A novel prevascularized tissue-engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas.

Although sites for clinical or experimental islet transplantation are well established, pancreatic islet survival and function in these locations remain unsatisfactory. A possible factor that might account for this outcome is local hypoxia caused by the limited blood supply. Here, we modified a prevascularized tissue-engineered chamber (TEC) that facilitated the viability and function of the seeded islets in vivo by providing a microvascular network prior to transplantation. TECs were created, filled with Growth Factor-Matrigel™ (Matrigel™) and then implanted into the groins of mice with streptozotocin-induced diabetes. The degree of microvascularization in each TECs was analyzed by histology, real-time PCR, and Western blotting. Three hundred syngeneic islets were seeded into each chamber on days 0, 14, and 28 post-chamber implantation, and 300, 200, or 100 syngeneic islets were seeded into additional chambers on day 28 post-implantation, respectively. Furthermore, allogeneic or xenogeneic islet transplantation is a potential solution for organ shortage. The feasibility of TECs as transplantation sites for islet allografts or xenografts and treatment with anti-CD45RB and/or anti-CD40L (MR-1) was therefore explored. A highly developed microvascularized network was established in each TEC on day 28 post-implantation. Normalization of blood glucose levels in diabetic mice was negatively correlated with the duration of prevascularization and the number of seeded syngeneic islets. Combined treatment with anti-CD45RB and MR-1 resulted in long-term survival of the grafts following allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry demonstrated that the frequency of CD4+Foxp3-Treg and CD4+IL-4+-Th2 cells increased significantly after tolerogenic xenograft transplantation, while the number of CD4+IFN-γ-Th1 cells decreased. These findings demonstrate that highly developed microvascularized constructs can facilitate the survival of transplanted islets in a TECs, implying its potential application as artificial pancreas in the future.

Introduction

Cellular transplantation represents an attractive treatment strategy for a variety of diseases, including diabetes, myocardial ischemia, and metabolic liver disease [1]. It has been reported that intrahepatic transplantation of donor-derived pancreatic islets is a realistic alternative cellular therapy for insulin-dependent diabetes mellitus [2]. Nevertheless, the procedure remains unsatisfactory due to inadequate glucose control. Although it has been proposed that the ‘Edmonton Protocol’ should constitute the standard guidelines for islet transplantation, having achieved high success rates [3], transplantation of islets via infusion through the portal vein results in massive islet loss within the first 2–4 d post-transplantation, and additional islets from 2–3 donors are required to achieve normoglycemia. The principal reason for this loss is islet apoptosis that occurs during the process of engraftment, which occurs following an instant blood-mediated inflammatory reaction (IBMIR) against the graft in the context of a lack of vascularity to supply oxygen [4, 5].

Anatomically, the pancreatic islets are immensely vascularized, allowing secretion of insulin and a rapid response to hyperglycemia [6]. A number of research studies have demonstrated that the islets lose their natural vascularization and extracellular matrix when isolated from the pancreas using traditional methods. These specialized characteristics render the islets highly susceptible to apoptosis due to insufficient partial pressure of oxygen and diffusion of nutrients [7, 8]. Therefore, the key to improving islet survival is to ensure rapid vascularization following transplantation and integration into the host’s systemic vasculature. In addition, it has been documented that the process of revascularization begins after 2–4 d, followed by creation of a network of vessels 10–14 d after transplantation [9]. However, the density of new blood vessels after transplantation is considerably lower than that observed in natural islets, regardless of whether the islets are delivered to the liver or to extrahepatic sites [10, 11].

Subcutaneous sites have been suggested as alternatives for islet transplantation, as the grafts can be easily monitored by imaging [12] and removed for retransplantation [13, 14]. However, unmodified subcutaneous sites for islet engraftment have not demonstrated a reversal of hyperglycemia in mice or humans [15]. Inadequate vascularization not only fails to supply sufficient oxygen tension but also impedes the secretion of insulin from the islets to accomplish glucose homeostasis [16, 17]. Therefore, the construction of a prevascularized network in advance is critical for successful subcutaneous islet transplantation. Additionally, islet transplantation into subcutaneous tissues has been dependent on methods using modified biomaterials, including oxygen generators, polymers, meshes, encapsulation devices, matrices, and growth factors [18, 19]. However, such strategies almost always fail due to the development of an inflammatory response caused by severe tissue incompatibility [20]. Hence, a successful subcutaneous graft should (i) have sufficient tissue capacity; (ii) facilitate minimally invasive methods of transplantation; (iii) establish a vascular network to ensure adequate nutrition for the graft following transplantation; (iv) allow dynamic connectivity between the graft and systemic circulation; and (v) elicit minimal inflammation to reduce the host response and promote long-term graft survival [21, 22].

The objective of this study was to demonstrate that a TEC filled with Growth Factor-Matrigel™ (Matrigel™) embedded into the groin of a mouse with streptozotocin-induced diabetes was capable of inducing local neovascularization to support the function of the allogeneic or xenogeneic islets transplanted inside. This is central to the advancement of clinical transplantation protocols because of the absence of donor organs. In addition, flow cytometry was used to characterize the inflammatory response, which is vital for determining whether a TEC can facilitate the long-term survival of islets following transplantation.

Materials and methods

Animals

Male BALB/c or C57BL/6J mice and Sprague-Dawley (SD) rats were used as pancreatic donors, and male C57BL/6J mice were used as recipients. All animals were 6–8 weeks old. Their cages were housed under a 12-h day/night cycle with ad libitum access to food and water. All procedures were compliant with the animal care principles approved by the Research Animal Protection Committee of Sichuan Provincial People’s Hospital.

Murine subcutaneous tissue engineering model

The tissue engineering model was established using a method similar to that described previously [23, 24]. Briefly, mice were anesthetized intraperitoneally using pentobarbital sodium (100 mg/kg) and placed in a supine position on gauze. The abdomen was shaved and wiped with an iodophor to sterilize the skin. A 7 mm incision was created in the groin, after which the site was prepared for insertion of a 5 mm-long silicone cylinder chamber (Dow-Corning, Harrodsburg, KY, USA) with an inner diameter of 3.35 mm (approximate volume: 44 μL). The silicone cylinder chamber was cut longitudinally and implanted adjacent to the superficial epigastric vessel in the groin, incorporating the inguinal fat pad into the distal end of the chamber. Matrigel™ (BD, Franklin Lakes, NJ, USA) and heparin (80 U/mL) (Pharmacia & Upjohn, Somerset, NJ, USA) were injected into the chamber, after which it was completely sealed with bone wax.

Transplantation

Diabetes was induced in C57BL/6 mice through intraperitoneal injection of streptozotocin (200 mg/kg, Sigma Aldrich, St. Louis, MO, USA), which was confirmed by blood glucose levels > 400 mg/dL (18.8 mmol/L) for at least 2 consecutive days using blood glucose monitoring (Roche, Mannheim, Germany). The mice were euthanized by CO2 asphyxiation. Islets (from C57BL/6 or Balb/c mice or SD rats) were isolated via cold digestion with 1.5 mg/ml collagenase (Roche, Mannheim, Germany) and then purified via discontinuous Ficoll gradients (densities: 1.11, 1.096, 1.066) of the pancreatic digests. The islets were transplanted into TECs as described previously [23, 24]. A functioning graft was defined as one in which a nonfasting blood glucose level <200 mg/dL was obtained, and rejection was defined as occurring when blood glucose levels of >200 mg/dL were observed on at least 2 consecutive days. The mice in all groups were monitored daily for the first two weeks and then once per week following transplantation until the mice were sacrificed. The TECs in diabetic mice that had remained normoglycemic for the whole 90 d were removed for evaluation of islet function.

