Reversal of Hyperglycemia by Subcutaneous Islet Engraftment Using an Atelocollagen Sponge as a Scaffold

Abstract

Type 1 diabetes mellitus (T1DM) affects 8.4 million people worldwide, with patients primarily relying on exogenous insulin injections to maintain blood glucose levels. Islet transplantation via the portal vein has allowed for the direct internal release of insulin by glucose-sensitive islets. However, this method might not be desirable for future cell therapy transplanting pluripotent stem cell-derived β cells, facing challenges including difficulties in cell retrieval and graft loss due to the instant blood-mediated inflammatory reaction (IBMIR). Here, we established a subcutaneous transplantation protocol using an atelocollagen sponge as a scaffold. While the subcutaneous site has many advantages, the lack of a vascular bed limits its application. To address this issue, we performed angiogenesis stimulation at the transplantation site using bFGF absorbed in a gelatin sponge (Spongel), significantly improving the microvascular area. Our in vivo experiments also revealed angiogenesis stimulation is crucial for reversing hyperglycemia in streptozotocin (STZ)-induced diabetic mice. In addition to the angiogenic treatment, an atelocollagen sponge is used to carry the islets and helps avoid graft leakage. With 800 mouse islets delivered by the atelocollagen sponge, the STZ-induced diabetic mice showed a reversal of hyperglycemia and normalized glucose intolerance. Their normoglycemia was maintained until the graft was removed. Analysis of the harvested islet grafts exhibited a high vascularization and preserved morphologies, suggesting that using an atelocollagen sponge as a scaffold helps maintain the viability of the islet grafts.

Graphical Abstract.

Introduction

Diabetes mellitus is a chronic health disease characterized by high blood glucose levels. Type 1 diabetes is characterized by insulin deficiency and hyperglycemia, mainly resulting from the autoimmune destruction of pancreatic β cells. About 8.4 million individuals worldwide are diagnosed with type 1 diabetes mellitus (T1DM) ¹ . T1DM patients maintain normal glycemia with daily insulin injections. Unlike glucose-responsive insulin secretion by islets, insulin injection results in suboptimal blood glucose regulation. Restoring lost pancreatic β cells through islet transplantation emerges as a curative solution but is limited by donor shortage.

Islet transplantation is shown to be successful through intraportal transplantation. However, intraportal transplantation might not be a desirable site for future cell therapy transplanting pluripotent stem cell–derived β cells. Cell loss attributed to the instant blood-mediated inflammatory reaction (IBMIR) occurs ² . The quest for alternative sites for islet transplantation has been unceasing, including the gastric submucosa and spleen^3,4, chosen for their physiological similarities to the pancreas. Immunologically privileged sites, such as the testis, eyes, and thymus^5–7, offer the potential for reduced graft rejection due to their suppressed immune responses. The submandibular gland, with its histological resemblance to the pancreatic gland, may support islet functionality ⁸ . The accessible subcutaneous and intramuscular sites provide practical and safe options for transplantation^9–13.

Subcutaneous space is undoubtedly a promising site for transplantation. The skin has an extensive capacity, enabling subcutaneous tissue to house a substantial quantity of donor cells. Subcutaneous transplantation is notably straightforward, comparatively safe, and easily accessible, obviating the need for open surgery. Subcutaneous transplantation is associated with minimal bleeding and a reduced likelihood of IBMIR, features that have garnered the interest of researchers in recent years.

However, the absence of a vascular bed within subcutaneous tissue presents a significant obstacle to early engraftment. Graft vasculature is essential for successful engraftment, precise blood glucose level monitoring, and rapid insulin release. Several studies aim to address this challenge, primarily focusing on three approaches. The first involves pre-vascularization, with or without growth factors, to induce angiogenesis^12,13. The second strategy is co-transplanting endothelial cells (ECs) and mesenchymal stem cells (MSCs) to accelerate vascularization^14,15. The third approach employs transplanting the islets with novel hydrogels to facilitate vascularization post-transplantation^11,16. Although the aforementioned approaches seem successful, the accessibility of the materials or techniques might be a barrier.

