Label-Free and High-Throughput Removal of Residual Undifferentiated Cells From iPSC-Derived Spinal Cord Progenitor Cells.

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.

Size-Based Microfluidic-Enriched Mesenchymal Stem Cell Subpopulations Enhance Articular Cartilage Repair.

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.

Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array.

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.

Recyclable Superhydrophobic Surface Prepared via Electrospinning and Electrospraying Using Waste Polyethylene Terephthalate for Self-Cleaning Applications.

Superhydrophobic surfaces, i.e., surfaces with a water contact angle (WCA) ≥ 150°, have gained much attention as they are multifunctional surfaces with features such as self-cleaning, which can be useful in various applications such as those requiring waterproof and/or protective films. In this study, we prepared a solution from recycled polyethylene terephthalate (PET) and fabricated a superhydrophobic surface using electrospinning and electrospraying processes. We observed that the fabricated geometry varies depending on the solution conditions, and based on this, we fabricated a hierarchical structure. From the results, the optimized structure exhibited a very high WCA (>156.6°). Additionally, our investigation into the self-cleaning functionality and solar panel efficiency of the fabricated surface revealed promising prospects for the production of superhydrophobic surfaces utilizing recycled PET, with potential applications as protective films for solar panels. Consequently, this research contributes significantly to the advancement of environmentally friendly processes and the progress of recycling technology.

Design of 3D Controller Using Nanocracking Structure-Based Stretchable Strain Sensor.

In this study, we introduce a novel design for a three-dimensional (3D) controller, which incorporates the omni-purpose stretchable strain sensor (OPSS sensor). This sensor exhibits both remarkable sensitivity, with a gauge factor of approximately 30, and an extensive working range, accommodating strain up to 150%, thereby enabling accurate 3D motion sensing. The 3D controller is structured such that its triaxial motion can be discerned independently along the X, Y, and Z axes by quantifying the deformation of the controller through multiple OPSS sensors affixed to its surface. To ensure precise and real-time 3D motion sensing, a machine learning-based data analysis technique was implemented for the effective interpretation of the multiple sensor signals. The outcomes reveal that the resistance-based sensors successfully and accurately track the 3D controller's motion. We believe that this innovative design holds the potential to augment the performance of 3D motion sensing devices across a diverse range of applications, encompassing gaming, virtual reality, and robotics.

Correction: Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics.

Correction for 'Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics' by Hyungkook Jeon et al., Lab Chip, 2020, 20, 3612-3624, https://doi.org/10.1039/D0LC00675K.

Rapid and Label-Free Classification of Blood Leukocytes for Immune State Monitoring.

A fully automated and label-free sample-to-answer white blood cell (WBC) cytometry platform for rapid immune state monitoring is demonstrated. The platform integrates (1) a WBC separation process using the multidimensional double spiral (MDDS) device and (2) an imaging process where images of the separated WBCs are captured and analyzed. Using the deep-learning-based image processing technique, we analyzed the captured bright-field images to classify the WBCs into their subtypes. Furthermore, in addition to cell classification, we can detect activation-induced morphological changes in WBCs for functional immune assessment, which could allow the early detection of various diseases. The integrated platform operates in a rapid (<30 min), fully automated, and label-free manner. The platform could provide a promising solution to future point-of-care WBC diagnostics applications.

Multi-dimensional-double-spiral (MDDS) inertial microfluidic platform for sperm isolation directly from the raw semen sample.

