Orbital Shaker vs. Static Transduction and Insulin Secretion.

(A) Confocal tilescan images of Ca²+ sensor Ad-CMV-GCaMP6m expression in whole human pancreatic tissue slices (nPOD 6558), transduced on an orbital shaker or in static culture with no aprotinin. Slices were transduced using 2.6 × 10L PFU of Ad-CMV-GCaMP6m per tissue slice, incubated for 24 hours at 37°C, and imaged 48 hours post-transduction. (B) Transduction efficiency was quantified using Mean Fluorescence Intensity (MFI), (± SEM, n = 5, students unpaired t-test, ** p < 0.01). (C) Dynamic insulin secretion of human pancreatic tissue slices from three to four nondiabetic donors were cultured on an orbital shaker (blue) or in static culture (red) after 24 and 48 hours, with and without aprotinin (apr). Insulin secretion traces from slices on Day 0 (gray) is repeated on all plots as a control. Slices were initially perfused with 5.5 mM glucose (5G) for 10 minutes, followed by stimulation with 16.7 mM glucose (16.7G) for 30 minutes. Data are normalized to the average baseline secretion at 5.5G. (D) Quantification of insulin secretion from perifusion assays in (C), shown as the area under the curve (AUC) for 16.7G stimulation at 0, 24 and 48 hours with or without aprotinin. AUC data from slices on Day 0 (gray) are provided as a control. (E) Same AUC data as in (D), but with aprotinin and no-aprotinin groups combined at 24 and 48 hours. (± SEM, n = 18, students unpaired t-test, * p < 0.05). The data are representative of the average of three to four donors, with each donor contributing four slices.

Endocrine and Exocrine Function Retained 72 Hours Post Adenoviral Transduction.

Realtime confocal time-lapse perfusion study of pancreatic tissue slices (nPOD 6559) expressing the adenoviral Ca2+ biosensor CMV-GCaMP6m, transduced using an orbital shaker (bottom row) or static culture (top row). Endocrine and exocrine tissues were stimulated using perfusion of either 16.7 mM high glucose (16.7G) or 10 μM carbachol, respectively. (A) Images were selected from the real-time perfusion study at the peak Ca2+ influx of the 16.7 mM stimulation, islets outlined in white. (B) Heat maps of realtime Ca2+ responses in individual cells in both islet and acinar tissues shown in (A), imaged 48 hours post-transduction. Baseline activity was first acquired by perfusion of low 3 mM glucose (3G) for five minutes, followed by 16.7G, eliciting Ca2+ influx within the pancreatic islet. High glucose was washed away by perfusion of 3G, followed by stimulation of acinar tissue using 10 μM carbachol. (C) Average real-time Ca2+ traces of (n=3) slices following the protocol outlined in (B). The X-axis is segmented to separately show Ca2+ influx in the islet (left) and acinar tissues (right). (D) Quantification of the Ca2+ traces in (C), shown as the area under the curve (AUC) for 16.7G stimulation in the islet or 10 μM carbachol stimulation in the acinar tissue. (± SEM, n = 5, students unpaired t-test, * p < 0.05, ** p < 0.01) (E) Histograms depicting the sum of pixel intensity values at peak Ca2+ influx in the islet during high glucose stimulation and in the acinar tissue during carbachol stimulation. (Kolmogorov-Smirnov nonparametric cumulative distributions test, **** p <.0001).

Targeted expression in specific cell types.

Confocal images of beta cell-targeted expression of adenoviral vectors INS-GRX1-roGFP2 (green) and INS-jRGECO1a (red). (A) Representative image of a human pancreas slice alongside higher magnification images of islets within the tissue (nPOD 6548). Slices were transduced using 2.6 × 10L PFU of INS-GRX1-roGFP2 per tissue slice, incubated for 24 hours at 37°C, and imaged 48 hours post-transduction. (B) Dual expression of INS-GRX1-roGFP2 (green) and INS-jRGECO1a (red) in an islet within a human pancreas slice. Dual expression was achieved in slices using 1.4 × 10L PFU of INS-GRX1-roGFP2 and 1.4 × 10L PFU of INS-jRGECO1a per tissue slice, incubated for 24 hours at 37°C. Confocal images were taken 48 hours post-transduction. (C) Perfusion of human pancreatic tissue slice in a Warner imaging chamber using 3 mM glucose for 3 minutes, followed by a 20 μM H2O2 stimulation. ROS detoxification was achieved by perfusing 1 mM DTT at 12 minutes.

Functional Ca²+ Biosensor, CaMPARI2, in Living Human Pancreas Tissue Slices (nPOD 6617 ND).