Here, we sought an accessible approach for angiogenesis stimulation and a commercially available scaffold for islet transplantation. We selected basic fibroblast growth factor (bFGF) for its cost-effectiveness and efficacy for angiogenesis stimulation. We implanted the islets using an atelocollagen sponge scaffold to the pre-vascularized subcutaneous site.

Materials and Methods

Animals

Ctl: CD1 (ICR), BALB/c (purchased from The Jackson Laboratory), and Rag-2/Jak3 double-deficient BALB/c (BRJ) ¹⁷ mice were used. Mice were maintained by crossing females with male mice or in vitro fertilization. Mice were allowed free access to food and water except when being fasted. Non-fasting blood glucose levels and body weights were measured from 8 weeks of age onwards in mice using a blood glucose meter ANTSENSE III (Horiba, Kyoto, Japan) by making an incision in the tail.

Scaffolds

Commercially available atelocollagen sponge (AteloCell^® Atelocollagen sponge, MIGHTY, ø5 mm × 3 mm, CSM-25, KOKEN, Tokyo, Japan) was manually cut into 1/6 size with a half-moon shape, 1 mm thick (Fig. 1A), to be used for carrying islets for transplantation.

Figure 1.

Subcutaneous transplantation of islets into a pre-vasculature stimulated site using an atelocollagen sponge scaffold. (A) A commercially available atelocollagen sponge scaffold is manually cut to a half-moon shape with a 5 mm diameter and 1 mm thickness (left). Blender (https://www.blender.org) was used to draw the 3D illustration (right). (B) Schematic representation of introducing islets into atelocollagen sponge. The islets were dispensed in 5μl of medium and then absorbed by an atelocollagen sponge. (C, D, E) The top panels in (C) and (D) show a bright field (BF) of islets. The islet’s distribution in the atelocollagen sponge (outlined by a broken line) (C) was revealed by Live (green) /Dead (red) staining (C). The control naked islets’ Live/ Dead staining result (D). Scale bars: 500 µm. (E) Quantitative analysis of live/dead assay. Data are presented as mean ± SD. The viability of islets absorbed in the atelocollagen sponge was high (>90%), not significantly different from the naked islets, analyzed by unpaired two-tailed Student’s t-test.

Live/Dead Assay

Freshly isolated mice islets were introduced to the atelocollagen sponge scaffold immediately before the Live/Dead assay. Calcein-AM (green, live) and ethidium homodimer (red, dead) were used to stain the islet-carried scaffold and naked islets according to the manufacturer’s protocol (R37601, ThermoFisher Scientific). Images were acquired with an Olympus BX51-FL (Olympus, Japan). Quantification of the cell viability was carried out by calculating the area of green (live) and red (dead) fluorescence using ImageJ. % cell viability: the green area versus the total islet area.

Diabetes Model Mice Generation

Streptozotocin (STZ) (195-15154, Wako, Japan) was freshly dissolved in 0.5 M citrate buffer (pH 4.5), kept on ice until use, and used within 15 min. Eight-week-old male BRJ or BALB/c mice received an intravenous injection of 140 mg/kg (BRJ) or 120 mg/kg (BALB/c). Their non-fasting blood glucose levels and body weights were measured 3, 7, and 14 days post-STZ injection. Mice with non-fasting blood glucose levels over 300 mg/dL and less than 700 mg/dL on day 14 post-injection were used.

