Comparison of New Preservation Solutions, HN-1 and University of Wisconsin Solution, in Pancreas Preservation for Porcine Islet Isolation

Abstract

For pancreatic islet transplantation, maintaining organ viability after pancreas procurement is critical and a major determinant for better graft function and survival. University of Wisconsin (UW) solution is currently the gold standard for abdominal organ preservation and the pancreas in particular. However, in the use of UW preservation solution for islet transplantation, there are disadvantages to be overcome, such as the inhibition of collagenase activity during pancreatic digestion. In this study, we compared UW solution with HN-1 solution in pancreas preservation for islet isolation. Islet yield was significantly greater in the HN-1 group than the UW group both before and after purification. In the in vitro assay, the adenosine triphosphate content in cultured islets was significantly higher in the HN-1 group than in the UW group. Furthermore, in streptozotocin-induced diabetic nude mice, the islet graft function of the HN-1 group was superior to that of the UW group. We concluded that the use of HN-1 solution is a promising approach for optimal pancreas preservation in islet transplantation.

INTRODUCTION

Islet transplantation potentially normalizes glucose metabolism in patients with type 1 diabetes (20,21). However, current isolation techniques usually require the transplantation of islets from two or more donor pancreata to establish normoglycemia. Although islet isolation techniques have been gradually improved in the past decade, further modification at each phase of islet isolation is required. Among them, the solution for organ preservation is critically important for maintaining its function and reducing ischemia–reperfusion injury, thus ultimately resulting in better β-cell function and withdrawal from insulin injection therapy. Donor pancreata for islet transplantation are usually preserved with University of Wisconsin (UW) solution, a colloid solution containing hydroxyethyl starch with a high potassium/sodium ratio. However, UW solution has several disadvantages: It is chemically unstable, it must be stored in the cold until use, and its short shelf-life makes it expensive. It is also highly viscous, which may complicate the initial organ flush (22). For islet isolation, it has been observed that UW inhibits the collagenase digestion phase of islet isolation, thus resulting in poor islet yields and islets of poor viability (2,19).

In this study, we compared a new preservation solution, HN-1, with UW solution for the success of the preservation and porcine islet isolation.

MATERIALS AND METHODS

Preservation Solution

We used HN-1 solution (Center for Promotion of Education and Science, Okayama, Japan) or UW solution (ViaSpan^®, DuPont Pharmaceuticals, Wilmington, DE, USA).

Porcine Islet Isolation

Three-year-old porcine pancreata (female, n = 10) were obtained at a local slaughterhouse. The operation was started about 10 min after the cessation of heartbeat. After removing the pancreas, we immediately inserted a cannula into the main pancreatic duct, infused each preservation solution (i.e., HN-1 or UW) for ductal protection (13), and put the pancreata into separate preservation containers. The time that elapsed between the start of the operation and the removal of the pancreas is considered the operation time. The time that elapsed between the stopping of the heart and the placement of the pancreas into the preservation solution is known as the warm ischemic time (WIT), whereas the cold ischemic time (CIT) is the time to the start of isolation following the placement of the pancreas into the preservation solution.

Islet isolation was conducted as previously described (6) in the standard Ricordi technique (18) with modifications introduced in the Edmonton protocol (16,20). In brief, after decontamination of the pancreas, the ducts were perfused in a controlled fashion with a cold enzyme blend of Liberase mammalian tissue free (MTF; 1.4 mg/ml) and thermolysin (0.075 mg/ml) (Roche Molecular Biochemicals, Indianapolis, IN, USA). The distended pancreas was then cut into seven to nine pieces, placed into a Ricordi chamber (Biorep Technologies, Miami, FL, USA), and shaken gently. The enzyme solution was recirculated through the Ricordi chamber at 37°C to aid pancreatic digestion, and dithizone staining (Sigma-Aldrich, St. Louis, MO, USA) of small extracted samples was used to monitor the extent of digestion. Once digestion was confirmed to be complete, dilution solution (Mediatech, Inc., Manassas, VA, USA) was introduced into the system. Then the system was cooled to stop further digestive activity. The digested tissue was collected in flasks and washed with fresh medium to remove the enzyme. The phase I period was defined as the time between placement of the pancreas in the Ricordi chamber and the start of collecting the digested pancreas. The phase II period was defined as the time between the start and end of collection. The digested tissue was incubated in UW solution prior to purification. Islets were purified with a continuous density gradient of iodixanol-based solution (Optiprep^®, Sigma-Aldrich) as previously reported (5,10,11).

