This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.
Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.
Type 1 diabetes mellitus (T1D) is an autoimmune endocrine disorder in which islet cells are ablated by the immune system, rendering patients dependent upon the injection of exogenous insulin for their whole lives. The Edmonton protocol represents a milestone in clinical studies of islet transplantation; islets were infused through the portal vein and transplanted at the intrahepatic site1. However, two main obstacles-inadequate sources of donor islets and poor islet engraftment-prevent the wide success of the islet transplantation2. Usually, islets need to be collected from three cadaveric donors to reverse the hyperglycemic condition of one patient; this is due to the low yield of islet isolation procedures and the islet loss after transplantation. In particular, although the post-transplantation islets were bathed in oxygen-rich blood, the direct contact with blood often evoked the instant blood-mediated inflammatory reaction (IBMIR), which could cause the acute loss of the islets. In the long term, it is thought that the gradual loss of islets in patients accounted for the drop of diabetes reversal rates in the clinical groups, which could reach 90% in the first year and declined to 30% and 10% by 2 and 5 years post-transplantation, respectively3.
Islet transplantation at extrahepatic sites has been an attractive strategy to reduce the direct contact of islets with blood while confining the transplants to more definable locations compared to intrahepatic infusion. Studies have been carried out in the kidney capsule, eye, muscle, fat pads, and subcutaneous spaces over the past years, showing that islets at these sites are able to survive and function to restore normoglycemia4. In addition, the islets at these sites are retrievable, making it possible for biopsy or even for further replacement procedures. Extrahepatic sites therefore demonstrate great potential for clinical transplantation5.
Biomaterial-based scaffolds have been intensively investigated for cell transplantation and tissue engineering. Three-dimensional (3D) scaffolds usually contain porous structures and can serve as cellular templates to generate spatial structure/organization of cells or as reservoirs to provide the controlled release of bioactive cues. Scaffolds have also been fabricated from polymeric materials, such as poly(glycolide-L-lactide)6, poly(dimethylsiloxane)7, and thermoplastic poly(urethane)8, to transplant islets in the EFP. Compared to the direct transplantation of islets, the use of scaffolds was found to reduce islet loss by preventing the leakage of islets into the intraperitoneal cavity9,10, providing mechanical protection and modulating the local inflammatory reaction. The scaffolds thus may be developed to promote islet engraftment at the transplantation sites7.
In this study, we intend to demonstrate a paradigm of islet transplantation in the EFP, carried out in mice models using a DCS. Scaffolds derived from extracellular matrices have attracted great interest in recent years due to the superior biocompatibility and more natural porous structures compared to synthetic products. Here, we describe a robust isolation protocol to obtain pancreatic islets at high yields from C57BL/6J mice. DCSs processed from the bovine pericardium were then seeded with islets, and the grafts were transplanted to the EFP in syngeneic diabetic models. Normoglycemia in mice was achieved within 10 days and was maintained for up to 100 days, until the removal of the grafts.
All experiments were approved by Peking University Institutional Animal Care and Use Committee (IACUC, IACUC no. COE-LuoY-1).
1. Islet Isolation
Figure 1: Photographs showing the cannulation of the bile duct and the perfusion of the pancreas with collagenase solutions. (A1) Pulling the duodenum until the bile duct is taut. (ampulla: the triangular, milky area on the surface of the duodenum; bile duct: the cord-like milky structure on the surface). (B1) Inserting the needle into the bile duct from the ampulla. (C1) Inflating the pancreas with the injection of enzyme. (A2, B2, and C2) Cartoon images of the procedures shown in A1, B1, and C1, respectively. Please click here to view a larger version of this figure.
Figure 2: Troubleshooting for the cannulation. (A1) The needle tip inserted in the lumen of the bile duct. (A2) The duct filled with enzyme solutions. (B1) The needle inserted in the lumen of the bile duct, and the duct filled with a blue dye. (B2) Due to inappropriate cannulation, the needle is beneath the bile duct, and only an inflated capsule is observed after dispensing the blue dye. (C1) A successful cannulation is evidenced by the distension of pancreas. (C2) Due to inappropriate clamping, blue dye enters the duodenum and causes distention. Please click here to view a larger version of this figure.