Cells that respond to glucose stimulation experience an influx of calcium ions. (A) An increase in intracellular calcium, along with exposure to ultraviolet (UV) light for photoconversion (PC), irreversibly converts the fluorescence of CaMPARI2 from green to red. (B) Schematic of CaMPARI2 setup including 24 well plate on 405 nm LED array controlled by pulse generator delivering 10 sec pulses for 5 min. (Inset) Single well containing transduced pancreatic tissue slice in 16.7G high glucose with photoconverted beta cells. (C) Slices transduced with the CaMPARI2 biosensor were incubated at 37°C in high or low glucose for ten minutes, followed by exposure or no exposure to PC light. PC light-treated groups were exposed using ten second pulses for five minutes. Tissue slices were then chemically fixed with paraformaldehyde for staining. (D, E) Glucose responsiveness in beta cells was quantified by calculating the CaMPARI2 red:green ratio. Only cells that were positive for insulin were quantified (n = 3 slices per group). Data were analyzed per beta cell or per islet. (± SEM, one-way ANOVA with Tukey’s post-hoc test, *** p < 0.001, **** p < 0.0001, outliers removed with ROUT Q = 1%).

Donor characteristics from nPOD cases.

Table summarizes donor information and applied figure.

Donor characteristics from Prodo Labs.

Table summarizes donor information and applied figure.

Functional Ca2+ Biosensor, CaMPARI2, in Living Human Pancreas Tissue Slices (nPOD 6622 ND Control).

(A) Slices transduced with the CaMPARI2 biosensor were incubated at 37°C in 3 mM and 16.7 mM glucose for ten minutes, or 30 mM KCl for one minute. Slices were then exposed to PC light using ten second pulses for five minutes before being washed with PBS. Tissue slices were then chemically fixed with paraformaldehyde for staining. Beta cells displayed strong calcium activity upon high glucose stimulation, resulting in significant photoconversion. (B) Glucose responsiveness in beta cells was quantified by calculating the CaMPARI2 Red:Green Ratio. Only cells that were positive for insulin were quantified. (n = 4) slices per group. (± SEM, one-way ANOVA with Tukey’s post-hoc test, *** p < 0.001, **** p < 0.0001, outliers removed with ROUT Q = 1%). (C) Perifusion analysis of pancreatic tissue slices from this non-diabetic donor demonstrated robust insulin secretion in response to high glucose stimulation, confirming the preserved glucose responsiveness of beta cells.

Functional Ca²+ Biosensor, CaMPARI2, in T2D nPOD Case 6619.

(A) Slices transduced with the CaMPARI2 biosensor were incubated at 37°C in 3 mM and 16.7 mM glucose for ten minutes or 30 mM KCl for one minute. Slices were then exposed to photoconversion (PC) using ten second pulses for five minutes before washing with PBS. Tissue slices were then chemically fixed with paraformaldehyde for staining. (B) Glucose responsiveness in beta cells was quantified by calculating the CaMPARI2 Red:Green ratio. We observed a dysregulation in beta cell glucose sensing, particularly elevated calcium influx in low glucose conditions. Only insulin-positive cells were quantified (n = 4 slices per group). Data were analyzed on a per-beta-cell or per-islet basis. (± SEM, one-way ANOVA with Tukey’s post-hoc test, *** p < 0.001, **** p < 0.0001, outliers removed with ROUT Q = 1%). (C) The T2D case exhibited an atypical response, with significantly elevated insulin secretion at low glucose levels, aligning with the dysregulated calcium influx observed using CaMPARI2.

Functional Ca²+ Biosensor, CaMPARI2, in T1D nPOD case 6618.

(A) Slices transduced with the CaMPARI2 biosensor were incubated at 37°C in 3 mM and 16.7 mM glucose for ten minutes. Slices were then exposed to PC light using ten second pulses for five minutes before washing with PBS. Tissue slices were then chemically fixed with paraformaldehyde for staining. (B) Glucose responsiveness in beta cells was quantified by calculating the CaMPARI2 red:green ratio. The residual beta cells were non-functional, showing no response to high glucose stimulation, resulting in no photoconversion of CaMPARI2. Only cells that were positive for insulin were quantified (n = 1 slice per group). Data were analyzed on a per-beta-cell basis. (C) Perifusion analysis of pancreatic tissue slices from this T1D case revealed a total absence of insulin release in response to both high glucose and KCl stimulations, confirming the non-functionality of the remaining beta cells (red). Perifusion trace from non-diabetic (ND) case 6622 (grey) provided as a control.

Functional Ca²+ Biosensor, CaMPARI2, Versus Islet Area in Human Isolated Islets and Slices (ND).

(A) CaMPARI2 Red:Green ratio of human isolated islets in response to increasing concentrations of glucose and KCl. Plots are separated by donor (n = 3) indicated by the different shades of red, with each point representing a single islet. (B) CaMPARI2 response of the three isolated islet donors in (A), plotted against islet area (µm²) for each glucose concentration (3, 5, 8, 11, 16 mM), with each point representing a single islet. (C) CaMPARI2 Red:Green ratio at 16 mM glucose versus islet area (µm²) in both isolated islets (red) and slices (blue) (n=3; nPOD6617, nPOD6618, nPOD6642). (D) Isolated islets (red) and islets in slices (blue) were categorized as Small (<8,000 µm²), Medium (8,000–20,000 µm²), or Large (>20,000 µm²), and plotted to show differences in islet size and function (± SEM, n = 18, Student’s unpaired t-test, *p < 0.05). (E) Histogram showing the relative frequency distribution of analyzed islet areas in isolated islets (red) and slices (blue).

Isolated islet donor characteristics from Integrated Islet Distribution Program (IIDP) cases.

Table summarizes donor information and applied figure.