Whipple's Disease Mimicking Sarcoidosis.

Whipple's disease is a rare systemic disease caused by a Tropheryma whipplei infection. Although older literature reports a low rate of incidence, case reports continue to rise due to increased awareness of the disease. Classic Whipple's disease presents as weight loss, diarrhea, and arthralgia and may involve the heart, central nervous system (CNS), or any other organ system. Some patients with Whipple's disease do not have the classic signs and symptoms of the disease.  We present a case of Whipple's disease in a patient with poor appetite, weight loss, and granulomatous inflammation of various organs, including the kidneys and spleen, mimicking sarcoidosis. She had presented three years earlier with acute kidney injury (AKI) and hypercalcemia. The renal biopsy revealed diffuse granulomatous interstitial nephritis. Both AKI and hypercalcemia resolved with prednisone; however, her weight loss and decreased appetite continued. The initial positron emission tomography (PET) scan showed increased fluorodeoxyglucose (FDG) avidity in the spleen and large intestine, and the splenic biopsy revealed non-caseating granulomas. A diagnosis of sarcoidosis was made, and she was started on methotrexate with prednisone. Nevertheless, the weight loss and poor appetite were relentless. A repeat PET scan showed increased FDG avidity in loops of the small and large intestines. A small intestinal biopsy revealed positive periodic acid-Schiff (PAS) and negative acid-fast bacilli (AFB) revealing the diagnosis of Whipple's disease. Whipple's disease should be considered in the differential diagnosis of sarcoidosis, especially in those patients worsening on standard immunosuppression.

Preseeding of Mesenchymal Stem Cells Increases Integration of an iPSC-Derived CM Sheet into a Cardiac Matrix.

Cell sheet technology has demonstrated great promise in delivering a large amount of therapeutic cells for tissue repair, including in the myocardium. However, the lack of host integration remains one of the key challenges in using cell sheets for cardiac repair. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been reported to facilitate tissue repair and regeneration in a variety of ways. It has been demonstrated that paracrine factors from MSCs could enhance scaffold recellularization and vascularization. In this study, we used an in vitro cardiac matrix mimic platform to examine the effects of hMSCs preseeding on the interactions between cell sheets and cardiac matrix. The fabricated human induced pluripotent stem cells-derived cardiomyocyte sheets were attached to a decellularized porcine myocardium slice with or without preseeding of hMSCs. The hMSCs preseeding significantly enhanced the interactions between cardiomyocyte sheets and cardiac matrix in terms of cell migration distance, cell distribution, and mature vascular and cardiomyocyte marker expressions in the matrix. Growth factor and matrix metalloproteinases array analysis suggested that hMSCs- induced vascularization and MMPs regulation are the two possible mechanisms that lead to the improved CMs and cardiac matrix interactions. Further examination of these two mechanisms will enable the development of new approaches to facilitate transplanted cells for tissue repair.

Enabling single cell electrical stimulation and response recording via a microfluidic platform.

Electrical stimulation (ES) has been recognized to play important roles in regulating cell behaviors. A microfluidic device was developed for the electrical stimulation of single cells and simultaneous recording of extracellular field potential (EFP). Each single cell was trapped onto an electrode surface by a constriction channel for ES testing and was then driven to the outlet by the pressure afterward. This design allows the application of ES on and detection of EFP of single cells continuously in a microfluidic channel. Human cardiomyocytes and primary rat cortex neurons were tested with specific ES with the device. Each cell's EFP signal was detected and analyzed during the ES process. Results have shown that after applying specific ES on the excitable single cells, the cells evoked electrical responses. In addition, increased secretion of glutamic acid was detected from the stimulated neurons. Altogether, these results indicated that the developed device can be used to continuously apply ES on and accurately determine cell responses of single cells with shorter probing time. The throughput of the measurement can achieve 1 cell per minute, which is higher than the traditional ES methods that need culturing cells or manually positioning the cells onto the electrode surface. Before and after the application of ES, the cell viability had no significant change. Such a device can be used to study the biological process of various types of cells under electrical stimulation.

