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Articles by Tricia Harvat in JoVE
JoVE General
A Perifusion جزيرة متعدد حدودي النظام داخل جهاز ميكروفلويديك Perifusion
1Department of Surgery, University of Illinois, Chicago, 2Department of Bioengineering, University of Illinois, Chicago
ووضعت جزيرة perifusion ميكروفلويديك الجهاز لتقييم افراز الانسولين من الجزر الحيوية متعددة ومضان التصوير المتزامن لتدفق الكالسيوم والتغيرات المحتملة الميتوكوندريا.
Other articles by Tricia Harvat on PubMed
Microfluidic Device for Multimodal Characterization of Pancreatic Islets
A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca(2+)](i)) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL min(-1), without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay.
Microfluidic Perifusion and Imaging Device for Multi-parametric Islet Function Assessment
A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple pancreatic islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The fanned out design of the second generation device optimized the efficient mixing and uniform distribution of rapid alternating solutions in the perifusion chamber and allowed for the generation of reproducible glucose gradients. Simultaneous imaging of calcium influx and mitochondrial potential changes in response to glucose stimulation showed high signal-noise ratio and spatial-temporal resolution. These results suggest that this system can be used for detailed study of the endocrine function of pancreatic islets with simultaneous imaging of intracellular ion fluxes and mitochondrial membrane potential changes. This tool can be used for quality assessment of islets preparation before transplantation and for in vitro studies of islet function.
Size-based Separation and Collection of Mouse Pancreatic Islets for Functional Analysis
Islet size has recently been demonstrated to be an important factor in determining human islet transplantation outcomes. In this study, a multi-layered microfluidic device was developed and quantified for size-based separation of a heterogeneous population of mouse islets. The device was fabricated using standard soft lithography and polydimethylsiloxane (PDMS). Size-based separation was first demonstrated via injection of a heterogeneous population of glass beads between 50-300 microm in diameter which were separated into five sub-populations based on their diameter. Next, a heterogeneous population of mouse pancreatic islets, between 50-250 microm in diameter was separated into four sub-populations. Throughout this process the islets remained intact without any signs of damage, as indicated by cell viability staining. Islet glucose-stimulated insulin secretion of each sub-population of islets was also evaluated demonstrating that islets smaller than 150 microm have superior stimulation indexes (SI) compared to islets larger than 150 microm. In this study, we found that islets between 100 microm and 150 microm in diameter had the greatest SI value in a heterogeneous population of islets.
Modified Gold Nanoparticle Vectors: a Biocompatible Intracellular Delivery System for Pancreatic Islet Cell Transplantation
Islet transplantation is an emerging therapy for type 1 diabetes mellitus with variable success. Molecular therapeutics is a promising approach to improve islet graft function and transplant outcomes. Traditional delivery vectors, however, have poor cell penetration and generally lead to compromised islet function. Modified gold nanoparticles represent a potential alternative in that they are taken up into cells efficiently and have unique binding properties. The objective of this study was to investigate whether gold nanoparticles can transfect islets uniformly without compromising cellular function.
Application of Microfluidic Technology to Pancreatic Islet Research: First Decade of Endeavor
β-cells respond to blood glucose by secreting insulin to maintain glucose homeostasis. Perifusion enables manipulation of biological and chemical cues in elucidating the mechanisms of β-cell physiology. Recently, microfluidic devices made of polydimethylsiloxane and Borofloat glass have been developed as miniaturized perifusion setups and demonstrated distinct advantages over conventional techniques in resolving rapid secretory and metabolic waveforms intrinsic to β-cells. In order to enhance sensing and monitoring capabilities, these devices have been integrated with analytical tools to increase assay throughput. The spatio-temporal resolutions of these analyses have been improved through enhanced flow control, valves and compartmentalization. For the first time, this review provides an overview of current devices used in islet studies and analyzes their strengths and experimental suitability. To realize the potential of microfluidic islet applications, it is essential to bridge the gap in design and application between engineers and biologists through the creation of standardized bioassays and user-friendly interfaces.
Highly Purified Versus Filtered Crude Collagenase: Comparable Human Islet Isolation Outcomes
This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n=52) with high-purified Serva NB1 collagenase (Serva NB1, n=42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to remove endotoxin contamination and impurities; in addition, this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The digestion efficacy between Sigma V and Serva NB1 was statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p=0.0014). However, the islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p=0.23) between groups. There was no significant purity difference observed in fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p=0.061) and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p=0.187) were also similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. The endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 EU/ml vs. 0.052 EU/ml, p=0.003). Additionally, in the Sigma V group there was minimal lot to lot variation and no significant loss of enzymatic activity after filtration. These findings indicate that the use of Sigma V or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all.
Dual Microfluidic Perifusion Networks for Concurrent Islet Perifusion and Optical Imaging
This study explores a new class of duplex microfluidic device which utilizes a dual perifusion network to simultaneously perform live-cell optical imaging of physiological activities and study insulin release kinetics on two islet populations. This device also incorporates on-chip staggered herringbone mixers (SHMs) to increase mixing efficiency and facilitate the generation of user-defined chemical gradients. Mouse islets are used to simultaneously measure dynamic insulin release, changes in mitochondrial potentials, and calcium influx in response to insulin secretagogues (glucose and tolbutamide), and show a high signal-to-noise ratio and spatiotemporal resolution of all measured parameters for both perifusion chambers. This system has many potential applications for studying β-cell physiology and pathophysiology, as well as for therapeutic drug screening. This dual perifusion device is not limited to islet studies and could easily be applied to other tissues and cells without major modifications.
Systematic Prevention of Bubble Formation and Accumulation for Long-term Culture of Pancreatic Islet Cells in Microfluidic Device
Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.
Islet Preconditioning Via Multimodal Microfluidic Modulation of Intermittent Hypoxia
Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations, while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1 min/1 min 5-21% cycling for 1 h), translating to improved insulin secretion. Moreover, blocking mitochondrial K(ATP) channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues.
