Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Summary

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.

Abstract

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.

Introduction

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 hyperglyc....

Protocol

All experiments were approved by Peking University Institutional Animal Care and Use Committee (IACUC, IACUC no. COE-LuoY-1).

1. Islet Isolation

1. Preparation of reagents and equipment.
   1. Reconstitute collagenase P powder (2 U/mg) in HBSS to make a 5 mg/mL solution and filter it through a 0.22 µm filter to remove the bacteria. Prepare 0.6 mL-aliquot solutions of collagenase P in 15-mL conical tubes and store at -20 °C.
      NOTE: During use, each aliquot is diluted with HBSS to give 6-mL working solutions with final concentrations of 0.5 mg/mL, or 1 U/mL (enough for treating 3 mice). The working solutio....

Results

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 is.......

Discussion

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 homog.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	0.45x15 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