ABSTRACT Background and Aims Adult pancreas-derived islet progenitor cells (IPCs) have recently been shown to expand in culture and differentiate into endocrine-like organoids. However, translation of this approach to a clinically compatible workflow requires cell enrichment strategies and validation using tissue obtained during real-world clinical procedures. Here, we adapted our previously described IPC platform to non-endocrine pancreatic tissue fractions generated during clinical islet isolation procedures and evaluated their capacity to generate functional islet organoids. Methods Non-endocrine pancreatic tissue fractions obtained during clinical islet isolation were expanded ex vivo and enriched using fluorescence-activated cell sorting (FACS) for CD81 and CD9, surface markers previously identified in IPC populations. Sorted cells were expanded, induced to form IPC clusters, and differentiated with ISX9 to generate islet organoids. Differentiation was assessed by gene expression analysis, flow cytometry, immunofluorescence, calcium flux assays, glucose-stimulated insulin and glucagon secretion, and single-cell RNA sequencing. Results Clinically derived non-endocrine cell fractions yielded expandable IPC populations expressing progenitor-associated markers. FACS-purified and expanded CD81 + /CD9 + IPCs were enriched with BMPR1A and P2RY1. Sorted cells generated three-dimensional BMPR1A + and RGS16 + IPC clusters. IPC clusters differentiated into islet organoids with upregulated expression of canonical beta-and alpha-cell transcription factors. Single-cell transcriptomic profiling revealed activation of coordinated endocrine gene programs and alignment with reference human islet endocrine signatures, while the undifferentiated IPC compartment was marked by enrichment of PTX3, FST, CEMIP, and GREM1. Terminally differentiated cells exhibited depolarization-induced calcium influx and glucose-regulated insulin and glucagon secretion. Conclusions These findings establish an adaptable workflow for expansion and production of functional islet organoids recovered from clinically derived pancreatic tissue. This strategy may provide an unlimited autologous source of adult progenitor-derived islets for future islet cell replacement therapies in diabetes.

Abstract The long-term simulation of biological systems at the cellular level presents a significant computational challenge due to pronounced multiscale behavior and severe numerical stiffness. While implicit solvers offer improved stability in solving stiff systems, they can incur prohibitively high computational costs. Alternative conventional methods, such as mass scaling or unit transformation, are often inadequate because model stiffness may arise from multiple causes beyond inertial effects. To address these limitations, this study proposes a model-reduction technique integrated with an adaptive Runge–Kutta solver, which substantially reduces computational time. The effectiveness of the proposed approach is demonstrated by successfully simulating a 14-day adipogenic differentiation process in less than 1 h and 9 min of computational time on a typical desktop computer. Numerical evaluations conducted on a linear spring–mass–damper benchmark, a nonlinear Duffing oscillator, and a detailed human mesenchymal stem cell model show that the proposed approach substantially outperforms conventional scaling techniques and stiff solvers, establishing it as a robust and efficient tool for the long-term simulation of large, multiscale, stiff systems.

This paper presents a high-speed approach to simulating the long-term mechanobiological development of stem cells during the adipogenesis process. A novel three-dimensional model of human bone marrow-derived mesenchymal stem cells (hMSCs) undergoing adipogenic differentiation is presented herein. The elements of the cellular model have minute masses in femtograms and dimensions in nanometers. The disproportionality between the force and mass terms of the system, yielding a multiscale dynamic model, requires the solution to be calculated in femto- and picosecond time-steps. This makes producing the two-week time history of the adipogenic differentiation process computationally infeasible with conventional methods, even with the aid of supercomputers. The scaling method, based on the method of multiple scales proposed in authors' previous works, has been shown to address these imbalances and yield fast computational time for long-term simulation of cell processes. Herein, a novel approach to the scaling formulation is proposed, and methods for choosing scaling factors are presented and examined. Employing the new formulation results in a computational time of less than 1 h and 9 min on a normal desktop computer for the simulation of the 3D cellular model for the two-week time history of the adipogenic differentiation process. This is faster than previous efforts, which modeled the cell in two dimensions.

