# Human assembloid of human blood vessel organoids with pancreatic islets improves insulin secretion over time ex vivo

Emily Tubbs, Mahira Mehanović, Mélanie Lopes, Clément Quintard, Stéphanie Combe, Clara Pirlian, Mathieu Armanet, Thomas Domet, Julia Sabatier, Sabrina Granziera, et al.

► To cite this version:

Emily Tubbs, Mahira Mehanović, Mélanie Lopes, Clément Quintard, Stéphanie Combe, et al. Human assembloid of human blood vessel organoids with pancreatic islets improves insulin secretion over time ex vivo. Cell Reports, 2025, 44 (10), pp.116378. 10.1016/j.celrep.2025.116378. hal-05311050

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Distributed under a Creative Commons Attribution 4.0 International License
**Cell Reports**

# Human assembloid of human blood vessel organoids with pancreatic islets improves insulin secretion over time ex vivo

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Emily Tubbs, Mahira Mehanović,
Mélanie Lopes, ..., Delphine Freida,
Josef M. Penninger, Xavier Gidrol

## Correspondence

emily.tubbs@cea.fr (E.T.),
xavier.gidrol@cea.fr (X.G.)

## In brief

Tubbs et al. report a human assembloid composed of self-organizing 3D blood vessel organoids with human pancreatic islets. This assembloid improved insulin secretion over time on five donors, suggesting new islet prevascularization that could potentially improve islet transplantation strategies and outcomes for type 1 diabetes patients.

## Highlights

* Co-culture of human pancreatic islets and BVOs leads to an auto-organized assembloid

* Increased angiogenesis cytokine secretion profiles of these assembloids

* Improved insulin secretion over time of islet and BVO assembloids (n = 5 donors)

<figure><img src="image_3.png" /></figure>
Report

Human assembloid of human blood vessel organoids
with pancreatic islets improves insulin secretion
over time ex vivo

Emily Tubbs,<fnref n="1" />,<fnref n="12" />,<fnref n="13" />,* Mahira Mehanović,<fnref n="1" />,<fnref n="12" /> Mėlanie Lopes,<fnref n="1" /> Clément Quintard,<fnref n="2" /> Stéphanie Combe,<fnref n="1" /> Clara Pirlian,<fnref n="1" /> Mathieu Armanet,<fnref n="3" />,<fnref n="10" />,<fnref n="11" /> Thomas Domet,<fnref n="3" />,<fnref n="10" />,<fnref n="11" /> Julia Sabatier,<fnref n="4" /> Sabrina Granziera,<fnref n="4" /> Karim Bouzakri,<fnref n="5" />,<fnref n="6" /> William Bietiger,<fnref n="5" /> Delphine Freida,<fnref n="1" /> Josef M. Penninger,<fnref n="2" />,<fnref n="7" />,<fnref n="8" />,<fnref n="9" /> and Xavier Gidrol<fnref n="1" />,.*

SUMMARY

Pancreatic islet transplantation is a promising treatment strategy for type 1 diabetes patients; however, there are still major challenges to overcome, including vascularization. Despite a substantial need to understand the islet graft decline observed in patients 5–10 years post transplantation, clinical translation remains challenging, probably because of species-specific features and the lack of *in vitro* vascularized models. Here, we report results of a human assembloid composed of self-organizing 3D blood vessel organoids (BVOs) with human pancreatic islets. We demonstrate that co-culture of islets and BVOs *ex vivo* leads to an auto-organized assembloid (by light-sheet fluorescence microscopy) and improves insulin secretion over time (functionality assays) on 5 independent human islet donors (from 3 different hospital facilities). Taken together, this study could accelerate our understanding of type 1 diabetes and facilitate therapeutic development for type 1 diabetes patients.

INTRODUCTION

Pancreatic islet transplantation is a promising treatment strat-
egy for type 1 diabetes; however, there are still major chal-
lenges to overcome, including vascularization. Physiologically,
pancreatic islets have a rich microvascular network and high
metabolic rate, receiving 5-fold higher blood flow than the
exocrine pancreas.<fnref n="1" /> However, the microvascular network,
consisting of arterioles, capillaries, and venules, is dissociated
during deceased donor islet isolation, contributing to poor
engraftment outcomes with 25%–50% of islet survival post
transplantation.<fnref n="2" /> Prompt and adequate blood supply during
the immediate post-transplantation period is obstructed, since
the graft revascularization process can take several days to
weeks.<fnref n="3" /> During this period, avascular islets depend on oxygen

and nutrients supplied by diffusion, leading to central islet
necrosis induced by ischemia upon intrahepatic islet trans-
plantation.<fnref n="4" /> Different strategies have emerged during the
quest to improve islet survival, including alternative sites of im-
plantation and, more recently, bioengineering of prevascular-
ized islets with endothelial and proangiogenic supporting
cells.<fnref n="5" />,<fnref n="6" /> Nevertheless, the latter approach often utilizes pre-
vascularized islets, including primary endothelial cells from
the human umbilical cord lacking mural cells that are needed
for proper blood vessel function. Novel strategies for the gen-
eration of prevascularized islets with native microvessels have
shown improved islet functionality, vascularization, and
engraftment, emphasizing the integral role of the microvas-
cular bed.<fnref n="7" />,<fnref n="8" /> Recently, a model of self-organizing 3D human
blood vessel organoids (BVOs) has been developed from

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Université Grenoble Alpes, CEA, Inserm, IRIG, UA13 BGE, Biomics, Grenoble, France</footnote>

<footnote n="2">²Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada</footnote>

<footnote n="3">³Cell Therapy Unit, AP-HP, Hôpital Saint-Louis, 75010 Paris, France</footnote>

<footnote n="4">⁴Cell Therapy Units from Laboratory of Cell Therapy for Diabetes (LTCD), PRIMS Facility, Institute for Regenerative Medicine and Biotherapy (IRMB), University Hospital of Montpellier, 34094 Montpellier, France</footnote>

<footnote n="5">⁵Research Unit of Strasbourg University “Diabetes and Therapeutics”, European Center for the Study of Diabetes (CeeD), UR7294, 67200 Strasbourg, France</footnote>

<footnote n="6">⁶ILONOV, 67200 Strasbourg, France</footnote>

<footnote n="7">⁷Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria</footnote>

<footnote n="8">⁸Eric Kandel Institute, Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria</footnote>

<footnote n="9">⁹Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany</footnote>

