HardwareX

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.

Altair-dvOPM is an open-access direct-view oblique plane microscope designed for large-field, three-dimensional imaging of cleared and expanded tissue sections. By combining photographic-lens-based detection, externally launched oblique illumination and precision-registered modular baseplates, the system achieves micrometer-scale lateral resolution over a ~5.4 mm field of view without custom objectives or highly specialized alignment procedures. We demonstrate imaging across scales, from subcellular structures in expanded cells to centimeter-scale expanded tissue sections, and provide documentation, CAD files, Zemax models and open-source control software to support replication and extension.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

Understanding plant growth dynamics requires imaging across day-and-night cycles to quantify growth, movement and development in the aerial plant body and to capture the rhythmic nature of these processes. This requires imaging in light during the day and in darkness at night without perturbing plant physiology. Nighttime imaging has typically depended on infrared (IR) illumination, producing monochrome datasets that require specialised hardware and separate analysis pipelines when combined with daytime RGB imaging. Here, we evaluated very low-intensity green (dimG) illumination from standard LEDs as a practical alternative for colour-consistent nighttime imaging and assessed its physiological impact in Arabidopsis thaliana and Lactuca sativa (lettuce). We show that high resolution colour images can be obtained under dimG using low- cost cameras, with sufficient consistency between full-spectrum and dimG images to allow direct comparison and unified image analysis. We show that very low-fluence green light (<0.5 mol m-2 s-1) does not sustain circadian oscillations of gene activity under continuous exposure and does not perturb rhythms when applied during the dark phase of diel cycles. DimG imaging enabled accurate detection of diel leaf movement profiles in Arabidopsis circadian mutants, revealing genotype-specific phase differences under varying photoperiods. In lettuce, dimG pulses and continuous dimG enabled accurate quantification of diel leaf movement without affecting growth, stomatal opening, electron transport rate or chlorophyll content. Motion profiles under continuous dimG mirrored those under darkness. Our findings establish dim green illumination as a cost-effective solution for night-time imaging, simplifying phenotyping workflows with minimal impact on physiology.

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.

Visual prostheses face a critical miniaturisation challenge: converting photoreceptor signals to biologically appropriate retinal ganglion cell (RGC) stimulation patterns within the spatial constraints of intraocular implants. Existing systems rely on external microcontrollers for signal processing, limiting scalability for high-density pixel arrays. This paper presents an integrated per-pixel circuit architecture that directly converts photocurrent into frequency-modulated current pulses that match RGC activation thresholds. The design targets are established through NEURON computational modelling of red-green colour-opponent midget RGCs, identifying stimulation thresholds of +0.1nA to +3.5nA for depolarisation and -0.1nA for repolarisation. The proposed circuit combines a transimpedance amplifier, a voltage-controlled oscillator with a Schmitt trigger, and a current-controlled output stage to generate biphasic pulses within these thresholds. A complementary output provides lateral inhibition, reducing crosstalk between adjacent RGC stimulation sites. Photoreceptor integration is achieved using P3HT:PCBM organic photodiodes for cone-associated RGCs and phototransistors for rod-associated RGCs, validated through OghmaNano finite element simulations. The photodiode circuit produces output frequencies of 2.5Hz (dark) to 600Hz (100 W/m2), matching reported RGC response ranges. This architecture eliminates external processing requirements, enabling scalable high-density retinal prostheses design.

Data-driven materials development requires large, well-characterized libraries of precisely defined formulations. While microfluidic platforms excel at generating highly controlled materials, their throughput is often limited by the challenge of efficiently interfacing device outputs with standard well plates. This bottleneck frequently necessitates manual transfer or non-microfluidic workflows, constraining both throughput and reproducibility. Here, we present LMNOP-bot (Libraries of Micro- and Nano-materials, OPen-source bot), an open-source robotic platform for the automated generation and collection of micro- and nanomaterial libraries from serial microfluidic outputs. Using synchronized, pressure-driven flow, LMNOP-bot enables continuous formulation and direct deposition into standard well plates. The system is low-cost (<$700, excluding pressure regulators), constructed from readily available or easily fabricated components, and designed for broad accessibility. LMNOP-bot collects [&ge;]30 {micro}L per formulation at a rate of one sample every four seconds, representing an approximately 50x increase in throughput over existing serial microfluidic workflows, and operates robustly for over 10,000 runs without maintenance. We demonstrate compatibility with both PDMS/glass and commercial polycarbonate devices, with seamless interfacing to 96- and 384-well plates. Repeated sampling confirms high precision and reproducibility. By removing a key bottleneck in microfluidic library generation, LMNOP-bot enables rapid, scalable, and accessible exploration of material design spaces.