Disruption of the blood–brain barrier (BBB) after stroke or traumatic brain injury (TBI) exacerbates cerebral edema, in?ammation, and neuronal damage. Tight junctions (TJs) between endothelial cells are critical for barrier function, yet few therapies directly target TJ reassembly. Poloxamer 188 (P188), an FDA-approved amphiphilic triblock copolymer, has demonstrated membrane-stabilizing and vascular-protective properties in diverse injury models. We hypothesized that P188 promotes BBB recovery by sealing compromised membranes and enhancing TJ reassembly. Using a 96-Transwell mouse brain endothelial cell model, barrier disruption was induced with 0.01% Triton X-100, and permeability assessed by 70 kDa fluorescent dextran flux and zonula occludens-1 (ZO-1) immunostaining. Triton treatment increased dextran flux and disrupted ZO-1 localization, whereas P188, applied before or after injury, significantly reduced permeability and restored ZO-1 intensity to control levels. In vivo, ?uorescently conjugated P188 (3 mg/kg) administered intravenously in mice accumulated in the brain within 6 h, suggesting the ability to reach sites of BBB injury. These findings support a dual role for P188 in BBB repair—mechanical stabilization of damaged membranes and structural restoration of TJs. Combined with its established safety, P188 emerges as a promising candidate for therapeutic intervention in acute neurovascular disorders, warranting further investigation in preclinical models of stroke and TBI.

Diabetes mellitus is a metabolic disorder that is rapidly growing across the world. Our laboratory has recently demonstrated that photobiomodulation (PBM) can couple to its metabolic pathways by modulating calcium dynamics in islet cells, including α- and β-cells. Using computer vision algorithms, changes in PBM-induced calcium dynamics can be verified, and, more importantly, this led us to propose hypotheses that will likely advance our understanding of photostimulatory effects in islet cells. In our previous paper, we determined changes in calcium spiking in response to PBM at 810 nm by manually segmenting the cells and the calcium spiking patterns. We have since developed a computer vison pipeline to automate cell segmentation and subsequent image analyses. By using automated methods for segmentation, registration, tracking, and statistical analysis, we were able to improve the accuracy of previously observed changes in calcium spiking in response to PBM in both cell types. Moreover, this pipeline was applied to elucidate the wavelength-dependent modulation of calcium dynamics at 1064 nm. The extent of increase in calcium spiking appears to have been overestimated by manual analysis, and the machine learning pipeline was able to capture and segment nearly 3-fold more cells, suggesting improved accuracy in the analysis of calcium spiking in islet cells. Detailed calcium analysis also indicates a biphasic dose response among α- and β-cells in response to PBM therapy at different wavelengths. The current findings offer a novel hypothesis and may facilitate the use of translational PBM as a potential therapy for diabetes mellitus.

Amphiphilic triblock poloxamer 188 (P188) has demonstrated its therapeutic potential for muscle, cardiac and neurological injuries. While this surfactant is thought to primarily reseal the disrupted cell membrane, the specific mechanisms that mediate the reparative effect of P188 remain to be fully elucidated. Here, we investigated the transport mechanisms of P188 cellular uptake by fluorescently conjugating P188 with the fluorophore, Rhodamine 110 (Rh110). Fluorescent conjugation did not alter the P188 structure as characterized by nuclear magnetic resonance, Fourier Transform infrared spectroscopy, and acid-base titration, and the hydrophobicity was also quantified. In mouse brain endothelial cells, Rh110 alone was unable to accumulate inside the cells, while the P188 + Rh110 was rapidly transported across the cell membrane and became saturated in less than 1 hour. The transport dynamics were determined to be clathrin-dependent endocytosis, which was significantly altered in saponin-damaged cells or in cells with disrupted actin cytoskeletal organization; this suggests that transport via vesicle trafficking may be involved. Reparative effects of P188 appear to remodel the membrane organization and restore the transport properties. Instead of relying on manual image analysis, we utilized a machine learning pipeline that was recently developed in our laboratory to more rapidly and accurately analyze the cellular images of fluorescent P188 dynamics. This computer vision pipeline significantly reduced the time needed to segment, analyze, and perform statistical analyses. Finally, when injected into the mouse tail vein following a traumatic injury to the brain, we report for the first time that the P188 + Rh110 was observed in the brain tissue, indicating that P188 can cross the blood-brain barrier (BBB). Taken together, the dual therapeutic effects of P188 should include (1) resealing the disrupted cell membrane and (2) modulation of the intracellular cell repair machinery that might be involved in response to traumatic brain injury.

Traumatic Brain Injury (TBI) is a major cause of severe disability and death, resulting in significant health care and economic burden. Poloxamer 188, a synthetic tri-block copolymer approved by the FDA, has been studied for its potential effects on traumatic brain injury (TBI). The neuroprotective abilities of P188 have attracted significant attention. This systematic review aims to compile evidence of P188's effect on the treatment of TBI. A comprehensive literature search was conducted using PubMed, SCOPUS, and Google Scholar databases, which yielded 20 articles that satisfied the inclusion criteria. These articles have shown direct protective effects of P188 on brain tissue following TBI, including restitution of the increase cell membrane permeability, attenuation of neuronal necrosis and apoptosis, improvement of mitochondrial viability, reduction in axonal disruption, and restoration of the blood brain barrier. In animals, P188 has been shown to improve sensorimotor functions, as well as spatial learning and memory.