<footnote n="10">¹⁰AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, INSERM Centre d'Investigation Clinique en Biothérapies CIC-BT, F-75010 Paris, France</footnote>

<footnote n="11">¹¹Université Paris Cité, INSERM U1342, F-75010 Paris, France</footnote>

<footnote n="12">¹²These authors contributed equally</footnote>

<footnote n="13">¹³Lead contact</footnote>

<footnote>*Correspondence: emily.tubbs@cea.fr (E.T.), xavier.gidrol@cea.fr (X.G.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2025.116378</footnote>
<figure><img src="image_5.png" /></figure>

human induced pluripotent stem cells (iPSCs).⁹ BVOs present a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, thus exhibiting the functional, morphological, and molecular features of human microvasculature.⁹,¹⁰

Recent advances in organoid and particularly in assembloid technologies hold promise for generating self-organizing systems formed by the integration of multiple organoids or cell types. These assembloid platforms are being developed mainly under neuronal conditions to study tissue and disease modeling.¹¹ In fact, an assembloid recapitulating the descending corticospino-musculo pathway has been developed, and recently a human assembloid has been generated recapitulating the ascending neural sensory pathway.¹²,¹³ Considering pancreas-specific structural organization and organoid assembly principles,¹⁴,¹⁵ here we report results of a human assembloid composed of self-organizing 3D BVOs with human pancreatic islets, a strategy to prevascularize islets, which could accelerate our understanding of type 1 diabetes and facilitate therapy development for type 1 diabetes patients.

# RESULTS

## Generation of assembloids

To generate assembloids, human islets were co-cultured with BVOs, with one individual islet paired with one individual BVO (Figures 1A and 1B). Initially, BVOs were generated from iPSCs in a 3D collagen I-Matrigel matrix and collected for co-culture with human islets, using a medium mix of each cell type. The co-culture kinetics were monitored daily by bright-field microscopy, and engulfment of the human islet and BVO was observed on day 3. The generation of assembloids composed of islets and BVOs was reproducible, as confirmed by quantification of the area and the minFeret, a measure of an object's size along a specific direction, of the islet, which significantly increase during 14 days due to the fusion with the BVO (\*\*p < 0.005 vs. day 0, n = 3 donors, 13-18 replicates; Figure 1C).

To understand the sequence of events during the generation of assembloids, an in-depth analysis and 3D observation of whole samples were performed with light-sheet fluorescence microscopy (LSFM) using the islet-specific marker glucagon and endothelial cell marker CD31 on day 3 and day 7 of co-culture. Upon antibody specificity validation (Figure S1), maximum-intensity projection of LSFM z stacks was used for 3D visualization of BVO and islet structures, which were distinguished with CD31 and glucagon labeling, respectively (Figure 2A). Representative z stack images of CD31 and glucagon immunostaining were used to detect islet engulfment within the BVOs on day 3 and day 7 of co-culture, corresponding to complete fusion (Figure 2B), which was further confirmed on day 14 (Figure S2). Single LSFM z stacks were used for detection of double-positive regions, indicating islet invasion and localization within BVOs. The 3D reconstruction of glucagon-positive islets and CD31-positive BVOs was also visualized in videos (Videos S1, S2, and S3). Taken together, these preliminary results of five different islet donors confirm the generation of assembloids composed of islets with BVOs as well as invasion of the islet with endothelial cells.

## Assembloids improve islet functionality

Following the generation of assembloids of islets and BVOs, we first performed live and dead cell labeling with SYTO13 and propidium iodide, showing that the co-culture conditions as well as the mix of media used had no negative impact on overall viability on day 3 and day 7 (data not shown, as viability of each structure could not be clearly discriminated). Then, we investigated the functionality of islets within the assembloids containing BVOs by performing a glucose-stimulated insulin secretion assay during the early days of co-culture (day 3 or 5), at mid-culture (on day 7), and late during culture (on day14). We observed similar insulin secretion profiles for the five pancreatic islet donors (Figure 3 and Table 1 for donor information).

Islets within assembloids containing BVOs had similar profiles compared to islets cultured alone, showing that BVOs had no negative impact on islet functionality (Figures 3A and 3B). Regarding the stimulation index, as expected, there is a significant decrease of functionality of islets alone between early (day 3–5) and late culture (day 14, *p* = 0.022; Figure 3B). Interestingly, no significant decrease was observed between early and late culture of assembloids, highlighting that, when co-cultured with BVOs, islet functionality was maintained over time. In fact, during late culture (day 14), there is a significant increase of islet functionality for islets within assembloids containing BVOs (*p* = 0.0008 vs. islet alone day 14). Interestingly, both the time and fusion had a significant effect on islet glucose responsiveness (*p* = 0.0103 and *p* = 0.0005, respectively; two-way ANOVA with Sidak's multiple comparison test).

Taken together, our results highlight the beneficial impact on islet functionality due to assembloid generation. To determine the effect of the remaining Matrigel surrounding the BVO on islet function, we investigated an additional condition, including human islets in contact with 10 µL of Matrigel (0.337 mg/mL), and performed glucose-stimulated insulin secretion (GSIS) assays, as previously mentioned, which showed no beneficial effect over time (n = 3 independent human islet donors, each condition performed in 3–6 replicates; Figure S3), highlighting that, alone, the matrix surrounding the BVO does not improve islet function.

Overall, our results highlight that the generation of human assembloid significantly maintains the insulin-secretory function of human islets until day 14 versus 3 days for isolated islets.

## Assembloids increase angiogenesis cytokines

To understand the underlying mechanisms of the benefit of the assembloid, a cytokine array was performed on the supernatants of islets, BVOs, and the assembloids (Figure 4, n = 3 donors corresponding to 3 independent experiments). A comparison of the protein expression profiles under these conditions revealed that cytokines related to angiogenesis (such as interleukin-8 [IL-8]) were expressed in higher quantities in the assembloids and islets cultured under BVO-conditioned medium compared to the other conditions. SDF-1α was expressed in higher quantities in the assembloids compared to the other conditions. Interestingly, these growth factors have previously been described as improving the survival and insulin secretion of pancreatic islets.¹⁶,¹⁷ As shown in Figure 4, BVOs alone produce angiogenic growth factors such as VEGF-A, MMP-9, GDF-15, and SDF1-α, which have been described as enhancing vascularization and basement
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Islet and BVO
assembloid

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 1. Auto-organization of human islet and BVO assembloids**</figcaption></figure>

(A) Schematic of the islet- and iPSC-derived blood vessel organoid (BVO) assembloid method. On day 1, a human islet and BVO are put in co-culture. On day 3, partial engulfment is observed. On day 5, the human islet is fully embraced by the BVO and remains within the assembloid until day 14.