Anigogenesis Stimulation

Under isoflurane anesthesia, an approximate 1.5 cm incision at the neck area of the diabetic mice was made. The skin and the subcutis were separated, and a pocket-like space was generated using a small scissor. For each mouse, 100 µl trafermin (Fiblast^®; bFGF 100 µg/ml; 2699710R2024, Kaken Pharmaceutical Co., Ltd., Japan) was soaked onto a freshly cut gelatin sponge (Spongel^®; 919100716, Astellas, Japan), cut into pieces of 1 cm (length) × 1 cm (width) × 0.5 cm (thick). Once the gelatin sponge absorbed all the solution and became soft, it was inserted into the generated subcutis space. The incision was closed by a Skin AutoClip (C-29, Natsume Seisakusho Co., Ltd., Japan).

Quantification of the Blood Vessel

To evaluate the angiogenesis stimulation (Fig. 2), one week after angiogenesis treatment, the subcutaneous site was opened, the inserted gelatin sponge was removed, and an image was taken. The image was converted to a black-white image using Adobe Photoshop software (Adobe 22.5.0), analyzed by ImageJ/ Fiji (version 2.14.0, https://imagej.nih.gov/ij/), and the areas below the threshold were outlined (Supplementary Figure S1, right panel). The sum was calculated as the blood vessel area. Blood vessel area percentage: blood vessel area versus the total area.

Figure 2.

Angiogenesis stimulation using bFGF and gelatin sponge (Spongel) enhanced the area of vessel blood. (A) A schematic drawing of the experiment is shown. (B) Images of mouse skin one week after angiogenesis stimulation, displaying the microstructure with microvascular formation. From left to right: (upper panels) representative images of the Blank control group (cut open), 100 µl PBS contained gelatin sponge group; (lower panels) 100 µl bFGF alone group, and the 100 µl bFGF containing gelatin sponge group, respectively. Scale bar: 1 cm. (C) The blood vessel (red) area percentage of the four treated groups (n = 3 mice in each group). Data are presented as mean ± SD. Significant differences are shown as ^*P < 0.05, ^**P < 0.01, analyzed by one-way ANOVA and Dunnett’s multiple comparisons test.

The harvested grafts were sectioned, and the blood vessels were visualized by anti-CD31 antibody staining (Fig. 3). The ratio of the CD31 areas versus the total islet area shows the percentage of the blood vessel area in the islets.

Figure 3.

Reversal of hyperglycemia by subcutaneous islet transplantation in BALB/c mice. BALB/c mice islets (mislet) subcutaneously transplanted to diabetic BALB/c. Recipients reversed diabetes and acted similarly to healthy mice after islet transplantation with angiogenesis, While islet transplantation without angiogenesis stimulation results in primary non-function. (A) A schematic diagram of the experimental design; (B–G) Magenta lines, With angiogenesis stimulation (angio-treated), n = 4; gray lines, without angiogenesis (non-angio-treated), n = 3; open circle plots with black lines, healthy mice, n = 6; (B, C) (B) Non-fasting blood glucose levels. Broken gray line: normoglycemia threshold of 200 mg/dL. (C) Body weight of diabetic BALB/c mice transplanted with 800 BALB/c mouse islets; (D, E, F, G) Intraperitoneal Glucose Tolerance Test (IPGTT) at post-operation week 8 (POW8)(D, E) or POW 16 (F, G). (D, F) Blood glucose of the recipients in the pre-angiogenesis group (Angio-treated group) exhibited responses resembling those of healthy mice. In contrast, recipients in the non-pre-angiogenesis group (Non-angio-treated group) showed typical diabetic reactions characterized by a delayed peak in blood glucose levels, higher glucose concentrations, and an inability to return to baseline levels within 120 min. (E, G) The Area under the curve (AUC) of the angio-treated and healthy mice was significantly lower than the non-angio-treated mice groups. Data are presented as mean ± SD. Significant differences are shown as *P < 0.05, ***P < 0.001, analyzed by one-way ANOVA and Tukey’s multiple comparisons test.