Intraperitoneal glucose tolerance test (IPGTT)

Recipient mice were subjected to an intraperitoneal glucose tolerance test (IPGTT) at 90 d post-transplantation to further assess metabolic capacity. The mice were fasted for 12 hours before receiving intraperitoneal D-glucose (2 mg/g in saline). Blood samples were obtained from the tail vein of the recipient mice 0, 15, 30, 60, 90 and 120 minutes after injection. Blood glucose levels were analyzed, and the different transplant groups were compared.

Histology

Intact tissue from the TEC was removed and fixed overnight in 4% formaldehyde prior to processing and embedding in paraffin. Five-micrometer-thick sections were cut and stained with a primary antibody against either vascular endothelial growth factor-positive (VEGF) (1:250; Abcam, Cambridge, MA, UK) or CD31 (1:250; Abcam, Cambridge, MA, UK) and counterstained with hematoxylin and eosin (H&E). After washing, the tissue sections were incubated with a secondary antibody, 488-Alexa anti-rabbit or anti-guinea pig IgG (Molecular Probes, Eugene, OR, USA), at room temperature. Photomicrographs were acquired using a digital camera (BA200, Xiamen, China) with an appropriate filter.

Quantitative real-time PCR

Relative mRNA expression levels of VEGF were quantified by qRT-PCR. Total RNA was extracted from the adipose tissue in the TEC with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Roche, Mannheim, Germany). Subsequently, the relative expression levels of mRNA transcripts of interest were quantified using a SYBR Green kit and normalized to the expression of β-actin. The change in relative expression compared with the control (mRNA levels defined as a value of 1) was calculated using the 2-△△Ct method. Primers for qRT-PCR were designed and synthesized by Qingke Biotech (Wuhan, China), and the sequences were as follows:

β-actin: forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′,

reverse 5′-ATGGAGCCACCGATCCACA-3′;

VEGF: forward 5′-AAGAGAAGGAAGAGGAGAGG-3′,

reverse 5′-GGTAGACATCCATGAACTTGA-3′

Western blotting (WB)

TEC adipose tissue was homogenized and lysed using high-efficiency RIPA buffer (Solarbio, Beijing, China) or an NE-PER nuclear and cytoplasmic extraction kit (Boster, Pleasanton, CA, USA) with protease and phosphatase inhibitors (Roche, Mannheim, Germany). Equal quantities of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) followed by transfer to PVDF membranes (Millipore, Burlington, MA, USA). The blots were blocked with 5% nonfat milk (BD, Franklin Lakes, NJ, USA) or 5% BSA (Amresco, Houston, TE, USA) dissolved in 1× TBST (blocking buffer). The blots were then probed with a primary antibody against VEGF (1:2000; Abcam, Cambridge, MA, UK) dissolved in blocking buffer, followed by alkaline phosphatase-conjugated mouse IgG (1:5000; Abcam, Cambridge, MA, UK) as the secondary antibody. HRP (Millipore, Burlington, Massachusetts, USA) chemiluminescence signals were detected using a chemiluminescence imaging analysis system (Tanon, Shanghai, China). Densitometry of the immunoblot images was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunotherapy

Mice were treated by intraperitoneal injection of 100 μg of an anti-mouse CD45RB antibody (Bio X Cell, West Lebanon, NH, USA) on days 0, 1, 3, 5, and 7 after transplantation. In addition, 500 μg of an anti-mouse CD40L antibody (MR-1, Bio X Cell, West Lebanon, NH, USA) was administered intraperitoneally on days 2, 4, 7, and 14 following transplantation.

Cell stimulation and flow cytometry

The method of mouse euthanasia was described previously. Single cells from the spleen were suspended in RPMI 1640 culture medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and Cell Stimulation Cocktail (eBioscience, San Diego, CA, USA) and then cultured in 6-well plates for 5 hours in a 37°C/5% CO2 incubator.

Lymphocytes were separated from the spleen by passing through a 70 μm nylon mesh. Erythrocytes were lysed with ammonium chloride buffer, after which the remaining cells were washed and counted using a hemocytometer. One million cells were suspended in staining buffer with the following fluorochrome-conjugated antibodies: CD4-FITC (mAb; BD, Franklin Lakes, NJ, USA), CD25-APC (mAb; BD, Franklin Lakes, NJ, USA), IL-4-PECy7 (mAb; BD, Franklin Lakes, NJ, USA), IFN-γ-PE (mAb; BD, Franklin Lakes, NJ, USA), and Foxp3-PE (mAb; BD, Franklin Lakes, NJ, USA). Intracellular Foxp3, IL-4, and IFN-γ within lymphocytes were stained using a fixation/permeabilization staining kit (eBioscience, San Diego, CA, USA). All samples were analyzed using a FACSCanto II flow cytometer (BD, Franklin Lakes, NJ, USA) and analyzed using Flow Jo analysis software (Tree Star Inc.).

Statistical analysis

Data were analyzed using GraphPad Prism software version 5 (GraphPad version 5.01). Graft survival in the experimental groups was compared using Kaplan–Meier survival curves. Other differences between experimental groups were evaluated using Student’s t-test. P < 0.05 was considered statistically significant.

Results

Significant prevascularization of the TECs suggests that it is a suitable site for islet transplantation

The procedure for construction of the TEC is presented in Fig 1A and 1B. Following its implantation in the groin, it exhibited good histocompatibility and safety during the study. None of the mice suffered leg paralysis, wound infection, chamber exposure, or sinus formation. The chamber grafts in each TEC (>28 d) were richly vascularized, as can be observed in the macroscopic images (Fig 1C). Based on our knowledge, we hypothesized that the scale of neovascularization in the TECs was related to the duration of prevascularization. Therefore, an extensive analysis of the chamber grafts from each TEC was undertaken after 7, 14, 28, and 35 d. As shown in Fig 2A, HE-stained adipose tissue from the TEC demonstrated significantly greater formation of vessels on days 28 and 35 than on 7 d and 14 d implantation. As expected, the TEC implants stained as VEGF+ and platelet endothelial cell adhesion molecule-1-positive (CD31+) on days 7, 14, 28 and 35 post-implantation. VEGF and CD31 are used as markers of neovascularization to represent the prevascularization of the TECs. The associated vasculature of the TECs had increased significantly by days 28 and 35 (Fig 2A).

Fig 1

Construction of tissue-engineered chamber and islet transplantation.

(A) Flow diagram of TEC construction and islet transplantation. (B) The silicone cylinder chamber, including Matrigel™ and heparin, was inserted and implanted adjacent to the superficial epigastric vessel. A fat pad and bone wax were used to close the ends of the silicone tube. (C) A microvascular network was established (50×).

Fig 2

Highly developed microvascularized network established in the TEC on day 28 post-implantation.

(A) HE images (1st row; scale bars: 10 μm) and histological analysis (2nd and 3rd rows; scale bars: 40 μm) of a prevascularized TEC on days 7, 14, 28 and 35. Red arrows indicate blood vessels in HE staining (1st row; scale bars: 10 μm). Microvessels were stained with anti-VEGF and anti-CD31, as marked separately by red arrows (2nd and 3rd rows; scale bars: 40 μm). (B) Levels of VEGF mRNA expression were quantified. The extent of prevascularization confirmed a significant increase at 28 d compared with 7 d and 14 d (p**<0.001, p***<0.001) and showed no significant difference between the 28 d and 35 d groups (p# = 0.0538). (C) Western blot analysis indicated VEGF protein expression levels at different time points in TECs. The control group had a chamber (but had no Matrigel™ and no heparin), and the sample came from the fat pad tissue near the superficial epigastric vessel. Data are shown as the mean ± SD and are representative of three separate experiments.