Insulin secretion from pancreatic β-cells is integral in maintaining the delicate equilibrium of blood glucose levels. Calcium is known to be a key regulator and triggers the release of insulin. This sub-cellular process can be monitored and tracked through live-cell imaging and subsequent cell segmentation, registration, tracking, and analysis of the calcium level in each cell. Current methods of analysis typically require the manual outlining of β-cells, involve multiple software packages, and necessitate multiple researchers—all of which tend to introduce biases. Utilizing deep learning algorithms, we have therefore created a pipeline to automatically segment and track thousands of cells, which greatly reduces the time required to gather and analyze a large number of sub-cellular images and improve accuracy. Tracking cells over a time-series image stack also allows researchers to isolate specific calcium spiking patterns and spatially identify those of interest, creating an efficient and user-friendly analysis tool. Using our automated pipeline, a previous dataset used to evaluate changes in calcium spiking activity in β-cells post-electric field stimulation was reanalyzed. Changes in spiking activity were found to be underestimated previously with manual segmentation. Moreover, the machine learning pipeline provides a powerful and rapid computational approach to examine, for example, how calcium signaling is regulated by intracellular interactions.

This paper presents a high-speed, long-term approach to simulating the mechanobiology of cells with an inhomogeneous mass distribution ranging from femtograms to picograms. An accurate representation of cellular processes necessitates the inclusion of subcellular structures characterized by minute masses and dimensions. The minute objects yield multiscale dynamic models with disproportionate terms, which require inordinate amounts of computational time to simulate. The computational requirements limit the time span of the simulation to time histories shorter than one second, even when employing supercomputers. This paper examines adipogenesis, the transformation of human bone marrow-derived mesenchymal stem cells (hMSC) into adipocytes, a process that spans two weeks. The proposed simulation techniques are based on a novel scaling technique that addresses differently sized masses. This work addresses unique challenges beyond the authors’ earlier work, which addressed disproportionality between large stiffness and damping forces in relation to small masses in the dynamic model. This new approach reduces computational time to less than 1 hour and 45 minutes on a standard desktop computer for the two-week duration of adipogenesis.

To achieve the high-resolution separation of particles in size, focusing particles into a single stream in the centerline of a microchannel is required. Inertial microfluidic focusing may not be effective for particles smaller than $\sim \boldsymbol{3\ \mu} \mathbf{m}$ . In this study, we used negative dielectrophoretic (nDEP) force generated by axisymmetric quadric electrodes to focus particles smaller than $\boldsymbol{3\ \mu} \mathbf{m}$ into a single stream. This requires fabrication of electrodes on both the top and the bottom surfaces of the microchannel. Unlike the device containing electrodes in one side of the channel, the device with axisymmetric electrodes poses much more complexity in its fabrication. This paper reports a simple and cost-effective fabrication method. Fluorescent polystyrene particles of $\boldsymbol{2\ \mu} \mathbf{m}$ diameter were successfully focused in the centerline of the microchannels with two different types of electrodes - chromium (Cr) and indium tin oxide (ITO). We also investigated frequency effects on direction of DEP force and the particles alignment.

Multibody dynamic simulations of the mechanobiology of cellular processes have not been obtained for time histories of orders larger than one second even with the employment of supercomputers. A mechanobiologically representative model of a cellular process includes subcellular structures with small masses and lengths. A key development in this work is the inclusion of a coarse-grained representation of the cytoskeleton, based on the tensegrity model, with masses from femtogram to picogram in size and lengths from nanometers to microns in size. A second key development is the inclusion in the model of bodies that increase in mass over time. The forces acting on these bodies will be orders of magnitude larger than the masses. The correspondingly large accelerations necessitate the use of small time steps to obtain an accurate solution. Adipogenic differentiation, adipogenesis, of a human bone marrow-derived mesenchymal stem cell (hMSC) develops over a time span of two weeks in the experiment. Numerically integrating this multiscale model for such a long time period is computationally infeasible with conventional methods. A novel scaling approach based on the method of multiple scales is used herein to accurately simulate this two weeks of time history on a desktop computer in less than 3.5 hours. This much faster than real time simulation facilitates the study of the time dependent elements of adipogenesis and the mechanobiology of cellular processes in general.

