microcarriers with stirred bioreactors promotes maturation marker expression compared to static controls comprising matrix-embedded organoids. Our method offers promise for generating epithelial organoids that serve as multicellular models and organ building blocks for scalable biofabrication.

In vitro-transcribed (IVT) messenger RNA (mRNA) therapeutics require rigorous quality control of 5' capping efficiency and 3' poly(A) tail length, both of which constitute critical quality attributes (CQAs). Current analytical workflows for these CQAs rely on hybridization with nucleic acid probes and biotin-based affinity purification, which constrain throughput and increase per-assay cost. Here, we report a rationally designed 5' untranslated region (5' UTR) that sustains high translational efficiency while enabling probe-free, one-step purification for rapid quantification of capping efficiency and poly(A) tail length distribution. This approach exploits a strategically engineered 5' UTR incorporating a purine-only RNase A-resistant fragment terminating at a single pyrimidine cleavage site, together with a translation-enhancing element comprising an N6-methyladenosine (m6A) deposition motif and an 18S rRNA complementarity sequence. This dual-function design accommodates GA-rich sequence segments without compromising mRNA translational output. Compared with conventional probe- and biotin-dependent workflows, the present method substantially reduces cost and processing time and is compatible with simultaneous detection of segmented AmGn poly(A) tail variants. This integrated platform provides a simplified, probe-free and construct-universal for high-throughput in-process control (IPC) of mRNA therapeutics.

Human pluripotent stem cells (hPSCs) are increasingly expanded in 3D suspension culture to support scalable cell culturing, yet how the transition from 2D to 3D cultures affects hPSCs remains poorly understood. Here, we compare hPSCs in 2D and 3D cultures using transcriptomics, proteomics, spent medium analysis, and stable-isotope tracing. hPSCs maintained pluripotency in both formats, although suspension culture showed altered growth dynamics and increased initial cell loss. Multi-omics analysis identified culture format as the dominant driver of molecular variation and revealed changes in adhesion, cytoskeletal organization, glycolysis, and oxidative phosphorylation. Functional analyses showed increased lactate production from glucose and stable-isotope tracing revealed increased glucose-derived pyruvate formation and altered partitioning of glucose-derived carbon within the TCA cycle in 3D suspension cultures. Glutamine contribution to the TCA cycle was reduced, whereas relative reductive carboxylation was increased in 3D suspension cultures. Together, these findings show that transition from 2D to 3D suspension culture induces metabolic rewiring in hPSCs.

Plate-based full-length single-cell RNA sequencing resolves transcript structure details but remains difficult to scale because each cell usually requires a separate library. Here we developed CHART-seq (Combinatorial Heteroduplex Assay via Recombinant Tn5), which uses orthogonally indexed Tn5 complexes to tagment RNA/cDNA heteroduplexes and permits early sample pooling. The workflow processed up to 96 cells per library, was compatible with 384-well expansion, and completed library preparation within 3 h at a reagent cost below US$1 per cell. At matched sequencing depth, CHART-seq detected more genes and annotated isoforms than Smart-seq2, Smart-seq3, Flash-seq and SHERRY2, while retaining broad genebody coverage and reproducible expression estimates. In the CHART-seq results of vascular smooth muscle cells, TGF-{beta}1 pretreatment before PDGF-BB exposure partly restored contractile features, suppressed a PDGF-associated inflammatory programme, and induced a distinct metabolic-matrix response with coordinated transcript-usage changes. These biological findings remain exploratory because independent biological replicates were unavailable. CHART-seq provides a rapid, scalable route to full-length single-cell transcript profiling with gene-programme and candidate isoform resolution.

Wastewater sequencing using probe-capture enrichment of public health-relevant viruses is a powerful, non-intrusive approach to monitor infectious diseases, but performance depends on sample type and processing approach. We compared two processing pipelines from wastewater influent samples collected at three timepoints across three wastewater treatment facilities. Each sample was split for parallel processing, with viruses concentrated (1) directly from whole influent using nano-sized affinity particles and (2) by settling and dewatering the solids fraction. Combined DNA/RNA extracts were prepared into libraries using a pre-commercial Illumina protocol, then probe-capture enrichment was performed with Illumina Viral Surveillance Panel (VSP) v1 (66 targets) and v2 (242 targets). Sequencing depth was increased for VSP v2 to accommodate the expanded target set. On average, 160 +/- 146 viral species were detected per sample, with wastewater fraction (sample type) explaining 19% of the variance in viral abundances. Solids contained a higher abundance of DNA viruses compared to RNA viruses, while influent exhibited the inverse. Metagenomic analysis of the VSP v2 sequencing data detected more viral species in influent (median 358) than in solids (median 184), but probe-capture enrichment produced more comparable target-virus readouts across fractions, supporting the use of either fraction for wastewater-based epidemiology.

Direct RNA nanopore sequencing offers the capacity to directly profile native RNAs, enabling detection of diverse epitranscriptomic modifications based on current changes as the RNA passes through a pore. Innovations in chemistry, hardware and bioinformatic tools have increased sequencing capacity and accuracy, creating demand for sequencing multiple samples within individual runs. Two recent solutions enable barcoding and demultiplexing of samples, but independent comparisons of their performance is limited. Here we benchmark WarpDemuX and SeqTagger using orthogonal ground truths derived from species identity and matched IVT spike-ins across four RNA004 flow cells. Both achieve high demultiplexing precision but differ in their trade-offs in precision and retention. SeqTagger provided strong performance at its recommended settings, whilst WarpDemuX performance varied by model, barcode and read quality. IVT-derived score cutoffs allowed dataset-specific calibration and increased WarpDeMux assignment yield, although precision achieved on IVTs was not consistently reproduced in species reads. These results support matched IVTs as internal controls for guiding choice of demultiplexing settings, while demonstrating the need to validate demultiplexing precision in biological RNA populations.