(B) Images obtained by bright-field microscopy.

(C) Quantification of these images for 3 donors and 13–17 replicates over 14 days, regarding the area, MinFeret, roundness, and solidity using ImageJ plugins.
Scale bar: 100 µm. **p** < 0.005 versus day 0.

membrane formation in islets.¹⁸,¹⁹ The close relationship between pancreatic islets and endothelial cells is crucial for islet function; in fact, islets secrete VEGF-A to activate endothelial cells, in turn facilitating vascularization.²⁰ In addition to their role in blood

flow within pancreatic islets, endothelial cells interact with various factors, including angiocrine factors like hepatocyte growth factor,²¹ which modulate the proliferation and function of pancreatic β cells, as well as extracellular matrix proteins.
<figure><img src="image_11.png" /><figcaption>**Figure 2. Assembloid of human islet- and iPSC-derived BVOs**</figcaption></figure>

(A and B) The maximum-intensity projection of z stacks (A) and a single z stack section (B), showing immunofluorescence labeling with CD31 (green) and glucagon (red) of BVOs alone or with islets on day 3 and day 7 of co-culture.

(C and D) Corresponding IMARIS analyses.

The islet and BVO fusion and penetration on day 7 are shown in orange boxes. Scale bar: 100 µm.

We detected highly secreted cytokines by BVOs that are related to inflammation (such as Serpin E1), and β cell survival (such as Dkk-1). Compared to islet alone, the assembloids showed increased expression of Dkk-1, an inhibitor of Wnt signaling.²² The Wnt/β-catenin pathway plays a significant role in the development and function of insulin-producing β cells.²³,²⁴ Moreover, stromal cell-derived factor 1 (SDF-1) is expressed in microvascular endothelial cells within the islets and surrounding interstitial stromal tissue, promoting pancreatic β cell survival.¹⁹

Previous studies have shown that the majority of IL-8 is produced by β cells themselves, suggesting that islet isolation and culture induce an inflammatory response.²⁵ In our study, it seemed that IL-8 was increased under assembloid conditions compared to islets alone. Interestingly, IL-8 has also been demonstrated to promote angiogenesis.²⁶ Osteopontin, SDF-1α, vascular cell adhesion molecule 1 (VCAM-1), and monocyte chemoattractant protein 1 (MCP-1) are also increased under assembloid conditions compared to islet alone. Interestingly, it seems that these cytokines are not induced by the sole
<figure><img src="image_12.png" /></figure>

effect of the BVO-conditioned medium but due to the direct contact between islets and BVOs. Remarkably, these cytokines have been shown to play a role in angiogenesis. In fact, osteopontin induces angiogenesis through activation of phosphatidylinositol 3-kinase/AKT and ERK1/2 in endothelial cells,²⁷ the SDF-1 and CXCR4 signaling pathway seems to be a molecular hub modulating neo-angiogenesis,²⁸ VCAM-1 regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization,²⁹ and MCP-1-induced angiogenesis is mediated by vascular endothelial growth factor A.³⁰

Adiponectin level reduced in islets cultured in BVO-conditioned medium compared to islet culture conditions. As shown previously, adiponectin is functionally active in human islets,³¹ and it seems to reverse β cell damage under some conditions.³² In fact, as shown previously, adiponectin exerts its beneficial effect on islets through the APN-HNF4α-PPARα cascade,³³ and it would be interesting to analyze this pathway in future studies.

<table><caption>**Table 1. Human islet deceased donor information**</caption><thead><tr><th></th><th>Age</th><th>Sex</th><th>BMI (kg/m²)</th><th>Islet isolation center</th></tr></thead><tbody><tr><td>Donor 1</td><td>55</td><td>M</td><td>25.1</td><td>LTCD Montpellier</td></tr><tr><td>Donor 2</td><td>52</td><td>F</td><td>25</td><td>AP-HP Paris</td></tr><tr><td>Donor 3</td><td>75</td><td>F</td><td>22.5</td><td>CeeD Strasbourg</td></tr><tr><td>Donor 4</td><td>71</td><td>M</td><td>29.8</td><td>CeeD Strasbourg</td></tr><tr><td>Donor 5</td><td>61</td><td>M</td><td>30.3</td><td>CeeD Strasbourg</td></tr></tbody></table>

M, male; F, female.

<caption>**Figure 3. Assembloids improve functionality of human islets**</caption>

Shown is a comparison of the insulin secretion of human islets co-cultured with iPSC-derived BVOs at early, mid, or late culture in response to low glucose (LG) solution ([2.8 mM]) and then to high glucose (HG) solution [16.7 mM]) on 5 independent human islet donors, with each condition performed in 3-6 replicates (A); relative stimulation index = [high glucose solution]/[low glucose solution] (B). *p* ≤ 0.05, **p** ≤ 0.01, ***p** ≤ 0.001.

These differences in protein expression profiles may contribute to the greater protective effects of BVOs on islet inflammation and insulin secretion. Taken together, the cytokine profiles and enhanced insulin secretion observed underline the benefits of islets and BVO assembloids.

## DISCUSSION

In this study, we present an islet prevascularization strategy where the co-culture of human islets and BVOs results in an assembloid with islet functionality maintained over time. The generation of as-

sembloids with BVOs prevented the pancreatic islet from the gradual decline of insulin secretion classically observed when islets are cultured alone.

Previous studies reported successful generation of assembloids of iPSC-derived brain organoids and BVOs, providing the brain organoids with robust vascular network-like structures simulating the *in vivo* developmental processes of the brain for further applications in various neurological diseases.³⁴⁻³⁷ This evidence suggests that BVOs represent a functional microvasculature model that can be used in multiple assembloid settings.

