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, intramuscular region, subcutaneous space, bone marrow, and omental pouch were explored for experimental islet transplantation^5,6,7. 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 supply^10,11 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 clinically^5,12. 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.

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 filter unit and a 60 mL syringe and precool in ice. A volume of 5 mL for each donor recipient is required.
2. Euthanize a 10-week-old C57BL/6 male mouse (23 ± 2 g) by cervical dislocation and spray the external part of the mouse with 75% ethanol for a few seconds. Meanwhile, fill a 5 mL syringe with collagenase solution and change the syringe needle with a bending blunt-pointed perfusion needle (32 G).
3. Put the donor mouse in the supine position on an ice bag under the dissecting scope, cut a transverse opening using straight pointed ophthalmic scissors in the skin of the pubic area, and completely incise the skin towards the head. Then completely open the abdomen via a V-incision from the pubic region to the xiphoid process.
4. Expose the gall bladder and the entire length of the common bile duct by repositioning the liver. Then clamp the duodenal opening of the common bile duct with a vascular clamp and cut a small opening in the gallbladder.
   NOTE: Optimal needle placement is important to prevent backflow into the liver and gallbladder.
5. Cannulate the common bile duct from the gall bladder opening using the perfusion needle in step 1.2, then inject ~2 mL of collagenase solution into the pancreas. After that, separate the perfused pancreas from the intestines, stomach, and spleen using two pairs of noninvasive microtweezers, and put it into a 50 mL conical tube on ice.
   NOTE: Repeat the process for all donor mice. Three consecutively perfused pancreases (no more than 40 min apart) can be combined into a 50 mL conical tube prefilled with 3 mL of collagenase solution for each pancreas.
6. Add 100 μL DNase I (10 mg/mL) per pancreas into the 50 mL conical tubes, and digest the pancreases in a water bath at 37 °C by shaking the conical tubes for 3–5 min.
   NOTE: Shake the tubes vigorously 40x in 10 s intervals to disassociate the tissue prior to digestion in the water bath, and then moderately shake the tubes during digestion.
7. Add stop solution (2.5 mg/mL BSA-HBSS solution) up to a final volume of 50 mL to block the digestion, and pulse centrifuge the tubes to a speed of 750 x g at 4 °C and quickly stop.
8. Pour off the supernatant and wash the pellets 2x by gently resuspending in 15–25 mL of BSA-HBSS. Pulse centrifuge the tube with the same speed conditions as outlined in step 1.7 for 1 min.
9. Pour off the supernatant and pool the pellets of the three conical tubes into a 50 mL conical tube. Resuspend the pellets in a total volume of 10 mL of histopaque-1119. Mix homogeneously, and sequentially add 5 mL of histopaque-1077 and 5 mL of HBSS by pipetting slowly along the side of the tube.
10. Spin the samples in the centrifuge at 750 x g at 4 °C for 10 min without brakes.
11. Aspirate the islets from the HBSS/histopaque-1077 interface into a 50 mL conical tube using a disposable 5 mL Pasteur pipette, add BSA-HBSS to a final volume of 50 mL, and pulse centrifuge at 750 x g at 4 °C.
12. Pour off the supernatant and wash the islets using 30 mL of BSA-HBSS. Centrifuge for 1 min at 750 x g at 4 °C.
13. Resuspend the islets using 30 mL of culture medium (10% FBS-1%P/S-CMRL-1066) per tube and pour in a 10 cm diameter light-tight culture dish. Under a stereomicroscope, using 200 μL gel-loading pipet tips, handpick the islets from the solution based on their morphology (i.e., spheroidal, white). Put into an untreated cell culture dish with 10 mL of culture medium.
    NOTE: A second handpicking can be carried out if the purity of first-purified islets is not optimal after the first picking.
14. Check the purity of the handpicked islets by dithizone staining under a light microscope and detect the viability of islets by fluorescein diacetate (FDA)-propidium iodide (PI) staining under a fluorescent microscope.
15. Culture the isolated islets in culture medium (as in step 1.13) in an incubator at 37 °C, 95% air-5% CO₂ before transplantation.