Furthermore, the qRT-PCR results demonstrated that VEGF mRNA expression levels were significantly higher on days 28 and 35 than on days 7 and 14. However, there was no difference in expression levels between 28 and 35 d (Fig 2B). Quantification of VEGF protein expression revealed trends consistent with the results of qRT-PCR (Fig 2C), and the extent of prevascularization closely correlated with the duration.

Euglycemia levels in diabetic mice correlated with the duration of prevascularization and the number of implanted islets in the TEC site

Because a highly developed microvascularized network had been established in the TEC on day 28 post-implantation, we hypothesized that the normalization of blood glucose levels in diabetic mice was correlated with the duration of prevascularization and the number of islets implanted. To prove this hypothesis, 300 syngeneic islets were transplanted into individual TECs on days 0, 14, and 28 post-TEC implantation. As shown in Fig 3A, the sham group did not exhibit a reversal of diabetes at any time point. However, the islets transplanted on day 28 post-TEC implantation reversed diabetes by 7.4±3.6 d. Conversely, islets transplanted on days 0 and 14 post-TEC implantation resulted in a reversal of diabetes after 32.3±11.7 d and 13.0±4.2 d, respectively. Thus, the results suggest that normalization of blood glucose levels in diabetic mice was negatively correlated with the duration of prevascularization.

Fig 3

The TEC represents a promising site for syngeneic islet transplantation.

The time required for blood glucose to return to normal levels (200 mg/dL) in diabetic mice was negatively correlated with the duration of prevascularization (A) and the quantity of seeded syngeneic islets (B). (C) Nonfasting blood glucose showed maintenance of euglycemia after 300 syngeneic islets were transplanted into the TEC, with hyperglycemia developing following TEC removal (black arrow). (D) Donor islets stained positive for insulin in a recipient TEC 90 d post-transplantation (shown brown), indicated by red arrow (Scale bars: 40 μm). Diabetic mice that had neither constructed chambers nor transplanted islets served as the sham group. Txpl: Transplantation.

The effect of the number of islets on blood glucose level normalization in diabetic mice after prevascularization was further investigated. A total of 300 (n = 5), 200 (n = 5), and 100 (n = 5) islets were transplanted on day 28 post-implantation, and the time required for blood glucose to recover was determined. As shown in Fig 3B, 300 islets were more effective at reversing hyperglycemia, which was achieved after 7.4±3.6 d, than were 200 (14.0±3.5 d) or 100 islets (20.4±4.7 d). This indicates that the return to normal levels of blood glucose in the diabetic mice was negatively correlated with the number of islets transplanted.

The long-term function of syngeneic islets within a TEC was then evaluated. Syngeneic islets transplanted into a TEC reversed diabetes in 100% of recipients over the long term (5/5) (Fig 3C). Patients with insulin-dependent diabetes mellitus also need to pay attention to weight in the clinic. Therefore, weight monitoring was conducted in mice both before and after transplantation. The results demonstrate that the body weight of diabetic mice increased gradually when blood glucose was maintained at normal levels (S1A Fig). In addition, histological analysis of grafts revealed that syngeneic islets stained strongly positive for insulin (Fig 3D). These images verified that the TEC is a suitable site for transplantation in future studies.

Feasibility of the TEC as a transplantation site for long-term survival of allografts and xenografts established by anti-CD45RB and anti-MR-1

Because implantation of allografts and xenografts is a potentially promising solution for insulin-dependent diabetes mellitus, short-term immunosuppression to protect the long-term survival of allografts and xenografts within a TEC was investigated. We first explored whether anti‐CD45RB or/and MR-1 therapy could prolong the survival of allografts from recipient C57 mice with STZ-induced diabetes. Three hundred allogeneic islets from 2 donors were transplanted into the TEC, and the nonfasting blood glucose level was monitored. Not surprisingly, treatment with anti‐CD45RB or anti‐MR-1 alone failed to control hyperglycemia in any recipient (n = 5) (Fig 4A). Conversely, long-term graft survivors (LTS) that were recipients of allografts survived >90 d following combined antibody treatment with anti-CD45RB and anti-MR-1 (Fig 4B). Groin grafts containing islets were removed at 90 d post-transplantation, and blood glucose levels rapidly exceeded 400 mg/dL (Fig 4B, black arrow). This indicates that the maintenance of euglycemia in recipient mice was dependent on the islets that were transplanted into the TEC. Histology of the grafts confirmed that implanted islets stained strongly positive for insulin (Fig 4F, left).

Fig 4

Long-term function of allogeneic and xenogeneic islet grafts transplanted into TECs.

(A) Islet allografts (Balb/c to C57BL/6) in the no treatment group were rejected within 22±2.2 d. Islet allografts treated with anti-CD45RB or anti-MR-1 alone were also rejected in recipients within 24±3.12 d or 26±1.74 d, respectively. (B) Islet allografts subjected to dual anti-CD45RB or anti-MR-1 treatment displayed long-term graft survival in all recipients (5/5, 100%), which became hyperglycemic following TEC removal (black arrow). (C) Islet xenografts (SD rat to C57BL/6 mouse) were rejected in the no treatment group, the CD45RB-treated group and the MR-1-treated group after 13±3.1 d, 16±3.12 d, and 18±1.74 d, respectively. Islet xenografts treated with dual anti-CD45RB and anti-MR-1 exhibited long-term graft survival (16/20, 80%) and recurrence of hyperglycemia following TEC removal (black arrows). (E) A total of 300 xenogeneic islets were retransplanted into diabetic mice that had failed to accept the xenograft. In three of four diabetic mice, hyperglycemia was ameliorated until the TEC was removed (black arrow). (F) Insulin-stained donor allografts and xenografts in a recipient TEC 90 d post-transplantation (shown in brown). Red arrows indicate islets (Scale bars: 40 μm).

Whether xenografts exhibited a similar function in the TECs was then examined. A total of 300 xenogeneic islets were implanted to explore their long-term performance in recipient C57 mice with STZ-induced diabetes. As shown in Fig 4C, treatment with anti‐CD45RB or anti‐MR-1 alone failed to control hyperglycemia in any xenograft recipient (n = 5). However, sixteen of the twenty recipients maintained euglycemia following immunotherapy (Fig 4C and 4D). We reimplanted xenogeneic islets into mice in which the reversal of hyperglycemia had failed and explored whether euglycemia could be maintained over the long term following dual anti-CD45RB plus anti-MR-1 antibody treatment. The results revealed that three of the four recipient mice experienced a reversal of diabetes and maintained euglycemia over the long term (Fig 4E).

Additionally, the body weight of diabetic mice in which allografts and xenografts had been implanted increased gradually when blood glucose was maintained at normal levels (S1B and S1C Fig). The allogeneic and xenogeneic islets implanted within TECs displayed intense positive staining for insulin (Fig 4F, right). After 90 d, an IPGTT was conducted. Both the allograft and xenograft groups had well-preserved glucose clearance profiles similar to those of the naive group (S2A Fig). These data reveal that allografts and xenografts implanted into TECs could maintain euglycemia over the long term.

Xenogeneic islets located in TECs induced long-term recipient-specific immune tolerance

The evidence suggests that the persistence of memory T cells (CD4 and CD8) after transplantation may lead to rejection, which destroys most insulin-producing β cells [25]. Therefore, inflammatory infiltration was evaluated by immunohistochemistry in xenografts on the 90th day after transplantation. Infiltration of CD4+ and CD8+ T cells decreased significantly after immunotherapy (anti-CD45RB plus MR-1) in comparison with the untreated group (Fig 5A). These results showed that xenogeneic islets transplanted in TECs were protected during immunotherapy.