Our study demonstrates the utilization of BVOs for islet prevascularization, a strategy that could potentially improve islet transplantation strategies and outcomes. Interestingly, previous studies have shown that co-transplantation of human islets with microvascular fragments (MVFs) isolated from adipose tissue significantly improves islet survival and function post transplantation.⁸ The MVFs have been shown to be a justified source for islet vascularization, as they provide functional fragments of arterioles, capillaries, and venules. Islets secrete VEGF-A to activate endothelial cells and vascularization, while the laminin-rich vascular basement membrane regulates insulin gene expression.³⁸ These interactions between the microvasculature and islets are crucial for islet vascularization and function. A study by Nalbach et al. showed that fusion of MVFs with human islets improves their vascularization, viability, and functionality post transplantation.⁷ However, the islet prevascularization with MVFs included human islet dissociation and re-aggregation, a common approach of prevascularization strategies that can often be detrimental to the islet
<figure><img src="image_13.png" /><figcaption>Figure 4. Effect of islet and BVO assembloids on the cytokine expression levels</figcaption></figure>

A heatmap analysis shows the levels of the top 47 cytokines collected in the supernatant across three different experimental (Exp.) conditions: islet alone, BVO alone, and islet and BVO assembloid, and islet cultured in BVO-conditioned medium (Islet cond), from three distinct islet donors, by cytokine array. Analysis based on the pixel density, as shown on the color bar on the right, indicating the normalized relative intensity of cytokine levels ranging from 0 to 1.

islet vascularization and function,⁴² but we have shown that, at our proportions, Matrigel surrounding the BVO does not improve islet function alone. Other matrices could certainly be used, but they require further investigations for applications in regenerative medicine.

Implementation of co-transplantation with time-effective approaches is opening possibilities, such as simple yet effective engraftment of a suspension containing a mixture of freshly isolated islets and MVFs.⁴³ Our results suggest a potential clinical co-transplantation strategy of deceased donor islets with BVOs following simple co-culture to allow the generation of assembloids a few days before implantation.

Recent advancements in stem cell-derived pancreatic islets are extremely encouraging as an alternative source of insulin-secreting cells to overcome human organ donor scarcity for islet transplantation.⁴⁴ Interestingly, the iPSC-derived BVOs could be generated from

either the donor or the recipient patient stem cell source, with the latter enabling hypoimmunogenic stem cell lines that may avoid the risk of rejection. This opens a path for a half-autogeneic iPSC-derived patient BVO and half-allogenic islet transplantation.

In this study, we have demonstrated that the assembloid composed of BVOs preserves the functionality of the islet *ex vivo*. However, the major impact of the blood supply on islet survival and functionality cannot be assessed. This major limitation should be addressed in future studies using *in vivo* experiments to determine the benefit on the diabetes phenotype and/or a microfluidic chip to visualize the perfusion of islets, similar to what we have done with BVOs.⁴⁵ We have recently shown that BVOs enable perfusion into an organ-on-chip model,⁴⁵ highlighting the tremendous possibilities of disease modeling using both assembloid generation and the perfusion capacity of BVOs. Vascularized organoid-on-chip systems replicate the complex microenvironment of living tissues, providing a versatile platform for studying vascular

physiology as well as labor intensive and time consuming.³⁹ In addition, the choice to re-aggregate islets with endothelial and stromal cells instead of microvasculature units can lead to their separation and migration out of islet aggregates.⁴⁰,⁴¹ During the first days of co-culture, we observed fusion in all tested wells, showing remarkable assembloid generation efficiency. Moreover, we did not observe any islet escape from BVOs during 2 weeks of co-culture. Our vascularization strategy does not require pseudoislet formation, nor does it utilize MVFs, but instead, it employs complete microvasculature units.

## Limitations of the study

A major limitation of the clinical application of BVO and islet assembloids so far is the need for a collagen I-Matrigel matrix to generate BVOs. Interestingly, we cannot exclude that the remaining collagen I-Matrigel matrix around the BVO could serve as an anchoring point for the islet before assembloid generation or influence vascularization. Other studies have shown a beneficial effect of collagen hydrogels and MVFs on
biology and disease modeling. By incorporating prevascularized organoids into these microfluidic devices, physiological blood flow and nutrient transport could be achieved, recapitulating the dynamic interactions between blood vessels and surrounding tissues. We believe that this innovative approach will enable the generation of vascularized organoids with enhanced functionality and relevance for drug screening, disease modeling, and personalized medicine. Taken together, the prevascularization of pancreatic islets seems to be a promising approach to improve islet transplantation for patients, and hopefully the assembloid strategy suggested in this paper for BVOs and islets will enable a swift and efficient approach applicable to islet transplantation for patients.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Emily Tubbs (emily.tubbs@cea.fr).

### Materials availability

This study did not generate new materials.

### Data and code availability

* Data are available from the corresponding author upon request. Bright-field images were analyzed using ImageJ/FIJI to assess the circularity, aspect ratio, minimum caliper diameter (MinFeret), roundness, and solidity of organoids.⁴⁶

* Pixel densities from the autoradiographs of cytokine arrays were analyzed using ImageJ/FIJI software with a custom script developed in our laboratory (available upon request) and were normalized to ensure comparability across samples. Finally, the heatmap was generated using GraphPad Prism software based on these normalized pixel densities, visually representing the cytokine expression levels across different samples, indicating the normalized relative intensity ranging from 0 to 1 with red, black, and green colors corresponding to high, median, and low cytokine levels, respectively. This visualization highlights the variation in cytokine expression among the different experimental conditions and donors. 3 donors were considered for each experimental group to generate 1 membrane per experimental group.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

The authors are grateful for technical assistance from Flora Clément and Patricia Obeid. This work was supported by ANR-JCJC "ATOP1" (ANR-24-CE52-7278-01) and the CEA "OOC inflexion" and "Focus organoids." We thank the microscopy facility MuLife of IRIG/DBSCI, funded by CEA Nanobio and GRAL LabEX (ANR-10-LABX-49-01) and financed within the University Grenoble Alpes graduate school CBH-EUR-GS (ANR-17-EURE-0003).

## AUTHOR CONTRIBUTIONS

E.T. and X.G. conceived and directed the project. E.T., M.M., S.C., and D.F. performed experiments. M.M., M.L., S.C., and C.P. performed analyses. C.Q. and J.P. developed and provided blood vessel organoids. M.A., T.D., J.S., S.G., K.B., and W.B. provided human pancreatic islets. E.T. and X.G. analyzed results and wrote the paper with contributions from M.M. All authors reviewed and edited the paper.

## DECLARATION OF INTERESTS

J.P. is one of the authors of the patent application US20200199541A1 relating to blood vessel organoid generation. Furthermore, J.P. is a

founder, shareholder, and chairman of the scientific advisory board of Angios Biotech, a company establishing BVOs for drug testing and vascular transplants.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* METHOD DETAILS

* Human islets

* BVOs

* Co-culture of human islets and BVOs

* Assembloid image analysis

* Immunofluorescence staining

* Acquisition with confocal and LSFM

* GSIS assays

* Cytokine array

* STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.116378.