2. Islet Culture on the Scaffold
NOTE: DCS has a porosity of about 79%, a thickness of about 0.6 mm, and a pore size ranging from 12 to 300 µm.
3. Islets Transplantation at the EFP Site
Our clamping method, performed using a microscopic hemostatic clamp, is straightforward and time-saving compared with the suture ligation technique. It took roughly 4 h to isolate and purify about 1,200 islets from 6 mice. The freshly isolated islets typically had a rough periphery under an optical microscope (Figure 3A). Once the islets recovered from the isolation process, they looked bright and tight and acquired a smooth surface. However, the stressful isolation could still induce cell death, resulting in the sloughing of cells from the islet surfaces, and unhealthy islets often contained a dark necrotic core (Figure 3B). We measured the diameters of 945 islets from 5 mice; the calculated mean islet diameter was 130.42 ± 41.75 µm (Figure 3C).
To avoid recipient immune rejection, we performed syngeneic transplantation in C57BL/6J mice. Typically, 500 islets laden DCS were transplanted to the site of the EFP and reversed the hyperglycemia within 10 days, compared with the 30 days observed in the free islet group. The normoglycemia was maintained for about 100 days, until the retrieval of the grafts (Figure 3D). The loaded islets were evenly spread on DCS and covered by EFP. The islet-laden DCS could also be easily handled using forceps (Figure 3E and 3F). The histological study showed that the evenly distributed islets were revascularized and surrounded by the EFP tissue and the DCS after transplantation for 60 days (Figure 3G). The immunostaining of insulin further confirmed the successful engraftment of the islets (Figure 3H).
Figure 3: Transplantation of scaffold-supported islets to the EFP site. (A) Representative picture of fresh islets isolated from mice. (B) Islets cultured for 12 h, with dead cells sloughing off the islet surface. Inset: unhealthy islets have a dark, necrotic core. (C) Size distribution of 945 islets from 5 mice. (D) Non-fasting blood glucose level of the diabetic mice transplanted with DCS-supported islets and free islets. The black arrow indicates that the graft was retrieved at this time point. (E) Photograph showing the transfer of islet-laden scaffold onto the surface of a spread EFP tissue. (F) Representative phase contrast image of the islet-laden DCS. Inset: optical picture of the DCS, held by forceps. (G) Representative histological H&E image of the transplanted islets, surrounded by DCS and EFP, after 60 days. (H) Immunostaining of the DCS-supported islets, explanted after 60 days. Scale bars = 150 µm (A, B), 100 µm (F), 500 µm (G), and 25 µm (H). Please click here to view a larger version of this figure.
Pancreas perfusion and digestion time are two key parameters that affect islet yield and quality. Moskalewski first reported the use of a crude collagenase mixture to digest minced guinea pig pancreas11. Lacy et al. reported the injection of enzymes into the duct system to perfuse the pancreas, which greatly increased the islet yield12. The ductal perfusion of enzyme allows for the maximal exposure of pancreatic surface area to the enzyme, resulting in a more homogeneous digestion and a greater release of intact islets in comparison to digesting minced pancreas13. In our experience, the successful cannulation of the bile duct and the perfusion of the whole pancreas were prerequisites for high islet yields. This is because the pancreatic tail (splenic lobe) actually contains most of the islets compared with the pancreatic tissue close to the duodenum. There are two ways to cannulate the bile duct that are reported in the literature: i) inserting the needle close to the liver site while blocking the entry of enzyme into the duodenum13,14 and ii) inserting the needle close to the duodenum while blocking the entry of enzyme into the liver15. Here, we adopted the latter technique, which does not require bending the needle or repositioning the mouse. A well-trained researcher can perform the cannulation of 10 mice within 40 min when following our protocol. The digestion time for the pancreas varies with the age and species of the mice. Over-digested pancreas produce small islets and under-digested pancreas have acinar cells attached to the islets. It is therefore important to optimize the digestion time to obtain high yields of healthy islets.
