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. 2026 May 23;28(2):43. doi: 10.1007/s10544-026-00826-w

Spatially controlled Islet delivery into a bioengineered vascular Niche improves subcutaneous transplantation

Elisa Balboni 1,2, Jocelyn Nikita Campa-Carranza 1,3, Tommaso Bo 1, Gabrielle E Rome 1, Henry Seaborne 1, Arya Venugopalan 1, Rim Ouni 1, Jesus Paez-Mayorga 1, Corrine Ying Xuan Chua 1, Alessandro Grattoni 1,4,5,✉
PMCID: PMC13198518  PMID: 42176117

Abstract

Pancreatic islet transplantation represents a promising therapeutic strategy for type 1 diabetes (T1D). In this context, the subcutaneous space offers a safer and more accessible alternative to the clinically established intraportal delivery route. However, a major unmet need remains the development of a site capable of supporting rapid vascularization and sustained islet function. In its current configuration, this approach is limited by poor vascularization, hypoxia-driven islet loss, loss of spatial control during graft delivery, and tissue disruption associated with delivery strategies. To address these challenges, the Neovascularized Implantable Cell Homing and Encapsulation (NICHE) device provides a prevascularized subcutaneous platform that supports islet viability and enables localized immunosuppressant delivery. In this study, we describe the incorporation of a 3D-printed removable placeholder into the NICHE platform to preserve a defined cavity within the device during prevascularization and enable subsequent islet delivery. The placeholder shapes the spatial organization of vascularized tissue, promoting uniform three-dimensional islet distribution upon transplantation. Surface characterization showed that polyamide and BioMed Clear resin exhibit distinct cell adhesion properties, enabling controlled tissue integration and atraumatic placeholder removal. Collectively, these results are consistent with preservation of vascularization, improved islet dispersion within the cell reservoir, reduced inflammatory cell infiltration, and support of metabolic function compared to injection-based delivery. These findings establish spatially controlled cell delivery within a prevascularized niche as a key design principle for enhancing engraftment efficiency and advancing the translational potential of subcutaneous islet transplantation for T1D and related cell-based therapies.

Graphical abstract

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Supplementary Information

The online version contains supplementary material available at 10.1007/s10544-026-00826-w.

Keywords: Cell encapsulation, Subcutaneous transplantation, Islet transplantation, Prevascularization, Spatial distribution, Type 1 diabetes

Introduction

Type 1 diabetes (T1D) arises from autoimmune destruction of pancreatic β-cells and is characterized by lifelong dependence on exogenous insulin therapy (Atkinson and Eisenbarth 2001; DiMeglio et al. 2018). Although insulin administration enables glycemic control, it does not fully replicate physiological regulation, leaving patients at risk of hyperglycemia and long-term complications (Forbes and Cooper 2013; Home 2012; Owens 2002; Yang and Cao 2017). Pancreatic islet transplantation (iTx) offers a promising therapeutic approach to restore endogenous insulin secretion (Gamble et al. 2018; Rickels and Robertson 2019). However, its clinical translation remains limited by key challenges, including limited donor availability, early islet loss following intraportal infusion, and the need for systemic immunosuppression (Bellin et al. 2012; Shapiro 2012).

Cell encapsulation has emerged as a strategy to overcome the immunological limitations of iTx (Dolgin 2016; Farina et al. 2019). This approach involves enclosing pancreatic islets within a semipermeable biomaterial that allows the selective diffusion of glucose, nutrients, and insulin while providing a physical barrier against immune cell infiltration (Veiseh et al. 2015a, b). However, these systems rely on passive diffusion, which can limit oxygen and nutrient transport (Ernst et al. 2022; Huan et al. 2024; Wang et al. 2024a, b). In addition, they are susceptible to foreign body reaction (FBR), leading to protein adsorption, immune cell recruitment, and fibrotic capsule formation, which collectively compromise graft survival and therapeutic efficacy (Capuani et al. 2022a, b; Veiseh et al. 2015a, b).

Given these limitations, the choice of implantation site plays a critical role in determining the success of islet engraftment and function. The subcutaneous space has emerged as a suitable extrahepatic site for iTx due to its accessibility, retrievability, and capacity to host implantable devices or biomaterials (Pepper et al. 2017; Veiseh et al. 2015a, b; Yu et al. 2020). Nevertheless, its limited vascularization and tendency toward fibrotic encapsulation create a hostile environment for oxygen-demanding islets, leading to early graft loss (Einstein et al. 2023a, b; Samuel A. Einstein et al. 2023a, b; Zhou et al. 2023). To address these challenges, biomaterial-based strategies have been designed to promote vascularization and enable controlled drug delivery, thereby providing localized and time-controlled immune protection to the graft (Dolgin 2016; Rickels 2019; Sneddon et al. 2018). Despite promising preclinical outcomes, translation remains limited (Einstein et al. 2023a, b; Zhou et al. 2023).