Received: May 21, 2025

Revised: August 2, 2025

Accepted: September 15, 2025

## REFERENCES

1. Brusko, T.M., Russ, H.A., and Stabler, C.L. (2021). Strategies for durable β cell replacement in type 1 diabetes. Science 373, 516-522. https://doi.org/10.1126/science.abh1657.

2. Citro, A., Moser, P.T., Dugnani, E., Rajab, T.K., Ren, X., Evangelista-Leite, D., Charest, J.M., Peloso, A., Podesser, B.K., Manenti, F., et al. (2019). Biofabrication of a vascularized islet organ for type 1 diabetes. Biomaterials 199, 40-51. https://doi.org/10.1016/j.biomaterials.2019.01.035.

3. Brissova, M., and Powers, A.C. (2008). Revascularization of transplanted islets: Can it be Improved? Diabetes 57, 2269-2271. https://doi.org/10.2337/db08-0814.

4. Giuliani, M., Moritz, W., Bodmer, E., Dindo, D., Kugelmeier, P., Lehmann, R., Gassmann, M., Groscurth, P., and Weber, M. (2005). Central necrosis in isolated hypoxic human pancreatic islets: Evidence for postisolation ischemia. Cell Transplant. 14, 67-76. https://doi.org/10.3727/000000005783983287.

5. Wassmer, C.-H., Lebreton, F., Bellofatto, K., Perez, L., Cottet-Dumoulin, D., Andres, A., Bosco, D., Berney, T., Othenin-Girard, V., Martinez De Tejada, B., et al. (2021). Bio-Engineering of Pre-Vascularized Islet Organoids for the Treatment of Type 1 Diabetes. Transpl. Int. 35, 10214-10217. https://doi.org/10.3389/ti.2021.10214.

6. Takahashi, Y., Sekine, K., Kin, T., Takebe, T., and Taniguchi, H. (2018). Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation. Cell Rep. 23, 1620-1629. https://doi.org/10.1016/j.celrep.2018.03.123.

7. Nalbach, L., Roma, L.P., Schmitt, B.M., Becker, V., Körbel, C., Wrublewsky, S., Pack, M., Später, T., Metzger, W., Menger, M.M., et al. (2021). Improvement of islet transplantation by the fusion of islet cells with functional blood vessels. EMBO Mol. Med. 13, e12616. https://doi.org/10.15252/emmm.202012616.

8. Aghazadeh, Y., Poon, F., Sarangi, F., Wong, F.T.M., Khan, S.T., Sun, X., Hatkar, R., Cox, B.J., Nunes, S.S., and Nostro, M.C. (2021). Microvessels support engraftment and functionality of human islets and hESC-derived
pancreatic progenitors in diabetes models. Cell Stem Cell 28, 1936-1949. e8. https://doi.org/10.1016/j.stem.2021.08.001.

9. Wimmer, R.A., Leopoldi, A., Aichinger, M., Kerjaschki, D., and Penninger, J.M. (2019). Generation of blood vessel organoids from human pluripotent stem cells. Nat. Protoc. 14, 3082-3100. https://doi.org/10.1038/s41596-019-0213-z.

10. Werschler, N., and Penninger, J. (2023). Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells. J. Vis. Exp. 2023, 1-14. https://doi.org/10.3791/64715.

11. Onesto, M.M., Kim, J.I., and Pasca, S.P. (2024). Assembloid models of cell-cell interaction to study tissue and disease biology. Cell Stem Cell 31, 1563-1573. https://doi.org/10.1016/j.stem.2024.09.017.

12. Andersen, J., Revah, O., Miura, Y., Thom, N., Amin, N.D., Kelley, K.W., Singh, M., Chen, X., Thete, M.V., Walczak, E.M., et al. (2020). Generation of Functional Human 3D Cortico-Motor Assembloids. Cell 183, 1913-1929.e26. https://doi.org/10.1016/j.cell.2020.11.017.

13. Kim, J., Imaizumi, K., Jurjut, O., Kelley, K.W., Wang, D., Thete, M.V., Huc-dacova, Z., Amin, N.D., Levy, R.J., Scherrer, G., et al. (2025). Human assembloid model of the ascending neural sensory pathway. Nature 183, 1913-1929.e26. https://doi.org/10.1038/s41586-025-08808-3.

14. Kim, M., Cho, S., Hwang, D.G., Shim, I.K., Kim, S.C., Jang, J., and Jang, J. (2025). Bioprinting of bespoke islet-specific niches to promote maturation of stem cell-derived islets. Nat. Commun. 16, 1430. https://doi.org/10.1038/s41467-025-56665-5.

15. Reza, H.A., Santangelo, C., Iwasawa, K., Reza, A.A., Sekiya, S., Glaser, K., Bondoc, A., Merola, J., and Takebe, T. (2025). Multi-zonal liver organoids from human pluripotent stem cells. Nature 641, 1258-1267. https://doi.org/10.1038/s41586-025-08850-1.

16. Liu, Z., and Habener, J.F. (2009). Stromal cell-derived factor-1 promotes survival of pancreatic beta cells by the stabilisation of beta-catenin and activation of transcription factor 7-like 2 (TCF7L2). Diabetologia 52, 1589-1598. https://doi.org/10.1007/s00125-009-1384-x.

17. Hubber, E.L., Rackham, C.L., and Jones, P.M. (2021). Protecting islet functional viability using mesenchymal stromal cells. Stem Cells Transl. Med. 10, 674-680. https://doi.org/10.1002/sctm.20-0466.

18. Guo, H., Li, R., Zucker, S., and Toole, B.P. (2000). EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface. Cancer Res. 60, 888-891.

19. Yano, T., Liu, Z., Donovan, J., Thomas, M.K., and Habener, J.F. (2007). Stromal Cell-Derived Factor-1 (SDF-1)/CXCL12 Attenuates Diabetes in Mice and Promotes Pancreatic β-Cell Survival by Activation of the Prosurvival Kinase Akt. Diabetes 56, 2946-2957. https://doi.org/10.2337/db07-0291.

20. Hogan, M.F., and Hull, R.L. (2017). The islet endothelial cell: a novel contributor to beta cell secretory dysfunction in diabetes. Diabetologia 60, 952-959. https://doi.org/10.1007/s00125-017-4272-9.