Fig 5

Anti-CD45RB and anti-MR-1 induced immune tolerance in TECs.

(A) CD4+ and CD8+ T lymphocytes were evaluated by immunohistochemistry after islet transplantation. The group with no treatment (right) demonstrated significant infiltration (brown region) compared with that of the group treated with anti-CD45RB and MR-1 (left). Red arrows indicate islets. Scale bars: 40 μm; The proportions of CD4+ FOXP3+ Treg, CD4+ IFN-γ+ Th1 and CD4+ IL-4+ Th2 cells were measured by FACS after transplantation for 7, 14 and 90 d (LTS) (n = 3 mice per group). (B) The percentage of Tregs in the LTS group was significantly higher than those in the 7 d and 14 d groups. (C) Numbers of CD4+ IFN-γ+ Th1 cells decreased in the 7-day and LTS groups. (D) The number of CD4+ IL-4+ Th2 cells increased significantly on days 14 and 90. FACS histograms are representative of at least three independent experiments examined on days 7, 14, 90 after transplantation (p*<0.05 versus naive, p**<0.005 vs. 7 d group, p***<0.005 vs. 14 d group).

T regulatory cells (Tregs) play a central role in maintaining immune homeostasis and peripheral tolerance to foreign antigens in the body. Therefore, it is important to explore the development of immune tolerance in response to variations in the numbers of Treg cells in xenograft recipients. The numbers of CD4+ Foxp3+ Tregs were measured at different time points after transplantation, including 7 d, 14 d, and 90 d (LTS), using flow cytometry. As displayed in Fig 5B, the percentage of Tregs in the LTS group increased significantly compared with that in the 7 d and 14 d groups. The proportion of CD4+ Foxp3+ Treg cells increased from a normal value of 3.1% to almost 7.9% on day 90 after transplantation. In addition, we attempted to gain insight into the distribution of functional helper T cell subsets in mice to illustrate the development of immune tolerance. Splenocytes were stimulated for 6 hours using a cell stimulation cocktail prior to staining for intracellular markers. Compared with those in the LTS group, the levels of IFN-γ-producing CD4+ T cells (Th1) were lower in the 7 d and 14 d groups (Fig 5C). The number of IL-4-producing CD4+ T (Th2) cells in the LTS group was higher than those in the 7 d and 14 d groups. A significant difference was observed between the LTS group and other groups (Fig 5D). Changes in the Th1/Th2 ratio, further indicating the T cell response, were skewed toward tolerance.

Discussion

In the present study, a silicone tube chamber with a rich blood vessel network was constructed using a tissue engineering method. The feasibility and safety of transplantation were considered in detail. Silicone is a safe material that induces a minimal tissue response in animals and humans [26]. The present study evaluated a 5 mm-long cylindrical silicone chamber filled with Matrigel™ matrix placed in the groins of mice surrounding the superficial epigastric vessel. Matrigel™ originated from the Engelbreth-Holm-Swarm mouse sarcoma and was found to be a substrate that induces angiogenesis in vivo [27]. In particular, major components, including laminin and collagen IV, modulate cell signaling interactions and are critical for pancreatic β-cell survival and differentiation [28]. A number of studies have reported that neovascularization can be observed as early as 2 weeks, with a network of vessels appearing in the islets by day 21 [23]. In our study, the results revealed that an abundant microvascularized system was established by day 28 in the TEC. Furthermore, following the transplantation of 300 syngeneic islets into TECs in diabetic mice, it is easy to observe that the blood glucose of the diabetic mice was restored to euglycemia after 7.4±3.6 d. The results are similar to those obtained with islets seeded into a kidney capsule in diabetic mice, which is the most common method of transplantation in basic research [13, 29]. Some studies have demonstrated that a microenvironment created from the integration of a vascular network could improve the survival of grafts in the long term [30]. In addition, the abundant prevascularization in the TEC supplies more nutrition to the islets, supporting enhanced survival and function compared with the transplantation site of the kidney capsule. However, we have yet to study autoimmune diabetes models, such as NOD or NRG Akita mice, which exhibit impaired vascularity associated with human diabetes [31]. It is essential to understand the relationship between islet viability within a TEC and revascularization in the future.

Intrahepatic transplantation not only activates an inflammatory reaction in the blood but also fails to support the survival of implanted islets [32]. Therefore, the liver can be considered a suboptimal site for islet transplantation. Many other sites have been reported, including the greater omentum [33, 34], kidney [35], spleen [36], gastric submucosa [37], epididymal fat [38], muscle tissue [39], and anterior chamber of the eye [40], and the level of blood glucose has been demonstrated to be controlled. Nevertheless, pancreatic islet survival and function in these locations remain unsatisfactory [41] due to invasive surgery, inadequate angiogenesis, and the corresponding lack of direct integration with the host. In our study, the TEC chamber was designed and placed in a subcutaneous site as the first step prior to subsequent islet transplantation in the second step. The strategies were effective in achieving prolonged glycemic control over the long term. In addition, the grafts were implanted, monitored, and removed quickly if local complications such as malignant transformation or unchecked hormone release were observed. In summary, subcutaneous sites can be regarded as an alternative for the transplantation of pancreatic islets [42].

Transplantation of pancreatic islets is a promising treatment for insulin-dependent diabetes mellitus. However, the limited availability of pancreatic islets limits their clinical use. In theory, islets derived from animals (porcine islets) can solve the problem of organ shortage, but problems associated with strong rejection need to be solved in the future [43]. The mechanism of rejection following xenogeneic transplantation is extraordinarily complicated, as shown by the small number of reports of successful induction of xenografts [44, 45]. In the present study, euglycemia was maintained in the TEC by an effective dual antibody treatment (anti-CD45RB plus anti-MR-1) after xenotransplantation from rats to C57 mice. CD45 and CD40 are both TCR surface receptors [46, 47]. The use of these two antibodies can significantly inhibit the proliferation of T cells. As shown in Fig 5A, dual antibody treatment prolonged islet survival and protected them from destruction by an inflammatory response, maintaining their initial shape, structure, and function of insulin secretion. In addition, the protective potential of CD4+ Foxp3+ Treg and CD4+ IL-4+ T cells in xenotransplantation has been identified previously [48, 49]. According to the findings of the present study, we believe that anti-CD45RB plus MR-1 significantly inhibits the cellular immune response in TECs, which decreases the proportion of CD4+ IFN-γ+ Th1 cells and increases the numbers of CD4+ IL-4+ Th2 cells and CD4+ Foxp3+ Treg cells for long-term tolerance.

Remarkably, islets transplanted into the TEC formed a pancreatic “organoid”. This construct remained intact and removable. The retrievability of the construct is an important clinical consideration for future work because it allows retransplantation or the insertion of alternative insulin-producing cells, such as stem cell-derived pancreatic progenitor cells, following complications. These results suggest that the TEC is a promising strategy for clinical application in islet transplantation.

In summary, the present study demonstrated the safety and feasibility of the TEC as a promising site that promotes the survival of islets. Tissue-engineered prevascularized chambers can be expected to become an attractive therapy for insulin-dependent diabetes mellitus.

Variation in weight from TEC implantation to islet transplantation.

Continued weight stability indicated the overall safety of the TEC 28 d post-implantation. Weight increased after transplantation, closely correlating with treatment regimens in the TEC.

(TIF)

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Comparison of IPGTTs of syngeneic, allogeneic, and xenogeneic islets 90 days after transplantation in a TEC.