(H, I, J) Immunohistochemical analysis of the harvested graft at POW 24 and 30. (H, I) Representative images are shown. Insulin (INS), green; glucagon (GCG), red; CD31, gray. The ratios of the CD31 areas versus total islet area in the angio-treated islets (each dot represents one islet, 13 islets from 4 mice) (H) are significantly higher than those in non-angio-treated islets (each dot represents one islets, 5 islets from 3 mice) (I). (J) Data are presented as mean ± SD. Significant differences are shown as *P < 0.05, analyzed by unpaired two-tailed Student’s t-test.

Islets Isolation

Wild-type (WT) 8-week-old male ICR or BALB/c mice were used as donors for islet transplantation. Islets were isolated with some modifications by the collagenase digestion of the pancreas, as described previously (Kikawa et al.). Briefly, donor mice were euthanized, and the pancreas was exposed by laparotomy. The common bile duct was exposed and clamped at Vater’s ampulla. 2–3 mL of cold DMEM low glucose media (11885-092, GIBCO, USA) containing 2 mg/mL of collagenase IV (GIBCO, USA) was injected into the pancreas through the common bile using a needle (Natsume Seisakusho, Tokyo, Japan). The pancreas was subsequently removed and incubated in a 37°C water bath for 35 minutes. The cell suspension was filtered through a 400 µm mesh. Followed by washing the filtrate three times in warmed DMEM low glucose media containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The islets were collected by density-gradient centrifugation at 900 g for 20 min using density-gradient reagent Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA). Gathered islets were washed three times in warmed DMEM (containing 1000 mg/L glucose, 1% penicillin-streptomycin, and 10% FBS). A 100 µm filtration was performed to remove single cells. The islets on the filter were carefully collected and handpicked under a stereomicroscope until a population of pure islets was obtained. Approximately 80~100 islets were obtained per mouse. The Islets were then cultured with RPMI (11875-093, Life Technologies, US) (containing 10% FBS and 1% penicillin-streptomycin) for no more than 8 hours.

Subcutaneous Islet Transplantation Using an Atelocollagen Sponge

One week after angiogenesis stimulation, an incision was made at the same site, and the gelatin sponge (Spongel) was carefully removed. Freshly isolated islets were transferred to a 1.5 ml tube and rinsed twice in RPMI without supplements. After the islets were settled, a 1 ml syringe (SS01-T, Terumo Corporation, Japan) with the outer soft needle of 24G indwelling needle (09-035, Nipro, Japan) attached was used to suck the islets. The islets were dispensed drop by drop into a 10-cm dish (SH90-20, AGC Techno Glass Co., Ltd., Japan). Each droplet was immediately covered with a half-moon-shaped atelocollagen sponge to absorb the islets. Islets carried by the atelocollagen sponge were then transferred to the pocket-like subcutaneous cavity left after the removal of the sponge.

Immunohistochemistry

Tissue samples were fixed in 4% paraformaldehyde (Nacalai Tesque, Japan) at 4°C for 16 h. Fixed samples were dehydrated and cleared. Then, paraffin was infiltrated by an automatic paraffin replacement machine (CT-Pro20, Genostaff, Japan). Paraffin-embedded tissue samples were cut into 8.5 μm thick sections. After deparaffinization and rehydration, the tissue slices were blocked by 20% of the Blocking one (Nacalai Tesque, Japan) and detected by antibodies. The following antibodies were used: guinea pig anti-insulin (A0564, Dako Denmark A/S, Denmark; 1:10), mouse anti-glucagon (G2564, Sigma-Aldrich, US; 1:1,000), and goat anti-CD31 (AF3628, R&D System, Inc., US; 1:50). Alexa Fluor 488 donkey anti-guinea pig IgG (Life Technologies, US; 1:1,000), Alexa Fluor 568 donkey anti-mouse IgG (Biotium, US; 1:1,000), and Alexa Fluor 647 donkey anti-goat IgG (Jackson ImmunoResearch, US; 1:1,000). Primary antibodies were not applied for the negative control staining, but a mixture of secondary antibodies was applied. Tissue sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Roche Diagnostics, Switzerland). Images were acquired with an Axio Observer Z1, Axio Zoom V16, and LSM 780 (Zeiss, German).