Cardiac tissue-derived extracellular matrix scaffolds for myocardial repair: advantages and challenges.

Decellularized extracellular matrix (dECM) derived from myocardium has been widely explored as a nature scaffold for cardiac tissue engineering applications. Cardiac dECM offers many unique advantages such as preservation of organ-specific ECM microstructure and composition, demonstration of tissue-mimetic mechanical properties and retention of biochemical cues in favor of subsequent recellularization. However, current processes of dECM decellularization and recellularization still face many challenges including the need for balance between cell removal and extracellular matrix preservation, efficient recellularization of dECM for obtaining homogenous cell distribution, tailoring material properties of dECM for enhancing bioactivity and prevascularization of thick dECM. This review summarizes the recent progresses of using dECM scaffold for cardiac repair and discusses its major advantages and challenges for producing biomimetic cardiac patch.

In Vivo Assessment of Decellularized Porcine Myocardial Slice as an Acellular Cardiac Patch.

Acellular cardiac patches made of various biomaterials have shown to improve heart function after myocardial infarction (MI). Extracellular matrix scaffold derived from a decellularized tissue has unique advantages to serve as an acellular cardiac patch due to its biomimetic nature. In this study, we examined the therapeutic outcomes of using a decellularized porcine myocardium slice (dPMS) as an acellular patch in a rat acute MI model. dPMSs with two different thicknesses (300 and 600 µm) were patched to the infarcted area of the rat myocardium, and their effects on cardiac function and host interactions were assessed. We found that the implanted dPMS firmly attached to host myocardium after implantation and prevented thinning of the left ventricular (LV) wall after an MI. A large number of host cells were identified to infiltrate into the implanted dPMS, and a significant number of vessel structures was observed in the dPMS and infarcted area. We detected a significantly higher density of M2 macrophages in the groups treated with dPMSs as compared to the MI group. Contraction of the LV wall and cardiac functional parameters (left ventricular ejection fraction and fractional shortening) was significantly improved in the treatment groups (300 and 600 µm dPMS) 4 weeks after surgery. Our results proved the therapeutic benefits of using dPMS as an acellular cardiac patch for the treatment of acute myocardial infarction.

A microfluidic device for noninvasive cell electrical stimulation and extracellular field potential analysis.

We developed a device that can quickly apply versatile electrical stimulation (ES) signals to cells suspended in microfluidic channels and measure extracellular field potential simultaneously. The device could trap cells onto the surface of measurement electrodes for ES and push them to the downstream channel after ES by increasing pressure for continuous measurement. Cardiomyocytes, major functional cells in heart, together with human fibroblast cells and human umbilical vein endothelial cells, were tested with the device. Extracellular field potential signals generated from the cells were recorded. We found that under electrical stimulation, cardiomyocytes were triggered to alter their field potential, while non-excitable cells were not triggered. Hence this device can noninvasively distinguish electrically excitable cells from electrically non-excitable cells. Results have also shown that increased cardiomyocyte cell number led to increased magnitude and occurrence of the cell responses. This relationship could be used to detect the viable cells in a cardiac tissue. Application of variable ES signals on different cardiomyocyte clusters has shown that the application of ES clearly boosted cardiomyocytes electrical activities according to the stimulation frequency. In addition, we confirmed that the device can apply ES onto and detect the electrical responses from a mixed cell cluster; the responses from the mixed cluster is dependent on the ratio of cardiomyocytes. These results demonstrated that our device could be used as a tool to optimize ES conditions to facilitate the functional engineered cardiac tissue development.

A Thin Layer of Decellularized Porcine Myocardium for Cell Delivery.

