Microfluidic perifusion and imaging device for multi-parametric islet function assessment
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- Adewola, A.F., Lee, D., Harvat, T. et al. Biomed Microdevices (2010) 12: 409. doi:10.1007/s10544-010-9398-1
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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.
KeywordsIslets physiologyImagingGlucose gradientMicrofluidics
Microfluidic devices have emerged as valuable tools for biomedical research (Hansen and Quake 2003) and in clinical settings (Maheswaran et al. 2008). The advantage of microfluidic devices over conventional perfusion devices is that they use relatively small amounts of reagents and analytes, significantly decreasing costs. The small scale also allows leveraging of microscale phenomena such as laminar flow (Taylor et al. 2006; Mosadegh et al. 2007; Liu et al. 2008) and fast diffusion (Higgins et al. 2007) for new experimental modalities not possible with macroscale tools. Additionally, multiple tasks or assays can be integrated onto a single device for improved experimental throughput.
Condensing multiple assays into one microfluidic device has the potential to distinguish good islet preparations from poor preparations which could potentially solve one of the limitations of islet transplantations. By directly measuring metabolic viability including calcium influx and mitochondrial potential changes in response to insulin secretagogues this could provide improved predictors of islet viability function and transplant outcomes. One of the standard methods to analyze islet function is through perifusion of pancreatic islets (Cabrera et al. 2008). This well established technique is used to study the kinetics of insulin secretion. The fundamental physiological role of pancreatic beta-cells is to monitor and respond to moment-to-moment changes of blood glucose levels through insulin secretion. Under physiological conditions, insulin secretion is tightly regulated and depends on intracellular dynamic calcium changes and mitochondrial membrane potential changes. The monitoring of dynamic insulin secretion versus static insulin secretion provides a distinct advantage for understanding islet physiology and Pathophysiology. It is also of great interest for the evaluation of isolated human islets prior to transplantation to avoid transplanting dysfunctional islets that would work poorly in the recipient. Since microfluidic devices have been demonstrated to provide an ideal and even superior extracellular environment for cells including islets, several attempts have been made to apply microfluidic devices to islet studies. These studies looked at continuous perifusion with online electrophoresis immunoassay of single and multiple mouse islets. Other studies looked at calcium communication and oscillation in single islets, and a droplet-based microfluidic device using a microelectrode for manipulating and observing molecular signals was also developed (Roper et al. 2003; Rocheleau et al. 2004; Chen et al. 2008). These specialized methods exemplify the challenges and difficulties involved in the islet field; however, the technical complexity of these devices such as the fabrication and hardware requirements made them less suitable for routine laboratory and clinical use.
In this work, our goal was to address these limitations and develop a chip that would become a practical research tool for quantitative monitoring of dynamic insulin secretion. To do this we further characterized the capability of the system through generating and maintaining a complex chemical gradient in the device. In addition, we also developed the device to be a multiplexed system capable of simultaneous monitoring of dynamic insulin secretion in combination with multiple fluorescence probes. We also demonstrated the ability of this device to generate various stimulation profiles such as constant ramps, ramping up and down, and steps in a completely automated setup.
2 Methods and materials
2.1 Software simulation
The microfluidic devices were designed using CFD-GEOM software. For the original device, a 3-D computational grid was designed with inlet and outlet as straight channels with dimension of 2 mm in width, 19 mm in length, and 500 µm in height. With a large circular well (perifusion chamber, 7 mm wide and 3 mm deep) located in the middle. The bottom-layer consists of an array of tiny circular wells (500 μm diameter and 150 μm depth). Fluorescence simulation experiments indicated that in the original perifusion chamber there was uneven distribution with a time-delay between different regions, especially during the wash out period. We speculated that this was caused by the straight inlet design of the microchannel. To improve reagent mixing in the perifusion chamber, we applied the simulation of a computational grid using CFD-GEOM so that the inlet mixing channel tapered to the circular well was radically fanned out (inwardly) to a final width of 5 mm. Similarly, the outlet channel was designed to be radically fanned out (outwardly). Another modification was to place the inlet and outlet microchannel at the same level as the large circular well instead of being on top of the wells, intending to reduce liquid vibration and sudden hydrodynamic pressure changes. The modeled grids were used for simulating exchange of solution in the circular well and velocity of flow at an inlet flow rate of 1 ml/min in CFD-ACE software. Simulation of mixing in the device was done by setting the initial condition of the channel to have a fluid with 0 molar concentration of water and then we flowed in a fluid with 1 molar concentration of dye. The simulation results were processed using CFD-View software. These simulations were done at steady state.