Here, we propose a fully-automated platform using a spiral inertial microfluidic device for standardized semen preparation that can process patient-derived semen samples with diverse fluidic conditions without any pre-washing steps. We utilized the multi-dimensional double spiral (MDDS) device to effectively isolate sperm cells from other non-sperm seminal cells (e.g., leukocytes) in the semen sample. The recirculation platform was employed to minimize sample dependency and achieve highly purified and concentrated (up to tenfold) sperm cells in a rapid and fully-automated manner (~ 10 min processing time for 50 mL of diluted semen sample). The clinical (raw) semen samples obtained from healthy donors were directly used without any pre-washing step to evaluate the developed separation platform, which showed excellent performance with ~ 80% of sperm cell recovery, and > 99.95% and > 98% removal of 10-µm beads (a surrogate for leukocytes) from low-viscosity and high-viscosity semen samples, respectively. We expect that the novel platform will be an efficient and automated tool to achieve purified sperm cells directly from raw semen samples for assisted reproductive technologies (ARTs) as an alternative to density centrifugation or swim-up methods, which often suffer from the low recovery of sperm cells and labor-intensive steps.

Engineering a deformation-free plastic spiral inertial microfluidic system for CHO cell clarification in biomanufacturing.

Inertial microfluidics has enabled many impactful high throughput applications. However, devices fabricated in soft elastomer (i.e., polydimethylsiloxane (PDMS)) suffer reliability issues due to significant deformation generated by the high pressure and flow rates in inertial microfluidics. In this paper, we demonstrated deformation-free and mass-producible plastic spiral inertial microfluidic devices for high-throughput cell separation applications. The design of deformable PDMS spiral devices was translated to their plastic version by compensating for the channel deformation in the PDMS devices, analyzed by numerical simulation and confocal imaging methods. The developed plastic spiral devices showed similar performance to their original PDMS devices for blood separation and Chinese hamster ovary (CHO) cell retention. Furthermore, using a multiplexed plastic spiral unit containing 100 spirals, we successfully demonstrated ultra-high-throughput cell clarification (at a processing rate of 1 L min-1) with a high cell-clarification efficiency of ∼99% (at the cell density changing from ∼2 to ∼10 × 106 cells mL-1). Benefitting from the continuous and clogging-free separation with an industry-level throughput, the cell clarification device could be a critical breakthrough for the production of therapeutic biologics such as antibodies or vaccines, impacting biomanufacturing in general.

Correction: Huang et al. Deep-Learning Based Label-Free Classification of Activated and Inactivated Neutrophils for Rapid Immune State Monitoring. Sensors 2021, 21, 512.

The authors wish to make the following correction to their paper [...].

Three-Axis Tension-Measuring Vitreoretinal Forceps Using Strain Sensor for Corneal Surgery.

Precise motion control is important in robotic surgery, especially corneal surgery. This paper develops a new tension-measurement system for forceps used in corneal surgery, wherein contact force is applied only to a specific location for precise control, with precise movements detected by attaching a nano-crack sensor to the corresponding part. The nano-crack sensor used here customizes the working range and sensor sensitivity to match the strain rate of the tip of the forceps. Therefore, the tension in the suture can be sufficiently measured even at suture failure. The printed circuit board attached to the bottom of the system is designed to simultaneously collect data from several sensors, visualizing the direction and magnitude of the tension in order to inform the surgeon of how much tension is being applied. This system was verified by performing pig-corneal suturing.

Fully Automated, Sample-to-Answer Leukocyte Functional Assessment Platform for Continuous Sepsis Monitoring via Microliters of Blood.

We report a fully automated, sample-to-answer, and label-free leukocyte activation analysis platform for monitoring immune responses in sepsis, by integrating the multidimensional double spiral (MDDS) and isodielectric separation (IDS) subplatforms. The integrated platform can provide rapid and fully automated identification of clinically diagnosed sepsis patients from only 50 µL of peripheral blood volume within 25 min. Many critical innovations were implemented in direct interconnection between the two subplatforms, such as intermediate sample storage and sample transfer, addressing flow rate mismatch (from mL/min to µL/min), and integration of a ridge array for upstream cell focusing in the IDS subplatform. The ridge array in the IDS subplatform can prevent the distortion of electrical profiling due to the residual red blood cells even after the MDDS process. We showed that the integrated platform can separate leukocytes (up to >99.9% red blood cell removal) in the MDDS subplatform and automatically transfer them to the downstream ridge-integrated IDS subplatform for their activation analysis without any apparent ex vivo cell activation and any human intervention. We also demonstrated that the integrated platform can identify differences between leukocytes from human sepsis and healthy subjects significantly (p = 0.0024, 95% confidence interval) by looking into differences in the intrinsic electrical properties of leukocytes. The integrated platform could enable monitoring of host leukocyte function daily or even hourly as a bedside assessment tool, which is currently a critical yet unmet need for managing many critical care patients.