21. Nakamura, T., and Mizuno, S. (2010). The discovery of Hepatocyte Growth Factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86, 588-610. https://doi.org/10.2183/pjab.86.588.

22. D'Amico, L., Mahajan, S., Capietto, A.H., Yang, Z., Zamani, A., Ricci, B., Bumpass, D.B., Meyer, M., Su, X., Wang-Gillam, A., et al. (2016). Dick-kopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer. J. Exp. Med. 213, 827-840. https://doi.org/10.1084/jem.20150950.

23. Wong, S.K., Mohamad, N.V., Jayusman, P.A., and Ibrahim, N.I. (2023). A Review on the Crosstalk between Insulin and Wnt/β-Catenin Signalling for Bone Health. Int. J. Mol. Sci. 24, 12441. https://doi.org/10.3390/ijms241512441.

24. Napolitano, T., Silvano, S., Ayachi, C., Plaisant, M., Sousa-Da-Veiga, A., Fofo, H., Charles, B., and Collombat, P. (2023). Wnt Pathway in Pancreatic Development and Pathophysiology. Cells 12, 565. https://doi.org/10.3390/cells12040565.

25. Negi, S., Jetha, A., Aikin, R., Hasilo, C., Sladek, R., and Paraskevas, S. (2012). Analysis of Beta-Cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isola-tion and culture. PLoS One 7, e30415. https://doi.org/10.1371/journal.pone.0030415.

26. Shi, J., and Wei, P.K. (2016). Interleukin-8: A potent promoter of angiogenesis in gastric cancer. Oncol. Lett. 11, 1043-1050. https://doi.org/10.3892/ol.2015.4035.

27. Dai, J., Peng, L., Fan, K., Wang, H., Wei, R., Ji, G., Cai, J., Lu, B., Li, B., Zhang, D., et al. (2009). Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells. Oncogene 28, 3412-3422. https://doi.org/10.1038/onc.2009.189.

28. Petit, I., Jin, D., and Rafii, S. (2007). The SDF-1-CXCR4 signaling pathway: a molecular hub modulating neo-angiogenesis. Trends Immunol. 28, 299-307. https://doi.org/10.1016/j.it.2007.05.007.

29. Kaur, G., Sharma, D., Bisen, S., Mukhopadhyay, C.S., Gurdziel, K., and Singh, N.K. (2023). Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization. Commun. Biol. 6, 516. https://doi.org/10.1038/s42003-023-04905-z.

30. Hong, K.H., Ryu, J., and Han, K.H. (2005). Monocyte chemoattractant protein-1-induced angiogenesis is mediated by vascular endothelial growth factor-A. Blood 105, 1405-1407. https://doi.org/10.1182/blood-2004-08-3178.

31. Staiger, K., Stefan, N., Staiger, H., Brendel, M.D., Brandhorst, D., Bretzel, R.G., Machicao, F., Kellerer, M., Stumvoll, M., Fritsche, A., and Häring, H.U. (2005). Adiponectin is functionally active in human islets but does not affect insulin secretory function or β-cell lipoapoptosis. J. Clin. Endocrinol. Metab. 90, 6707-6713. https://doi.org/10.1210/jc.2005-0467.

32. Munhoz, A.C., Serna, J.D.C., Vilas-Boas, E.A., Caldeira da Silva, C.C., Santos, T.G., Mosele, F.C., Felisbino, S.L., Martins, V.R., and Kowaltow-ski, A.J. (2023). Adiponectin reverses β-Cell damage and impaired insulin secretion induced by obesity. Aging Cell 22, e13827. https://doi.org/10.1111/acel.13827.

33. Onodera, T., Kim, D.S., Ye, R., Wang, M.Y., Chen, S., Field, B.C., Straub, L., Sun, X.N., Li, C., Lee, C., et al. (2023). Protective roles of adiponectin and molecular signatures of HNF4α and PPARα as downstream targets of adiponectin in pancreatic β cells. Mol. Metab. 78, 101821. https://doi.org/10.1016/j.molmet.2023.101821.

34. Song, L., Yuan, X., Jones, Z., Griffin, K., Zhou, Y., Ma, T., and Li, Y. (2019). Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues. Sci. Rep. 9, 5977. https://doi.org/10.1038/s41598-019-42439-9.

35. Kong, D., Park, K.H., Kim, D.H., Kim, N.G., Lee, S.E., Shin, N., Kook, M.G., Kim, Y.B., and Kang, K.S. (2023). Cortical-blood vessel assembloids exhibit Alzheimer's disease phenotypes by activating glia after SARS-CoV-2 infection. Cell Death Discov. 9, 32. https://doi.org/10.1038/s41420-022-01288-8.

36. Ahn, Y., An, J.H., Yang, H.J., Lee, D.G., Kim, J., Koh, H., Park, Y.H., Song, B.S., Sim, B.W., Lee, H.J., et al. (2021). Human blood vessel organoids penetrate human cerebral organoids and form a vessel-like system. Cells 10, 2036. https://doi.org/10.3390/cells10082036.

37. Sun, X.Y., Ju, X.C., Li, Y., Zeng, P.M., Wu, J., Zhou, Y.Y., Shen, L.B., Dong, J., Chen, Y.J., and Luo, Z.G. (2022). Generation of Vascularized Brain Organoids to Study Neurovascular Interactions. eLife 11, e76707. https://doi.org/10.7554/eLife.76707.

38. Nikolova, G., Jabs, N., Konstantinova, I., Domogatskaya, A., Tryggvason, K., Sorokin, L., Fässler, R., Gu, G., Gerber, H.P., Ferrara, N., et al. (2006). The vascular basement membrane: A niche for insulin gene expression and β cell proliferation. Dev. Cell 10, 397-405. https://doi.org/10.1016/j.devcel.2006.01.015.

39. Wassmer, C.H., Bellofatto, K., Perez, L., Lavallard, V., Cottet-Dumoulin, D., Ljubicic, S., Parnaud, G., Bosco, D., Berishvili, E., and Lebreton, F. (2020). Engineering of Primary Pancreatic Islet Cell Spheroids for
<figure><img src="image_14.png" /></figure>

Three-dimensional Culture or Transplantation: A Methodological Comparative Study. Cell Transplant. 29, 963689720937292. https://doi.org/10.1177/0963689720937292.

