Jongyoon Han

Na-rich Prussian Blue (PB) is an attractive sodium-ion battery cathode owing to its high capacity and low cost, yet approximately 10 wt.% of crystal water in its framework induces electrolyte side reactions and Fe dissolution. Conventional thermal dehydration has been used to remove crystal water, but it consistently results in capacity fading and poor air stability. Here, the primary cause of degradation is experimentally demonstrated to be surface oxidation driven by Fe-O bond formation during heat treatment. Guided by this insight, a liquid-phase, low-temperature dehydration strategy based on nitrogen bubbling is introduced, which simultaneously eliminates crystal water and stabilizes the surface. The approach can be seamlessly integrated into electrode fabrication to minimize air exposure and improve cycling stability. Overall, this work clarifies the mechanistic role of surface oxidation in moisture-sensitive PB cathodes and highlights the critical importance of surface chemistry control in achieving stable electrochemical performance.

We present an efficient scheme to randomize a spin-state ensemble in a nonlinear spin-1 system by tuning chaos with an external periodic drive. Without modulation, the system exhibits a mixed phase space featuring regular islands embedded in a chaotic sea, where global mixing is inhibited by energy conservation. Using numerical simulations, we demonstrate that weak modulation of a linear Zeeman field not only facilitates transport between different energy shells but also drives ensembles toward a Haar-random distribution over spin states. Under optimized conditions, complete randomization is achieved on a timescale set by the inverse nonlinear interaction energy. In the overdriven regime, randomization is unexpectedly suppressed at specific modulation amplitudes, accompanied by the formation of sticky regions in phase space. We attribute this behavior to the dynamical cancellation of the leading low-order harmonic component of the periodic drive. These results illustrate how time-periodic driving can be used to engineer chaotic systems and achieve controllable randomization in nonlinear spin systems.

We present an efficient scheme to randomize a spin-state ensemble in a nonlinear spin-1 system by tuning chaos with an external periodic drive. Without modulation, the system exhibits a mixed phase space featuring regular islands embedded in a chaotic sea, where global mixing is inhibited by energy conservation. Using numerical simulations, we demonstrate that weak modulation of a linear Zeeman field not only facilitates transport between different energy shells but also drives ensembles toward a Haar-random distribution over spin states. Under optimized conditions, complete randomization is achieved on a timescale set by the inverse nonlinear interaction energy. In the overdriven regime, randomization is unexpectedly suppressed at specific modulation amplitudes, accompanied by the formation of sticky regions in phase space. We attribute this behavior to the dynamical cancellation of the leading low-order harmonic component of the periodic drive. These results illustrate how time-periodic driving can be used to engineer chaotic systems and achieve controllable randomization in nonlinear spin systems.

Thermophilic composting of sewage sludge under subzero temperatures remains a significant operational barrier to sustainable waste management. In this study, we report a full-scale composting process that treats 180 t of dewatered sewage sludge per day, achieving complete compost maturation within 10 days, even during freezing winter conditions. The system integrates a pre-fermentation reactor equipped with internal heating and aeration, enabling rapid thermophilic activation and maintaining aerobic stability despite ambient temperatures falling below −1.8 °C. Microbial community profiling revealed structured thermophilic succession even during the winter process, with Ureibacillus , Planifilum , and Mycolicibacterium enriched during peak activity. Short-chain fatty acid analysis indicated rapid acetate degradation during the reactor phase and a transient accumulation of butyrate following raw sludge remixing in winter, suggesting brief anaerobic microenvironments that were quickly resolved under resumed aerobic conditions. All major short-chain fatty acids were depleted by the maturation stage, reflecting a stable and sustained aerobic environment throughout the later composting phases. The final compost satisfied agronomic and safety criteria, including organic matter stabilisation, neutral pH, pathogen elimination, and heavy metal concentrations. These findings demonstrate the feasibility of year-round, thermophilic, and aerobically stable composting of dewatered sewage sludge at industrial scale. Furthermore, by minimizing anaerobic conditions during early decomposition, the system presents a strong potential for methane avoidance. We highlight its applicability as a scalable composting strategy for cold-climate regions, contributing to the development of sustainable sludge valorisation infrastructure within circular bioresource and climate-aligned waste management frameworks. • Industrial-scale composting system processed 180 tons/day of sewage sludge in 10 days, validated year-round. • Pre-fermentation reactor ensured thermophilic activation under subzero winter conditions. • Seasonal runs showed stable aerobic fermetation throughout composting. • Modeled carbon losses showed significant methane mitigation year-round. • Final composts met agronomic NPK and safety standards for soil application.