The naive mice were nondiabetic, nontransplanted C57 mice (black, n = 3), which are more tolerant of metabolic tests than transplant recipients. Blood glucose measurements were monitored at t = 0, 15, 30, 60, 90, and 120 minutes. Data points represent the mean ± S.E.M. of blood glucose values. No difference in the tolerance of mice to glucose challenge was observed in mice that received syngeneic, allogeneic, or xenogeneic islets in a TEC (n = 3) compared to naive animals (p*>0.01 vs. syngeneic group, p**>0.01 vs. allogeneic group, p***>0.01 vs. xenogeneic group).

(TIF)

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(PDF)

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22 Jun 2020

PONE-D-20-16218

A novel pre-vascularized tissue engineered chamber as a site for allogenic and xenogeneic islet transplantation to establish a bioartificial pancreas

PLOS ONE

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Reviewer #1: This is in principle an honest and straightforward study on implantation of a longitudinally cut silicone tube filled with matrigel to induce pre-vascularization. Subsequently, islets from autologous, allo or xeno origin are then transplanted and outcome monitored. It was found that the longer pre-vascularization went on, the better outcomes tended to be. Since the device is essentially open, both allo and xeno islets required immune suppression (here by anti-CD45 and anti-CD40L). Finally, in these longterm surviving islet recipients, some evidence of tolerized immunity was observed.

The problem is not data quality or clarity of the report but rather my impression of lack of novelty. Did we know vascularization matters greatly to survival? Yes. Will an open device like this accommodate protection against alloreactivity? No. And then the clinical question is whether immune suppression as in this paper is acceptable in the future. Likely not. So the paper fails to show an improved safety efficacy-perspective in my mind.

Minor comment: the authors state that 'the principle concern in clinic is weight'. I see more problems that that so best omit this statement.

Reviewer #2: The introduction summarizes well the shortcomings of intra-hepatic islet infusions and captures nicely the main challenges of extra-hepatic islet transplantation, including tissue capacity, kinetics, and adequate oxygen and nutrient delivery to the graft.

The paper hypothesises that the ability and timing of transplanted islets to normalize blood glucose in diabetic mice correlates to the degree of pre-vascularization of the tx site and the number of transplanted islets, respectively. By employing a tissue engineering technique developed in the late 80’s and combing with Matrigel, the authors examine the degree of vascularisation in the tissue engineered chamber (TEC) 7d, 14d, 28d and 35d after implantation and the effect on blood glucose when 300 syngeneic islets are transplanted into the TEC after 0, 14 and 28 days of pre-vascularization of the TEC. The effect of islet numbers on blood glucose lowering is examined by transplanting 100, 200 and 300 syngeneic islets after 28 days of pre-vascularisation. The authors examine if allo- and xenogeneic islets can survive and function for prolonged time in the TEC by treating the diabetic mice with anti-CD45RB treatment for 7 days +/- anti-CD40L for up to 14 days.

The authors show, that a 28d pre-vascularised TEC is permissive for long term (90d) islet function and survival in syngeneic transplantations and that increasing islet numbers decreases the time interval from islet injection to blood glucose normalisation. Furthermore, the authors provide data indicating, that combination treatment with anti-CD45RB (7d) + anti-CD40L (14d) is permissive for allo-and xenogeneic islet transplantations in this model.

The observed effect of pre-vascularization is in accordance with numerous studies on the effect of oxygen on islet survival and function in vivo and the protective effect of anti-CD45RB (7d) and anti-CD40L has also been demonstrated previously.

The introduction section is relatively well written and addresses relevant background information.

The result section, however, appears to have been written in haste, with little proof-reading. Graphs are mixed up, legends flawed, there are incomplete references and both linguistic and argumentative flaws. The discussion section appears written in haste as well, with several errors and lacks perspective.

I have listed some of the issues below, but the list is by no means exhaustive. I recommend the authors to revise the language to improve readability and correct the flaws before the manuscript can be properly reviewed. I have therefore chosen not to follow the otherwise helpful outline of how to review, since it does not make sense at this stage in my opinion. In its current form, I am inclined to reject the article.

Issues to clarify/correct:

1. The authors refer to allo- or xeno islet transplantation as a potentially promising solution for insulin-independent diabetes mellitus both in the abstract and on p 16, p 21. Do the authors mean insulin-dependent?

2. On page 16, the authors write that they explored whether anti-CD45RB (7d) and anti-CD40L could prolong the survival of the mice. Do they mean survival of the graft?

3. Page 16 bottom, the authors state that the data in Fig 4B indicates that glucose tolerance was dependant on the number of transplanted islets. This does not make sense in that context. Please correct.

4. On page 20-21, the paper lists several sites that have “never been used clinically”, including omentum, muscle and anterior chamber of the eye. It the authors mean to state, that these sites have never been tested clinically, this is incorrect.

5. Fig 3: Fig 3A and 3B has been switched. Thus, the text and the figure legend refer to the wrong graph. The x-axis legend on current fig 3B must be corrected (not 28d). In Fig 3C, please state the number of islets (300?)

6. P15, referral to suppl. Fig 1 for bodyweight. However, this is Suppl. Fig 2. Please correct.

7. Please double check references; the method of creating the TEC is referred to 23 and 24, Hussey et al. and Menger et al. but the method is only described in Cronin et al. which is mentioned in the text but not on the reference list.

8. Fig 4: in the legends of the graphs (red line), what is meant by “uncontrol”?. In the text legend to fig 4, how can 5/5=80%?

9. Fig 3: The 5 animals in the group given 300 islets on d 28 are the same in the two graphs (A & B). Were these two experiments run simultaneously?

10. Since the paper concludes that the TEC is a promising strategy for islet transplantation due to the vascularisation induced by Matrigel, it would be of interest to hear the authors view on the usability of Matrigel in the clinic and the scalability of this technology?

11. The method of creating the TEC and transplanting the islets is unclear and should be clarified, since the referenced papers do not. Are the islets inserted into the tube through the fat pad/ bone wax used to seal the tube? Does this require reopening of the surgical incision made to insert the silicone tube?

12. Histology: Please check if 5mm thick sections are correct. Also, please clarify the spatial origin of the tissue shown in Fig 2: is this from the periphery, the centre or the end of the tube? Is the vascularisation evenly distributed throughout the TEC? Please show HE stained sections from the tissue surrounding the TEC to support the statement that the silicone is well tolerated. Are the images in row 1, 2 and 3 in Fig 2 from the same/adjacent sections?

13. Fig 2. Please clarify if sham is control silicone tube with no Matrigel and no Heparin as in Fig 3. If it is inguinal adipose tissue with no tube, please show sections from the no Matrigel control.

14. The inner diameter of the silicone tube is said to be 3.7 mm. Please show histology of islets in the centre of the tube/tissue chamber.1.85 mm is a long diffusion distance for oxygen to reach the central tissue and may significantly impact the capacity of this technology by only allowing survival of islets in the periphery of the tube.

15. In the discussion, the paper mentions that grafts were removed if teratoma or malignant transformation was observed. From where did teratomas arise? Adult islets do not contain pluripotent cells. Does the Matrigel induce uncontrolled growth of the islets or native cells? Please discuss how this affects the safety and relevance of this model.

16. It would be of interest if the authors would discuss the scalability of this approach to clinically relevant numbers of islets (in the range of 400K-800K IEQs based on portal vein injections in 60-80 kg patients)

17. It is concerning, that only 5 animals are included in each group, and I am surprised to see so little variation, since there normally is significant variation among animals. Raw data should be reviewed. Statistical analysis of samples from 5 animals is not sufficiently robust to draw solid conclusions from. Has power analysis been performed?