To overcome these barriers, we developed the Neovascularized Implantable Cell Homing and Encapsulation (NICHE) device, a subcutaneous platform engineered to establish a prevascularized space to support subsequent islet engraftment (Paez-Mayorga et al. 2022). The NICHE integrates two critical features for islet engraftment: (i) in situ pre-vascularization of a central cell reservoir, supported by mesenchymal stem cell-mediated angiogenesis, and (ii) localized immunosuppressant delivery from an adjacent U-shaped drug reservoir, which reduces systemic drug exposure while preventing graft rejection (Campa-Carranza et al. 2025; Capuani et al. 2022a, b, 2025; Paez-Mayorga et al. 2020). The spatial separation of drug and cell compartments enables sustained, controlled mass transport of immunosuppressants from the drug reservoir to the adjacent cell space, ensuring prolonged local immunoprotection with minimal systemic toxicity. Prior work with the NICHE demonstrated long-term survival of allogeneic islets and reversal of diabetes without the need for systemic immunosuppression (Paez-Mayorga et al. 2022).

Despite these advances, ensuring homogeneous islet distribution within the cell reservoir remains a key limitation. Single-port injection aggregates and confines cells along the needle track, restricting three-dimensional dispersion and resulting in local hypoxia and impaired vascularization. Furthermore, this procedure disrupts newly formed tissue, compromising the integrity of the prevascularized microenvironment. The resulting inflammatory cell infiltration poses an additional threat to transplanted islet viability and function, underscoring the need for strategies that precondition the implantation site prior to cell delivery.

Recent strategies have focused on temporary scaffolds, spacers, or degradable hydrogels to precondition implantation sites by forming vascularized niches for subsequent cell delivery (Pepper et al. 2015a, b; Saito et al. 2024; Wang et al. 2024a, b). These space-forming approaches promote angiogenesis and establish a well-perfused microenvironment, enhancing subsequent graft survival and integration. The preformed three-dimensional space also facilitates more homogeneous cell distribution, improving oxygen and nutrient diffusion and functional coupling with the surrounding vasculature (Saito et al. 2024; Wang et al. 2024a, b).

In this study, we present a refined placeholder-based strategy within the NICHE platform designed to enable atraumatic retrieval at the time of transplantation and to minimize disruption of the prevascularized microenvironment. This configuration aims to support uniform, three-dimensional distribution of transplanted islets within a defined vascularized cavity, thereby reducing cell aggregation and associated transport limitations. We hypothesized that preserving a predefined cavity during prevascularization would enable atraumatic islet loading, improve graft distribution, reduce procedural inflammation, and ultimately enhance engraftment, advancing the translational potential of subcutaneous islet transplantation and related cell-based therapies.

Methods

NICHE fabrication

Placeholder and stopper fabrication

The placeholder and stopper components were designed in SolidWorks (v2022, Dassault Systèmes) and fabricated via stereolithography (SLA) using a Form 4B printer (Formlabs) with a biocompatible photopolymer resin (BioMed Clear). The placeholder consists of a single-piece structure incorporating a central lumen aligned with a 1.2 mm access port to facilitate cell delivery, and lateral ports aligned with the DR. Devices were printed at 50 μm resolution and post-processed by isopropanol washing (20 min), UV curing (60 °C, 60 min), and support removal. The stopper component was fabricated using the same process to ensure material compatibility and sealing performance.

Device assembly

The NICHE device core was fabricated from biocompatible polyamide 12 (PA220; hereafter referred to as PA) using Selective Laser Sintering (SLS) additive manufacturing performed by Sculpteo. The architecture comprises a dual-compartment configuration, consisting of a U-shaped drug reservoir (DR) surrounding a central cell reservoir (CR). Devices were assembled following a previously established protocol (Paez-Mayorga et al. 2022). Briefly, two nanoporous polyethersulfone (PES) membranes (Sterlitech) were positioned between the DR and CR to permit controlled diffusion of immunosuppressive agents. The membranes (pore size: 30 nm, thickness: 150 μm) were bonded between the reservoir interfaces using implantable-grade silicone adhesive (MED3-4213) to ensure uniform interfacial sealing. The device was sealed on both sides with a dual-layer nylon woven mesh (Elko Filtering; outer layer 100 × 100 μm, inner layer 300 × 300 μm) providing mechanical containment while maintaining permeability for host tissue integration and vascular ingrowth. DR access ports were sealed using the previously described silicone adhesive to maintain structural integrity and prevent leakage under physiological conditions. All components were autoclaved prior to assembly. Device assembly was performed under a laminar flow hood to maintain sterility. Final sterilization was performed using ethylene oxide gas at the Houston Methodist Research Institute Current Good Manufacturing Practice (cGMP) core facility.

Two device configurations were generated: (i) NICHE devices without a placeholder and (ii) NICHE devices incorporating a removable placeholder within the CR. Both configurations were fabricated, assembled, and sterilized identically to ensure procedural consistency. When applicable, the placeholder was inserted into the CR and mechanically secured on the proximal side to ensure sealing integrity.