Long-term, automated tracking of group-housed social animals using RFID (radio frequency identification) is a promising approach in ethological neuroscience. However, low-frequency (LF) RFID, while long-established in the field, is constrained by its inherent low data rates, which lead to two critical limitations: (1) compromised spatiotemporal resolution, and (2) the inability to identify multiple tags (animals) simultaneously. To address these limitations, we developed eeeHive, a high-frequency (HF) RFID-based animal tracking system with a fully custom hardware architecture that enables high-speed, multiplexed antenna polling and concurrent multi-tag reading. The polling time per antenna in eeeHive was 5.9 ms, with an additional 8.2 ms read time per tag. We applied the system to track 24 mice for one week, and six common marmosets for seven weeks. The system successfully tracked individuals even within dense clusters, revealing complex behavioral traits characterized by spatial utilization, temporal dynamics, behavioral regularity, and inter-individual relationships. Additional tests with Japanese fire-bellied newts and Nile tilapia juveniles demonstrated comparable tracking performance in aquatic environments. Taken together, eeeHive overcomes the inherent limitations of conventional LF RFID, establishing a powerful HF RFID-based platform for fine-scale behavioral tracking of group-housed animals across terrestrial and aquatic species.

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

Chlamydomonas reinhardtii is a model green alga extensively used to study photosynthesis and cilia using molecular biology and genetics. Electroporation is a very common technique to transform DNA into the nuclear genome, which is essential to generate mutant collections and express transgenes. Here, we describe a simple, fast, and efficient protocol to transform strains with an intact cell wall. It achieves a good transformation efficiency without cell wall digestion or use of commercial kits and is compatible with the widely available Gene Pulser electroporation system. Key featuresO_LIHigh transformation efficiency of Chlamydomonas reinhardtii strains with an intact cell wall. C_LIO_LIFaster than currently available electroporation protocols. C_LI

Species-level Candida identification can inform antifungal management, but reliable identification platforms remain inaccessible in many clinical microbiology laboratories, whereas phase contrast microscopy -- a common feature of routine laboratory microscopes -- is widely available. We asked whether this ubiquitous optical tool, combined with a consumer smartphone and deep transfer learning, could provide a feasible low-cost approach for preliminary Candida species discrimination. Fifteen clinical isolates of four species (C. albicans, C. glabrata, C. tropicalis, C. krusei) were collected from a single clinical microbiology laboratory and imaged using a consumer-grade smartphone coupled to a standard phase contrast microscope. Suspensions in human serum were imaged immediately after preparation (T0) and after 2-hour incubation at 37{degrees}C (T2). Pretrained vision backbone architectures were evaluated as fixed feature extractors under strict Leave-One-Strain-Out cross-validation. The best-performing model -- EfficientNet-B0 embeddings with a Linear Support Vector Machine applied to T2 images -- achieved an apparent internally cross-validated strain-level balanced accuracy of 0.833 and an overall strain accuracy of 86.7% (13/15 strains correctly classified). C. albicans, C. glabrata, and C. tropicalis were each identified with 100% recall. Both misclassified strains belonged to C. krusei -- the species with the smallest panel representation (n=3 strains) -- with misclassification attributable to limited strain diversity and suboptimal image quality. These findings demonstrate promising feasibility for preliminary image-based Candida species discrimination from smartphone-acquired phase contrast microscopy images, and support further evaluation in larger, externally validated strain collections.

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1

High-resolution microscopy techniques are used across research and industry to analyse biological systems, from biomolecules to subcellular organelles, multicellular models and tissues. As multimodal imaging workflows and quantitative analysis of bioimaging data become increasingly widespread, there is a growing need for materials and methods to calibrate imaging systems and evaluate the fidelity of generated image data. Here, we present three-dimensional microscopy phantoms fabricated using two-photon photolithography from transparent resins that exhibit both broadband visible autofluorescence and Raman scattering across the fingerprint and C-H stretching regions. Suitable for analysis using optical profilometry, the phantoms were dimensionally calibrated with SI traceability using a metrological confocal microscope. Immersible in air and common aqueous imaging media, the phantoms are compatible with a wide variety of optical microscopy techniques, including one and two-photon excited fluorescence and coherent Raman scattering microscopy. We employed a forked wedge design to validate image deconvolution results and a stacked lattice phantom to recover image distortion matrices under realistic biological imaging conditions. We demonstrate the impact of correcting chromatic offsets and axial scaling errors for a representative application: analysis of a cell seeded scaffold using confocal laser scanning fluorescence microscopy. These phantoms provide a versatile platform for calibration, quality control and validation of multimodal imaging pipelines and improved quantitative optical microscopy.

For decades, the photon counting histogram (PCH) was used as the sole method to quantify fluorophore numbers in a diffraction-limited focal volume. This technique combines spatial excitation profiles, and the distribution of photon counts to register the photon emission statistics of individual fluorophores. However, this approach has not yet been transferred to widefield fluorescent imaging due to the lack of fast and single photon sensitive camera sensors which can capture the photon emission statistics of a single fluorophore. Here, we explore avenues towards quantitative analysis of the active fluorophore number by leveraging recent advancements in single photon avalanche diode (SPAD) array technology. Binary exposures of a SPAD array can be synchronized with picosecond laser pulses to measure the PCH in a widefield setting. Then, by modeling the statistical relationship between the active fluorophore number and the PCH in a region of interest following a laser pulse, we can perform Bayesian inference of this number. The model is demonstrated experimentally by counting quantum dots and various numbers of fluorescent dye molecules bound to DNA origamis. We find that this method has several important applications in widefield microscopy, including enhanced localization microscopy and constrained fitting of multiple unresolvable fluorescent emitters.