It has been observed that a nanoparticle can exhibit underdamped motion while moving toward the focal point of an optical trap. It is unclear whether this motion is caused by laser or fluid forces. Dielectrophoretic forces can trap nanoparticles as an alternate approach to optical trapping. The electrical trap uses no laser, so we can determine which force causes the underdamped motion. A microchannel with a quad-electrode arrangement on its ceiling and floor was designed to explore this question. Supplying an oscillating voltage to these electrodes generates an oscillating electric field resulting in the dielectrophoretic force that traps the particle. However, matching common characteristics, such as trap stiffness, is difficult between the two methods. This paper compares the two approaches for a 2 μm diameter particle. Instead of matching the trapping characteristics, the next step in this work is to use the dielectrophoretic device to explore the effect of the particle’s momentum on its motion, which can explain the underdamped motion. Combining optical and dielectrophoretic trapping will offer new insights into the dynamic behavior of small particles in a fluid medium.

Insulin secretion from pancreatic β-cells is integral in maintaining the delicate equilibrium of blood glucose levels. Calcium is known to be a key regulator and triggers the release of insulin. This sub-cellular process can be monitored and tracked through live-cell imaging and subsequent cell segmentation, registration, tracking, and analysis of the calcium level in each cell. Current methods of analysis typically require the manual outlining of β-cells, involve multiple software packages, and necessitate multiple researchers - all of which tend to introduce biases. Utilizing deep learning algorithms, we have therefore created a pipeline to automatically segment and track thousands of cells, which greatly reduces the time required to gather and analyze a large number of sub-cellular images and improve accuracy. Tracking cells over a time-series image stack also allows researchers to isolate specific calcium spiking patterns and spatially identify those of interest, creating an efficient and user-friendly analysis tool. Using our automated pipeline, a previous dataset used to evaluate changes in calcium spiking activity in β-cells post-electric field stimulation was reanalyzed. Changes in spiking activity were found to be underestimated previously with manual segmentation. Moreover, the machine learning pipeline provides a powerful and rapid computational approach to examine, for example, how calcium signaling is regulated by intracellular interactions in a cluster of β-cells.

Significant efforts have been committed to better understand and regulate insulin secretion as it has direct implications on diabetes. The first phase of biphasic insulin secretion in response to glucose lasts about 10 minutes, followed by a more sustained release persisting several hours. Attenuated insulin release in the first phase is typically associated with abnormal beta-cells. While near-infrared photobiomodulation (PBM) demonstrates potential for multiple therapeutic applications, photostimulatory effects on alpha- and beta-cells remain to be further elucidated. Herein, we demonstrate that 810 nm PBM exposure at fluence of 9 J/cm(2) can elevate the intracellular reactive oxygen species within 15 minutes following photostimulation. In addition, calcium spiking showed an approximately 3-fold increase in both ATC1 (alpha-cells) and BTC6 (beta-cells) and correlates with hormone secretion in response to PBM stimulation. Our findings could lay a foundation for the development of non-biologic therapeutics that can augment islet transplantation.

The cell plasma membrane suffers structural disruptions from both daily environmental stresses and trauma. Rapid loss of cell viability occurs if membrane integrity is not rapidly restored. Physiological membrane sealing involves alteration of local intermolecular thermodynamics that is manifested by changes in membrane tension which precede reassembly of the membrane planar bilayer structure. Certain block copolymer surfactants, including poloxamer 188 (P188), have been proven to seal-disrupted cell membranes. However, the specific molecular mechanics of poloxamer-mediated membrane sealing remains a target of investigation. A decrease in membrane tension precedes membrane sealing by natural intrinsic cell sealing processes. The effect of P188 on the quasistatic membrane tension of Madin-Darby canine kidney (MDCK) and Swiss 3T3 fibroblasts cells under normal and saponin-permeabilized conditions was measured using laser optical tweezer (LOT)-extracted membrane tethers. The tether trap length of saponin-permeabilized MDCK cell membranes decreased from an uninjured control of 11.28 ± 1.1 μm to 6.43 ± 0.67 μm. Treatment with P188 (0.2 mM) significantly increased the tether trap length to 9.69 ± 1.0 μm (p < 0.05) while the control polymer, polyethylene glycol (0.2 mM) resulted in tether trap length of 7.02 ± 0.73 μm that was not significantly different. Similar observations were made in the saponin-permeabilized fibroblasts. Corresponding fluorescence cell viability assays revealed that P188-treated cells had a higher survival rate. Thus, surfactant copolymer membrane sealing restores the membrane integrity by decreasing the membrane tension. Cells quickly become non-viable when plasma membrane integrity is lost. Restoring or healing the cell membrane requires alteration in the forces that stabilize the membrane structure. The membrane defect healing processes are preceded by a decrease in membrane interfacial tension. This study demonstrated that amphiphilic block copolymer surfactants, such as poloxamer 188 (P188), reduce membrane tension and promote cell survival. Using laser optical tweezers, this work quantified the effect of surfactant copolymer-catalyzed sealing. Our work utilized laser optical tweezers to demonstrate that copolymer-catalyzed membrane sealing corresponds with a decrease in membrane tension, much like the physiologic response to membrane injury.