2.2 Device fabrication
The device fabrication has been described previously (Mohammed et al. 2009). In brief, a 3-inch silicon wafer was first cleaned with acetone, methanol and isopropanol and dried with nitrogen gas. The silicon wafer was further treated with an oxygen plasma at 100 W for 30 s (Plasma Preen) to oxidize any residual organics. SU-8 photoresist was then spin coated on the wafer, patterned and developed using standard protocols. These steps were repeated for the second layer. PDMS was poured over each photoresist mold and cured at 80° for 1 h and 45 min. The inlet, outlet and large circular wells were formed on the appropriately cured PDMS using syringe needles or a cork borer. All of the layers were then bonded together using oxygen plasma to form the final perifusion device.
2.3 Mixing experiments
The efficiency of solution mixing in the perifusion chamber of the modified device was first characterized experimentally by using a fluorescence dye. In brief, DI water was perfused through the device for 30 s, followed by the dye (2 mM FITC, Sigma, MO) for 60 s. The dye was then flushed out with DI water. Time-lapse images were collected for different positions in the device with a high-speed, high-resolution charge-couple device (CCD, Retiga,SRV, Fast 1394 QImaging) and analyzed using image processing software (Slidebook,4.2, Olympus).
Complex glucose gradient profiles were characterized by mixing two different concentrations of glucose solutions delivered by two syringe pumps (Harvard Apparatus 22, Harvard Apparatus). One pump supplied a low glucose solution and another pump supplied a high glucose solution. The glucose solutions were mixed using a Y-shape connector (En-30726-41, Cole Palmer, Size 1/16 in. of ID) plus 10 cm tubing attached to it to ensure proper mixing before entering the inlet mixing microchannel. The flow rate and injection volume of both pumps were controlled by custom-made software developed through LabView system (Supporting Information, SI). The profiles of linear, bell-shape, and square-shape glucose gradients were assessed and compared to expected values. Glucose concentration was measured using a glucometer (Ascensia, Elites XL).
2.4 Isolation and culturing of islets of langerhans
The pancreatic islets of 8–12 weeks old C57/B 6 mice (Jackson Laboratory, Main) were isolated and cultured as previously described (Avila et al. 2006). Briefly, the pancreas was injected in a retrograde manner through the pancreatic duct with 0.375 mg/dl Collagenase P. The distended pancreas was incubated at 37°C for 15 min and shaken vigorously for 5 s to dissociate the pancreatic acinar tissue and then washed twice with HBSS. Discontinuous Ficoll (Mediatech, VA) gradients were used for islet purification. The islets were then cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone Inc, MA) and antibiotics at 37°C.
2.5 Simultaneous islet perifusion and fluorescence imaging
For all perifusion and imaging experiments, 25–30 mice islets were incubated with 5 µM Fura-2/AM (a calcium indicator, Molecular probes, CA) and/or 2.5 µM Rhodamin 123 (Rh123, a mitochondrial potentials indicator, Sigma, MO) for 30 min at 37°C in Krebs-Ringer buffer (KRB) containing 2 mM glucose. Then islets were introduced into the temperature-controlled microfluidic device through the inlet microchannel and mounted on an inverted epifluorescence microscope (Leica DMI 4000B). The loaded islets were then perifused by a continuous flow of KRB2 at 37°C (pH 7.4) for 10 min. KRB containing different concentrations of glucose and other stimulators were administered to the islets using a peristaltic pump or syringe pumps. Multiple islets could be simultaneously observed with 10–20× objectives. Dual-wavelength Fura-2 was excited ratiometrically at 340 and 380 nm, and changes in [Ca2+]c are expressed as F340/F380 (%). Rh123 is a lipophilic cation that partitions selectively into the negatively-charged mitochondrial membrane. Hyperpolarization of the mitochondrial membrane causes uptake of Rh123 into the mitochondria and a decrease in fluorescence is observed due to intermolecular crowding and quenching. Rh123 is excited at 495 nm; Excitation wavelengths were controlled by means of suitable excitation filters (Chroma Technology) mounted in a Lambda DG-4 wavelength switcher. Emission of Fura-2/AM and Rh123 fluorescence was filtered using a Fura2/FITC polychroic beamsplitter and double band emission filter (Chroma Technology. Part number: 73,100bs). SimplePCI software (Hamamatsu Corp) was used for imaging acquisition and analysis. These images were collected with a high-speed, high-resolution charge-coupled device (Retiga-SRV, Fast 1394, QImaging). In addition, aliquots were collected by a fraction collector from the device every minute to measure insulin secretion.
2.6 Insulin ELISA method
The perfusate samples were frozen at -80°C until quantification with the ELISA kit. Insulin was measured using a Mammalian or Mice Insulin ELISA (Mercodia AB, Uppsala, Sweden) kit, according to the protocol provided by the manufacturer.