Inflammation resolution circuits are uncoupled in acute sepsis and correlate with clinical severity.

Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized proresolving mediators are agonists for antiinflammation and for promoting resolution, and they are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized proresolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and patients with sepsis by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte proresolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for antiinflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.

Deep-Learning Based Label-Free Classification of Activated and Inactivated Neutrophils for Rapid Immune State Monitoring.

The differential count of white blood cells (WBCs) is one widely used approach to assess the status of a patient's immune system. Currently, the main methods of differential WBC counting are manual counting and automatic instrument analysis with labeling preprocessing. But these two methods are complicated to operate and may interfere with the physiological states of cells. Therefore, we propose a deep learning-based method to perform label-free classification of three types of WBCs based on their morphologies to judge the activated or inactivated neutrophils. Over 90% accuracy was finally achieved by a pre-trained fine-tuning Resnet-50 network. This deep learning-based method for label-free WBC classification can tackle the problem of complex instrumental operation and interference of fluorescent labeling to the physiological states of the cells, which is promising for future point-of-care applications.

Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics.

A fully-automated and portable leukocyte separation platform was developed based on a new type of inertial microfluidic device, multi-dimensional double spiral (MDDS) device, as an alternative to centrifugation. By combining key innovations in inertial microfluidic device designs and check-valve-based recirculation processes, highly purified and concentrated WBCs (up to >99.99% RBC removal, ∼80% WBC recovery, >85% WBC purity, and ∼12-fold concentrated WBCs compared to the input sample) were achieved in less than 5 minutes, with high reliability and repeatability (coefficient of variation, CV < 5%). Using this, one can harvest up to 0.4 million of intact WBCs from 50 µL of human peripheral blood (50 µL), without any cell damage or phenotypic changes in a fully-automated operation. Alternatively, hand-powered operation is demonstrated with comparable separation efficiency and speed, which eliminates the need for electricity altogether for truly field-friendly sample preparation. The proposed platform is therefore highly deployable for various point-of-care applications, including bedside assessment of the host immune response and blood sample processing in resource-limited environments.

Modulation of saliva pattern and accurate detection of ovulation using an electrolyte pre-deposition-based method: a pilot study.

We developed an electrolyte pre-deposition-based saliva pattern modulation method to detect ovulation with high accuracy and reliability. Ovulation tests using human saliva have advantages in terms of the earlier ovulation detection and more convenient sample collection procedure; however, accuracy is low, which is a critical limitation given that the concentrations of salivary constituents can vary depending on the health status of the tested individual and subjective user judgement of the test result. In this study, we quantitatively analyzed saliva patterns according to the concentrations of electrolytes and proteins in the ovulation test and found that changes in the saliva pattern during the ovulatory period can be controlled by sodium chloride (NaCl) pre-deposition, which directly affects the accuracy of ovulation detection. The 100 nmol NaCl pre-deposition condition proved optimal, being two-fold more sensitive to changes in saliva pattern versus the non-pre-deposition condition (accuracy of ovulation detection = 66.6% and 33.3%, respectively). Although accuracy remained insufficient for actual applications compared to the urine-based ovulation detection method, we expect that the electrolyte pre-deposition method will greatly contribute to enhancing the performance of saliva-based ovulation detection tests, toward a commercially satisfactory level of accuracy.

Development of a Waterproof Crack-Based Stretchable Strain Sensor Based on PDMS Shielding.

