Fig 1.
Mouse pancreatic islets adhere to silk matrices, with a preference for foam with RGD.
(A) Photographs of the various formats; fiber (left), foam (middle) and film (right) and micrographs together with adhered islet (lower panel, asterisks). Scale bars = 50μm. (B) Islet adherence after different time points (days). Islets adhered better to foam, fiber and film, compared to the control (n = 16, in duplicates, ***p<0.001). (C) Schematic descriptions of the spider silk proteins used; wild type 4repCT (WT) and 4repCT with different cell binding motifs (RGD, IKVAV and YIGSR) incorporated N-terminus of 4repCT. The non-functional peptide RGE was incorporated as control for RGD. (D) Islet adherence at day 1–2 (white bars) and day 5 (black bars) on foam with different motifs (n = 6, in duplicates). Islets adhered better to all silks compared to control (*** p≤0.001 at day 2, * p≤0.05 for WT and RGE at day 5, and **p≤0.001 for RGD, IKVAV and YIGSR at day 5). Islets adhered better to RGD compared to WT (*** p≤0.001 for day 2 and day 5) and RGE (*** p≤0.001 for day 2 and *p≤0.05 for day 5).
Fig 2.
Mouse pancreatic islets are maintained functional when adhered to silk foam.
Insulin release (ng/10 islets*hour) from adhered islets after 2 days (A) and 5 days (B) in culture on silk matrices (WT, RGD, IKVAV, YIGSR and RGE), compared to non-adhered islets in control wells. Insulin released after low glucose concentration (3mM) is shown in white bars and insulin released after stimulating glucose concentration (16.7mM) is shown in black bars. At day 2, adhered islets on RGD and YIGSR released more insulin per islet after glucose stimulation compared to control islets (n = 6, triplicates *p≤0.05). (C) Representative pattern of F340/F380 traces due to glucose and KCl induced changes in [Ca2+]i in single mouse islets cultured for 1–2 days on WT, RGD or free floating. (D) Mean value of increase in ratio due to stimulation by 11 mM glucose. (E) Mean value of islet oscillations per minute for slow (light gray) and fast (dark gray) oscillations in [Ca2+]i after high glucose stimuli. (F) Mean value of increase in ratio due to stimulation by 25 mM KCl. (>17 islets from each condition).
Fig 3.
Adherence to silk matrices leads to improved in vitro islet maintenance with preserved endothelial cells.
(A) Representative (n = 5) micrographs of islets after 2 weeks in control wells (left), on WT foam (middle) and on RGD foam (right). Scale bars = 50 μm. (B) Upper graph: Percentage of amount of islets with intact islet architecture after 2 weeks free floating in control wells or adhered to silk foam (WT, RGD, IKVAV, YIGSR and RGE). Lower graph: Mean value of necrotic bodies per islets after 2 weeks in control wells or on foam. Islets on WT, RGD and IKVAV foam showed less necrotic bodies compared to control (n = 5, in duplicates **p≤0.01). (C) Representative (n = 3) maximum intensity projection (MIP) micrographs of pancreatic mouse islets at the day of isolation (upper panel) and after 2 weeks in culture (lower panel). Insulin staining is shown in green and the endothelial marker CD31 is shown in red. Scale bars = 50 μm. (D) Percentage of islets with preserved endothelial cells (CD31-positive stained islets) after 2 weeks in culture (data pooled from 3 experiments, in total 22 islets *p≤0.05).
Fig 4.
Islets maintained on RGD foam show improved in vivo survival with vascularization.
