In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.
Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.
The worldwide incidence and prevalence of type 1 diabetes mellitus (T1DM) are quickly rising, according to the statistical data of the International Diabetes Federation (IDF)1. Islet transplantation is one of the most promising approaches for treating T1DM4. Since the great breakthrough made in clinical islet transplantation using the Edmonton protocol2 was reported, functioning islet graft survival in T1DM recipients after 5 years now reaches about 50%3.
In the past, several transplantation sites, such as the liver, kidney capsule, spleen, intramuscular region, subcutaneous space, bone marrow, and omental pouch were explored for experimental islet transplantation5,6,7. Some of the above sites have been tested in clinical settings8. Although islet transplantation into the liver remains the most widely used method in clinical application at present9, there are several important problems to address when using this site. For instance, how to reduce early loss of the transplanted islets caused by the instant blood mediated inflammatory reaction (IBMIR) and poor oxygenation supply10,11 and how to retrieve the islet grafts if necessary, because they diffusely localize in the liver. The renal capsule may be an ideal site for rodent recipients. However, the tight kidney capsule limits the transplantation of sufficient allogeneic islets in humans, although it may be a better fit for islet xenotransplantation due to the highly purified porcine islet preparations used clinically5,12. Therefore, the search for a more suitable site for islet transplantation is in progress.
The subcutaneous space may be used as a clinically applicable site for islet transplantation due to its accessibility. However, the efficiency of islet transplantation into the subcutaneous space is extremely low, thus requiring a relatively large number of islets to reverse hyperglycemia13. Recently, a Japanese research team found the ISWAT, a novel subcutaneous site superior for islet transplantation in a murine model when compared to the liver14. The ISWAT contains the epigastric artery and vein, so the rich blood supply may ensure islet graft revascularization. In this manuscript, we propose an easy implantation method using a basement membrane matrix hydrogel to fix syngeneic murine islets in the ISWAT. This protocol proves effective for islet transplantation.
All procedures in this protocol followed the principles of animal welfare of the Ethics Review Committee of Shenzhen Second People's Hospital. Islet graft recipients and donors were 8- to 10-week-old C57BL/6 male mice purchased from the Medical Animal Center of Guangdong Province. The procedure of the harvesting, isolation, culture, or administration of the harvested cells were carried out in aseptic conditions.
1. Islet preparation
2. ISWAT islet transplantation
Two procedures are introduced in this protocol: murine islet preparation and islet transplantation into the ISWAT site. In the first procedure, after perfusing and digesting with Type V collagenase solution, purifying with Histopaque-1119 and Histopaque-1077 and an additional hand-picking step, the isolated murine islets will be sufficiently pure for transplantation (as shown in Figure 1) and the isolated islets that have a high viability will be used for transplantation (as shown in Figure 2). In the second procedure, diabetes induction with STZ chemical is critical. The optimal dose of STZ depends on mouse strain and age, and successful induction of diabetes is defined by non-fast blood glucose levels of more than 16.7 mmol/L at the same time on two consecutive days. The diabetic mice can survive without islet transplantation for few weeks. The islet grafts should be fully mixed with hydrogel before being transplanted into the ISWAT site, and a few minutes need to pass for the grafts to be fixed into the IWSAT (Figure 3). When transplanted into the ISWAT, the islet grafts reversed hyperglycemia for about one month, and the body weight of the recipient mice gradually increased (Figure 4). At 100 days after transplantation, the islet grafts were retrieved for histological analysis (Figure 5). As shown in Figure 4, the non-fasting blood at ~10 AM on the test days rapidly rose after the grafts were removed. H&E staining demonstrated that the islet grafts remained intact. Insulin and glucagon immunofluorescence staining showed the transplanted islets functioned well (Figure 5).
Figure 1: Pancreas perfusion and digestion and islet purification. (A) The process of pancreas perfusion (A1–A6). (B) The endpoint of pancreas digestion, with particles suspended. (C) Purified islets observed under the light microscope. Please click here to view a larger version of this figure.
Figure 2: Purity and viability of purified islets. (A) Islet purity determined by dithizone staining. Islet viability assessed by FDA-PI double staining. (B) Light microscopy image of islets. (C) FDA staining shows viable cells in green. (D) PI fluorescence red indicates dead cells. Please click here to view a larger version of this figure.
Figure 3: Islet transplantation. (A) After anesthesia, shave the hair at the transplantation site with a shaver blade and fix recipient limbs in the abdominal upward position with surgical tape. (B) After laparotomy, the ISWAT transplantation site is exposed. (C) Islets mixed in hydrogel are slowly transplanted into the ISWAT site. Please click here to view a larger version of this figure.
Figure 4: Blood glucose levels and body weight posttransplantation. Non-fasting blood glucose levels (red line) and body weights (blue line) of the recipient mice transplanted with syngeneic islets (n = 7). The black arrow indicates that the grafts were removed 100 days after transplantation. Some variations in blood glucose levels comparing the various recipients was observed, reflecting differences in islet quality and function. Please click here to view a larger version of this figure.
Figure 5: Histology and immunofluorescence. Graft sections were stained with H&E, DAPI (nuclei), anti-mouse insulin antibody, and anti-mouse glucagon antibody. Please click here to view a larger version of this figure.
Pancreas islet transplantation is a promising therapy to treat T1DM. The effect of this therapy is affected by many factors and choosing an optimal site for islet implantation is extremely important. The ideal anatomical site for islet transplantation should have the following characteristics: accessibility for simple transplantation, biopsy, and graft retrieval procedures; reduced complications; high success rate of blood glucose control; and long-term survival of the islet grafts15,16.