Regeneration can involve the coordination of pattern formation in an outgrowth with the spatial pattern of pre-existing tissues, such as along body axes. Planarian adult axis patterning serves as a robust context for uncovering the mechanisms of such pattern integration. We investigated how the dorsal-ventral boundary (DVB), which surrounds the animal periphery at the dorsal-ventral (DV) median plane, regulates anterior-posterior (AP) axis growth and patterning. We define a spatial DVB gene expression atlas that includes genes encoding signaling, adhesion, and transcription factors. Wnt inhibition results in anterior positional information induction and ectopic head formation that is restricted to the DVB. DVB can be transplanted, and DVB identity can be experimentally induced at ectopic locations. Ectopic DVB is competent for anterior positional identity induction following Wnt inhibition, enabling the generation of animals with ectopic heads at experimentally dictated locations. DVB removal blocks the anteriorization that normally follows Wnt inhibition and prevents anterior positional information expression during head regeneration. Anterior positional information induction at the DVB after Wnt inhibition occurs independently from anterior pole formation, which promotes head patterning in regeneration. Our findings reveal a hierarchical model of pattern integration across body axes in which DV patterning is central by producing a DVB with competence to direct formation of large AP axis regions. This mechanism enables coordination of orthogonal positional information in the context of regeneration.

Microbial Contaminations Live microbial contamination threatens cell and gene therapies. In article number 2500253, Springs, Yu, and co-workers introduce a rapid, sensitive method that integrates electrostatic microfiltration (EM)-based enrichment with digital loop-mediated isothermal amplification (dLAMP) for universal and strain-specific detection of live microbial contaminants down to 1 CFU/mL. This method also has broad applications in diagnostics, biomanufacturing, food safety, and environmental surveillance. Image credit: Betsy Skrip, MIT.

Abstract Acute myocardial infarction (AMI), a leading cause of death globally, triggers complex inflammatory responses critical to patient outcomes. However, rapid tools for profiling immune responses at the single‐cell level are lacking. The integrated Single‐cell Enzyme and Antigen Quantification (iSEAQ) system addresses this gap by enabling high‐throughput, single‐cell analysis of immune cell activity using just 20 μL of blood. This novel tool processes live CD66b and CD3 cells to quantify the secretion of Granzyme B, Neutrophil Elastase, and CD31 within minutes. Longitudinal studies on nine AMI patients revealed that CD66b + cells are major contributors (up to 95%) to key inflammatory enzymes, including the unexpected secretion of Granzyme B. iSEAQ achieves unparalleled sensitivity (0.4 fg/cell) and predictive accuracy (>90%) for patient profiling across AMI onset, treatment, and discharge. This innovation provides clinicians with a rapid, precise method to monitor immune responses, unveiling new insights into AMI inflammation and therapy.

Conventional iron determination methods, such as colorimetric assays, often lack sensitivity at low iron levels and are susceptible to interference from complex compositions in serum and plasma. In this study, we developed a rapid, sensitive, and accurate method for iron determination in 1 min utilizing an inexpensive benchtop 0.5-T micromagnetic resonance relaxometry (μMRR) system with acidification treatment of samples (pH < 1). The method yielded a highly linear calibration curve (R 2 > 0.999) between the transverse relaxation rate R 2 and iron concentration from 0.5 to 1000 μM, with a limit of detection (LOD) of 0.25 μM and a minimal assay volume of 5 μL. The accuracy of this method was validated by inductively coupled plasma mass spectrometry (ICP-MS) across a diverse range of biological samples. This μMRR-based approach offers a rapid and convenient alternative for serum and plasma iron measurements, which can substantially reduce clinical diagnostic time and support real-time iron monitoring for patients.