Hematoxylin and Eosin Staining

Paraffin section slides were used for hematoxylin and eosin (H&E) staining after paraffin removal. The sections were stained with hematoxylin (30001, Muto Pure Chemicals Co., Ltd., Japan) and eosin (32053, Muto Pure Chemicals Co., Ltd., Japan). Images were acquired with a Keyence all-in-one BZ-X800 (Keyence Corporation, Japan).

Intraperitoneal Glucose Tolerance Test

Intraperitoneal Glucose Tolerance Test (IPGTT) was performed at post-operation weeks (POW) 8, 12, or/and 16. Mice fasted for 6 hours (h) were used. Blood glucose levels were measured before (0 min) and at 15, 30, 60, 90, and 120 min post-intraperitoneal administration of 10% glucose solution (Ootsuka, Tokyo, Japan) at 2 mg/g body weight.

Statistics

All data are presented as mean± SD) standard deviation). Data were analyzed by one-way analysis of variance (ANOVA), Tukey’s multiple comparisons test, or unpaired two-tailed Student’s t-test using GraphPad Prism (GraphPad Software, version 10.2.1). Significance differences are shown as *P < 0.05, **P < 0.01, ***P < 0.001, and are indicated in the figure legends. All data were obtained from more than three independent experiments, except where indicated.

Data Availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Results

The Use of an Atelocollagen Sponge Scaffold to Support the Viability of the Islets

We first tested if an atelocollagen sponge could be used as a scaffold to support mouse islets for transplantation. An atelocollagen sponge with a 5 mm diameter was manually cut into six small half-moon shapes (ø5 mm × 1 mm in thickness) was used (Fig. 1A, left panel). With its porous structure (Fig. 1A, right panel), the atelocollagen sponge is expected to serve as a scaffold to support resident spheroids and thus facilitate engraftment. Approximately 100 freshly isolated islets were collected in a drop of up to 5 µl, and then a piece of atelocollagen sponge was placed onto the islet drop to absorb the liquids along with the islets (Fig. 1B). Live/dead assay of islets immediately after absorbed in the atelocollagen sponge demonstrates the localization of the islets within the scaffold and high viability (>90%) of absorbed islets with no significant difference compared to the naked islets (Fig. 1C, D, E). The result demonstrated that an atelocollagen sponge is suitable for carrying islets without damaging them.

Pre-Stimulation of Angiogenesis Using a bFGF-Soaked Gelatin Sponge Promoted Blood Vessel Formation in the Subcutis

We then attempted to stimulate angiogenesis to enhance the vascularization before transplanting the islets into the diabetes mouse model. A subcutaneous site for islet transplantation is undeniably promising but confronts the unavoidable challenge of lacking a vascular bed. We tested using a gelatin sponge (Spongel^®) soaked with bFGF (trafermin; Fiblast^®), inserted it into the subcutis, and compared its effect on stimulating angiogenesis with control experiments (blank, treated with 100 µl trafermin [bFGF sol], or gelatin sponge soaked with phosphate-buffered saline) by evaluating the total area of the blood vessel, 1-week post-stimulation (Fig. 2A, Supplementary Figure S1). Our observation showed that the induced new microvasculature emerged from existing blood vessels (Fig. 2). We observed significantly more prominent areas of blood vessel formation 1 week after treating the recipients with a bFGF-soaked gelatin sponge (Fig. 2).

