Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells - PubMed (original) (raw)
Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells
Krishana S Sankar et al. PLoS One. 2011.
Abstract
Pancreatic islets are heavily vascularized in vivo with each insulin secreting beta-cell associated with at least one endothelial cell (EC). This structure is maintained immediately post-isolation; however, in culture the ECs slowly deteriorate, losing density and branched morphology. We postulate that this deterioration occurs in the absence of blood flow due to limited diffusion of media inside the tissue. To improve exchange of media inside the tissue, we created a microfluidic device to culture islets in a range of flow-rates. Culturing the islets from C57BL6 mice in this device with media flowing between 1 and 7 ml/24 hr resulted in twice the EC-density and -connected length compared to classically cultured islets. Media containing fluorescent dextran reached the center of islets in the device in a flow-rate-dependant manner consistent with improved penetration. We also observed deterioration of EC morphology using serum free media that was rescued by addition of bovine serum albumin, a known anti-apoptotic signal with limited diffusion in tissue. We further examined the effect of flow on beta-cells showing dampened glucose-stimulated Ca(2+)-response from cells at the periphery of the islet where fluid shear-stress is greatest. However, we observed normal two-photon NAD(P)H response and insulin secretion from the remainder of the islet. These data reveal the deterioration of islet EC-morphology is in part due to restricted diffusion of serum albumin within the tissue. These data further reveal microfluidic devices as unique platforms to optimize islet culture by introducing intercellular flow to overcome the restricted diffusion of media components.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. A microfluidic device to treat pancreatic islets with laminar flow.
(A) A schematic of a microfluidic channel is shown to demonstrate how pancreatic islets are held in laminar flow. Pancreatic islets (oval) are brought into microfluidic channels (outlined) and trapped when the channel height drops from 125 to 25 µm. This stops the islet from moving down the channel while allowing fluid to flow past the islet (arrows). (B) A representative image of islets held at the ‘pea pod’ shaped dam in a channel. In this experiment, the islets were cultured for 24 hr prior to loading. The islets are sitting in the tall channel (125 µm) against the dam wall to the right. Flow in the device is past the islets from left to right. (C) An image of the three channel microfluidic device used for varying fluid flow-rates past islets. The device shown is filled with bromophenol blue solution to highlight the channels and tubing. The device has three individual inputs (tubing) with a common output (not shown). The three microfluidic channels (125 µm) each have a dam for islet retention (spherical region, 25 µm) followed by varying lengths of output channels (125 µm). These varied channel lengths allowed media flow to be controlled with one syringe pump, and media flow at three different flow-rates relative to length. Flow rates were ultimately determined by measuring the height of the input reservoirs before and after incubation.
Figure 2. The effect of fluid-flow on EC area and connected length.
Pancreatic islets were cultured in either the microfluidic device with flow (device-treated) or in non-flowing media in an incubator (control) followed by anti-PECAM-1 immunolabelling. (A) A representative Day 0, freshly isolated islet shows heavy vascularization of the tissue prior to culture. (Scale bar 125 µm) (B) A representative control islet cultured in no-flow for 24 hr displaying characteristic coalescence of EC. (Scale bar 100 µm) (C) A representative islet cultured for 24 hr in a microfluidic device displaying increased EC density and connected length compared to control islets (Cntl(0)). (Scale bar 150 µm) (D) The total EC connected length relative to the circumference (EC Length L/c) of freshly isolated islets (hatched bar), control islets (Cntl(0)) at 24 (black bars) and 48 hr (open bars), and device-treated islets exposed to fluid flow (1–3, 3–5, and 5–7 ml/24 hr). (E) The EC area relative to islet area (EC percent area) of freshly isolated islets (hatched bar), control islets (Cntl(0)) at (24 (black bars) and 48 hr (open bars), and device-treated islets exposed to fluid flow (1–3, 3–5, and 5–7 ml/24 hr). The EC length and fractional areas were measured from the islets isolated from at least 3 different mice on independent days. The data shown is the mean ± sem for the following number of pooled islets: 43 (Day0), 14 (Control, 24 hr), 12 (24 hr, 1–3 ml/24 hr), 19 (24 hr, 3–5 ml/24 hr), 10 (24 hr, 5–7 ml/24 hr), 16 (Control, 48 hr), 10 (48 hr, 1–3 ml/24 hr), 7 (48 hr, 3–5 ml/24 hr), and 7 (48 hr, 5–7 ml/24 hr). (F) The EC fractional area for the data shown in Fig. 2E re-grouped into large and small islets based on the median diameter (178 µm). Data shown are the mean ± sem for the following number of pooled islets: 21 (Large Islets, Day0), 22 (Small Islets, Day(0)), 9 (Large Islets, Cntl(0)), 8 (Small Islets, Cntl(0)), 12 (Large Islet, 1–7 ml/24 hr), and 12 (Small Islets, 1–7 ml/24 hr). The * and ** indicate p<0.05 and p<0.01 compared to control (Cntl(0)) or where otherwise indicated.