BACKGROUND: The zonal organization of articular cartilage is critical for the biphasic mechanical properties of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, compromising the functional efficacy of the repaired tissue, which could account for tissue failure in the long term. Autologous chondrocyte implantation (ACI) still suffers from inconsistent efficacy and a long recovery period stemming from implantation of a heterogeneous chondrocyte mixture. HYPOTHESIS: Stratified implantation of zonal chondrocytes would facilitate the recapitulation of articular cartilage zonal properties and improve the repair efficacy of ACI treatment. STUDY DESIGN: Controlled laboratory study. METHODS: Autologous chondrocytes extracted from porcine articular cartilage were subjected to dynamic microcarrier expansion followed by size-based segregation using a spiral microfluidic device for the enrichment of zonal chondrocytes. Zonal chondrocytes were implanted into a chondral defect as a bilayered hydrogel construct consisting of superficial zone chondrocytes overlaying middle/deep zone chondrocytes (n = 6). Twelve months after implantation, the repair efficacy was compared against implantation of full-thickness cartilage-derived heterogeneous chondrocytes expanded on tissue culture plates (n = 5) or microcarriers (n = 6). RESULTS: CT), compression modulus, and surface lubrication analysis, at 12 months demonstrated statistically significant improvement in cartilage and subchondral bone repair with zonal chondrocyte bilayered implantation. Magnetic resonance imaging (MRI) T2 mapping indicated progressive improvement in graft maturation as early as 3 months, reaching normalcy at 9 months. CONCLUSION: This study demonstrates that with appropriate expansion and isolation of zonal chondrocytes, stratified zonal chondrocyte implantation is able to facilitate restoration of articular cartilage zonal architecture and significantly enhance the functional repair as compared with current ACI treatment. CLINICAL RELEVANCE: With appropriate expansion and enrichment of zonal chondrocytes, stratified zonal chondrocyte implantation could represent a significant advancement over current ACI-based cartilage repair, with the potential to support quicker and better recovery.

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However, CAR T cell products exhibit batch-to-batch variability in cell number, quality, and in vivo efficacy due to donor-to-donor heterogeneity, and pre/post-manufacturing processes, and the manufacturing of such products necessitates careful testing, both post-manufacturing and pre-infusion. Here, we introduce the Cell Trajectory Modulation (CTM) assay, a microfluidic, label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.e., size, deformability). CTM assay correlates with phenotypic metrics, including CD4:CD8 ratio, memory subtypes, and cytotoxic activity. Validated across multiple donors and culture platforms, the CTM assay requires fewer than 10,000 cells and delivers results within 10 minutes. Compared to labeled flow cytometry processing, the CTM assay offers real-time data to guide adaptive manufacturing workflows. Thus, the CTM assay offers an improvement over existing phenotypic assessments, marking a step forward in advancing CAR T cell therapy manufacturing. CAR T cell manufacturing faces significant challenges that impact cell quality and in vivo efficacy. This necessitates reliable cellular characterization methods. Here the authors present a real-time, label-free, microfluidic method that profiles cellular biophysical properties and correlates them to activation state and CAR T potency, facilitating the rapid phenotypic cell assessment during production.

Nuclear magnetic resonance (NMR) measurements of moving samples are limited by the depletion of the sample during long experiments, making it difficult to apply NMR relaxometry techniques to moving fluid samples for various manufacturing and reaction monitoring applications. Traditional techniques for compensating for flow-induced artificial relaxation in postprocessing do not work for samples with unknown or variable flow rates, which is a significant barrier to the widespread adoption of NMR monitoring systems for existing processes. In this work, we present a compact NMR coil geometry that removes the effect of sample depletion during long experiments on moving samples, making it possible to perform flow-agnostic measurements on samples with variable or uncontrolled flow rates. We evaluate this coil geometry using relaxometry experiments on fast-flowing water samples and demonstrate its potential for process monitoring with real-time measurements of the dairy concentration.

assays used for cell line qualification. Most short-read sequencing assays, however, still require over a week to obtain a final test result since the sequencing must be completed before bioinformatic analysis can begin, which is still too long for some cell and gene therapy products that must be released as soon as possible to reach critically ill patients. Oxford Nanopore sequencing can address these issues, as it provides real-time basecalling and sequence alignment, which can reduce the overall assay time. Still, as with any sequencing platform, the abundance of background nucleic acid from the human or mammalian host can mask the signal from a low-level viral contaminant. To address this, we have developed a sensitive sample preparation workflow using concentration, nuclease treatment, and agnostic PCR methods to eliminate background signals and amplify viral contaminant reads, leading to a 3-log improvement in the limit of detection that is comparable to or better than short-read sequencing approaches. This approach will lead to more rapid and improved detection of viral contaminants in cell and gene therapy manufacturing.