**********

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21 Sep 2020

Dear editorial-lmanager and Reviewers,

Thank you for providing the valuable suggestions and comments for our manuscript (ID: PONE-D-20-16218). We have carefully revised the manuscript according to the reviewer comments. The answers to the comments are listed as following.

Reply to Reviewer #1

Comment. This is in principle an honest and straightforward study on implantation of a longitudinally cut silicone tube filled with matrigel to induce pre-vascularization. Subsequently, islets from autologous, allo or xeno origin are then transplanted and outcome monitored. It was found that the longer pre-vascularization went on, the better outcomes tended to be. Since the device is essentially open, both allo and xeno islets required immune suppression (here by anti-CD45 and anti-CD40L). Finally, in these long-term surviving islet recipients, some evidence of tolerized immunity was observed.

Comment 1. The problem is not data quality or clarity of the report but rather my impression of lack of novelty.

1. Answer. In many study, various strategies were designed to per-vascularization before islet transplantation[1]. However, we first proposed the feasibility about the allogeneic and xenogeneic islet transplantation in the subcutaneous. If the rejection occurs after transplantation, we can still re-transplanted islets in the TECs. Therefore, the TECs can be designed as an artificial pancreas organ and seeded fresh islet repeatedly. In theory, this study is important for development biomaterials on xenotransplantation. Meanwhile, this principle is very meaningful for exploration artificial pancreas organ.

Comment 2. Did we know vascularization matters greatly to survival? Yes. Will an open device like this accommodate protection against alloreactivity? No.

2. Answer. Based on our knowledge, the encapsulation of pancreatic islets allows for transplantation in the absence of immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane[2]. The TECs alone cannot avoid rejection in our study, so we used immunosuppressive agents (anti-CD45RB and anti-CD40L) to protection against alloreactivity. The CD45 and CD40 are both TCR surface receptors [3, 4]. The use of these two antibodies can significantly inhibited the proliferation of T cell and the occurrence of immune rejection.

Comment 3. And then the clinical question is whether immune suppression as in this paper is acceptable in the future. Likely not.

3. Answer. I agree with you, the immunologic therapy (anti-CD45RB and anti-CD40L) in the article have not applied in the clinic. Although, these two antibodies have potent resistance to immune rejection in animal studies[5], there is still a lack of feasibility and safety research in large animals. Therefore, the application of anti-CD45RB and anti-CD40L is a little far away in the clinic.

Comment 4. So the paper fails to show an improved safety efficacy-perspective in my mind. Minor comment: the authors state that 'the principle concern in clinic is weight'. I see more problems that that so best omit this statement.

4. Answer. In our study, we evaluated the safety in construction of TECs and immunologic therapy. We discovered that none of the mice suffered leg paralysis, wound infection, chamber exposure, or sinus after the pre-vascularization chamber was constructed. It shows that the TECs has good safety and biocompatibility. Subsequently the normalization of blood glucose levels in the diabetic mice when allogeneic and xenogeneic islets transplanted into the TECs, and the weight also showed an upward trend. Histology of the grafts (>90d) showed that islets stained strongly-positive for insulin. I have deleted the statement which are incorrect in the study.

Reply to Reviewer #2

Comment. The introduction summarizes well the shortcomings of intra-hepatic islet infusions and captures nicely the main challenges of extra-hepatic islet transplantation, including tissue capacity, kinetics, and adequate oxygen and nutrient delivery to the graft.The paper hypothesises that the ability and timing of transplanted islets to normalize blood glucose in diabetic mice correlates to the degree of pre-vascularization of the tx site and the number of transplanted islets, respectively. By employing a tissue engineering technique developed in the late 80’s and combing with Matrigel, the authors examine the degree of vascularisation in the tissue engineered chamber (TEC) 7d, 14d, 28d and 35d after implantation and the effect on blood glucose when 300 syngeneic islets are transplanted into the TEC after 0, 14 and 28 days of pre-vascularization of the TEC. The effect of islet numbers on blood glucose lowering is examined by transplanting 100, 200 and 300 syngeneic islets after 28 days of pre-vascularisation. The authors examine if allo- and xenogeneic islets can survive and function for prolonged time in the TEC by treating the diabetic mice with anti-CD45RB treatment for 7 days +/- anti-CD40L for up to 14 days. The authors show, that a 28d pre-vascularised TEC is permissive for long term (90d) islet function and survival in syngeneic transplantations and that increasing islet numbers decreases the time interval from islet injection to blood glucose normalisation. Furthermore, the authors provide data indicating, that combination treatment with anti-CD45RB (7d) + anti-CD40L (14d) is permissive for allo-and xenogeneic islet transplantations in this model. The observed effect of pre-vascularization is in accordance with numerous studies on the effect of oxygen on islet survival and function in vivo and the protective effect of anti-CD45RB (7d) and anti-CD40L has also been demonstrated previously. The introduction section is relatively well written and addresses relevant background information. The result section, however, appears to have been written in haste, with little proof-reading. Graphs are mixed up, legends flawed, there are incomplete references and both linguistic and argumentative flaws. The discussion section appears written in haste as well, with several errors and lacks perspective. I have listed some of the issues below, but the list is by no means exhaustive. I recommend the authors to revise the language to improve readability and correct the flaws before the manuscript can be properly reviewed. I have therefore chosen not to follow the otherwise helpful outline of how to review, since it does not make sense at this stage in my opinion. In its current form, I am inclined to reject the article.

Comment 1. The authors refer to allo- or xeno islet transplantation as a potentially promising solution for insulin-independent diabetes mellitus both in the abstract and on p 16, p 21. Do the authors mean insulin-dependent?

1. Answer. yes, it means the “insulin-dependent diabetes mellitus”. I have corrected the errors of typing in the original text.

Comment 2. On page 16, the authors write that they explored whether anti-CD45RB (7d) and anti-CD40L could prolong the survival of the mice. Do they mean survival of the graft?

2. Answer. Immunologic therapy (anti-CD45RB and anti-CD40L) could prolong the survival of the graft. We have revised it in the original text.

Comment 3. Page 16 bottom, the authors state that the data in Fig 4B indicates that glucose tolerance was dependent on the number of transplanted islets. This does not make sense in that context. Please correct.

3. Answer. We have been revised in the original text.

Comment 4. On page 20-21, the paper lists several sites that have “never been used clinically”, including omentum, muscle and anterior chamber of the eye. It the authors mean to state, that these sites have never been tested clinically, this is incorrect.

4. Answer. Other studies have also explored seeding islets into different locations, including omentum, muscle and anterior chamber of the eye, which could maintain the normal level of blood glucose in the clinic. However, pancreatic islet survival and function in these locations remain unsatisfactory[6, 7]. The expression about “never been used clinically” is incorrectand we have been revised in the original text.

Comment 5. Fig 3: Fig 3A and 3B has been switched. Thus, the text and the figure legend refer to the wrong graph. The x-axis legend on current fig 3B must be corrected (not 28d). In Fig 3C, please state the number of islets (300?)

5. Answer. We have been revised in the original text.

Comment 6. P15, referral to suppl. Fig 1 for bodyweight. However, this is Suppl. Fig 2. Please correct.

6. Answer. The original text has been revised.

Comment 7. Please double check references; the method of creating the TEC is referred to 23 and 24, Hussey et al. and Menger et al. but the method is only described in Cronin et al. which is mentioned in the text but not on the reference list.

7. Answer. The original text has been revised.

Comment 8. Fig 4: in the legends of the graphs (red line), what is meant by “uncontrol”?. In the text legend to fig 4, how can 5/5=80%?

8. Answer. Un-control is the group of no treatment by anti-CD45RB or/and anti-CD40L. The description of “unconrtol” was incorrectly stated in the original manuscript. We have been corrected it.

