Supporting Information
Supplemental Materials and Methods
Mice
Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, Maine) were housed in virus antibody–
free rooms in microisolator cages and exposed to a 12-h light/dark cycle with ad libitum access
to autoclaved food and water and were used at 11-13 wk of age as islet recipients and at 11-13
wk as islet donors. Animal studies were performed under protocols reviewed and approved by
the University of Miami Institutional Animal Care and Use Committee. Isolation and
transplantation of murine pancreatic islets were performed at the DRI Preclinical Cell Processing
and Translational Models Core.
Pancreatic Islet Isolation
Islets were obtained from donor pancreata of 11-13 wk old male C57BL/6 mice by a
mechanically enhanced enzymatic digestion using collagenase (Sigma-Aldrich, St. Louis, MO)
followed by separation on discontinuous density gradients (Mediatech; Herndon, CA), as
previously described (Pileggi et al. 2005). Islets were cultured overnight (37°C, 5% CO2) in
CMRL-1066 medium (Gibco-Invitrogen; Carlsbad, CA) supplemented with 10% fetal bovine
serum (Gibco-Invitrogen), 2 mM L-glutamine (Gibco-Invitrogen), 50 U/ml penicillin (GibcoInvitrogen), 50 µg/ml streptomycin (Gibco-Invitrogen) and 25 mM HEPES (Gibco-Invitrogen)
in non tissue culture-treated Petri dishes. Before transplantation, islets were counted as islet
equivalents (IEQ) and aliquoted in non-tissue culture-treated Petri dishes.
Diabetes Induction
Mice were rendered diabetic by a single intravenous injection of streptozotocin (STZ, 200
mg/kg; Sigma-Aldrich). Only mice that were overtly diabetic (non-fasting blood glucose levels
≥350 mg/dl for three consecutive days before transplantation) were used as recipients.
Ex-vivo Culture of Islets in Engineered Fibrin Gels
In parallel to islet transplantation in mice, 450 IEQ islets per experimental condition were
cultured ex vivo and imaged at 2, 3, 5 and 11 days after seeding to evaluate sprouting of isletresident cells (including endothelial and fibroblastic cells). Islets were collected and transferred
to a low-binding 0.5ml microfuge tube. Islet residual supernatant was removed and islets were
seeded on tissue culture-treated glass bottom Petri dishes (‘ISL AL’) or resuspended within 80 µl
of engineered fibrin gels with bound FNIII9-10/12-14 (without GFs for ‘ISL+FIB mix’, and with
hVEGF-A165 and hPDGF-BB for ‘ISL+FIB+GF mix’ and ‘ISL+FIB+GF w/o APR mix’
experimental groups) and immediately transferred to tissue culture-treated glass bottom Petri
dishes for polymerization to occur. Phase contrast images were taken with a Zeiss inverted
microscope at low magnification (5x) at each time point (2, 3, 5 and 11 days after seeding).
Images were quantified with ImageJ to quantify sprouting of islet-resident cells (by measuring
area occupied by single cells outgrowing from islets and dividing them by the total area, at least
5 different fields containing 2-5 islets).
Assessment of Graft Function
Graft function was monitored by measuring non-fasting glycemic values on whole blood samples
obtained from the tail of the animals using portable glucometers (OneTouch Ultra 2; LifeScan)
daily or twice per week. Mice with stable non-fasting glycemic levels <200 mg/dl were
considered euglycemic. To exclude residual function of the native pancreas after long-term
follow-up, the graft-bearing EFPs were explanted and non-fasting glycemic values monitored in
the subsequent days to observe prompt return to hyperglycemia.
Graft Histology, Immunofluorescence and Imaging
Subcutaneous grafts were embedded in Tissue-Tek OCT (Sakura; Torrance, CA) and snapfrozen. Frozen samples were sectioned (75 µm thick sections for analysis at day 7 after
transplant and 5 μm thick sections for analysis at day 21 after transplant) and either processed for
standard H&E histology or fixed in cold acetone and processed for immunofluorescence. Graftbearing EFPs were fixed in 10% buffered formalin and embedded in paraffin blocks. Samples
were sectioned (5 µm) and either processed for standard H&E histology or processed through
antigen retrieval (sodium citrate buffer) before proceeding with immunofluorescence processing.
For immunostaining, samples were permeabilized in 0.2% Triton X-100 (Sigma) at RT for 5-10
min, then blocked in 2% bovine serum albumin (Sigma) and 10% serum (Sigma) of the host of
the secondary antibody in phosphate buffered saline (PBS, Invitrogen) for 90 min at RT. Primary
antibodies were prepared at working concentrations in 2% BSA in PBS and incubated with the
tissue sections overnight at 4°C. Secondary antibodies were purchased from Molecular Probes
(Eugene, OR) and prepared at 1:200 working concentration in 2% BSA in PBS and incubated
with the tissue sections for 1 hr at RT. Nuclear staining was obtained with 4’,6 diamino-2phenilindole (DAPI; Molecular Probes). Islets were identified by insulin+ beta cells (guinea pig,
1:100; Dako, Carpinteria, CA) and glucagon+ alpha cells (rabbit, 1:100; Biogenex, Fremont,
CA). Blood vessels were identified by CD31+ (frozen sections: rat, 1:50, BD Biosciences, San
Jose, CA; formalin/paraffin sections: rabbit, 1:20, Abcam, Cambridge, MA) and Lyve-1- (rabbit,
1:100, Reliatech, Angio-Proteomie, Boston, MA) structures.
Images were acquired at the DRI Imaging Core with a Leica DMIRB microscope for histology
(Leica, Richmond, IL) or a Leica SP5 inverted confocal microscope (for fluorescence imaging).
Density of islets and blood vessels within islets were quantified with ImageJ (NIH). Briefly, islet
density was quantified by dividing the area of insulin+ cells (obtained by threshold of the
fluorescent staining signal over the negative background) by the total area of the graft. Blood
vessel density within islet grafts was quantified by dividing the area of CD31+Lyve-1- blood
vessels (obtained by threshold of the fluorescent staining signal over the negative background)
by the area of insulin+ cells.
Statistics
Unless otherwise noted, data are presented as mean ± SD. Statistical comparisons were based on
Student’s t-test or analysis of variance (ANOVA) with Tukey post hoc test for pairwise
comparisons. A confidence level of 95% was considered significant. Actuarial survival curves
and log-rank test were used to compare diabetes reversal amongst experimental groups. Prism
5.0 for Macintosh software (Graphpad, San Diego, CA) was used for analysis.
References
Pileggi A, Molano RD, Berney T, Ichii H, San Jose S, Zahr E, Poggioli R, Linetsky E, Ricordi C,
Inverardi L. 2005. Prolonged allogeneic islet graft survival by protoporphyrins. Cell
Transplant 14(2-3):85-96.