Decellularized porcine myocardium has shown many benefits as a cell delivery scaffold for cardiac therapy. However, using full thickness decellularized myocardium as cardiac patch may lead to poor viability and inhomogeneous distribution of delivered cells, due to perfusion limitations. In this study, we explored the feasibility of decellularized porcine myocardial slice (dPMS) to construct a vascularized cardiac patch for cell delivery. Decellularized porcine myocardium was sliced into thin layers (thickness~300 µm). Adipose-derived Stem cells (ASCs) obtained from rat and pig were seeded on dPMS. The viability, infiltration, and differentiation of seeded ASCs were examined. The mechanical properties of dPMSs of various thickness and native myocardium were tested. We noticed dPMS supported attachment and growth of rat and pig ASCs. Both rat and pig ASCs showed high viability, similar patterns of proliferation and infiltration within dPMS. Rat ASCs showed expression of early-endothelial markers followed by mature-endothelial marker without any additional inducers on dPMS. Using rat myocardial infarction model, we delivered ASCs using dPMS patched to the infarcted myocardium. After 1 week, a higher number of transplanted cells were present in the infarcted area when cells were delivered using dPMS versus direct injection. Compared with MI group, increased vascular formation was also observed.

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection.

We report a high-sensitivity cell secretome detection method using competitive immuno-aggregation and a micro-Coulter counter. A target cell secretome protein competes with anti-biotin-coated microparticles (MPs) to bind with a biotinylated antibody (Ab), causing decreased aggregation of the functionalized MPs and formation of a mixture of MPs and aggregates. In comparison, without the target cell secretome protein, more microparticles are functionalized, and more aggregates are formed. Thus, a decrease in the average volume of functionalized microparticles/aggregates indicates an increase in cell secretome concentration. This volume change is measured by the micro-Coulter counter, which is used to quantitatively estimate the cell secretome concentration. Vascular endothelial growth factor (VEGF), one of the key cell secretome proteins that regulate angiogenesis and vascular permeabilization, was used as the target protein to demonstrate the sensing principle. A standard calibration curve was generated by testing samples with various VEGF concentrations. A detection range from 0.01 ng/mL to 100.00 ng/mL was achieved. We further demonstrated the quantification of VEGF concentration in exogenous samples collected from the secretome of human mesenchymal stem cells (hMSCs) at different incubation times. The results from the assay agree well with the results of a parallel enzyme-linked immunoabsorbent assay (ELISA) test, indicating the specificity and reliability of the competitive immuno-aggregation assay. With its simple structure and easy sample preparation, this assay not only enables high sensitivity detection of VEGF but also can be readily extended to other types of cell secretome analysis as long as the specific Ab is known.

Development and Comparison of Two Immuno-disaggregation Based Bioassays for Cell Secretome Analysis.

Cell secretome analysis has gained increasing attention towards the development of effective strategies for disease treatment. Analysis of cell secretome enables the platform to monitor the status of disease progression, facilitating therapeutic outcomes. However, cell secretome analysis is very challenging due to its versatile and dynamic composition. Here, we report the development of two immuno-disaggregation bioassays using functionalized microparticles for the quantitative analysis of the cell secretome. Methods: We evaluated the feasibility of our developed immuno-disaggregation bioassays using antibody-conjugated MPs and protein-conjugated MPs for the detection of target cell secretome protein. The vascular endothelial growth factor (VEGF)-165 protein was tested as a model cell secretome protein in the serum and serum-free conditions. The status of MP aggregates was examined with a light microscopy and AccuSizerTM 780 Optical Particle Sizer. The accuracy of our bioassays measurement was compared with standard ELISA method. Results: The developed bioassays successfully detected target VEGF protein present in serum-free buffer and serum-containing complete cell culture medium with high sensitivity and specificity. Additionally, the immuno-disaggregation bioassays using antibody-conjugated MPs and protein-conjugated MPs have a wide detection range from 0.01 ng/mL to 100 ng/mL and 0.5 ng/mL to 100 ng/mL, respectively. The sensitivity of the bioassay using antibody-conjugated MPs was approximately one order of magnitude higher than the bioassay using protein-conjugated MPs. Conclusion: Our promising results indicate the potential of the developed bioassays as powerful platforms for the quantitative analysis of cell secretome.