Figure 3. Media access in a microfluidic device measured using fluorescent dextran.
To explore the nature of the fluid flow inside treated islets, real-time fluorescent imaging was applied to 24 hr device-treated islets (3 ml/24 hr). (A) We flowed fluorescent dextran (3,000 MW) into the microfluidic device and imaged penetration of this dye into the tissue. The dye fills the channel and intercellular spaces of the islet. Connected (path-like) regions that resemble islet-EC morphology (arrow) were immediately evident (black scale bar = 40 µm). (B) A magnified image shows that fluorescent dextran surrounds each cell in the islet (Scale bar = 15 µm). (C) These same islets were immunofluorescently labelled for ECs using PECAM-1 immunofluorescence. The connected regions identified by fluorescent dextrans co-labelled as ECs (compare arrows in A and C) (Scale bar = 40 µm). (D) To measure the kinetics of fluorescent dextran exchange with the intercellular space, we examined the arrival of the dye as it was introduced into the channel. A typical time series is shown with media flowing 3 ml/24 hr from the right to left. The images shown were acquired at 0, 12, and 101 s after the arrival of fluorescent dextran to the islet until it eventually fills both the channel and intercellular space. In the absence of dye, the channel and islet are dark (0 s). At 12 s after the dye arrival, the channel had filled with dye except for a tail of non-labelled solution pushed from the unlabelled islet. At 101 s after the dye arrival, the intercellular spaces were clearly visible and the dye completely filled the channel. (E) To determine the dependence of media exchange on flow rate, we measured the time required to penetrate the islet at various flow rates. These data were collected from the islets of 4 separate mice on different experimental days. The data is plotted as mean clearance time ± sem for the pooled islet data. (F) Islets were cultured in non-flowing media for less than 24 hr prior to imaging. Fluorescent dextran images were collected in a sequence similar to (D), but with spatial resolution similar to (A). This imaging sequence allowed us to measure the intensity of fluorescent dextran at EC structures (EC, black line) or neighbouring intercellular spaces (Intercellular, grey line). The arrow indicates the arrival of fluorescent dextran at the islets. The fractional intensity was determined from the intensity at time zero and the maximal intensity reached at each region at >100 s. Data are shown from the average ± sem for 90 neighbouring pairs of regions from 7 different islets. No difference in arrival rate was measured between EC and intercellular spaces indicating no preference for flow through the lumen of the ECs.
Figure 4. Effect of serum albumin on EC morphology.
Islets were cultured for 24 hr in normal culture (Control, 10% FBS) and the microfluidic device (3 ml/24 hr flow rate). The media used in the device was either normal media (10% FBS), serum free media (Serum Free) or serum free media containing 4% bovine serum albumin (4% BSA). The islets were then fixed and immunofluorescently labelled for PECAM-1. (A) Representative maximum projection image of PECAM-1 immunofluorescence for each culture. (B) EC Percent Area of control islets (open bar) and device treated islets (black bars) cultured as indicated. (C) The relative EC length of control islets (open bar) and device treated islets (black bars) cultured as indicated. Each bar consists of the data collected from 10 to 17 islets collected from 3 mice, with the islets from each mouse isolated on independent days. The * and ** indicate p<0.05 and p<0.01 compared to control (open bar, 10% FBS), respectively by two-tailed student's t-test.
