Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells.

Microfluidic Device Fabrication Devices were fabricated using elastomer polydimethylsiloxane (PDMS) (Dow-Corning) as described previously [16] , [17] . The cured mould was irreversibly bonded to a 24×50-mm coverslip (VWR Scientific) by oxygen plasma treatment (Harrick Scientific, Ossining, NY) and Tygon tubing was inserted directly into the cored-out port holes.

Microfluidic Device Design

The microfluidic device design and process are described in detail in the Supplemental Material ( Text S1 & Fig. S1 , S2 , and S3 ). Briefly, islets were brought into 125 µm tall ×300 µm wide microfluidic channels through inlet port tubes. Islets moved along these channels by media flow until they reached a dam wall. This dam structure was 25 µm tall, 1,800 µm wide, and 0.13 cm long, and allowed solution to flow past but blocked the movement of islets. Microfluidic devices provide only non-turbulent or laminar flow at the flow rates used in these studies [14] . Two main designs were used for these studies: a three-channel microfluidic device and an independent-channel microfluidic device ( Fig. S1 ). The three-channel device placed islets in three different flow-rates based on varied channel lengths and by using a single syringe pump. The independent-channel device provided a single flow-rate in parallel channels to facilitate subsequent live cell imaging or effluent collection.

Ethics Statement

Animal procedures were approved by the Animal Care Committee of the University Health Network, Toronto, Ontario, Canada in accordance with the policies and guidelines of the Canadian Council on Animal Care (Animal Use Protocol #1531).

Pancreatic Islet Isolation and Treatment

Pancreatic islets were isolated from 8- to 12- week-old C57BL6 male mice by using collagenase digestion (Roche) [18] , [19] . Islets were cultured in RPMI medium 1640 containing 11 mM glucose, 10% FBS, 5 U/ml penicillin-streptomycin, and 20 mM HEPES. Control islets were incubated in a non-treated culture dishes in a humidified incubator at 37°C and 5% CO 2 . Device-treated islets were loaded into the microfluidic devices shortly after isolation and incubated in a custom-built desk-top incubator described in detail in the Supplemental Material ( Fig. S2 ). Briefly, the microfluidic device was submerged in a stirred water bath at 37°C with flow driven by a syringe pump. The reservoir media was also submerged in a separate water-bath maintained just above 37°C to reduce formation of air bubbles in the microfluidic channel. A cap of mineral oil was placed on top of the media in the reservoir to reduce evaporation and drift in pH.

Immunofluorescent Detection

Control and device-treated islets were labelled within the microfluidic device. Islets were fixed (2% PFA, PBS, 1 hr, 100 µL/hr), blocked (PBS, 0.1% TritonX-100, 4°C, 10% Normal Goat Serum, 4 hr, 15 µL/hr), incubated with primary antibody (PBS, 0.1% TritonX-100, 4°C, 1% Normal Goat Serum, 1∶100 dilution rat anti-mouse PECAM-1, 3 hr, 3 µL/hr), rinsed (PBS, 0.1% TritonX-100, 4°C, 10% Normal Goat Serum, 1 hr, 15 µL/hr) and incubated with 1∶500 goat anti-rat Alexa 633 (PBS, 0.1% TritonX-100, 4°C, 10% Normal Goat Serum, 3 hr, 15 µL/hr). Islets were imaged using a 20×0.50 NA objective lens in 3 µm steps for 16–20 slices (dependant on the size of the islet) on an Olympus FluoView 300 microscope. Images were analyzed using ImageJ 1.41o (Wayne Rasband, NIH, USA). EC fractional area was measured by calculating the area of PECAM-1 labelling divided by the total area of the islet (6 slices/islet). Total connected length was determined using Simple Neurite Tracer throughout the entire stack of images. PECAM-1 labelled regions were tracked through the stack and the resulting summation was normalized to the circumference of the islet. The average of individual islets was used for statistical analysis. A two tail, unpaired, two sample student's t-test was used and significance was determined with p