BACKGROUND: There is a current, absence of reliable, blood-sparing, diagnostic tools to measure and trend real-time changes in the levels of inflammation and its effects on the immune cells in the infant. METHODS: We deployed the BiophysicaL Immune Profiling for Infants (BLIPI) system in the neonatal intensive care unit to describe immune cell biophysical profiles using 50 microliters of blood per sample from term and preterm infants. RESULTS: A total of 19 infants (8 term, 11 preterm) were recruited and 24 blood samples were collected in their first month. Based on the profiles of immune cells' size and deformation, there was a clear distinction between term and preterm infants, with 48/50 markers significantly different. A preterm infant with late-onset bacterial sepsis had notable size and deformability differences compared to the rest of the preterm cohort. There was a significant correlation between immune cell biophysical profiles and clinical markers such as C-reactive protein, white blood cell counts, and immature-to-total neutrophil (I:T) ratios, with Pearson correlation coefficients for linear regression models of 0.98, 0.97 and 0.94 respectively. CONCLUSION: This study highlights the potential for the biophysical immune cell profiling system to provide an overview of the infant's current immune activation and response. IMPACT: We present a novel, minimally invasive diagnostic system that leverages the physical properties of immune cells to provide a rapid and direct assessment of the immune status, requiring 20 times less blood volume than standard tests. This study demonstrates the potential of a compact, deployable system that is capable of performing biophysical profiling to assess immune cell activation in term and preterm infants, by revealing distinct differences in cell size and deformation between groups. The system's sensitive, quantitative measures were correlated with routine clinical biomarkers, highlighting its ability to provide a rapid, minimally invasive, real-time monitoring of neonatal immune status.

Live microbial contamination poses high risks to cell and gene therapies, threatening manufacturing processes and patient safety. Rapid, sensitive detection of live microbes in complex environments, such as CAR-T cell cultures, remains an urgent need. Here, an innovative sample-to-result workflow is introduced using digital loop-mediated isothermal amplification (dLAMP), enhanced by Electrostatic Microfiltration (EM)-based enrichment, for rapid sterility testing. By rationally designing primers targeting 16S and 18S rRNA, dLAMP assay enables both universal detection (covering >80% of known species) and strain-specific identification of bacterial and fungal contaminants in CAR-T cell spent medium and final products, directly from microorganism lysates. Enhanced by EM-based enrichment of low-abundance live microbes, the workflow achieves unparalleled sensitivity and speed, detecting contamination levels as low as 1 CFU/mL in complex CAR-T cell cultures within 6 h. Compared to qPCR and 14-day compendial methods, the approach demonstrates superior accuracy and significantly faster turnaround times. This workflow holds transformative potential for real-time monitoring in cell therapy manufacturing and rapid safety assessments of CAR-T cell products prior to patient infusion. Beyond cell therapy, the method is broadly applicable to infectious disease diagnostics, biomanufacturing monitoring, food safety, and environmental surveillance.

Monitoring immune cell function is increasingly being recognized as a more relevant biomarker than traditional white blood cell counts, yet the need for repeated relatively large blood volumes still continues to pose a significant challenge. To overcome this, we developed a sample-sparing platform using inertial microfluidics that can process as little as 10 μL of blood to isolate leukocytes for downstream functional analysis. Our platform isolates leukocytes with ∼80% purity, >90% in-device recovery, and >95% viability. Neutrophils were our primary focus due to their sensitivity to external stimuli and their critical role in immune responses. Neutrophils isolated through our new method did not show inadvertent activation, as evidenced by unchanged expression of activation markers CD62L and CD11b, with phenotypes comparable to control cells in whole blood. We conducted a range of functional assays, including phagocytosis, ROS production, and NETosis with all tests confirming that neutrophils maintained their functionality after isolation. These assays were performed using standard laboratory workflows, demonstrating the platform's compatibility with techniques such as flow cytometry and cell culture assays. Furthermore, we showed the versatility of our platform by successfully isolating leukocytes from challenging samples, including mouse blood from the vena cava or tail vein, as well as human capillary blood obtained by fingerstick. This adaptability highlights the potential of this platform for clinical and research applications, particularly in frequent immune monitoring or cases where sample volume is limited.