In situ single cell detection via microfluidic magnetic bead assay.

We present a single cell detection device based on magnetic bead assay and micro Coulter counters. This device consists of two successive micro Coulter counters, coupled with a high gradient magnetic field generated by an external magnet. The device can identify single cells in terms of the transit time difference of the cell through the two micro Coulter counters. Target cells are conjugated with magnetic beads via specific antibody and antigen binding. A target cell traveling through the two Coulter counters interacts with the magnetic field, and have a longer transit time at the 1st counter than that at the 2nd counter. In comparison, a non-target cell has no interaction with the magnetic field, and hence has nearly the same transit times through the two counters. Each cell passing through the two counters generates two consecutive voltage pulses one after the other; the pulse widths and magnitudes indicating the cell's transit times through the counters and the cell's size respectively. Thus, by measuring the pulse widths (transit times) of each cell through the two counters, each single target cell can be differentiated from non-target cells even if they have similar sizes. We experimentally proved that the target human umbilical vein endothelial cells (HUVECs) and non-target rat adipose-derived stem cells (rASCs) have significant different transit time distribution, from which we can determine the recognition regions for both cell groups quantitatively. We further demonstrated that within a mixed cell population of rASCs and HUVECs, HUVECs can be detected in situ and the measured HUVECs ratios agree well with the pre-set ratios. With the simple device structure and easy sample preparation, this method is expected to enable single cell detection in a continuous flow and can be applied to facilitate general cell detection applications such as stem cell identification and enumeration.

Prevascularization of Decellularized Porcine Myocardial Slice for Cardiac Tissue Engineering.

Prevacularization strategies have been implemented in tissue engineering to generate microvasculature networks within a scaffold prior to implantation. Prevascularizing scaffolds will shorten the time of functional vascular perfusion with host upon implantation. In this study, we explored key variables affecting the interaction between decellularized porcine myocardium slices (dPMSs) and reseeded stem cells toward the fabrication of prevascularized cardiac tissue. Our results demonstrated that dPMS supports attachment of human mesenchymal stem cells (hMSCs) and rat adipose derived stem cells (rASCs) with high viability. We found that cell seeding efficiency and proliferation are dPMS thickness dependent. Compared to lateral cell seeding, bilateral cell seeding strategy significantly enhanced seeding efficiency, infiltration, and growth in 600 µm dPMS. dPMS induced endothelial differentiation and maturation of hMSCs and rASCs after 1 and 5 days culture, respectively. These results indicate the potential of dPMS as a powerful platform to develop prevascularized scaffolds and fabricate functional cardiac patches.

Microfluidic Magnetic Bead Assay for Cell Detection.

We present a novel cell detection device based on a magnetic bead cell assay and microfluidic Coulter counting technology. The device cannot only accurately measure cells size distribution and concentration but also detect specific target cells. The device consists of two identical micro Coulter counters separated by a fluid chamber where an external magnetic field is applied. Antibody-functionalized magnetic beads were bound to specific antigens expressed on the target cells. A high-gradient magnetic field was applied to the chamber closer to the second counter via an external cylindrical magnet. Because of the magnetic interaction between the magnetic beads and the magnetic field, target cells were retarded by the magnetic field; transit time of a target cell (bound with magnetic beads) passing through the second counter was longer than that through the first counter. In comparison, transit times of a nontarget cell remained nearly the same when it passed through both counters. Thus, from the transit time delay we can identify target cells and quantify their concentration in a cell suspension. The transit time and the size of each cell were accurately measured in terms of the width and amplitude of the resistive pulses generated from the two Coulter counters. Experiments demonstrated that for mixed cells with various target cell ratios, the transit time delay increased approximately linearly with the increasing target cell ratio. The limit of detection (LOD) of the assay was estimated to be 5.6% in terms of target cell ratio. Cell viability tests further demonstrated that most cells were viable after the detection. With the simple device configuration and easy sample preparation, this rapid and reliable method is expected to accurately detect target cells and could be applied to facilitate stem cell isolation and characterization.