AbstractHypertrophic adipocytes have been implicated in the progression of obesity‐induced complications. These enlarged fat cells cause release of free fatty acids and inflammatory cytokines, promote hypoxia and fibrosis and limit insulin sensitivity. Photobiomodulation (PBM), or the influence of light on biological tissues, has previously been demonstrated to reduce lipid accumulation in stem cells undergoing adipogenesis. Here, we characterize the effect of PBM on an in vitro hypertrophic obesity model and its influence on reduction of lipids and restoration to normal adipocyte function. Adipose‐derived stem cells were induced to hypertrophy with the addition of palmitic acid (PA) and was confirmed with fluorescent imaging of lipid content, and functional changes such as glucose uptake. Glucose transport into the cell was diminished and the expression of glucose transporter GLUT4 was downregulated. PBM‐treated hypertrophic cells (1064 nm and 17.6 J/cm2 every day for 7 days following addition of PA) decreased the lipid levels in hypertrophic adipocytes, restored the GLUT4 protein expression and enhanced glucose transport. Taken together, PBM is shown capable of restoring the cellular morphology and function of hypertrophic cells. This could have important clinical implication for the development of laser‐based potential therapeutic treatment of complications due to metabolic syndrome.

The importance of cell mechanics has long been recognized for the cell development and function. Biomechanics plays an important role in cell metabolism, regulation of mechanotransduction pathways and also modulation of nuclear response. The mechanical properties of the cell are likely determined by, among many others, the cytoskeleton elasticity, membrane tension and cell-substrate adhesion. This coordinated but complex mechanical interplay is required however, for the cell to respond to and influence in a reciprocal manner the chemical and mechanical signals from the extracellular matrix (ECM). In an effort to better and more fully understand the cell mechanics, the role of nuclear mechanics has emerged as an important contributor to the overall cellular mechanics. It is not too difficult to appreciate the physical connection between the nucleus and the cytoskeleton network that may be connected to the ECM through the cell membrane. Transmission of forces from ECM through this connection is essential for a wide range of cellular behaviors and functions such as cytoskeletal reorganization, nuclear movement, cell migration and differentiation. Unlike the cellular mechanics that can be measured using a number of biophysical techniques that were developed in the past few decades, it still remains a daunting challenge to probe the nuclear mechanics directly. In this paper, we therefore aim to provide informative description of the cell membrane and cytoskeleton mechanics, followed by unique computational modeling efforts to elucidate the nucleus-cytoskeleton coupling. Advances in our knowledge of complete cellular biomechanics and mechanotransduction may lead to clinical relevance and applications in mechano-diseases such as atherosclerosis, stem cell-based therapies, and the development of tissue engineered products.

Brain injuries caused by an explosive blast or blunt force is typically presumed to associate with mechanical trauma to the brain tissue. Recent findings from our laboratory suggest that shockwaves produced by a blast can generate micron-sized bubbles in the tissue. The collapse of microbubbles (i.e., microcavitation) may induce a mechanical trauma and compromise the integrity of the blood-brain endothelium (BBE). To test our hypothesis, we engineered a BBE model to determine the effect of microbubbles on the structural and functional changes in the BBE. Using monolayers of mouse primary brain microvascular endothelial cells, the permeability coefficient was measured following simulated blast-induced microcavitation. This event down-regulated the expression of tight junction markers, disorganized the cell-cell junction, and increased permeability. Since poloxamers have been shown to rescue damaged cells, the cells were treated with the FDA-approved poloxamer 188 (P188). The results indicate P188 recovered the permeability, restored the tight junctions, and suppressed the expressions of matrix metalloproteinases. The biomimetic interface we developed appears to provide a systematic approach to replicate the structure and function of BBE, determine its alteration in response to traumatic brain injury, and test potential therapeutic treatments to repair the damaged brain endothelium.