Roughness assessment

Surface roughness of PA and resin components was characterized by laser scanning confocal optical profilometry (VK-X1000, Keyence). Measurements (n = 15 per material) were acquired using a 4x objective over a 500 × 500 µm2 area with a lateral resolution of 0.1 μm. Three-dimensional surface maps were processed using Keyence MultiFile Analyzer, including plane correction and noise filtering. The arithmetical mean height (Sa) was used to quantify surface roughness. Additional parameters, including maximum height (Sz) and the developed interfacial area ratio (Sdr), were also evaluated.

Cell adhesion assay

Cell adhesion was evaluated using F344 rat-derived mesenchymal stem cells (MSCs; Cyagen). Samples were placed on a 1% agarose-coated 6-well plate to minimize nonspecific cell attachment to the culture plate. MSCs (P3–P5) were seeded at 1.5 × 105 cells/well and cultured in Dulbecco’s Modified Eagle Medium (DMEM) for 48 h under standard conditions (37 °C, 5% CO2). Samples were fixed in 10% formalin, dehydrated through a graded ethanol series (50–100%), and dried using hexamethyldisilazane to preserve cell morphology prior to SEM imaging.

Scanning electron microscopy (SEM) imaging

Dried samples obtained from the cell adhesion assay were sputter-coated with a 7 nm platinum–iridium conductive layer using a Q150R S sputter coater (Quorum Technologies). Imaging was performed using a field-emission scanning electron microscope (FE-SEM) (Sigma 300, Zeiss) operated at an accelerating voltage of 5 kV. Micrographs were acquired at magnifications ranging from 200x to 2000x to assess cell morphology and cell–material interactions.

Animal model

Male Fischer F344 rats (6 weeks old; Charles River Laboratories, Houston, TX, USA) were used in this study. All procedures were performed at the Houston Methodist Research Institute (HMRI) animal facility in accordance with the Animal Welfare Act, the Public Health Service Policy, and the NIH Guide for the Care and Use of Laboratory Animals. Protocols were approved by the HMRI Institutional Animal Care and Use Committee (IACUC; protocol IS00007362). Animals were housed under standard conditions with ad libitum access to food and water.

NICHE implantation

NICHE devices were loaded with mesenchymal stem cells (MSCs) to promote prevascularization, following previously established protocols (Campa-Carranza et al. 2025; Capuani et al. 2025; Paez-Mayorga et al. 2020, 2022). Briefly, bone marrow–derived MSCs from F344 rats (Cyagen) were used at passages 3–5 and suspended in a thermosensitive Pluronic F-127 hydrogel (20% v/v in DMEM; Sigma-Aldrich), which remains liquid at low temperature and undergoes gelation at physiological temperature. The cell–hydrogel suspension (5 × 10⁵ MSCs in 400 µL) was introduced into the CR through the dual-layer nylon mesh.

Both device configurations (with and without placeholder) were loaded using identical procedures. Devices were implanted immediately after loading to preserve cell viability and subsequent vascularization.

For implantation, animals were anesthetized with inhaled isoflurane, and devices were placed subcutaneously in the dorsal region through a small incision, as previously described (Paez-Mayorga et al. 2022).

Vascularization analysis

NICHE devices with and without the placeholder were loaded with MSCs and implanted subcutaneously in F344 rats for 6 weeks (n = 4 per configuration). Following explantation, tissues were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Prior to embedding, the NICHE framework was removed to facilitate sectioning. Tissue sections (5 μm) were stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome (MT) at the HMRI Research Pathology Core. For blood vessel labeling, antigen retrieval was performed using rodent decloaker solution (Biocare Medical), followed by blocking in 5% normal goat serum. Sections were incubated overnight at 4 °C with biotinylated Bandeiraea simplicifolia lectin (10 µg/mL), followed by incubation with streptavidin–alkaline phosphatase (1:100). Signal detection was performed using Warp Red chromogen according to the manufacturer’s instructions.

Sections corresponding to the CR were imaged using a BZ-X800 microscope (Keyence), and whole-reservoir images were reconstructed by stitching fields acquired at 20x magnification.

Vascularization within the CR was quantified from lectin-stained sections by measuring (i) blood vessel density (vessels/mm²) and (ii) vessel area normalized to CR area. Quantitative analysis was performed using a custom semi-automated Python-based algorithm for vessel identification and morphometric analysis.

Rat Islet isolation

Pancreatic islets were isolated from syngeneic Fischer 344 donor rats using a collagenase digestion protocol as previously described (Seaborne et al. 2026). Briefly, the pancreas was perfused via the common bile duct with collagenase solution (CIzyme RI, Vitacyte) supplemented with DNase I in Hanks’ Balanced Salt Solution (HBSS; Gibco), excised, and digested at 37 °C. Islets were purified by density gradient centrifugation (OptiPrep, Sigma-Aldrich) and cultured for 72 h prior to transplantation under standard conditions (25 °C, 5% CO2).

