Microwell culture platform maintains viability and mass of human pancreatic islets

Abstract

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures.

Method: Human islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested.

Results: The microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions.

Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

Keywords: cell death; diabetes; human pancreatic islets; hypoxia; islet transplantation; long-term culture; microwells; type 1 diabetes.

Figure 1

Overview of the culture method of human islets. Isolated human islets were seeded on two different types of culture dishes (flat or microwell) and cultured for two weeks. (A) A schematic of culture method using flat-bottomed, conventional dishes. (B) A schematic of culture method using microwell-bottomed dishes.

Figure 2

Islet death induced by the fusion during the culture. (A) A schematic of molecular diffusion: single islets (left panel) vs. fused islets (right panel). (B) A representative live (green color)/dead (red color) staining image of cultured human islets. Single islet (white arrowhead) and fused islets (yellow arrowhead).

Figure 3

Strategy to culture various-sized human islets using two different-sized microwells. Islet size distribution was obtained from 48 human islet isolations, demonstrated in our previous study; ~70% of islets were 50 – 150 µm in diameter and ~30% in >150 µm. EZSPHERE 901SP-300 (140 µm [minor axis] × 300 µm [major axis]) was used for 50 – 150 µm-islets. EZSPHERE 901SP-400 (200 µm [minor axis] × 370 µm [major axis]) was used for >150 µm-islets. Microscopic laser scanning 3D (inverted image of microwells) and 2D bright-field images are shown.

Figure 4

Islet morphology, viability and mass in a two week-culture of human islets. (A) A bright-field image of two week-cultured islets on the conventional flat-bottomed dishes. Scale bar: 1 mm. (B) Combined islets from two flat dishes and stained with DTZ. Overview (upper, scale bar: 500 µm) and enlarged picture (bottom, scale bar: 100 µm). (C) A bright-field image of two week-cultured islets in microwell dishes. Upper picture: >150 µm-islets in EZSPHERE 901SP-400. Bottom picture: 50 – 150 µm-islets in EZSPHERE 901SP-300. Scale bar: 1 mm. (D) Combined islets from two microwell dishes, stained with DTZ. Overview (upper, scale bar: 500 µm) and enlarged picture (bottom, scale bar: 100 µm). (E) Live/dead staining of two week-cultured islets from flat dishes (upper) and microwell dishes (bottom). (F) Viability assessment of islets from three donors. Data of each donor is plotted as different color dots (red, blue and green for Donors A, B and C, respectively). Pre-culture data is presented in yellow bar and post-culture in purple. *P < 0.05 in Student’s t-tests. (G) Islet mass analysis shown as % relative value to that of pre-culture. n = 3 donors. *P < 0.05 in Student’s t-tests. (H) Islet purity analyzed by DTZ staining. n = 3 donors.

Figure 5

Cell composition of key cell types in two week-cultured human islets. (A) Representative immunofluorescence images of pre- and post-cultured (flat dish and microwell dish) islets with major endocrine components (insulin, glucagon and somatostatin. Scale bar: 100 µm. (B) Representative immunohistochemistry images of pre- and post-cultured islets for area quantification analyses. Anti-glucagon for alpha cells (upper row), anti-insulin for beta cells (middle row) and anti-vWF for endothelial cells (bottom row). (C) The percent (%) of cell composition to islet area pre- and post-culture time points. n = 3 donors. Data of each donor is plotted in different color dots (red, blue and green for Donors A, B and C, respectively). (D) Analyses of % changes normalized to pre-culture islets.

Figure 6

Insulin-secreting function and metabolism of two week-cultured human islets. (A) Glucose-stimulated insulin secretion assay was performed with 1 hour of low glucose exposure followed by 1 hour of high glucose exposure. Insulin secretion was normalized by the islet number (IEQ) applied. Data of pre- and post-cultured (flat dish and microwell dishes) islets are presented. n = 3 donors. Data of each donor is plotted in different color dots (red, blue and green for Donors A, B and C, respectively). (B) Stimulation index calculated as the ratio of high insulin secretion over low insulin secretion. (C) OCR assay with 4 solution phases with 1) baseline at 3 mM glucose, 2) glucose stimulation at 20 mM, 3) 5 µM oligomycin, and 4) 5 µM rotenone and 5 µM antimycin. n = 3 donors. Absolute OCR data was normalized by the islet number (IEQ) applied. Data plots are the average of 3 donors. Data of individual donor islets is available in Supplementary Figure 6 . (D) OCR assay data analysis normalized to the baseline OCR. (E–H) Analyses of baseline OCR, response to high glucose, ATP dependency, and non-ATP respiration dependency in pre- and post-cultured (flat dish and microwell dishes) islets. Detailed method for the analysis is described in Supplementary Figure 2 . Data of each donor is plotted in different color dots (red, blue and green for Donor A, B and C, respectively).