Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Summary

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Abstract

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

Introduction

Islet transplantation is a cellular replacement therapy for patients with severe diabetes mellitus. Recent reports have shown that rates of insulin-independence at three years after transplantation improve up to 44%¹ and that approximately 80% of recipients who receive more than 600,000 total islet equivalents achieve insulin independence². Furthermore, in the most recent Collaborative Islet Transplant Registry report, it was revealed that fasting blood glucose levels were maintained at 60-140 mg/dL for over a period of 5 years in over 70% of patients who underwent islet transplant alone. The study also determined that around 90% of the patients who received islet transplant alone or islet transplantation after kidney transplant did not develop any severe hypoglycemic events for over 5 years³.

Although the clinical outcomes of this treatment have been improving, some limitations must still be addressed, including the necessity of establishing an optimal transplant site. The liver is a typical transplant site for clinical islet transplantation because it is the largest organ that can accommodate a high volume of islets. However, in some patients the liver is unavailable (e.g., due to portal hypertension, hepatitis, and/or cirrhosis⁴) and therefore other sites, including the renal subcapsular space^5,6, omental pouch^7,8,9,10, mesentery¹¹, gastrointestinal tract¹², skeletal muscle¹³, subcutaneous tissue¹³, bone marrow¹⁴, and spleen^15,16,17, have been considered as alternative transplant sites.

Although intraperitoneal islet transplantation can be performed easily under local anesthesia, making the intraperitoneal cavity an appealing site for clinical islet transplantation, upon transplant, the islets are dispersed throughout the entire intraperitoneal cavity, making islet engraftment detection and successful engraftment confirmation difficult. Therefore, the intraperitoneal cavity is not widely recognized as an ideal clinical transplant site. Instead, it is frequently utilized as a control model for preclinical studies to investigate the effectiveness of transplanted encapsulated¹⁸ and bioengineered islets¹⁹. However, an exact comparison between bioengineered and control islets is difficult to achieve due to the challenges in performing an accurate engraftment assessment.

In contrast, the use of intraperitoneal white adipose tissue in the omental pouch⁸, mesentery, and other extrahepatic locations has been well reported^{10,20,21,22,23} and many of the studies investigating the function of bioengineered islets transplanted using white adipose tissue were able to report promising therapeutic outcomes^20,24,25,26. As the use of epididymal adipose tissue facilitates the detection of transplanted islets, the "fat-covered islet transplantation method", utilizing epididymal adipose tissue, was developed to overcome the limitations of intraperitoneal islet transplantation. In this paper, fat-covered islet transplantation using epididymal adipose tissue is described.

Protocol

The following procedure is performed in three steps. The first step includes the induction of diabetes in the recipient mice and the isolation of donor islets. The second step involves the preparation of islets before transplantation. In the third step, islet transplantation onto epididymal adipose tissue and covering of the islets using the adipose tissue is performed. After that, the therapeutic effects were assessed. The handling of the mice and the experimental procedures performed in this study comply with the ''Principles of Laboratory Animal Care'' (Guide for the Care and Use of Laboratory Animals, National Institutes of Health publication 8^th edition, 2011), and the experimental protocol was approved by the Animal Care and Use Committee of Fukuoka University (approval number: 186018).

1. Surgical preparation

1. Induction of diabetes: Induce diabetes in 20-25 g body weight, 8-12-week-old recipient male mice through intravenous injection of 18 mg/mL streptozotocin solution prepared in 0.1M citrate buffer (180 mg/kg body weight). Mice with blood glucose levels exceeding 400 mg/dL are considered to be diabetic. Use diabetic mice within 1 week after diabetes induction before excessive atrophy of the epididymal white adipose tissue for covering islets.
2. Islet isolation: Perform murine islet isolation one day before transplantation following Gotoh's method²⁷ for islet isolation.
3. In brief, digest pancreatic tissue using collagenase solution. Isolate islets by density gradient centrifugation using an appropriate cell separation solution. Then culture islets overnight in an incubator at 22 °C and 5% CO₂ (culture at <37 °C has been reported to prevent islet death^28,29,30,31).
   NOTE: Handle the purified islet cultures in a safety cabinet. Filter-sterilize all solutions used for islet isolation and culture using a 0.22 µm filter.