40. Nguyen-Ngoc, K.-V., Jun, Y., Sai, S., Bender, R.H.F., Kravets, V., Zhu, H., Hatch, C.J., Schlichting, M., Gaetani, R., Mallick, M., et al. (2022). Engineered vasculature induces functional maturation of pluripotent stem cell-derived islet organoids. Preprint at bioRxiv. https://doi.org/10.1101/2022.10.28.513298.

41. Jun, Y., Kang, A.R., Lee, J.S., Park, S.-J., Lee, D.Y., Moon, S.-H., and Lee, S.-H. (2014). Microchip-based engineering of super-pancreatic islets supported by adipose-derived stem cells. Biomaterials 35, 4815-4826. https://doi.org/10.1016/j.biomaterials.2014.02.045.

42. Salamone, M., Rigogliuso, S., Nicosia, A., Campora, S., Bruno, C.M., and Ghersi, G. (2021). 3D Collagen Hydrogel Promotes in Vitro Langerhans Islets Vascularization Through Ad-Mvfs Angiogenic Activity. Biomedicines 9, 739. https://doi.org/10.3390/biomedicines9070739.

43. Wrublewsky, S., Weinzierl, A., Hornung, I., Prates-Roma, L., Menger, M.D., Laschke, M.W., and Ampofo, E. (2022). Co-transplantation of pancreatic islets and microvascular fragments effectively restores normoglycemia in diabetic mice. NPJ Regen. Med. 7, 67. https://doi.org/10.1038/s41536-022-00262-3.

44. Hogrebe, N.J., Ishahak, M., and Millman, J.R. (2023). Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes. Cell Stem Cell 30, 530-548. https://doi.org/10.1016/j.stem.2023.04.002.

45. Quintard, C., Tubbs, E., Jonsson, G., Jiao, J., Wang, J., Werschler, N., Laporte, C., Pitaval, A., Bah, T.S., Pomeranz, G., et al. (2024). A microfluidic platform integrating functional vascularized organoids-on-chip. Nat. Commun. 15, 1452. https://doi.org/10.1038/s41467-024-45710-4.

46. Cui, H., Wang, X., Wesslowski, J., Tronser, T., Rosenbauer, J., Schug, A., Davidson, G., Popova, A.A., and Levkin, P.A. (2021). Assembly of Multi-Spheroid Cellular Architectures by Programmable Droplet Merging. Adv. Mater. 33, e2006434. https://doi.org/10.1002/adma.202006434.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td><strong>Antibodies</strong></td><td></td><td></td></tr><tr><td>anti-human CD31</td><td>Abcam</td><td>Cat# ab134168, RRID:AB_2890012</td></tr><tr><td>anti-human glucagon</td><td>Abcam</td><td>Cat# ab10988, RRID:AB_297642</td></tr><tr><td>FITC goat anti-rabbit</td><td>Interchim</td><td>Cat# 111-095-144, RRID:AB_2337978</td></tr><tr><td>goat anti-mouse Cy3-conjugated secondary antibodies</td><td>Interchim</td><td>Cat# 115-165-003, RRID:AB_2338680</td></tr><tr><td>nuclear counter-staining using Hoechst 33342</td><td>ThermoFisher Scientific</td><td>Cat# H1399, RRID:AB_33342</td></tr><tr><td><strong>Critical commercial assays</strong></td><td></td><td></td></tr><tr><td>STELLUX Chemiluminescence Human Insulin ELISA</td><td>ALPCO</td><td>Cat# 80-INSHU-CH01, RRID:AB_2894946</td></tr><tr><td>Proteome Profiler Human XL Cytokine Array Kit</td><td>R&amp;D Systems</td><td>Cat# ARY022B, RRID:AB_3662112</td></tr><tr><td><strong>Experimental models: Cell lines</strong></td><td></td><td></td></tr><tr><td>BVO</td><td>NC8 cell line</td><td>M. Boehm (National Institutes of Health)</td></tr><tr><td><strong>Experimental models: Organisms/strains</strong></td><td></td><td></td></tr><tr><td>Human pancreatic islets</td><td>Islet isolation centers</td><td>LTCD Montpellier, AP-HP Paris, CeeD Strasbourg</td></tr></tbody></table>

### METHOD DETAILS

#### Human islets

The human pancreatic islets were provided by the Laboratory of Cell Therapy for Diabetes (LTCD), PRIMS facility, Institute for Regenerative Medicine and Biotherapy (IRMB), University Hospital of Montpellier, Montpellier, France (authorization no.:AC-2019-3555); as well as the Cell Therapy Unit, AP-HP, Saint Louis Hospital, Paris, France, (authorization no.:AC-2020-4101), and the Research Unit of Strasbourg University "Diabetes and Therapeutics", European Center for the Study of Diabetes (CeeD), Strasbourg, France (authorization no.:DC-2019-3439). Human islets were isolated from pancreas from five brain-dead donors for therapeutic purposes. The procedure was approved by the National Agency for Medicines and Health Products Safety in accordance with the French bioethics law (Act No.2004-800; Act No.2011-814). After confirmation by the Agence de la Biomédecine, islets not suitable for transplantation are redirected to the cell therapy units' scientific platform. The use of human islets for scientific research was appointed to our laboratory by the French Ministry of Education and Research (DC-2024-6399).

Islets were cultured in complete medium composed of CMRL 1066 medium, 1 g/L Glucose (Gibco, ThermoFisher Scientific, 11530037) supplemented with 10% FBS, 10 mM HEPES, 1 mM Sodium Pyruvate, 2 mM L-Glutamine. 100 mg.U/mL-1 Penicillin, 100 µg mL-1 streptomycin (ThermoFisher Scientific, 15140-122), in a 5% CO2 humidified atmosphere at 37°C.

#### BVOs

3D human blood vessel organoids were generated from human iPSCs, in particular the NC8 cell line (kindly provided by M. Boehm (National Institutes of Health) as previously described.⁹

#### Co-culture of human islets and BVOs

Native human islets were individually placed into a U-shaped 96-well microplate with an ultra-low attachment surface (Corning, 4520), and one single BVO was added per well. Islets and BVOs were cultured in complete islet medium mixed with the differentiation BVO medium (ratio 1:1). On the day of the co-culture, control islets medium was also changed with this same medium mix (ratio 1:1) to circumvent the sole effect of medium. To go further and determine if BVO secretions had an effect on islet function another condition was tested in parallel: islets were cultured in BVO conditioned medium during 48h. The cell culture supernatants were collected every 48h and stored at -20°C for cytokine array. Fresh medium or conditioned medium was added. To determine the effect of the remaining Matrigel surrounding the BVO, we cultured islets in 10µL droplets of Matrigel (Corning, 354277) diluted to 0.337mg/mL and incubated for 1h at RT prior loading islets.