(A) Percentage of islets that showed a stable or positive increase in size over the 4 weeks period in the eye. (5 mice, 1–6 islets per eye). (B) Percentage of control islets (white bars) and islets from RGD foam (black bars) that showed clear vascularization by transmitted light imaging 4 weeks post transplantation. (n = 3 separate transplantation experiments, 12 recipient mice in total, 1–6 islets per eye). (C) Representative (n = 3) bright field MIP micrographs of control islet (left) and islet from RGD foam (right) during in vivo imaging of the eye, where vasculature can be seen as grey areas. Scale bars = 50 μm. (D) Morphology by H/E (left panel) and insulin (green, right panel) staining of eye sections showing representative (n = 3) control islet (upper graphs) and islet from RGD foam (lower graphs). Vasculature was seen in islets from both culture conditions (white arrowhead) although vessels with erythrocytes were more common in islets from RGD foam. Areas of visual cell death were sometimes present in control islet (white lined circle). Scale bars = 50 μm. (E) Vasculature with erythrocytes were found within all (100%) of the islets from RGD foam (black bar), compared to in only 50% of the control islets (white bar)(mean % of islets found per eye ± SEM). (n = 3, 5–10 islets per eye **p≤0.01). (F) Cell death (light grey) and insulin negative cells (dark grey) was seen to a higher degree in control islets compared to islets cultured on RGD foam (mean per islets found per eye ± SEM). (n = 3, 5–10 islets per eye *p≤0.05, **p≤0.01).
Fig 5.
Human pancreatic islets adhere to silk matrices with maintained in vitro function for 3 months.
(A) Islet adherence to silk matrices with various motifs (WT, RGD, IKVAV, YIGSR and RGE) after 1 to 5 days in culture. Islets adhered better to all silks compared to control (n = 6, triplicates **p≤0.01, ***p≤0.001). More islets adhered to RGD foam compared to RGE (***p≤ 0.001). (B) Stimulation index of adhered human islets after cultured for 5 days on foam compared to non-adhered islets in control well. (n = 6, triplicates) (C) Islet adherence after 1 month of culture. No adhered islets were observed in the control wells. Islets on RGD foam showed improved adherence compared to WT and RGE (n = 4, in triplicates ***p≤0.001). (D) Islets remaining after 1 month in culture had maintained function of insulin release. The stimulation index of islets in control wells (non-adhered) and adhered to foam were all above 2 (n = 4, in triplicates). (E) Insulin produced after stimulation of all remaining islets within a well after 1 month of culture. Islets cultured on RGD foam showed increased insulin release compared to control islets (n = 4, in triplicates, **p≤0.01). (F) Upper graph: Percentage of islets with intact islet architecture after 1 month in control wells or on foam. Islets were better maintained when cultured on WT and RGD compared to the islets in the control wells (n = 5, triplicates, *p≤0.05, **p≤0.01). Lower graph: Mean value of necrotic bodies per islet after 1–3 months in control wells or on foam. Islets adhered to WT and RGD foam showed less necrosis compared to control islets (n = 4, triplicates **p≤0.01). (G) Representative micrographs of islets after 3 months in control wells (top), wells with WT foam (middle) and RGD foam (bottom). Scale bars = 100 μm.
Fig 6.
Human pancreatic islets are well established with connecting cell growth following the shapes of the silk matrices.
(A) Human islets adhered preeminent to the RGD foam (bright field) and were connected in a network by cell outgrowth following the foam structure (cell nuclei seen in blue). (B) The islets maintained viable and produced insulin (green) after culture for 4 weeks (cell nuclei seen in blue). (C) Mean value of number of ordinary cell outgrowths (black) and angiogenic sprouts (white) found per human islets cultured for 4–12 weeks on different matrices. Islets adhered on RGD foam showed more cell outgrowth and sprouts compared to control, WT and RGE (n = 4, triplicates *p≤0.05 and **p≤0.01). (D) Example of angiogenic sprouting (white arrowhead). Endothelial cells (red) emanating from one insulin positive (green) islet and connecting to another (cell nuclei seen in blue). (E) Except for maintained islets (asterisks) some newly formed insulin-positive (green) islet-like clusters (arrow head) were found, and these were also connected by sprouting endothelial cells (red) guided by the foam structure. Upper left: the same picture in bright field (cell nuclei seen in blue). (F) Mean value of islet-like clusters after 4–12 weeks from young (white bars) and old (black bars) islet donors. Most islet-like clusters were found on the RGD foam. Islets from young donors showed increased number of islet-like clusters compared to the old islet donors (2 young donors, islet wells in triplicates and 3 old donors, islet wells in triplicates **p≤0.01). Scale bars = 50 μm.