Figure 5. Measuring beta-cell [Ca2+]-activity using Fura-2.
Islets subjected to 48 hr of fluid-flow (3 ml/hr) followed by labelling with Fura-2 to evaluate beta-cell [Ca2+]-responses. (A) Typical control and device-treated islets at 2 mM glucose displayed a consistent flat base-line [Ca2+]-response. After the addition of 11 mM glucose, control islets displayed normal [Ca2+]-oscillations while the device-treated response was small and flat. Data is representative of the response from a total of 11 islets from N = 3 mice, with the islets from each mouse isolated on independent days. (B) Control islets typically displayed fast Ca2+-oscillations at 11 mM glucose while the response of device treated islets was flat. (C) The change in 340∶380 nm ratio is the transition from 2 to 11 mM glucose in control and device-treated islets. (D) The change in 340∶380 nm ratio transitions from 11 mM glucose to 30 mM KCl in control and device-treated islets. The similar 30 mM KCl response delta indicates similar electrical activity in response to membrane depolarization. Each bar consists of the data collected from a total of at least 6 islets from 3 mice, with the islets from each mouse isolated on independent days. The * indicates p<0.05 compared to control by two-tailed student's t-test.
Figure 6. Monitoring synchronous beta-cell [Ca2+]-response in pancreatic islets using Fluo-4.
Islets were cultured in control or microfluidic device flow (3 ml/24 hr) for 48 hr prior to labelling with Fluo-4. The dye labels cells as the periphery of the islet as observed using confocal microscopy by a ring of labelled cells. Representative control (left, A) and device-treated (right, B) islets are shown, each with three regions of interest (ROI) that are subsequently plotted in (C–F) (Scale bar 120 µm, 90 µm respectively). (C & D) At 2 mM glucose, the [Ca2+]-response is flat as expected in both the control (left, C) and device-treated islets (right, D). (E & F) At 11 mM glucose, [Ca2+]-oscillations were synchronous in functioning control islets (E, compare ROI 1, 2 and 3); however, no [Ca2+]-oscillations were observed in the device-treated islets (F) suggesting moderate damage to the periphery of the islet.
Figure 7. Glucose-stimulated NAD(P)H and insulin responses.
Islets were cultured in control conditions or in microfluidic device flow (3 ml/24 hr) for 48 hr. (A) Control and flow treated islets were subsequently treated with 2 and 20 mM glucose and imaged using two-photon excitation of NAD(P)H. Scale bar in top left panel is 50 µm. (B) The fold NAD(P)H intensity from control and device treated islets. Data shown is the mean ± sem for 21 and 24 islets collected from 3 separate mice on independent days. The * indicates p<0.05 compared to control by two-tailed student's t-test. (C) Islets were cultured in control or in microfluidic device flow (device, 3 ml/24 hr) for 48 hr. The control islets were placed in a microfluidic device and both control and flow treated islets were subsequently treated with 2 mM glucose followed by 11 mM glucose in imaging buffer. The paired effluent was collected from each channel containing 4–6 islets. Insulin levels were then normalized to total insulin per channel using effluent collected with Triton X-100 permeabilization. A comparison between control and device-treated islets shows similar basal insulin levels with an increase in secretion at the higher glucose concentration. The data shown are from duplicate channels from 4 mice collected on independent days.
References
- Olsson R, Carlsson PO. The pancreatic islet endothelial cell: Emerging roles in islet function and disease (Reprint vol 38, pg 492–497, 2005). International Journal of Biochemistry & Cell Biology. 2006;38:710–714. - PubMed
- Nyqvist D, Kohler M, Wahlstedt H, Berggren PO. Donor islet endothelial cells participate in formation of functional vessels within pancreatic islet grafts. Diabetes. 2005;54:2287–2293. - PubMed
- Bretzel RG, Jahr H, Eckhard M, Martin I, Winter D, et al. Islet cell transplantation today. Langenbecks Archives of Surgery. 2007;392:239–253. - PubMed
- Zoellner H, Hofler M, Beckmann R, Hufnagl P, Vanyek E, et al. Serum albumin is a specific inhibitor of apoptosis in human endothelial cells. J Cell Sci. 1996;109(Pt 10):2571–2580. - PubMed
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