Reversal of Hyperglycemia in Angiogenesis-Stimulated Recipients by Islet Transplantation in BALB/c Mice

We then compared transplanting 800 BALB/c islets carried in several pieces of atelocollagen sponge subcutaneously into the dorsal neck area of STZ-induced diabetic BALB/c recipients, with or without one-week angiogenesis stimulation before transplantation (Fig. 3A). Two weeks before transplantation (–2 weeks), STZ was injected to induce β cell destruction. Then, one week later (–1 week), angiogenesis stimulation was conducted. Then, one-week post angiogenesis stimulation, mouse islet transplantation was performed (Fig. 3A, 0 week). As a result, the recipients that received 800 islets transplantation with angiogenesis stimulation (angio-treated) showed gradually decreasing non-fasting blood glucose levels and finally achieved normoglycemia (Fig. 3B). Their normoglycemia was sustained until graft removal. In contrast, the recipients not treated with angiogenesis stimulation (non-angio-treated) did not exhibit reversal of hyperglycemia (Fig. 3B). Simultaneously, angio-treated recipients exhibited rapid body weight gain. Conversely, non-angio-treated recipients demonstrated slower or negligible body weight gain (Fig. 3C).

We then performed intraperitoneal glucose tolerance tests (IPGTT) at POW 8 and 16 (Fig. 3D, E, POW 8; Fig. 3F, G, POW 16) to assess the functionality of the engrafted islets. After a 6-hour fast, the fasting blood glucose levels of angio-treated recipients were similar to those of healthy control mice, whereas the non-angio-treated recipients exhibited higher fasting blood glucose levels. The angio-treated recipients and healthy mice showed peak blood sugar levels within 30 minutes after intraperitoneal glucose injection. In contrast, the glucose levels did not decrease within 60 min in most non-angio-treated recipient mice. Although blood glucose levels in all mice dropped after 120 minutes post high-glucose challenge, only those in the angio-treated recipients and healthy mice were lower than 200 mg/dL (Fig. 3D, F). The areas under the curve (AUC) in the angio-treated and healthy mice group were significantly lower than those in the non-angio-treated group, and there is no significant difference between AUC in the angio-treated group and healthy mice (Fig. 3E, G).

We tested the effect of 500 or 290 mouse islet transplantation, which was revealed to be suboptimal (Supplementary Fig. S2). We have performed 500 mouse islet transplantation twice, and only one mouse eventually reached normoglycemia out of five mice. However, the mice who received transplantation gained non-fasting body weights (Supplementary Figure S2B, C). The mice receiving 500 islet transplantation but not reaching normoglycemia exhibited glucose intolerance (Supplementary Figure S2B). The results showed that 500 mouse islets were not sufficient to rescue the hyperglycemia of our diabetes model mice.

We removed the grafts of the angio-treated recipient mice and non-angio-treated recipient mice at POW 24 or POW 30 (Supplementary Figure S2D). Upon graft removal, the non-fasting blood glucose of angio-treated recipient mice quickly reverted to hyperglycemia, and body weight decreased. In contrast, non-angio-treated mice showed no marked change in body weight or blood glucose levels (Fig. 3B, C). The grafts were harvested and subjected to immunohistochemistry staining of insulin (INS) glucagon (GCG) and endothelial marker CD31. The result revealed a dramatic difference in angiogenesis pretreatment after approximately six months of engraftment [Fig. 3H, I], while negative controls (without primary antibody) showed no non-specific staining (Supplementary Figure S2E). The numbers and sizes of the engrafted islets in the angio-treated group were significantly larger than those in the non-angio-treated group (Fig. 3H, I). We could hardly find engrafted islets in the non-angio-treated group. Almost the largest remaining islets we observed are shown (Fig. 3I). Although there was a considerable difference in the remaining size of the engrafted islets, the ratios of the CD31 areas versus the remaining islet areas in angio-treated mice are still significantly higher than those in the non-angio-treated mice (Fig. 3J).