This paper details the design of a poly(dimethylsiloxane) (PDMS)-shielded waterproof crack-based stretchable strain sensor, in which the electrical characteristics and sensing performance are not influenced by changes in humidity. This results in a higher number of potential applications for the sensor. A previously developed omni-purpose stretchable strain (OPSS) sensor was used as the basis for this work, which utilizes a metal cracking structure and provides a wide sensing range and high sensitivity. Changes in the conductivity of the OPSS sensor, based on humidity conditions, were investigated along with the potential possibility of using the design as a humidity sensor. However, to prevent conductivity variation, which can decrease the reliability and sensing ability of the OPSS sensor, PDMS was utilized as a shielding layer over the OPSS sensor. The PDMS-shielded OPSS sensor showed approximately the same electrical characteristics as previous designs, including in a high humidity environment, while maintaining its strain sensing capabilities. The developed sensor shows promise for use under high humidity conditions and in underwater applications. Therefore, considering its unique features and reliable sensing performance, the developed PDMS-shielded waterproof OPSS sensor has potential utility in a wide range of applications, such as motion monitoring, medical robotics and wearable healthcare devices.

An underwater superoleophobic nanofibrous cellulosic membrane for oil/water separation with high separation flux and high chemical stability.

Oil spills and an increasing demand for the treatment of industrial oily wastewater are driving the need for continuous large-scale oil/water separation processes. Herein, we report a nanofibrous cellulosic membrane (NFC membrane) for the continuous high-flux separation of large amounts of oil/water mixtures. The NFC membrane was fabricated using wet electrospinning, a facile yet effective method for stacking nanofibrous membranes with uniform porous structures on a substrate. Owing to its cellulosic nature, the membrane showed excellent underwater superoleophobicity along with robust chemical stability and was able to separate oil/water mixtures at efficiencies exceeding 99%. Repetitive oil/water separations could be performed using a single membrane, during which the oil content in the filtrate remained extremely low (<29 ppm). The nanofibrous membrane exhibited a fine porous structure that was interconnected throughout the membrane, resulting in a high oil intrusion pressure (>30 kPa) that allowed not only gravity-driven but also pressure-driven separation of oil/water mixtures. The separation flux reached 120 000 L m-2 h-1 during pressure-driven separations, which is a very promising feature for actual applications such as the large-scale treatment of industrial oily wastewater.

Omni-Purpose Stretchable Strain Sensor Based on a Highly Dense Nanocracking Structure for Whole-Body Motion Monitoring.

Here, we report an omni-purpose stretchable strain sensor (OPSS sensor) based on a nanocracking structure for monitoring whole-body motions including both joint-level and skin-level motions. By controlling and optimizing the nanocracking structure, inspired by the spider sensory system, the OPSS sensor is endowed with both high sensitivity (gauge factor ≈ 30) and a wide working range (strain up to 150%) under great linearity (R2 = 0.9814) and fast response time (<30 ms). Furthermore, the fabrication process of the OPSS sensor has advantages of being extremely simple, patternable, integrated circuit-compatible, and reliable in terms of reproducibility. Using the OPSS sensor, we detected various human body motions including both moving of joints and subtle deforming of skin such as pulsation. As specific medical applications of the sensor, we also successfully developed a glove-type hand motion detector and a real-time Morse code communication system for patients with general paralysis. Therefore, considering the outstanding sensing performances, great advantages of the fabrication process, and successful results from a variety of practical applications, we believe that the OPSS sensor is a highly suitable strain sensor for whole-body motion monitoring and has potential for a wide range of applications, such as medical robotics and wearable healthcare devices.

Development of an Integrated Evaluation System for a Stretchable Strain Sensor.

Recently, much research has been focused on stretchable or flexible electronic sensors for the measurement of strain or deformation on movable and variably shaped objects. In this research, to evaluate the performance of stretchable strain sensors, we have designed an integrated evaluation system capable of simultaneously measuring the change in stress and conductance of a strain sensor. Using the designed system, we have successfully evaluated the deformation characteristics, sensing range and sensing sensitivity of a stretchable strain sensor. We believe that the developed integrated evaluation system could be a useful tool for performance evaluation of stretchable strain sensors.