Our team previously described a protocol for islet transplantation in the omentum in a murine model17. In the omentum, normal blood glucose was achieved at a later time compared to islet transplantation under the kidney capsule. As a result, other sites for implanting islets were explored.
The ISWAT was reported as an alternative site to the liver, as it needs fewer islets to reverse hyperglycemia of the recipients compared to transplantation at the liver14. Moreover, transplanting the islets is easy, as is visualizing and retrieving the grafts14. In our study, recipient mice have reduced hyperglycemia within one month after islet transplantation, suggesting that the ISWAT requires longer than the kidney capsule to produce sufficient insulin. These results therefore indicate that the ISWAT site may not offer better conditions for diffusion of oxygen, nutrients, and for revascularization of the graft.
High purity and activity of isolated islets are of vital importance to reverse hyperglycemia of diabetic recipients in the allo-islet transplantation setting, and islet isolation protocols are not the same among different researchers18,19,20. Digestion time is very crucial for obtaining high quality islets. In our experience, it should not exceed 5 min. Isolated islets can be cultured in a 37 °C incubator overnight to allow recovery from the mechanical stress of the digestion procedure21.
The hydrogel used here is a basement membrane matrix that contains laminin, collagen IV, and growth factors17. It solidifies when it reaches body temperature and helps keep the islet grafts in the ISWAT site. While it is not toxic to the islets, whether the presence of the hydrogel affects islet engraftment and function remains to be determined.
Taken collectively, the ISWAT is a novel subcutaneous space for islet transplantation in rodent models and potentially for clinical use. For full evaluation, additional studies using larger mammal preclinical models are required.
The authors have nothing to disclose.
This work was supported by grants from National Key R&D Program of China (2017YFC1103704)Special Funds for the Construction of High Level Hospitals in Guangdong Province (2019), Sanming Project of Medicine in Shenzhen (SZSM201412020), Fund for High Level Medical Discipline Construction of Shenzhen (2016031638), Shenzhen Foundation of Science and Technology (JCJY20160229204849975, GJHZ20170314171357556), Shenzhen Foundation of Health and Family Planning Commission (SZXJ2017021SZXJ2018059), Medical Scientific Research Foundation of Guangdong Province of China (A2019218), China Postdoctoral Science Foundation (2018M633218).
0.22 μm Syringe-driven Filter Unit | Merck Millipore | SLHV033RB | |
1.5 mL centrifuge tube | Axygen | MCT-150-C | |
5 mL Pasteur pipette | JingAn Biological, China | J00085 | |
5 mL syringe | Szboon, China | 20170829 | |
50 mL conical tube | Corning | 430829 | |
5-0 surgical suture | sh-Jinhuan, China | CR537 | |
60 mL syringe | Szboon, China | 20170623 | |
75% Ethanol | LIRCON, China | 9180527 | |
Alexa Fluor 488 donkey anti-mouse IgG(H+L) | Invitrogen | A21202 | Dilution (1:200) |
anti-mouse Glucagon antibody | Abcam | ab10988 | Dilution (1:100) |
anti-mouse insulin antibody | Cell Signaling Technology | 3014s | Dilution (1:100) |
blunt-pointed perfusion needle | Oloey, China | 005 | 32G, yellow |
BSA | Meilune, China | MB4219 | |
C57BL/6 Mice | Medical Animal Center of Guangdong Province | 8~10 weeks | |
cell culture dish | BIOFIL, China | TCD000100 | General,Non-treated,87.8 mm diameter |
centrifuge | Thermo Scientific | ST16R | |
cephalosporin | Lukang medical, China | 150303 | |
CMRL-1066 | Sigma-Aldrich | C0422 | |
Codos Pet Clipper | Szcodos, China | CP-8000 | |
collagenase Type V | Sigma | C9262 | |
DAPI | Thermo Fisher | D1306 | |
D-hank's buffer | Coolaber, China | PM5140-10 | |
dithizone | Sigma-Aldrich | D5130 | |
Dnase I | Sigma-Aldrich | D4263 | |
Eosin staining media | Beyotime Biotech, China | C0109 | |
FBS | GE Healthcare Life Sciences | SH30084 | |
fluorescein diacetate (FDA) | Thermo Fisher | F1303 | |
fluorescent microscope | Leica | DMIL | |
gel-loading pipet tips | Corning | CLS4884 | |
HBSS | Coolaber, China | PM5150-10 | |
hematoxylin staining media | Cell Signaling Technology | 14166S | |
HISTOPAQUE-1077 | Sigma-Aldrich | RNBG0522 | |
HISTOPAQUE-1119 | Sigma-Aldrich | RNBG0536 | |
Hydrogel | BD Biosciences | 356234 | Basement Membrane Matrix |
Iodophor | LIRCON, China | 5190313 | |
light-tight culture dish | DVS, China | AN-5058548 | self-made, glass dish sprayed with black paint |
Medical Adhesive Tape | Cofoe, China | K12001 | |
non-invasive microtweezers | RWD Life Science | F11033-11 and F12016-15 | |
One Touch ultraeasy Basic blood glucose monitoring system | Johnson & Johnson | 33391713 | |
ophthalmic scissors | RWD Life Science | S12012-12 and S11001-08 | |
P/S (penicillin / streptomycin) | Gibco | 15140-122 | |
pentobarbital sodium | Sigma-Aldrich | P-010 | |
Propidium iodide | Sigma-Aldrich | P4864 | |
STZ (streptozotocin) | Sigma-Aldrich | S0130 | |
Test Strip | GenUltimate | 100-50 | |
TRITC-conjugated Goat anti-Rabbit IgG(H+L) | proteintech | SA00007-2 | Dilution (1:200) |
vascular clamp | RWD Life Science | R31006-04 |