Reversal of Hyperglycemia by Islet Transplantation in Rag-2/Jak3 Double-Deficient BALB/c (BRJ) Mice

Having confirmed the efficacy of angiogenesis in syngeneic transplantation, we then applied this protocol to an immunodeficient mouse model, laying the groundwork for cell therapy using stem-cell-derived β cells. Rag-2/Jak3 double-deficient BALB/c (BRJ) severe immunodeficiency mice were used as recipients. STZ was intravenously administered two weeks before islet transplantation. Angiogenesis stimulation was performed on day 7 post-STZ injection, and then 800 Ctl: CD1 (ICR) mouse islets were transplanted (Fig. 4A).

Figure 4.

Reversal of hyperglycemia by islet transplantation in Rag-2/Jak3 double-deficient BALB/c (BRJ) mice.

800 mouse islets from wild-type ICR mice, carried in the atelocollagen sponge scaffold, were transplanted into the pre-angiogenesis-treated subcutaneous pocket of immunodeficient diabetic BRJ mice. This transplantation facilitated a gradual reversal of diabetic symptoms in the recipients, eventually leading to a state resembling that of healthy mice. (A) Schematic diagram of the experimental design; (B–E) Magenta lines, transplanted mice, n = 4; open circle plots with black lines, healthy mice, n = 3; (B, C) (B) Non-fasting blood glucose levels. Broken gray line: normoglycemia threshold of 200 mg/dL. (C) Body weight of diabetic BRJ mice transplanted with 800 ICR mouse islets. Grafts were removed at POW 21, 26 (arrows); (D, E) Intraperitoneal glucose tolerance test (IPGTT) at 16 weeks post-operation (POW) revealed that (D) blood glucose levels of the transplanted mice exhibited a curve trend similar to that of healthy mice. (E) No significant difference in the area under the curve (AUC) was observed between the transplanted and healthy mice. Data are presented as mean ± SD. Analyzed by unpaired two-tailed Student’s t-test.

Similar to the results observed in BALB/c mice, subcutaneous islet transplantation improved the condition of BRJ mice, evidenced by a gradual decrease in blood glucose levels and a steady increase in body weight. At POW6, all recipients experienced a reversal of hyperglycemia, maintained normoglycemia, and achieved a long-term survival of over 140 days. Upon graft removal at POW 21 or 26, recipients resumed hyperglycemia, and their body weights decreased rapidly (Fig. 4B, C). The recipients were subjected to IPGTT at POW16 to examine the function of the implanted islets.

As expected, the IPGTT results of the angio-treated BRJ mice mirrored those seen in the angio-treated BALB/c mice. Overall, the BRJ mice that received ICR islets were glucose tolerant, similar to healthy mice; they peaked in blood glucose within 30 minutes and returned to baseline levels within 90 minutes post-glucose injection. In addition, their AUC showed no significant difference compared to the healthy mice (Fig. 4D, E).

High Vasculatures and Preserved Morphologies of the Recovered Islet Grafts

We terminated the experiment and analyzed the grafts after a long-term subcutaneous transplantation. Immunohistochemistry (Fig. 5A) and H&E staining (Fig. 5B) of the harvested grafts showed that the cellular morphology of the islets within the atelocollagen sponge was preserved (Fig. 5A, B). The cell composition of islets in the atelocollagen sponge showed no significant difference between the before and after transplantation (Tx) groups (Supplementary Figure S3). Interestingly, the islets showed zigzag borders in the before-Tx but not in the harvested group, suggesting the elimination of the unhealthy outer layer cells during the long-term transplantation (Supplementary Figure S3). Immunohistochemical staining for insulin (green), glucagon (red), CD31 (gray), and DAPI (blue) collectively suggested that using atelocollagen as a scaffold could help to keep the viability of the islet grafts (Fig. 5A). This preservation is attributed to the atelocollagen sponge, which acts as a pressure-resistant scaffold and maintains their morphologies and function under the skin (Fig. 5B).

Figure 5.