#### Assembloid image analysis

Islet and BVO co-cultures were observed daily with brightfield microscope (Axio Observer, Zeiss). Images were analyzed using ImageJ/FIJI to assess circularity, aspect ratio, minimum caliper diameter (MinFeret), roundness and solidity of organoids⁴⁶ during 14 days of co-culture.
## Immunofluorescence staining

For immunofluorescent staining, the cells were fixed by Image-iT Fixative Solutions (ThermoFisher Scientific, R377814) for 30min to 2h at room temperature at 600 rpm, rinsed with PBS and stored at 4°C. The cells were permeabilized by a blocking buffer containing FBS (ScienCell Research Laboratories, 0010), sodium deoxycholate solution (Sigma-Aldrich, D6750), Triton X-100 (Sigma-Aldrich, T8787), Tween 20 (Sigma-Aldrich, P1379) and Bovine Serum Albumin (Sigma-Aldrich, A9647) overnight at RT, 600 rpm. Primary antibodies rabbit anti-human CD31 (Abcam, ab134168) and mouse anti-human glucagon (Abcam, ab10988) were diluted at 1:1000 in blocking buffer incubated 48h at 4°C, 600 rpm. After 3 washes in PBST (0.05% Tween, Sigma-Aldrich, P1379), FITC goat anti-rabbit (Interchim, 111-095-144) and goat anti-mouse Cy3-conjugated secondary antibodies (Interchim, 115-165-003) were diluted at 1:1000 in blocking buffer and incubated for 48h at 4°C, 600 rpm. After 3 washes in PBST, nuclear counter-staining using Hoechst 33342 (ThermoFisher Scientific, H-1399) was carried out according to a routine protocol for 2h at room temperature.

## Acquisition with confocal and LSFM

Antibodies specificity was validated by immunofluorescence using confocal fluorescence microscopy (LSM 880, Zeiss). Then, after immunofluorescence staining, the cells were embedded into a cylinder of agarose low melt gel (ROTH, 6351.1) then transparentized using solution of CUBIC R2 (4.16M urea (Sigma-Aldrich, U5378), 1.46M sucrose (Sigma-Aldrich, S7903) and 0.67M triethanolamine (Sigma-Aldrich, 90279) overnight, and visualized by LSFM (Z1 ZEISS); detection = EC Plan-Neofluar 5×/0.16 foc (WD = 10.5 mm); illumination = 2 LSFM 5×/0.1 foc objectives; space between different Z stacks = 1.869-4.110 µm; pixel size = 0.37-0.91 µm; Light sheet thickness = 3.91-9.90 µm; using ZEN 3.1 software for Light sheet. The 3D reconstructions were done with Arivis vision 4D software (v2.12.6 × 64). Images were reconstructed in 3D for day 3 and day 7 using IMARIS v10.2 software (Oxford Instrument). For this, image pre-processing was performed and fluorescent signal were converted in computer-generated surface rendering. 3D rendered images were captured using coronal clipping plane to reveal the composition and structure of the assembloid.

## GSIS assays

Native human islets, or co-cultures with BVO individually placed in a 96-well plate with ultra-low attachment surface (Corning, 4520) were incubated at 37°C, 95% O2, 5% CO2 for 1h in 60 µL KREBS buffer (125 mM NaCl (Sigma-Aldrich, 014), 4.74mM KCl (Sigma-Aldrich, P-5405), 5 mM NaHCO3 (Sigma-Aldrich, S-8875), 1 mM CaCl2 (Sigma-Aldrich, C-8106) 1.2 mM MgSO4 (Merck, M-2643), 1.2 mM K2HPO4 (Sigma-Aldrich, P3786), 25mM HEPES (Sigma-Aldrich, H-3375), 0.1% free fatty acid Bovine Serum Albumin, pH 7.4) containing, 2.8 mM glucose (Sigma-Aldrich, 49163) as pre-incubation for 30min. Thereafter, cells were incubated for 1hr with 2.8 mM glucose solution (low glucose solution) and for 1hr with 16.7 mM glucose solution (high glucose solution). After each incubation step, supernatants were collected and stored at -80°C. Insulin concentration in each collected supernatant was measured in duplicate using STELLUX Chemiluminescence Human Insulin ELISA (ALPCO, 80-INSHU-CH01). A stimulation index (SI) was obtained by calculating the ratio of insulin measured between high and low glucose stimulation [high glucose solution]/[low glucose solution].

## Cytokine array

A cytokine array was conducted using a Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, ARY022B) following the manufacturer's guidelines. Collected cell culture supernatants from 9 to 15 replicates were pooled for each of three donors for sufficient detection signal and diluted in Array Buffer 6 (R&D systems, 893573) in 1:1.6 to 1:2.4 ratio. 4 groups were analyzed in parallel: islets cultured alone, islet cultured with BVO-conditioned media, BVOs cultured alone, and islets and BVOs assembloids. The 4 membranes were stained according to manufacturer's protocol using Chemi Reagent Mix, and imaged with ChemiDocTM Imaging System (Bio-Rad) to visualize the cytokine levels. Pixel densities from the autoradiographs were analyzed using ImageJ/FIJI software with a custom script developed in our laboratory and were normalized to ensure comparability across samples. Finally, the heatmap was generated using GraphPad Prism software based on these normalized pixel densities, visually representing the cytokine expression levels across different samples, indicating the normalized relative intensity ranging from 0 to 1 with red, black, and green colors correspond to high, median, and low cytokine levels, respectively. This visualization highlights the variation in cytokine expression among the different experimental conditions and donors. n = 3 donors were considered for each experimental group to generate one membrane per experimental group.

## STATISTICAL ANALYSIS

Results are expressed as mean values ±SD. Statistical analysis was performed using Graph Pad Prism (GraphPad Software Inc., San Diego, CA, USA). Statistically significant differences were assessed by ordinary two-way ANOVA with Sidak's multiple comparison test. The outliers' exclusion was performed with Grubbs test. Statistical significance was defined as a value of *p* ≤ 0.05, **p** ≤ 0.01, ***p** ≤ 0.001.