2. Preparation of islets for transplantation

1. Gather the appropriate instruments and materials as indicated in Figure 1A.
2. As digestive enzymes such as amylase and lipase may result in injury to the isolated and transplanted islets and a loss of islets can occur from being trapped in contaminating fibrous tissues within the culture dish, prior to transplantation, use forceps to handpick any extra-islet components from the pancreas, including acinar and fibrous tissues (Figure 1B), under a dissecting microscope. After picking, use a cell strainer to filter out single acinar cells.
3. Transfer the filtered islets to a new culture dish containing any appropriate culture medium or buffer solution (e.g., DMEM with low glucose, RPMI1640, CMRL1066, or HBSS) supplemented with bovine serum or albumin to prevent islet attachment to plastic and swirl the dish to position the islets in the center of the dish (Figure 1C). Using a P200 micropipette and the microscope, pick the individual islets into an appropriate collection tube (Figure 1D).
4. Place a new, 40 μm cell strainer on top of a 50 mL plastic tube (Figure 1E left and center) and wash the filter with fresh medium (Figure 1E right).
5. Use a 1000 μL pipette to add the islets to the strainer to separate the islets and single acinar cells (Figure 1F1 and Figure 1F2).
   NOTE: The purified islets on cell strainer will be approximately 100% pure.
6. Use forceps to invert the strainer on a new 60- or 100-mm sized non-treated culture dish containing culture medium or an appropriate buffer solution supplemented with bovine serum or albumin (Figure 1F3 and Figure 1F4). Use fresh medium/buffer to flush the islets into a new culture dish. Then add enough medium/buffer to the culture dish to reach a total volume of approximately 20 mL.
7. Count the islets under a microscope and divide the number of islets equally between individual 1.5 mL plastic centrifuge tubes according to the number of donor animals (Figure 1G). For example, two hundred, 100-200 µm islet equivalents (IEQ) from two mice would be added to each of two tubes.
8. Centrifuge the islets at 2,100 x g within 1 minute at room temperature and discard the supernatant. Around 20-30 µL of residual solution will typically remain in the tube (Figure 1H).

3. Islet transplantation onto epididymal adipose tissue and covering with epididymal white adipose tissue

1. Before the surgery, collect an anesthesia machine for small animals, stereo microscope, light source, 50-200 µL micropipette with 200 µL micropipette tips, cotton swabs, a 4-0 suturing set, and disinfected surgical instruments (Figure 2A). Autoclave the cooper scissors, ophthalmic scissors, Pean forceps, tweezers, and needle holders. After autoclaving, immerse the equipment in a 1% povidone-iodine solution (Figure 2A). Use cotton swabs for mobilization of the epididymal white adipose tissue and for hemostasis in cases of bleeding. Use a micropipette with a 50-200 µL tips for islet transplantation.
2. Deliver anesthesia to the diabetic recipient mouse using an inhaled anesthetic agent (2% isoflurane in oxygen). Apply ophthalmic lubricant to both eyes to prevent drying. Then place the mouse in the supine position (Figure 2B left) and remove the hair from the abdomen to prevent infection using hair clippers and/or depilatory cream. Disinfect the abdomen and the inguinal region using at least three alternating rounds of a povidone-iodine solution followed by 70% ethanol (Figure 2B right). Before the surgery, confirm anesthesia depth via the absence of a toe pinch reflex. Provide intra-operative thermal support using a heating pad and use a surgical drape to secure the sterile surgical area.
3. Incise the skin at the lower median area (Figure 2C left). A skin incision that is approximately 2 cm in length is recommended. Clamp the left abdominal wall with the Pean forceps (atraumatic forceps or retractor can also be used) and pull the tissue to the left side of the mouse to secure the surgical field (Figure 2C right). After laparotomy, decrease the percentage of isoflurane to 1.0-1.5% for anesthesia maintenance.
4. Use a cotton swab to mobilize the small and large intestine to the right side of the mouse (i.e., left side of the operator). The left epididymal white adipose tissue in the abdominal cavity are located in the left inguinal area. Mobilize the epididymal white adipose tissue and the left testis to outside of the abdomen (Figure 2D left) and stretch out the tissue (2D right).
5. Use a P200 micropipette equipped with a 200 µL pipette tip to collect the entire volume of islets from one 1.5 mL tube with gentle pipetting (Figure 2E left), taking care that no islets are left in the tube upon collection. Allow the collected islets to settle to the tip of the pipette by gravity (Figure 2E right).
6. Place the micropipette tip lightly onto the distended adipose tissue. Taking care to prevent excessive flushing of the medium/buffer in the tip, carefully seed the islets onto the tissue (Figure 2F left). After seeding, confirm a correct placement of the islets under a dissecting microscope (Figure 2F right).
7. Cover the islets with the epididymal white adipose tissue (Figure 2G). The use of sutures or biobinding agents is not needed.
8. Place the left testis under the epididymal white adipose tissue and return the tissues to the intraperitoneal cavity (Figure 2H). Close the skin in two layers (peritoneum, then muscle and skin) using a 4-0 suture (any sutures such as nylon or absorbable sutures can be used) (Figure 2I). Inject acetylsalicylic acid (300 mg/kg; SQ) near the wound for postoperative analgesia. Then place the mouse under a heat lamp and monitor until full recovery.