Understanding the biophysical properties of cells is essential for biological research, diagnostics, and therapeutics. Microfluidics enhances biophysical cell characterization by enabling precise manipulation and real-time measurement at the microscale. However, the high-throughput nature of microfluidic systems generates vast amounts of data, complicating analysis. Integrating artificial intelligence (AI) methods, including machine learning and deep learning, with microfluidic technologies addresses these challenges. AI excels at analyzing large, complex datasets, improving the accuracy and efficiency of microfluidic experiments and facilitating new biological discoveries. This review examines the synergy between microfluidics and machine learning for biophysical cell characterization, categorizing existing methods based on the types of input data used for machine learning analysis, highlighting recent advancements, and discussing challenges and future directions in this interdisciplinary field.

The emergence of induced pluripotent stem cells (iPSCs) offers a promising approach for replacing damaged neurons and glial cells, particularly in spinal cord injuries (SCI). Despite its merits, iPSC differentiation into spinal cord progenitor cells (SCPCs) is variable, necessitating reliable assessment of differentiation and validation of cell quality and safety. Phenotyping is often performed via label-based methods including immunofluorescent staining or flow cytometry analysis. These approaches are often expensive, laborious, time-consuming, destructive, and severely limits their use in large scale cell therapy manufacturing settings. On the other hand, cellular biophysical properties have demonstrated a strong correlation to cell state, quality and functionality and can be measured with ingenious label-free technologies in a rapid and non-destructive manner. In this study, we report the use of Magnetic Resonance Relaxometry (MRR), a rapid and label-free method that indicates iron levels based on its readout (T 2 ). Briefly, we differentiated human iPSCs into SCPCs and compared key iPSC and SCPC cellular markers to their intracellular iron content (Fe 3+ ) at different stages of the differentiation process. With MRR, we found that intracellular iron of iPSCs and SCPCs were distinctively different allowing us to accurately reflect varying levels of residual undifferentiated iPSCs ( i.e. , OCT4 + cells) in any given population of SCPCs. MRR was also able to predict Day 10 SCPC OCT4 levels from Day 1 undifferentiated iPSC T 2 values and identified poorly differentiated SCPCs with lower T 2 , indicative of lower neural progenitor (SOX1) and stem cell (Nestin) marker expression levels. Lastly, MRR was able to provide predictive indications for the extent of differentiation to Day 28 spinal cord motor neurons (ISL-1/SMI-32) based on the T 2 values of Day 10 SCPCs. MRR measurements of iPSCs and SCPCs has clearly indicated its capabilities to identify and quantify key phenotypes of iPSCs and SCPCs for end-point validation of safety and quality parameters. Thus, our technology provides a rapid label-free method to determine critical quality attributes in iPSC-derived progenies and is ideally suited as a quality control tool in cell therapy manufacturing.

Frequent, low-latency measurements of bioreactor culture growth are critical for achieving maximum culture efficiency and productivity. Typical cell density and viability measurements are made by manually removing a sample from the culture, but this approach is both slow and unsuitable for small culture volumes, which cannot support frequent destructive sampling. In this work, automated magnetic resonance relaxometry measurements of a sealed bioreactor system are used to estimate the cell density and provide qualitative information about the culture in near real-time. The system detects variations in cell density in minutes, enabling rapid intervention that would be impossible with the once-daily measurements taken by a traditional sampling-based culture analysis system.

Motivation: Bioreactor cell density measurements currently require removing samples from the culture. This limits how many samples can be taken in a day and precludes real-time culture monitoring. Goal(s): We sought to take nondestructive cell density measurements every ten minutes without removing cells from the bioreactor. Approach: We built a system that takes T2 relaxometry measurements through the bioreactor&amp;rsquo;s silicone tubing every ten minutes and compared these to measurements taken by a commercial cytometry system. Results: The T2 relaxation data closely tracks the true cell density. Changes made to the culture are detected much more quickly by the relaxometer. Impact: Magnetic resonance relaxometry can be used to track bioreactor culture growth in real time, and makes it possible to identify and correct problems with the culture before the culture fails.

