Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Summary

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.

Abstract

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.

Introduction

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, intra....

Protocol

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

1. Prepare a collagenase Type V working solution. Weigh collagenase Type V and dissolve with D-Hank's buffer to a final concentration of 1 mg/mL, filter using a 0.22 μm syringe-driven filte....

Results

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

Discussion

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 grafts^15,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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