Comment 9. Fig 3: The 5 animals in the group given 300 islets on d 28 are the same in the two graphs (A & B). Were these two experiments run simultaneously?

9. Answer. The group of Txpl D28 in Fig3 A and the group of 300 islets in Fig B is same experiment. In the study, we first transplanted 300 islets into the TECs on days 0, 14 and 28 post-chamber implantation. In order to explore the relationship between the time of pre-vascularization and blood glucose. Subsequently, 100, 200, and 300 islets were injected into the TECs on 28 day of pre-vascularization. In order to explore the relationship between the number of islets and blood glucose.

Comment 10. Since the paper concludes that the TEC is a promising strategy for islet transplantation due to the vascularisation induced by Matrigel, it would be of interest to hear the authors view on the usability of Matrigel in the clinic and the scalability of this technology?

10. Answer. MatrigelTM is used for lab research only. It originated from the Engelbreth-Holm-Swarm mouse sarcoma and has been found to be a substrate that induces angiogenesis in vivo. In particular, major components, including laminin and collagen IV, modulate cell signaling interactions, is critical for pancreatic β-cell survival and differentiation. Extracellular matrix (ECM) bio-scaffolds (similar to materigelTM) prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications.[8, 9]. The matrix may be modified to induce neo-vascularization by biology.

Comment 11. The method of creating the TEC and transplanting the islets is unclear and should be clarified, since the referenced papers do not. Are the islets inserted into the tube through the fat pad/ bone wax used to seal the tube? Does this require reopening of the surgical incision made to insert the silicone tube?

11. Answer. The image about constructing TECs and islet transplantation have been added in the original text. The referenced papers about transplantation is same as the construction of TECs, I have been corrected it in the original text. The surgical incision was reopened where the silicone tube was insert, and the scalp needle containing the islets inserted into the tube through the bone wax.

Comment 12. Histology: Please check if 5mm thick sections are correct. Also, please clarify the spatial origin of the tissue shown in Fig 2: is this from the periphery, the centre or the end of the tube? Is the vascularisation evenly distributed throughout the TEC? Please show HE stained sections from the tissue surrounding the TEC to support the statement that the silicone is well tolerated. Are the images in row 1, 2 and 3 in Fig 2 from the same/adjacent sections?

12. Answer. The slice thickness is 5μm which has been corrected in the original text. The image of HE in figure 2 is from the center which close to the fat pad. The image of immunohistochemistry is from the center of TECs. The method of pre-vascularization is implanted adjacent to the superficial epigastric vessel in the groin. Therefore, the extension of blood vessels is based on the superficial epigastric vessel and the pre-vascularization are not completely evenly distributed.

We understand that the tissue surrounding of TECs may better reveal the tolerated and bio-compatibility. However, we mainly focused on the feasibility of the TECs of pre-vascularized as a site to islet transplantation in the present study. We observed that none of mice have leg paralysis, wound infection, exposed cavities after TECs was constructed. It should be sufficient to draw a conclusion that the TECs is well bio-compatibility.

Comment 13. Fig 2. Please clarify if sham is control silicone tube with no Matrigel and no Heparin as in Fig 3. If it is inguinal adipose tissue with no tube, please show sections from the no MatrigelTM control.

13. Answer. In the figure 2B and 2C, the sham group should be the group of control which have a chamber (but have no matrigelTM and no heparin). I have corrected the errors of typing in the original text. In figure3A, the diabetic mice, which have neither constructed chamber nor transplanted islets, were acted as sham group. The sample is derived from the fat pad tissue near the superficial epigastric vessel, the image of HE was showed.

Comment 14. The inner diameter of the silicone tube is said to be 3.7 mm. Please show histology of islets in the centre of the tube/tissue chamber.1.85 mm is a long diffusion distance for oxygen to reach the central tissue and may significantly impact the capacity of this technology by only allowing survival of islets in the periphery of the tube.

14. Answer. The study is to explore the feasibility of islet transplantation by constructing a tissue engineering pre-vascularization chamber in the subcutaneous. Meanwhile, we also consider the factor of oxygen diffusion. The islets are transplanted near the superficial epigastric vessel where have abundant neo-vascularization.

Comment 15. In the discussion, the paper mentions that grafts were removed if teratoma or malignant transformation was observed. From where did teratomas arise? Adult islets do not contain pluripotent cells. Does the Matrigel induce uncontrolled growth of the islets or native cells? Please discuss how this affects the safety and relevance of this model.

15. Answer. The matrigelTM is a safe basement membrane to islets and would not induce mature cell again. Nowadays, stem cells are co-transplanted with islet cells that can improve the outcome of islet transplantation[10]. Therefore, the islets are co-cultured with stem cell which caused teratomas due to over-differentiation. However, stem cells were not involved in our study and the teratomas would not happened. We have revised relevant incorrect information in original text.

Comment 16. It would be of interest if the authors would discuss the scalability of this approach to clinically relevant numbers of islets (in the range of 400K-800K IEQs based on portal vein injections in 60-80 kg patients)

16. Answer. The question is very interesting. There are still many obstacles, including the uncertainty of the location and size of the chamber. Islets are transplanted into the liver via the portal vein that causes a large number of islet apoptosis due to local hypoxia as a result of the limited blood supply. If we find a suitable location to implantation a chamber of vascularization in the clinic, we believe that the number of transplantation islets will not exceed 400k-800k IEQs. Meanwhile, chamber was transplanted in the subcutaneous, which has the advantage of multiple transplantation islets.

Comment 17. It is concerning, that only 5 animals are included in each group, and I am surprised to see so little variation, since there normally is significant variation among animals. Raw data should be reviewed. Statistical analysis of samples from 5 animals is not sufficiently robust to draw solid conclusions from. Has power analysis been performed?

17. Answer. The fluctuation of blood glucose have similar among the normal mice (100mg/dL-200mg/dL) [11, 12]. Data were analyzed using Graph-Pad Prism software version 5 (Graph-Pad version 5.01), and the process of data analysis is scientific and reasonable. Under the condition of SPF, their cages were housed within a 12-h day/night cycle and with ad libitum access to food and water. Meanwhile, mice were transplanted and treated under the same experimental conditions. Therefore, there is little individual difference between mice. Based on past experience and other relevant study revealed that the statistical analysis of samples from 5 animals is sufficiently robust [13-15]. Furthermore, we focus on the survival of xeno-graft (n=20) to confirm the accuracy of the results.

The reviewer’s comments are very helpful, and we have revised the previous manuscript accordingly. For more detailed revisions, please refer to the revised manuscript.

Sincerely yours,

Zhao Gaoping

Department of University of Electronic Science and Technology of China

No.4, Jian She Road, chengdu, 610000, China

Email: gzhao@uestc.edu.cn

Reference

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2. de Vos P, Hamel AF, Tatarkiewicz K. Considerations for successful transplantation of encapsulated pancreatic islets. Diabetologia. 2002;45(2):159-73. Epub 2002/04/06. doi: 10.1007/s00125-001-0729-x. PubMed PMID: 11935147.

3. Chang VT, Fernandes RA, Ganzinger KA, Lee SF, Siebold C, McColl J, et al. Initiation of T cell signaling by CD45 segregation at 'close contacts'. Nature immunology. 2016;17(5):574-82. Epub 2016/03/22. doi: 10.1038/ni.3392. PubMed PMID: 26998761; PubMed Central PMCID: PMCPMC4839504.

4. Unutmaz D, Baldoni F, Abrignani S. Human naive T cells activated by cytokines differentiate into a split phenotype with functional features intermediate between naive and memory T cells. International immunology. 1995;7(9):1417-24. Epub 1995/09/01. doi: 10.1093/intimm/7.9.1417. PubMed PMID: 7495749.