3.1 Microfluidic device design and modification
The modified microfluidic device and experimental setup is shown in Fig. 1. The schematic depicts the cross-section view and experimental setup (left part) and an isometric view of the perifusion chamber is shown in the insert image (right part). The bottom layer of the device consists of an array of small circular (150 μm deep, 500 μm diameter) wells that help immobilize the islets exposed to flow. The next layer is a large circular well also called the perifusion chamber (3 mm deep, 7 mm diameter) that encompasses the array of the tiny wells. The modified mixing microchannel and outlet channel were placed at the same level as the perifusion chamber. A radically inward design (fanned out shape) tapered to the perifusion chamber was fabricated so that vortex fluid flowed downward into the perifusion chamber was accelerated and the flow of the liquid from the mixing chamber was unrestricted. The work energy of the liquid introduced into the perifusion chamber was substantially spent when the liquid reached the lower end of the perifusion chamber. An off-chip Y-shape mixer was also used to further help reagent mixing for the creation of a chemical gradient.
3.2 Software simulation and experimental simulation
3.3 The creation of glucose gradients in the microfluidic network
3.4 Spatial-temporal resolution of the islet perifusion system
3.5 Simultaneous islet perifusion and multiple-channel fluorescence imaging
With our modified design we also found that immediately after perifusion begins, a stable baseline release of insulin is established in a much shorter time (less than 10 min) when compared to the original design which took 20 min (data not shown) and no initial spike of insulin was recorded. The dramatic initial response of islets to a sudden increase of the glucose concentration has been previously described as a result of very sharp and rapid transient insulin peak followed by a return to or below baseline (Sweet et al. 2002). The modified design consisting of the fanned out inlet and outlet microchannel in addition to relocation of position could potentially be involved in dramatically reducing the hydrodynamic pressure changes experienced during the medium changes and mixing.
Previously, we presented a microfluidic perifusion system capable of simultaneously imaging multiple islets with a single fluorescence probe. This manuscript details a second generation of this device along with the added capability of a controlled glucose ramping with networked syringe pumps. Our results demonstrate the inlet channels have a significant influence on the flow dynamics and reagent mixing in our microfluidic system. Simultaneous and uniform distribution of the glucose solution in the perifusion chamber to all islets is very important for the application of islet perifusion technique. An important factor when studying the islet metabolism during transition between steady states is the time required for equilibration of the tested substance in the extracellular space of tissue. Our results indicate that the modified microfluidic system has a very quick and uniform mixing and reagent exchange in the perifusion chamber with no delay in the different regions inside the chamber. This was also confirmed in software simulation and experimental setup incorporating the fanned out channel design.
The second generation microfluidic perfusion system is capable of generating a complex concentration profile with simple control of syringe pumps. The underlying principle is to combine the simple geometry of design to construct composite stimulation profiles with a higher level of complexity and consistency. Our approach was to create a practically relevant system that is able to generate user-prescribed chemical gradients and profiles. The results indicate that this system can produce a linear glucose concentration profile, symmetric bell, and step profiles with a simple Y-connector in combination with our modified fanned out channel in agreement with expected values.
The system can also impose these profiles on islets while assessing multiple metabolic parameters simultaneously. The high spatial-temporal resolution of these parameters in response to glucose is well resolved by this system. This modified device uses a relatively small chamber volume (100μL) capable of measuring a characteristic biphasic insulin secretory pattern or other hormones using different flow rates without losing spatial-temporal resolution. Although, we observed minimal deviations of the experimental profile from the theoretical profiles in the creation of our chemical gradient and also in the flow dynamics at different flow rates. Attempts to optimize the spatial-temporal resolution of one parameter often interfered with the other parameters that we are simultaneously measuring; therefore, the current geometry of our device is a compromise for all three of our measured parameters. Thus, these small deviations were acceptable for our proposed semi-quantitative evaluation of human islet function. However, future applications of this device requiring a more precise and specific application may need to address these deviations. It is also worth noting that islet recovered from the chamber after experiments were morphologically intact, confirming that this device does not generate any physical stress to the islets.
Human islet transplantation has emerged as a promising alternative for Type I diabetes treatment with varying success rates. Currently, there is a lack of a comprehensive, quantitative, and robust method to assess human islets prior to transplantation, limiting the success rate. This perifusion system presented here could facilitate the development of a systematic, non-invasive approach to assessing islet metabolic and functional integrity in a real time manner prior to transplantation. In addition, this new device outlined here has other useful applications. The functional and anatomic integrity of islets maintained with continuous flow in the perifusion chamber suggests that this modified system could be used also for other tissues and cell types.
The flow characteristics of this microfluidic perifusion system allow for high spatial-temporal resolution experiments to assess islet endocrine function and metabolic parameters in whole islets. The present perifusion system is intended to provide a controlled experimental condition to correlate the dynamics of insulin release to metabolic events such as calcium influx and mitochondrial potential changes. These studies have demonstrated that our microfluidic device is an ideal tool for studying islet physiology in real time, allowing us to evaluate multiple islets in a very small volume with a rapid response. Future studies will focus this device to evaluate human islet potency prior to transplantation which could potentially lead to predicting islet transplantation outcomes.
Work was supported by American Association of University Women International fellowship to Adeola Adewola, NIH/NCRR (U42RR023245) to Jose Oberholzer and The Chicago Diabetes Project.