High vasculatures and preserved morphologies of the recovered islet grafts. Immunohistochemistry and hematoxylin & eosin staining of the harvested islet graft (POW 21) being carried in the atelocollagen sponge grafts. Representative images are shown. (A) Islets inside the atelocollagen sponge preserved their insulin and glucagon expression. A large number of the CD31-positive cells indicates successful engraftment. N = 4 mice were analyzed. Green, insulin; red, glucagon; gray, CD31; blue, DAPI. Scale bars: 100 µm (left); 50 µm (right).

(B) Hematoxylin and eosin staining showed the distribution of islets inside the atelocollagen sponge scaffold, illustrating islets that maintained healthy morphology. Left panel: a yellow broken line marks the atelocollagen sponge scaffold. A yellow box marks the magnified region (right panel). Right panel: light blue broken lines mark the islets inside the atelocollagen sponge scaffold. Scale bars: 500 µm (left), 100 µm (right).

Discussion

Here, we established a procedure for subcutaneous islet transplantation using a commercially available material for pre-vasculature and another commercially available material as an islet scaffold. We report that pre-vasculature improved the outcome of subcutaneous islet transplantation. It was reported that a pre-vascularized subcutaneous site could be generated using a device-less procedure^12,13. We believe the procedure we report here using the commercially available materials is technically easily accessible. We showed that an atelocollagen sponge scaffold is a suitable carrier for subcutaneous islet transplantation. We observed a reversal of hyperglycemia between 2 and 7 weeks after subcutaneous transplantation of 800 islets using two different mouse strains, BALB/c mice and a severe immune-deficient BRJ (BALB/c Rag2/jak3 -/-) mice. We found that pre-stimulation for angiogenesis for 1 week before transplantation led to an enhanced vasculature, which improved the outcome. The pre-stimulating period is short compared to the described procedure recently. It was reported that a 6-week pre-stimulation for angiogenesis gave the best cure rate compared to 2, 4, 6, and 8 weeks of pre-treatment ^18,19. Therefore, varying the period for vasculature could further improve the outcome of islet transplantation.

We used the BRJ (Rag2/jak3 double-deficient in BALB/c background) mice, which are severely immune-deficient, lacking mature T and B lymphocytes and natural killer (NK) cells, thereby demonstrating a pronounced immunodeficiency, resulting in enhanced receptivity to cell transplantation. BRJ mice would be a suitable recipient for subcutaneous transplantation of iPS-derived β cells. We also established Kuma mutant mice of the BRJ background, which could be used for cell transplantation ²⁰ .

Here, we show that using a commercially available atelocollagen sponge as a scaffold in cell therapy is feasible for treating diabetes. We observed long-term euglycemia achieved by islet transplantation without apparent immune rejection. The atelocollagen sponge can endure compressive stress up to 40 kPa ²¹ . This feature makes it well-suited for both 3D culture and cell transplantation.

The atelocollagen sponge stands as a promising open device with advantages, not only allowing it to resist specific physical pressures but also withstand the compressive forces from the recipient, better support the cells, and easy retrieval of the transplanted cells. Compared to closed devices, it ensures a high viability of the transplanted cells. Therefore, we consider it to be a promising scaffold. In islet transplantation, successful engraftment heavily depends on the vascularization and inflammation levels at the transplant site ²² . Thanks to its relatively large pore size, islet implantation using the atelocollagen sponge yielded good vascularization, which is crucial for engraftment ²³ . With telopeptides removed from collagen by enzymes, the likelihood of inflammatory responses by atelocollagen is reduced, which positions the atelocollagen sponge as a promising candidate for subcutaneous islet transplantation ²⁴ . However, it remains to be investigated whether there is a lower risk for IBMIR with subcutaneous islet transplantation versus the gold standard site via the portal vein. Atelocollagen sponge can be modified in shape and size according to the experimental purpose. This characteristic makes the atelocollagen sponge a quantifiable and combinable bio-block. It opens up possibilities for in vivo experiments involving various types of cells, including human iPS cell-derived β cells, for future cell replacement therapy or pharmaceutical research.