In modern bioprocessing, cell culture media is one of the most significant cost drivers, yet the nutrients and other critical factors in the media are often not fully utilized. With the renewed emphasis on reducing the cost of bioprocessing, there is much interest in reducing the overall use of cell culture media. In this work, we introduce a mesoscale microfluidic separation device based on the ion concentration polarization (ICP) process to regenerate the spent media for reuse by removing critical waste products from the cell culture that are known to inhibit the growth of the cells. We demonstrated that up to 75% of spent culture media can be regenerated and reused without affecting the cell viability. A detailed analysis of the materials consumed during antibody production indicated that one could improve the water process mass intensity by up to 33% by regenerating and recycling the media. Given that ICP separation systems have already been scaled up to support large-volume processing, it would be feasible to deploy this technology for manufacturing scale bioreactors (e.g., 50 L perfusion culture of CHO cells), reducing the overall operation cost and water use.

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.

Abstract Disclosure: A.T. Alves: ; Co-Founder. ; Self. ; LarmorBio. S.S. Thamarath: ; Co-Founder. ; Self. ; LarmorBio. S. Fjordside: Employee; Self; Novo Nordisk. S. Sassower, MS.EE: ; Co-Founder. ; Self. ; LarmorBio. J. Han: None. L. Bouchard: Advisory Board Member; Self; LarmorBio. R. Rohr, BS.EE: ; Co-Founder, CEO. ; Self. ; LarmorBio. A.P. Chambers: Employee; Self; Novo Nordisk. The declining age of onset of T2D is a vital factor influencing its prospective burden. An early manifestation of the disease leads to an extended duration, which amplifies and accelerates the risk of micro and macrovascular complications. Oxidative stress is a major driver in the pathogenesis of T2D. Early detection and prevention of oxidative stress overload can improve and potentially modify the long-term harmful outcomes of metabolic diseases, including chronic hyperglycaemia.A novel µNMR assay was developed to monitor oxidative stress from minimally invasive plasma samples at the point of care. This approach is highly sensitive to redox changes in the blood microenvironment, including increased ferric iron (Fe3+), protein oxidation, and lipid peroxidation[1]. We hypothesized that oxidative stress would increase significantly faster in the disease group, enabling disease progression monitoring throughout the study and near real-time subject risk stratification based on the core pathophysiology of the diabetic phenotype.This 15-week longitudinal study (6th - 21st week of age) quantified oxidative stress in db/db and db/+ mice (n = 15 per group), accompanied by HbA1c, blood glucose (BG), and body weight (BW). The assay detailed separation (P ≤ 0.01) between the db/db and db/+ mice from the onset of the diabetic phenotype of db/db at 10 weeks of age (mean HbA1c = 6.5%). The oxidative stress monitored continued to increase in the subsequent weeks, where the HbA1c of db/db mice reached maximum (mean HbA1c = 8.8%). The definition of good (HbA1c ≤ 8) and poor (HbA1c &amp;gt; 8) glycaemic control was defined where sub-stratification (P ≤ 0.05) was achieved. Furthermore, the assay highlights a positive correlation with BW (ρ+/db = 0.52, P ≤ 0.0001, ρdb/db = 0.57, P ≤ 0.0001). In the two groups of mice, a positive correlation of oxidative stress with HbA1c (ρ = 0.58, P ≤ 0.0001) and BG (ρ = 0.47, P ≤ 0.0001) was reported. During the 15-week study, the control db/+ group exhibited a 13% mean increase in oxidative stress. In contrast, the db/db group demonstrated a cumulative 20% increase. Notably, considering the diabetic phenotype of the db/db mice, we quantified oxidative stress attributed to aging at 13%.In view of changing diabetes demographics, a multimodal approach of targeted sub-phenotype treatment combined with advanced metabolic monitoring may provide holistic, clinically relevant information to reduce the micro and macrovascular burden of diabetes. In summary, the µNMR assay is an effective method for real-time monitoring of oxidative stress assessment in metabolic disease research and opens new possibilities for cardiometabolic risk assessment and targeted interventions.(1) Peng, W.K. et.al., npj Aging Mech Dis 2020, 6, 11 Presentation: 6/1/2024

Abstract Background Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy. Methods In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T 2 ) to characterize the effects of AA on improved MSC expansion and chondrogenic potential. Results Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a &gt; 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T 2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair. Conclusions Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion.