Placeholder strategy efficacy study in diabetic rats

To evaluate the effect of spatially controlled transplantation on graft performance, two NICHE configurations were compared: devices incorporating a solid placeholder (SPH) and standard devices without placeholder (CTRL). Devices were loaded with MSCs and implanted subcutaneously in male Fischer 344 rats (n = 10 per group) as described above. Both healthy and diabetic controls were included. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 50 mg/kg), and animals were considered diabetic after three consecutive blood glucose measurements > 300 mg/dL.

Following a 5-week prevascularization period, syngeneic pancreatic islets were transplanted into the cell reservoir (6500 IEQ/kg). In CTRL devices, islets were delivered via percutaneous injection through the access port, as previously described (Campa-Carranza et al. 2025; Capuani et al. 2022a, b, 2025; Paez-Mayorga et al. 2020, 2022). In SPH devices, the placeholder was surgically exposed and removed, and islets were delivered directly into the preformed cavity, followed by insertion of a stopper to reseal the device.

Devices were explanted at 2 weeks (n = 3 per group) for early-stage analysis, with samples allocated for flow cytometry and histological assessment. Remaining animals (n = 7 per group) were maintained until 45 days post-transplantation for endpoint evaluation. For assessment of vascular perfusion and islet engraftment, a subset of animals received intravenous lectin labeling prior to perfusion fixation. Explanted devices were processed for either light-sheet (n = 3 per group) microscopy or histological analysis (n = 4 per group). For light-sheet microscopy, the whole device was processed for tissue clarification.

Blood glucose measurement, intraperitoneal glucose tolerance test (IPGTT), and C-peptide quantification

Blood glucose (BG) levels were measured via tail vein sampling using a veterinary glucometer (AlphaTrack, Zoetis). Intraperitoneal glucose tolerance tests (IPGTTs) were performed prior to islet transplantation and 7 days post-transplantation. Rats were fasted for 3 h and injected intraperitoneally with 3 g/kg dextrose solution. BG levels were recorded at 0, 15, 30, 60, 90, and 120 min.

For functional assessment, plasma C-peptide levels were measured 10 days post-transplantation. Blood samples were collected via tail vein, and plasma was isolated by centrifugation (2,000 ×g, 12 min). C-peptide concentrations were quantified using a rat ELISA kit (Crystal Chem) according to the manufacturer’s instructions.

Flow cytometry

Explanted NICHE devices were processed to obtain single-cell suspensions from the cell reservoir (CR). Tissue was mechanically dissociated and enzymatically digested in RPMI containing collagenase/hyaluronidase and DNase I at 37 °C. The resulting suspension was filtered (40 μm), centrifuged, and treated with ACK lysis buffer to remove red blood cells. Cells were resuspended in FACS buffer and counted by Trypan Blue exclusion.

For flow cytometry, 1 × 106 cells were stained with a viability dye followed by surface markers for CD45, CD11b, CD103, CD86, CD163, RT1-B (MHCII), and CD43. Intracellular staining for CD68 was performed after fixation and permeabilization. Unstained and fluorescence-minus-one (FMO) controls were included. A detailed list of antibodies, including sources and catalog numbers, is provided in Supplementary Table S1.

Data were acquired after exclusion of debris, doublets, and dead cells. Leukocytes were defined as CD45+ cells, and myeloid populations as CD45+CD11b+ subsets. Macrophages were identified as CD45+CD11b+CD68+ and further characterized based on CD86 and CD163 expression. Antigen-presenting cell activation was assessed by RT1-B expression, and dendritic cell–associated populations by CD103. CD43 expression and side scatter (SSC-A) were used to further characterize neutrophil and monocyte populations.

Histological analysis

Tissue fixation, paraffin embedding, sectioning (5 μm), H&E/MT staining, image acquisition, and lectin-based quantitative vascular analysis were performed as described above. Whole-slide scans and high-resolution images were acquired using a BZ-X800 microscope (Keyence) at 20x magnification.

Tissue clarification and light sheet microscopy

Explanted tissues were cleared using the EZ Clear protocol (Hsu et al. 2022) and immunolabeled for insulin (anti-rat insulin, C27C9; Cell Signaling) and vascular structures (lectin-DyLight649). Cleared samples were equilibrated in refractive index–matching solution (EZ View), embedded in agarose, and imaged using a Zeiss Lightsheet Z.1 microscope (5x objective, 0.5× zoom). Volumetric datasets were acquired as tiled Z-stacks (20% overlap) at a voxel resolution of 1.829 × 1.829 × 7.03 μm³, with acquisition parameters maintained constant across samples.

Image stitching was performed using Stitchy, and three-dimensional reconstruction and quantitative analysis were conducted in Imaris. Insulin-positive (islet) and lectin-positive (vascular) volumes were segmented using automated intensity thresholding. Islet engraftment efficiency was calculated as the ratio of measured islet volume to theoretical transplanted volume (1 IEQ assumed as a 150 μm diameter sphere).