5. Molano RD, Pileggi A, Berney T, Poggioli R, Zahr E, Oliver R, et al. Prolonged islet allograft survival in diabetic NOD mice by targeting CD45RB and CD154. Diabetes. 2003;52(4):957-64. Epub 2003/03/29. doi: 10.2337/diabetes.52.4.957. PubMed PMID: 12663467.

6. Cantarelli E, Piemonti L. Alternative transplantation sites for pancreatic islet grafts. Current diabetes reports. 2011;11(5):364-74. Epub 2011/07/27. doi: 10.1007/s11892-011-0216-9. PubMed PMID: 21789599.

7. Shapiro AM, Pokrywczynska M, Ricordi C. Clinical pancreatic islet transplantation. Nature reviews Endocrinology. 2017;13(5):268-77. Epub 2016/11/12. doi: 10.1038/nrendo.2016.178. PubMed PMID: 27834384.

8. Saldin LT, Cramer MC, Velankar SS, White LJ, Badylak SF. Extracellular matrix hydrogels from decellularized tissues: Structure and function. Acta biomaterialia. 2017;49:1-15. Epub 2016/12/05. doi: 10.1016/j.actbio.2016.11.068. PubMed PMID: 27915024; PubMed Central PMCID: PMCPMC5253110.

9. Abbas Y, Carnicer-Lombarte A, Gardner L, Thomas J, Brosens JJ, Moffett A, et al. Tissue stiffness at the human maternal-fetal interface. Human reproduction (Oxford, England). 2019;34(10):1999-2008. Epub 2019/10/04. doi: 10.1093/humrep/dez139. PubMed PMID: 31579915; PubMed Central PMCID: PMCPMC6809602.

10. Oh BJ, Oh SH, Jin SM, Suh S, Bae JC, Park CG, et al. Co-transplantation of bone marrow-derived endothelial progenitor cells improves revascularization and organization in islet grafts. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013;13(6):1429-40. Epub 2013/04/23. doi: 10.1111/ajt.12222. PubMed PMID: 23601171.

11. Pepper AR, Gala-Lopez B, Pawlick R, Merani S, Kin T, Shapiro AM. A prevascularized subcutaneous device-less site for islet and cellular transplantation. Nature biotechnology. 2015;33(5):518-23. Epub 2015/04/22. doi: 10.1038/nbt.3211. PubMed PMID: 25893782.

12. Coronel MM, Geusz R, Stabler CL. Mitigating hypoxic stress on pancreatic islets via in situ oxygen generating biomaterial. Biomaterials. 2017;129:139-51. Epub 2017/03/28. doi: 10.1016/j.biomaterials.2017.03.018. PubMed PMID: 28342320; PubMed Central PMCID: PMCPMC5497707.

13. Hamilton DC, Shih HH, Schubert RA, Michie SA, Staats PN, Kaplan DL, et al. A silk-based encapsulation platform for pancreatic islet transplantation improves islet function in vivo. Journal of tissue engineering and regenerative medicine. 2017;11(3):887-95. Epub 2015/01/27. doi: 10.1002/term.1990. PubMed PMID: 25619945.

14. Lee HS, Lee JG, Yeom HJ, Chung YS, Kang B, Hurh S, et al. The Introduction of Human Heme Oxygenase-1 and Soluble Tumor Necrosis Factor-α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2016;16(1):44-57. Epub 2015/10/03. doi: 10.1111/ajt.13467. PubMed PMID: 26430779.

15. Yin N, Han Y, Xu H, Gao Y, Yi T, Yao J, et al. VEGF-conjugated alginate hydrogel prompt angiogenesis and improve pancreatic islet engraftment and function in type 1 diabetes. Materials science & engineering C, Materials for biological applications. 2016;59:958-64. Epub 2015/12/15. doi: 10.1016/j.msec.2015.11.009. PubMed PMID: 26652453.

Submitted filename: Response to Reviewers.docx

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13 Oct 2020

PONE-D-20-16218R1

A novel pre-vascularized tissue engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #2: Partly

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Reviewer #2: Yes

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Reviewer #1: All comments addressed. All comments addressed. All comments addressed. All comments addressed. All comments addressed.

Reviewer #2: Thank you for addressing previous comments.

Regarding statistical analysis, the Authors argue to my previous comment 17 (group size too small) that the use of GraphPad Prism ensures scientific and reasonable data analysis. This is not a valid argument for defending a small group size. A power analysis supporting n=5 in the experimental groups should be shared, since this data is the backbone of the manuscript.

Regarding the language, there are still numerous grammatical errors and unclear sentences throughout the manuscript. As an example, on p 17 top and mid:

“This indicates that maintain euglycemia in recipient mice was dependent on the islets which transplanted into the TEC”

“We could re-implanted xenogeneic islets to the mice which reversal of hyperglycemia had failed, and to explore whether could maintain euglycemia over the long-term following dual anti-CD45RB plus anti-MR-1 antibody treatment.”

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17 Nov 2020

Dear editorial-lmanager and Reviewers,

Thank you for providing the valuable suggestions and comments for our manuscript (ID: PONE-D-20-16218). We have carefully revised the manuscript according to the reviewer comments. The answers to the comments are listed as following.

Comment 17. It is concerning, that only 5 animals are included in each group, and I am surprised to see so little variation, since there normally is significant variation among animals. Raw data should be reviewed. Statistical analysis of samples from 5 animals is not sufficiently robust to draw solid conclusions from. Has power analysis been performed?

Answer: This prevascularized chamber model in murine is really technically demanding and time consuming. And the same model has been also used in islet transplantation in murine groin by different researches [1]. Cronin KJ et.al. used this model to test effects of different materials on revascularization. However, the innovation point in our study is not repeat the previous work which have been proved a successful method to build a prevascularized chamber for islet transplantation, but deepened the work and used this prevascularized chamber model to xenograft transplantation combined with the immunotherapy regime exploited by our research group. Thus, as to a well-documented prevascularized chamber model for islet transplantation, we allocated a small number of valuable model to prove this method could work in allograft transplantation just as that have been well proved by Hussey AJ and Hussey AJ [1], and distributed large number of precious model to test the effect of xenograft transplantation (n=20 in our study). In addition, compared to other works (n=6 in Hussey AJ’s work, and even n=5 in Andrew R Pepper’s work---fig.5 b)[2], the sample size (n=5 in our research) can also prove the efficient of prevascularized chamber in syngeneic and allogeneic transplantation. Thank you again for your careful review and sincere suggestions.

1. Cronin KJ, Messina A, Knight KR, Cooper-White JJ, Stevens GW, Penington AJ, et al. New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials. Plastic and reconstructive surgery. 2004;113(1):260-9. Epub 2004/01/07. doi: 10.1097/01.prs.0000095942.71618.9d. PubMed PMID: 14707645.

2. Pepper AR, Gala-Lopez B, Pawlick R, Merani S, Kin T, Shapiro AM. A prevascularized subcutaneous device-less site for islet and cellular transplantation. Nature biotechnology. 2015;33(5):518-23. Epub 2015/04/22. doi: 10.1038/nbt.3211. PubMed PMID: 25893782.

Submitted filename: Response to Reviewers.docx

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19 Nov 2020

A novel prevascularized tissue-engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas

PONE-D-20-16218R2

Dear Dr. Zhao,

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Academic Editor

PLOS ONE

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24 Nov 2020

PONE-D-20-16218R2

A novel prevascularized tissue-engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas

Dear Dr. Zhao:

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PLOS ONE