2. ISWAT islet transplantation

1. Induce diabetes in the 8-week-old male C57BL/6 mice (22 ± 2 g) by a single injection of streptozotocin (STZ). On day -4, fast the mice for ~4–6 h, then prepare a 2% STZ solution using 0.1 M citrate buffer (pH 4.4). Weigh, resuspend in buffer, and inject the mice intraperitoneally at a dose of 180 mg/kg.
   NOTE: STZ solution needs to be freshly prepared before use and covered with aluminum foil due to its light sensitivity. It should be used immediately, because it will lose activity within 15–20 min.
2. Puncture the caudal veins of the STZ-injected mice to collect some blood at around 10 AM of day -1 and day 0 and measure the non-fasting blood glucose level using a test strip and a basic blood glucose monitoring instrument. The mice will be used as islet transplantation recipients if the blood glucose levels of the 2 consecutive test days are both at least 20 mmol/L.
3. On day 0, weigh and mark all the recipient mice. Anesthetize each recipient by injecting 60 mg/kg pentobarbital sodium intraperitoneally.
   NOTE: Administer a toe pinch to check the depth of anesthesia. If the recipient mouse has no withdrawal reflex, the level of anesthesia is enough for surgery. If not, administer an additional 10 mg/kg pentobarbital sodium. The pentobarbital sodium used was diluted in sterile physiologic saline at the concentration of 2% (m/v).
4. Take the hydrogel out from a -20 °C freezer and keep it on ice to allow thawing. The hydrogel is liquid at 4–10 °C and will solidify at a higher temperature.
5. Pick ~450–500 islet equivalents (IEQ) for each recipient into a sterile 1.5 mL centrifuge tube under the stereomicroscope (as in step 1.13) with 200 µL of culture medium and keep on ice until ready for transplantation.
6. Swab the left inguinal area of the recipient using 75% ethanol. Place it in the supine position and fix the four limbs using surgical tape. Shave off the hair around the surgical site with electrical clippers and swab the area with Iodophor.
7. Make a vertical skin incision in this area using the ophthalmic scissors and noninvasive microtweezers, identify the inferior epigastric artery and vein in the ISWAT, and create a small pocket above the vessels.
8. Spin the islets tube for 30 s at 200 x g and remove the supernatant as much as possible. Aspirate 20 µL of completely thawed hydrogel and load it into the tube with the islets. Resuspend the islets gently, avoiding bubbles.
9. Deliver the entire islet-hydrogel mixture in the tube into the pocket (step 2.7) of the recipient with a 200 µL pipette tip.
   NOTE: This process requires two technicians, one for picking up the edges of the pocket using noninvasive microtweezers, and the other for delivering the islet mixture into the pocket.
10. Add 20 µL of cephalosporin (~5–10 mg) into the transplantation site after the islets-hydrogel mixture is completely solidified, then use a 5-0 surgical suture to close the muscle and skin with the continuous suture method.
    NOTE: The hydrogel needs about 3 min to solidify due to the body temperature.
11. Place the recipient into a clean cage and keep warm using a thermal pad. Keep monitoring until the recipient fully recovers and starts to move autonomously. Then administer 0.03 mg/ml Buprenorphine with sterile physiological saline intraperitoneally, according to 0.1 mg/Kg dose.
12. Repeat steps 2.3–2.11 for each recipient mouse.
13. Measure the non-fasting blood glucose levels of the recipient mice (as in step 2.2) 3–4x a week for the first month, and 1x a week thereafter.
14. At the end of the follow-up (100 days after transplantation), under anesthesia (carried out as in step 2.3) excise the ISWAT bearing the graft from the recipients following the sterile steps described in step 2.6. Fix the tissue in formalin and paraformaldehyde according to the histological protocols for hematoxylin and eosin (H&E) staining and immunofluorescence of insulin and glucagon.
15. Add cephalosporin and close the incision of the recipients as in step 2.10, then prepare for recovery as in step 2.11.

Representative 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 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.

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^15,16.

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 researchers^18,19,20. 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.

Materials

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