Statistical analysis

Statistical analyses were performed using GraphPad Prism (version 10.2.3). Comparisons between two groups were conducted using an unpaired two-tailed Student’s t-test, while multiple group comparisons were analyzed using two-way ANOVA. Data are presented as mean ± standard deviation (SD). Statistical significance thresholds are indicated in the respective figure legends.

Results

Material choice validation

The NICHE device consists of a polyamide (PA) core structure subdivided into the DR and CR, and a removable placeholder component that is replaced by a stopper following transplantation (Fig. 1A, B).

Fig. 1.

Fig. 1

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Structural configuration and surface characterization of NICHE device materials. (A) CAD renderings of the placeholder component and assembled NICHE device with placeholder inserted, representing the pre-transplant configuration. (B) Post-transplant configuration showing the device with the stopper after placeholder removal and cell loading. (C) Quantitative comparison of areal surface roughness (Sa) between PA and resin substrates (mean ± SD; ***p < 0.001). (D) Representative 3D optical profilometry reconstructions of PA and resin surfaces. Color scale represents surface height, from lowest (blue) to highest (red). (E–F) Corresponding surface height maps of (E) PA and (F) resin over a 500 μm × 500 μm area. (G–H) Scanning electron microscopy (SEM) images of PA and resin surfaces prior to cell seeding. (I–J) SEM images after cell seeding, showing differential cell adhesion on PA and resin substrates. Scale bars: (E–F) 500 μm; (G, I) 20 μm; (H, J) 50 μm

Quantitative surface roughness analysis showed that PA exhibited significantly higher arithmetical mean height (Sa) values compared to the resin placeholder (PA: 7.85 ± 1 μm vs. resin: 6.42 ± 0.86 μm; Fig. 1C), consistent with a rougher surface profile. Additional surface parameters, including maximum height (Sz) were consistent with Sa findings, while the developed interfacial area ratio (Sdr) was higher for the resin (Supplementary Fig. S1), reflecting its more finely structured surface.

Optical profilometry revealed distinct surface topographies (Fig. 1D–F). The PA surface exhibited a heterogeneous morphology with pronounced peak–valley variations, whereas the resin surface displayed a more uniform and linearly patterned profile. These qualitative observations are consistent with the measured Sa values, with PA samples showing greater surface heterogeneity relative to the smoother, more uniform resin surface.

MSC adhesion on PA and resin surfaces

Scanning electron microscopy showed that, in the absence of cells, both PA and resin surfaces exhibited clean surface morphologies (Fig. 1G–H). After 48 h of MSC culture, PA surfaces exhibited adherent cells with a flattened morphology and visible cytoplasmic extensions (Fig. 1I). In contrast, resin samples showed no detectable cell attachment, and the surface morphology remained largely comparable to unseeded controls (Fig. 1J), consistent with reduced cell adhesion and surface profile favorable for atraumatic placeholder removal.

Vascularization and foreign body response in the NICHE

Histological evaluation of explanted NICHE devices after 6 weeks of subcutaneous implantation are consistent host tissue infiltration and vascular development within the device (Fig. 2A, D). Lectin staining revealed integration of host tissue surrounding the implant and supported the presence of well-defined capillary structures characterized by erythrocyte-filled lumens within the surrounding connective tissue (Fig. 2B, E).

Fig. 2.

Fig. 2

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Histological evaluation of vascularization and fibrosis after implantation. (A) Representative hematoxylin and eosin (H&E)–stained section of the CTRL device, highlighting microvascular structures within the cell reservoir (scale bars: 50 μm). (B) Higher-magnification lectin-stained section of the CTRL device. (C) Quantification of capillary density (vessels/mm²) (blue: CTRL; green: SPH). (D) Representative H&E-stained section of SPH device, showing the defined cavity generated by the placeholder removal, with body-facing and skin-facing interfaces visible (scale bars: 500 μm). (E) higher magnification of SPH device. (F) Quantification of capillary area fraction (%). (G) Representative Masson’s trichrome–stained sections illustrating collagen deposition and fibrotic capsule formation surrounding the implanted device (scale bar: 500 μm). (H) Higher-magnification views of the fibrotic capsule and internal fibrotic layer; arrowheads indicate the internal fibrotic layer (scale bar: 50 μm). (I) Quantification of fibrotic capsule thickness, distinguishing the external capsule surrounding the device and the internal fibrotic layer formed within the cavity during implantation

Capillary area fraction did not differ between groups (CTRL: 13.64 ± 2.44%; SPH: 11.17 ± 5.04%; Fig. 2F), consistent with preservation of vascularization in the presence of the placeholder during preconditioning.