4. Monitoring after Islet transplantation (Summary)

1. Assess the therapeutic effects of islet transplantation by monitoring blood glucose, glucose tolerance test and histological assessment at postoperative day (POD) 28.
   1. Monitor the blood glucose, including the measurements of blood glucose at glucose tolerance test, using a small glucose meter.
   2. Collect the blood samples (a little microliters) from tail vein. Regarding histological assessment, murine insulin (for detecting engrafted islets) and von Willebrand factor (for detection of vessels, which is an evidence for islet engraftment) were detected in transplanted islets in the recovered epididymal adipose tissue by immunohistochemistry.

Results

To compare the transplant efficacy of fat-covered islet transplantation to that after intraperitoneal islet transplantation, the same number of islets was implanted onto the peritoneum at the left paracolic space of control recipient diabetic animals. The blood glucose levels of mice with fat-covered islet transplantation were observed to gradually and significantly decrease compared to intraperitoneal islet transplanted mice (p = 0.0023; Figure 3A). One month after transplantation, the bloo...

Discussion

The fat-covered islet transplantation method incorporates techniques from two different transplant techniques: intraperitoneal islet transplantation and intra-adipose tissue islet transplantation. As the surface membrane of epididymal white adipose tissue is considered to be the white adipose tissue that is covered by the peritoneum and that is attached to the epididymis, the fat-covered islet transplantation method can be anatomically categorized as a type of intraperitoneal islet transplantation. The technique by which...

Materials

Name	Company	Catalog Number	Comments
4-0 Nylon	Alfresa	ER2004NA45-KF2	Closing abdomen
Alexa 488-conjugated donkey anti-guinea pig	Jackson Immunoresearch	706-546-148	Secondary antibody for insulin antibody
Alexa 647-conjugated donkey anti-rabbit	Jackson Immunoresearch	711-606-152	Secondary antibody for von Willebrand factor antibody
DMEM, low glucose, pyruvate	ThermoFisher Scientific	11885084	Culturing islets, transplanting islets
Eosin	Fujifilm Wako Chemicals	051-06515	Using for staining tissue by eosin
Eppendorf Safe-Lock Tubes, 1.5 mL	Eppendorf	30120086	Collecting islets
Falcon 15 mL Conical Centrifuge Tubes	Corning	352095	Collecting islets
Falcon 40 µm Cell Strainer	Falcon	352340	Using for separating islets from other pancreatic tissue
Falcon 50 mL Conical Centrifuge Tubes	Corning	352070	Discarding excessive medium/buffer
Guinea pig anti-insulin	Agilent Technologies Japan, Ltd. (Dako)	IR002	Primary antibody for murine insulin
Hematoxylin	Muto Pure Chemicals Co., Ltd.	30002	Using for staining tissue by hematoxylin
Isodine solution 10%	Shionogi&Co., Ltd.	no catalog number	Using for disinfection
Isoflurane	Fujifilm Wako Chemicals	095-06573	Using for anesthesia
Labcon 1000 µL ZapSilk Low Retention Pipette Tips	Labcon	1177-965-008	Using for separating islets from other pancreatic tissue
Labcon 200 µL ZapSilk Low Retention Pipette Tips	Labcon	1179-965-008	Using for seeding islets onto epididymal white adipose tissue
Mintsensor	Sanwa Kagaku Kenkyusho Co. Ltd.,	8AEB02E	Using for monitoring blood glucose
Pipetteman P-1000	Gilson	F123602	Using for separating islets from other pancreatic tissue
Pipetteman P-200	Gilson	F123601	Using for seeding islets onto epididymal white adipose tissue
Rabbit anti-vWF	Abcam	ab6994	Primary antibody for murine von Willebrand factor