Research on machine learning and neural network classification models for detecting leaks or anomalies in water distribution pipelines has been actively conducted. Detecting leaks using “leakage noise” transmitted to the road surface is traditionally one of the most commonly used methods by human inspectors. However, the characteristics of these road surface leakage noises vary significantly over time across various environmental conditions, posing a challenge in finding a model that maintains consistent classification performance despite these variations. This study focuses on the premise that data preprocessing methods have a greater impact on improving classification performance than model selection and hyperparameter tuning. To effectively extract features from highly variable leakage noise, Fourier transform and Mel-frequency cepstral coefficients were used. Considering the possibility of redundant information, a tree-based model, which is less sensitive to multicollinearity, was employed to evaluate the classification performance of the leakage noise. Through this approach, we aim to propose a data preprocessing method that provides stable classification performance despite the variability in leakage noise, thereby contributing to the development of robust machine learning models.

Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time-consuming and labor-intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash-free, and target-responsive protein detection for single-cell IFN-γ cytokine secretion analysis within 30 min at room temperature is presented. By incorporating a DNA proximity assay (DPA) into a multifunctional microfluidic system, one-pot homogenous cytokine signal amplification, with a limit of detection of ≈50 secreted molecules per cell is achieved. iSECRETE can robustly handle various sample types that are shown. Two distinct immune activation assay modalities are demonstrated on iSECRETE. Finally, the detection of single-cell IFN-γ secretion as an activation hallmark of chimeric antigen receptor T cells within 6 h of exposure to cancer targets is shown. iSECRETE represents the fastest single-cell sample-to-result cytokine secretion assay to date, providing a powerful tool for advancing the understanding of biological phenotypes, functions, and pathways under in vivo-like conditions.

Abstract Regenerative therapies, including the transplantation of spinal cord progenitor cells (SCPCs) derived from induced pluripotent stem cells (iPSCs), are promising treatment strategies for spinal cord injuries. However, the risk of tumorigenicity from residual iPSCs advocates an unmet need for rapid SCPCs safety profiling. Herein, a rapid (≈3000 cells min ‐1 ) electrical‐based microfluidic biophysical cytometer is reported to detect low‐abundance iPSCs from SCPCs at single‐cell resolution. Based on multifrequency impedance measurements (0.3 to 12 MHz), biophysical features including cell size, deformability, membrane, and nucleus dielectric properties are simultaneously quantified as a cell is hydrodynamically stretched at a cross junction under continuous flow. A supervised uniform manifold approximation and projection (UMAP) model is further developed for impedance‐based quantification of undifferentiated iPSCs with high sensitivity (≈1% spiked iPSCs) and shows good correlations with SCPCs differentiation outcomes using two iPSC lines. Cell membrane opacity (day 1) is also identified as a novel early intrinsic predictive biomarker that exhibits a strong correlation with SCPC differentiation efficiency (day 10). Overall, it is envisioned that this label‐free and optic‐free platform technology can be further developed as a versatile cost‐effective process analytical tool to monitor or assess stem cell quality and safety in regenerative medicine.

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process, we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study, we used a sized-based, label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie, >3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the "downstream processing" of cell manufacturing workflow, ensuring better quality and safety of transplanted cells.

BACKGROUND: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing. HYPOTHESIS: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair. STUDY DESIGN: Controlled laboratory study. METHODS: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro-computed tomography, and mechanical analysis. RESULTS: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro-computed tomography of the subchondral bone. CONCLUSION: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs. CLINICAL RELEVANCE: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes.

, 10 CFU in 500 mL). Furthermore, EM-enabled sample preparation facilitates digital amplification techniques sensitively detecting broad pathogens, including bacteria, fungi, and viruses from various samples, in a rapid (≤3 h) sample-to-result workflow. Notably, the operational ease, portability, and compatibility/integrability with various downstream detection platforms highlight its great potential for widespread applications across diverse settings.

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.