Masson’s Trichrome staining showed the presence of a fibrotic capsule surrounding the implants in both conditions (Fig. 2G, H). Fibrotic capsule thickness was not significantly different between CTRL and SPH NICHE devices (CTRL: 210.41 ± 73.01 μm; SPH: 301.21 ± 83.85 μm; Fig. 2I). Quantification of the inner fibrotic layer adjacent to the device in the SPH NICHE group indicated a thickness of 28.27 ± 4.44 μm (Fig. 2I).

Islet transplantation outcomes in NICHE devices

Metabolic outcomes following islet transplantation were compared between the current NICHE design (CTRL) and the placeholder-based SPH NICHE configuration. Devices were implanted to allow a prevascularization phase prior to transplantation. Diabetes was induced with streptozotocin (STZ), followed by an insulin control phase to manage hyperglycemia before islet transplantation (day 0). Animals were monitored through serial metabolic assessments and tissue analyses up to day 45.

Fasting C-peptide levels increased from 0.05 ± 0.072 ng/mL (pre-transplant) to 0.24 ± 0.027 ng/mL (post-transplant) in CTRL animals (p = 0.0005), and from 0.02 ± 0.011 ng/mL to 0.22 ± 0.008 ng/mL in SPH animals (p < 0.0001) (Fig. 3A). Blood glucose levels (BGL) increased following STZ administration in both groups and were normalized during the insulin control phase. After islet transplantation (day 0), BGL remained below 200 mg/dL with comparable glycemic control between groups. Body weight decreased post-STZ and progressively recovered after transplantation, stabilizing by day 45. These data, along with additional metabolic assessments, are reported in Supplementary Figs. S2–S3; interpretation of IPGTT results was limited due to residual effects of insulin pellets.

Fig. 3.

Fig. 3

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In vivo functional and immunological assessment in a syngeneic transplantation model. (A) Fasting C-peptide levels measured pre- and post-transplantation for CTRL and SPH groups. (B) Representative flow cytometry contour plots of SSC-A/CD43⁺ populations showing neutrophils and monocytes. (C) Quantification of CD11b⁺ immune cell subsets, including neutrophils, monocytes, and dendritic cells (DCs). Data are presented as mean ± SD; statistical significance as indicated in the respective panels

Inflammatory Cell Infiltration Following Islet Transplantation

A syngeneic transplantation model was used to isolate the inflammatory response associated with device implantation and the transplant procedure, minimizing confounding immune responses to the islets. Innate immune cells, including neutrophils and macrophages, are key mediators of this procedural inflammatory response. Neutrophils act as first responders and facilitate the subsequent recruitment of monocytes involved in resolving the inflammatory response. The 14-day time point was selected to evaluate residual immune cell presence following resolution of the acute inflammatory phase and to allow tissue remodeling within the cavity generated by placeholder removal.

Immune cell populations within the NICHE were analyzed by flow cytometry 14 days post-transplantation (n = 3 per group; Fig. 3B-C). The percentage of neutrophils was 11.94 ± 6.54% in CTRL devices and 4.38 ± 0.83% in SPH devices (p = 0.0236). Monocyte levels were 9.54 ± 3.64% in CTRL and 5.07 ± 0.38% in SPH (p = 0.1026), and dendritic cells accounted for 4.52 ± 1.19% in CTRL and 1.82 ± 0.58% in SPH (p = 0.0253), aligned with a reduced inflammatory cell presence in SPH devices.

Histological Evaluation of Islet Engraftment and Revascularization

Histological and quantitative tissue analyses were performed at days 14 and 45 post-transplantation to characterize tissue organization and vascularization within the CR of CTRL and SPH devices. Representative H&E-stained cross-sections of the CR are shown in Fig. 4A. At both time points, tissue infiltration throughout the reservoir was observed in both device configurations.

Fig. 4.

Fig. 4

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Longitudinal histological and functional assessment of islet engraftment. (A) Representative H&E-stained sections of explanted CTRL and SPH NICHE at day 14 (rows 1–2) and day 45 (rows 3–4); arrowheads indicate engrafted islets within the cell reservoir (scale bars: 500 μm). (B–C) Higher-magnification lectin-stained sections showing peri-implant vasculature and intra-islet morphology (scale bars: 50 μm). (D) Quantification of vessel density (vessels/mm2) at day 14 and day 45. (E) Capillary area fraction (AF) within peri-implant tissue at day 14 and day 45. (F) Islet capillary area fraction at day 14 and 45. Data are presented as mean ± SD; statistical significance as indicated in respective panels. (G–H) Representative light-sheet fluorescence microscopy images of explanted CTRL and SPH NICHE showing lectin-labeled vascular structures (white) and insulin-positive islets (green) (scale bars: 1000 μm). Images were brightness-adjusted in post-processing to enhance visualization of islet distribution. (I) Quantification of engrafted islets based on insulin signal. Data are presented as mean ± SD; ns, not significant