STZ is an antibiotic compound that specifically destroys beta cells and induces diabetes in mice within 3 days16. The dosing varies with the specific mouse strain and age and should be determined by pre-experiments. To our knowledge, C57BL/6J mice require a lower dose of STZ than Balb/C mice. An overdose of STZ would cause severe hyperglycemia and lead to the death of the animals within a week, while an inadequate STZ dose lowers the diabetes incidence rate.
EFPs are highly vascularized tissues and conveniently accessible to surgery through minimally invasive procedures. The transplantation of islets to EFP is generally more facile and safer compared to the kidney capsule, another commonly reported site for islet transplantation in mouse models. In particular, the kidney is an essential organ and is delicate to handle; the transplantation of islets may fail or the animals may not survive the surgery4. The EFPs in mice are also similar to the omental pouch in humans. The transplantation study in EFP can not only facilitate our understanding of the prerequisite tissue environment for islet survival/function, but also lay the foundation for developing clinical transplantation procedures17.
The DCS used in this study was derived from bovine pericardium and was mainly made of collagen. The decellularized materials may not show immunogenicity and may only induce mild inflammatory responses in vivo18. When islets were seeded within the pores of the DCS, the scaffold offered mechanical protection and prevented the islets from clumping together, which could lead to the necrosis of the islets. The DCS scaffold containing the islets could be handled directly using forceps, allowing for the facile transfer of the transplant. Embedding the scaffolds within the EFP also reduced the leakage of islets into the peritoneum, unlike in the free islets transplanted without a scaffold8. Therefore, the DCS provides distinct advantages for islet transplantation.
The authors have nothing to disclose.
The authors would like to thank Wei Zhang from Guanhao Biotech for providing the decellularized scaffolds. We thank Xiao-hong Peng for the helpful discussions. This research was financially supported by the National Natural Science Foundation of China (Project No.31322021).
Dissecting scissor | Ningbo Medical | ||
Forceps | Ningbo Medical | ||
0.5 mm diameter wire mesh | Ningbo Medical | ||
70 μm cell strainer | Falcon | 352350 | |
Artery hemostatic clamp | Ningbo Medical | ||
Microscopic hemostatic clamp | Ningbo Medical | ||
Hemostatic forceps | Ningbo Medical | ||
Absorbable 6-0 PGLA sutures | JINHUAN | With needle | |
Wound clip | Ningbo Medical | ||
Cotton swab | Ningbo Medical | ||
Gauze | Ningbo Medical | ||
Sterile drapes | Ningbo Medical | ||
10mL syringe | JINGHUAN | ||
1 mL syringe | JINGHUAN | ||
27G intravenous needle | JINGHUAN | 0.45×15 RWSB | |
1.5 mL Eppendorf tube | Axygen | ||
15mL conical tube | Corning | 430791 | |
50mL conical tube | Corning | 430829 | |
35mm Non-treated Peri-dishes | Corning | 430588 | |
Transwell | Corning | 3422 | |
0.22 μm filter | Pall | PN4612 | |
10 mL serological pipet | Corning | 4488 | |
Pipet filler S1 | Thermo Scientific | 9501 | |
Pipette (2-20μL) | Axygen | AP-20 | AXYPETTM |
Dissecting microscope | Olympus | SZ61 | |
Centrifuge | Eppendorf | 5810R | |
Hank’s balanced salt solution | Gibco | C14175500CP | |
Collagenase P | Roche | COLLP-RO | |
Histopaque 1077 | Sigma | 10771 | |
RPMI 1640 | Gibco | 11879-20 | |
FBS | Gibco | 16000-044 | |
D-glucose | Gibco | A24940-01 | |
Glucose meter | Roche | ACCU-CHEK | |
Penicillin-streptomycin | Gibco | 15140-122 | |
Streptozotocin | Sigma | V900890 | VetecTM |
Chloral hydrate | J&K | C0073 | |
Sodium citrate | Sigma | 71497 | |
Citric acid | Sigma | C2404 | |
Iodophors | Ningbo Medical | ||
C57BL/6J, 10-12 weeks old | VitalRiver | Beijing, China | |
Decellularized scaffold | Guanhao Biotec | 131102 | Guangzhou, China |