The patch-clamp experimental technique is widely used to study the electrical properties of ion channels in biological and artificial lipid membranes. The key to the high quality of the experiments is the manufacturing of glass pipettes that provide highly electrically resistant contact between the edge of the pipette tip and the lipid bilayer. Preparation of the pipettes is particularly challenging for studies of the mitochondrial membranes due to the need for very small pipette tip sizes. Here, we present a robust procedure for producing pipettes suitable for experiments with native mitochondrial membranes. This procedure involves a two-step approach: initial fabrication of relatively large glass micropipettes using a standard micropipette puller, followed by tip refinement using a microforger to achieve smooth glass surface and reduced opening size. Pipette tip diameters and surface structure were examined using field emission - scanning electron microscopy (FE-SEM) imaging to assess the effects of variable parameters on pipette geometry and size. The resulting pipettes were validated in patch-clamp recording of the mitochondrial inner membranes. This approach enables the reproducible production of optimized pipettes for mitochondrial patch-clamp experiments, improving the quality and throughput of electrophysiological recordings of the mitochondrial ion channels.

Tracking gastrointestinal (GI) transit in preclinical models is essential for assessing gut motility and drug delivery. Current preclinical methods rely on end-to-end transit measurements or emptying studies that require terminal endpoints and organ explanation. Clinically, radiopaque "Sitz" markers are administered orally and their position in the GI tract is assessed through radiography. Sitz markers have been in use since 1969 and are typically mass-produced using industrial molding or extrusion, resulting in a single, fixed geometry with limited tunability. We present a stereolithography (SLA)-based method to fabricate customizable radiopaque markers using additive manufacturing with a barium sulfate (BaSO4)-doped resin. We demonstrate precise control over marker geometry, a key advantage over existing markers. Furthermore, we apply this method in vivo, tracking markers in a live rat model from ingestion to excretion using serial CT imaging. We systematically investigate how changes in marker geometry impact GI residency and transit time. Our results show that 3D printed markers provide a flexible and tunable platform for radiopaque marker fabrication and enable investigation of the fundamental relationship between a markers physical properties and its performance in a dynamic biological environment. This work establishes a novel, tunable platform for GI motility evaluation and drug delivery studies.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein mass range (17-800 kDa). We analysed holo-myoglobin (17 kDa) over a range of concentrations (10 M-10 nM) and capillary voltages to determine limits of detection and define a gentle operating regime. We additionally observe reduced Na+ adduction and preservation of the Zn2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin ([~]44 kDa), malate dehydrogenase ([~]70 kDa), glutamate dehydrogenase ([~]350 kDa), {beta}-galactosidase ([~]465 kDa), and GroEL ([~]800 kDa), were readily detected using nanopipette emitters. Compared with conventional 1-2 m internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of clarified bacterial lysate expressing -synuclein yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

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

Human milk contains structurally diverse glycans with key roles in shaping infant development, yet analytical constraints limit characterisation from low-volume samples. Glycosaminoglycans (GAGs), including chondroitin sulphate (CS), are understudied due to existing protocols requiring sample volumes of at least 5 mL and lengthy extraction steps prior to instrumental analysis. This study establishes a workflow for quantifying CS disaccharides from 25 {micro}L of human milk, enabling analysis of samples previously inaccessible to GAG profiling, such as those collected as salvage samples from neonatal intensive care units. For CS quantification, the CS is first enzymatically depolymerised using chondroitinase ABC to release repeating disaccharide units. Matrix complexity is reduced via two rounds of acetonitrile-based protein and lipid precipitation. Disaccharides are separated by hydrophilic interaction liquid chromatography and detected using a Triple Quadrupole Mass Spectrometer, providing robust sensitivity for all CS disaccharides. Method development and validation were performed using pooled mature human milk from term infants. This workflow facilitates detection of all CS disaccharides, with low but reproducible recoveries for total CS. Low- and high-level spike recoveries were 41.3% (RSDr 7.5%, RSDiR 15.9%) and 43.7% (RSDr 24.4%, RSDiR 27.9%), respectively. Despite modest absolute accuracy, precision remained sufficient to make relative comparison of CS concentrations between samples. This method expands the analytical toolkit for human milk glycomics, enabling same day preparation and CS profiling from sample volumes that are 200 times smaller than prior work, supporting future investigations into GAG-mediated functions in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723732v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@176dffborg.highwire.dtl.DTLVardef@16ae4ccorg.highwire.dtl.DTLVardef@d333c2org.highwire.dtl.DTLVardef@1eb3216_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Schematic of sample preparation protocol 25 L of human milk is combined with lyase enzymes and TRIS buffer containing the internal standard prior to incubation. Samples then undergo multiple rounds of centrifugation and refrigeration before analysis via LC-MS/MS. Made using BioRender.com. Glycan nomenclature following Varki et al., 2015. C_FIG