Lectin staining was used to visualize vascular structures within the CR at day 14 and day 45 post-transplantation (Fig. 4B). Vessel density at day 14 was 1465.82 ± 468.6 mm⁻2 (CTRL) and 1343.56 ± 222.3 mm⁻2 (SPH). At day 45, vessel density was 1345.66 ± 73.2 mm⁻2 (CTRL) and 1261.53 ± 323.0 mm⁻2 (SPH), showing no statistically significant differences (Fig. 4D). Capillary area fraction was 19.89 ± 5.26% (CTRL) and 20.07 ± 8.27% (SPH) at day 14, and 19.27 ± 6.03% (CTRL) and 16.22 ± 5.49% (SPH) at day 45, with no significant differences observed between groups at either time point (Fig. 4E), suggesting that vascular organization within the CR is maintained over time following placeholder removal. Engrafted islets were identified within the CR at day 14 and day 45 in both CTRL and SPH groups. In CTRL devices, islets presented as discrete clustered structures, whereas SPH devices showed a more distributed pattern of islet localization across the CR (Fig. 4A), suggesting improved spatial dispersion with the placeholder approach. Islet-associated vascularization, quantified as capillary coverage of islet structures, was 18.25 ± 5.06% (CTRL) and 19.48 ± 4.85% (SPH) at day 14, and 16.53 ± 3.56% (CTRL) and 15.51 ± 1.53% (SPH) at day 45, with no significant difference between groups at either time point (Fig. 4E–F), indicating comparable vascular support and engraftment over time.

Spatial distribution of engrafted islets

Three-dimensional organization and spatial distribution of engrafted islets within the CR were assessed by light-sheet fluorescence microscopy. Insulin-positive islets and lectin-labeled vascular structures were visualized throughout the device, enabling evaluation of islet localization relative to the surrounding vascular network.

Light-sheet imaging revealed distinct spatial distribution patterns of engrafted islets between device configurations (Fig. 4G–H). In CTRL devices, insulin-positive islets were predominantly localized in clustered specific regions of the CR, with limited presence toward peripheral areas. In contrast, SPH devices showed a more uniform distribution of insulin-positive islets across the entire CR volume, with islets detected in both distal and port regions and at multiple depths within the reservoir, suggesting improved spatial dispersion with the placeholder approach.

Lectin-positive vascular structures were observed in proximity to insulin-positive islets in both groups. Volumetric segmentation of insulin-positive structures revealed 382 ± 86 engrafted islets in SPH devices compared to 262 ± 64 in CTRL devices (p = 0.1272), representing a 46% greater islet count in the placeholder group (Fig. 4I), consistent with enhanced graft retention and more complete engraftment within the prevascularized CR.

Discussion

The present study demonstrates that the incorporation of a removable placeholder within the NICHE platform enables preservation of a defined transplantation cavity during prevascularization, promotes more uniform spatial organization of engrafted islets, reduces delivery-associated tissue disruption, and is associated with a lower local inflammatory response.

In the original NICHE configuration, islets are delivered through direct needle injection, which can lead to clustering of the graft along the injection track, suboptimal use of the available CR volume, and local tissue disruption associated with the transplant procedure. By contrast, the placeholder-based strategy preserves a predefined cavity during prevascularization, enabling atraumatic cell loading and more controlled graft placement.

This design builds upon previous NICHE studies demonstrating that the subcutaneous space, when properly conditioned, can support stable vascularization and sustained function of implanted cell therapies, while also offering advantages in accessibility, retrievability, and clinical translatability. Within this framework, material selection was guided by the need to enable controlled spatial organization of the transplantation site, allowing prevascularization while preserving a defined cavity for subsequent islet delivery. The PA core promotes a controlled foreign body response that supports implant stabilization without impairing vascular infiltration, as previously reported (Capuani et al. 2025; Paez-Mayorga et al. 2022). In contrast, the placeholder component was designed as a removable, low-adhesion element to preserve niche integrity upon extraction.

BioMed Clear resin was selected for this purpose due to its compatibility with high-resolution stereolithography fabrication, which enables the generation of relatively smooth surfaces compared to the PA core (Palivela Bhargav et al. 2026). By combining a rougher PA structure with a smoother resin surface, the device enables tissue integration where desired while limiting adhesion to the placeholder, consistent with established material strategies (Capuani et al. 2022a, b; Gomes et al. 2025; Kim et al. 2021; Nitti et al. 2023).

Preservation of vascularization during preconditioning represents a critical requirement for this approach. Prior NICHE studies have shown that adequate capillary density within the CR is essential for supporting islet survival and function (Campa-Carranza et al. 2025; Capuani et al. 2025; Paez-Mayorga et al. 2022). In this study, vascularization was maintained in the presence of the placeholder, indicating that the introduction of a transient, space-defining element does not impair the angiogenic response. This result is consistent with emerging strategies in tissue engineering in which temporary scaffolds or placeholders are used to guide tissue organization while allowing vascularization to proceed in adjacent regions (A. R. Pepper et al. 2015a, b; Saito et al. 2024; L. H. Wang et al. 2024a, b).

From a biological perspective, these findings are particularly relevant given the high oxygen demand of pancreatic islets. Islets are highly sensitive to hypoxia, and clustered graft configurations can exacerbate local oxygen and nutrient gradients, leading to impaired survival and function. In conventional injection-based delivery, heterogeneous cell distribution and local tissue disruption can further limit vascular access. By enabling a more homogeneous three-dimensional distribution of islets within a prevascularized space, the placeholder strategy promotes more comparable vascular proximity across the graft and attenuates oxygen and nutrient diffusion gradients.

Evaluation of the fibrotic response revealed the formation of a thin fibrotic layer lining the cavity generated by the placeholder. This layer remained limited in thickness and did not appear to restrict tissue reorganization or islet integration following placeholder removal.

To assess the functional relevance of this design, the redesigned NICHE device was evaluated against the original configuration in a transplantation setting, with a focus on how the delivery strategy influences the local inflammatory response. Direct injection has been associated with tissue injury and prolonged immune cell recruitment (Chen et al. 2024; Ramot et al. 2019). In contrast, the placeholder-based approach enables cell delivery into a preformed cavity, avoiding additional tissue penetration at the time of transplantation and thereby reducing procedural trauma. Consistent with this, a reduction in innate immune cell infiltration was observed in the SPH configuration, suggesting improved microenvironmental conditions for early engraftment. Although not all metrics reached statistical significance, the consistent directional trends observed across vascularization and graft integration endpoints are in line with a reduced inflammatory burden from the procedure and preserved vascular niche integrity in the SPH configuration.

Volumetric imaging further demonstrated that the placeholder design promotes a more uniform distribution of engrafted islets throughout the CR, compared to the clustered and injection-dependent localization observed in CTRL devices. This spatial organization reflects more uniform graft dispersion across the available CR volume and may contribute to enhanced retention, as indicated by the higher number of insulin-positive structures observed in the SPH configuration. From a translational perspective, these findings highlight the importance of spatial control in the design of implantable cell therapy platforms. The subcutaneous space offers clear advantages in accessibility and retrievability but is inherently limited by poor vascularization and susceptibility to fibrotic encapsulation. By combining prevascularization with controlled spatial delivery, the placeholder strategy addresses key barriers to subcutaneous transplantation, including heterogeneous graft distribution within the available volume, elevated procedural inflammatory burden, and destabilization of the prevascularized niche.

More broadly, this work suggests that preservation of a preconditioned microenvironment, coupled with controlled graft placement, represents a mechanistically grounded design principle for optimizing use of the transplantation volume, attenuating procedural inflammation, and preserving vascular niche integrity in implantable cell therapies.

Despite these strengths, several limitations should be acknowledged. The study was conducted in a syngeneic rodent model, which isolates device-related effects but does not fully capture the immune complexity of clinical transplantation. Interpretation of metabolic outcomes was limited by the presence of background exogenous insulin, which may have masked the independent contribution of the graft. In addition, the islet dose used was subtherapeutic and not expected to achieve full glycemic correction. Accordingly, this study primarily relies on structural and spatial metrics, and future studies incorporating longitudinal functional readouts without exogenous insulin confounding will be required to establish functional superiority. Finally, findings are limited to a defined post-transplantation time window, and longer-term studies will be necessary to evaluate sustained graft function and tissue remodeling.

In conclusion, this study establishes that spatial control of graft placement within a prevascularized niche is a key determinant of engraftment efficiency. By preserving a defined transplantation cavity and minimizing procedural disruption, the placeholder strategy provides a framework for improving the performance and translational potential of subcutaneous islet transplantation and related cell-based therapies. These findings highlight spatial control of the transplantation site as a critical design principle for enhancing the reliability and reproducibility of implantable cell therapy platforms.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary file 1 (802.1KB, pdf)

Acknowledgements

The author thanks Dr. Nicola Di Trani for insightful discussions and Yuelan Ren from the Research Pathology Core at Houston Methodist Research Institute for technical assistance. This work is funded by Breakthrough T1D from JDRF 2-SRA-2021-1078-S-B (A.G). The graphical abstract was created with BioRender.com.

Author contributions

E.B.: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing – Original draft. J.N.C.C.: Methodology, Investigation, Formal analysis. T.B.: Methodology, Investigation. G.E.R.: Investigation. H.S.: Investigation. A.V.: Investigation. R.O.: Investigation, Writing – Review & Editing. J.P.M.: Conceptualization, Supervision, Writing – Review & Editing. C.Y.X.C.: Supervision, Writing – Review & Editing. A.G.: Conceptualization, Supervision, Project administration, Writing – Review & Editing, Funding acquisition.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

J.P.M., C.Y.X.C., and A.G. are inventors of intellectual property licensed by Continuity Biosciences. AG is a co-founder and scientific advisor of Continuity Biosciences. The other authors declare no conflict of interest.

Footnotes

Publisher’s note

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Supplementary Materials

Supplementary file 1 (802.1KB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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