Islet Evaluation

Embedded islets were determined by dithizone staining (2 mg/ml final concentration, Sigma-Aldrich). The crude number of islets in each diameter class was determined by counting islets after dithizone staining (2 mg/ml final concentration) by means of an optical graticule (Olympus, Tokyo, Japan). The crude number of islets was then converted to the standard number of islet equivalents (IEQs; diameter standardizing to 150 µm) (17). The islet recovery was defined as the percentage of IEQs recovered after purification divided by the IEQs before purification. Islet viability after purification was assessed using a double fluorescein diacetate/propidium iodide (FDA/PI; Sigma-Aldrich) staining to visualize living and dead islet cells simultaneously (17,20,21). Fifty islets were inspected and their individual viability was determined visually, followed by calculation of their average viability.

Islet function was assessed by monitoring the insulin secretory response of the purified islets during glucose stimulation using a procedure described by Shapiro and colleagues (20,21). Briefly, 1200 IEQs were incubated with either 2.8 or 25 mM glucose in Roswell Park Memorial Institute medium (RPMI 1640; Sigma) for 2 h at 37°C and 5% CO₂. The supernatants were collected and insulin levels were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (ALPCO Insulin ELISA kit; ALPCO Diagnostics, Windham, NH, USA). The stimulation index was calculated by determining the ratio of insulin released from islets in high glucose to the insulin released in low glucose. The data were expressed as mean ± SE.

Determination of Adenosine Triphosphate Production

To measure adenosine triphosphate (ATP) production, isolated islets in each group were cultured overnight with Connaught Medical Research Laboratories (CMRL) medium (GIBCO-Invitrogen, Carlsbad, CA, USA) plus 5% fetal bovine serum (FBS, GIBCO-Invitrogen), washed twice with ice-cold PBS, and solubilized. The amount of ATP was then measured using an ATP assay system (ATP-lite, Perkin Elmer, Groningen, Netherlands) according to the manufacturer’s instructions. The data were normalized to IEQs and expressed as mean ± SE.

In Vivo Assessment

Mouse studies were approved by the Institutional Animal Care and Use Committee of Okayama University. Six-week-old male nude mice (Charles River Laboratories Japan, Inc., Kanagawa, Japan) (n = 20) were rendered diabetic by a single intraperitoneal injection of streptozotocin (STZ, Sigma-Aldrich) at a dose of 220 mg/kg. Hyperglycemia was defined as a glucose level of >350 mg/dl detected twice consecutively (Accu-Chek^® Compact Plus; Roche Diagnostics K.K., Tokyo, Japan) after STZ injection. The 1,500 IEQ pig islets obtained from each group were transplanted into the renal subcapsular space of the left kidney of diabetic nude mice, as previously described (7,8). During the 30-day posttransplantation period, the nonfasting blood glucose levels were monitored three times per week. Normoglycemia was defined when two consecutive blood glucose level measurements showed less than 200 mg/dl. No statistical differences in either pretransplantation blood glucose levels or pretransplantation body weight were observed between two groups of mice.

Statistics

Two groups were compared by Student’s t test or the Kaplan–Meier log-rank test. The differences between each group were considered significant if p < 0.05.

RESULTS

Porcine Islet Isolation Characteristics

The characteristics of porcine islet isolation protocols are shown in Table 1. There were no significant differences in pancreas size, operation time, WIT, or CIT between the two groups. Phase I and phase II periods were also similar in both groups.

Table 1.

Porcine Islet Isolation Characteristics

	UW (n = 5)	HN-1 (n = 5)
Pancreas size (g)	90.9 ± 7.5	91.6 ± 4.9
Operation time (min)	6.2 ± 1.6	8.2 ± 1.8
Warm ischemic time (min)	19.2 ± 2.1	22.8 ± 1.5
Cold ischemic time (min)	150.6 ± 8.0	157.2 ± 5.1
Phase I period (min)	12.8 ± 0.5	12.0 ± 0.6
Phase II period (min)	62.8 ± 4.0	59.0 ± 1.6

Data are expressed as mean ± SE. UW, University of Wisconsin solution; HN-1, Center for Promotion of Science Education, Japan solution.

Islet yield before purification was significantly higher in the HN-1 group (n = 5) than the UW group (n = 5) (UW: 433,149 ± 47,386 IEQ, 4,822 ± 522 IEQ/g, HN-1: 681,614 ± 52,354 IEQ, 7,588 ± 860 IEQ/g, p < 0.05) (Fig. 1A, B). The islet yield after purification for the HN-1 group was also higher than that for the UW group (UW; 344, 798 ± 39,750 IEQ, 3,912 ± 625 IEQ/g, HN-1; 600,428 ± 65,436 IEQ, 6,664 ± 880 IEQ/g, p < 0.05) (Fig. 1C, D). Other porcine islet characteristics are shown in Table 2. There were no other significantly different characteristics between the two groups.

Figure 1.

Islet yield before purification and after purification. The HN-1 solution (Center for Promotion of Science Education) group (n = 5) had significantly better islet yield both before (A, total islet yield; B, islet yield/g) and after purification (C, total islet yield; D, islet yield/g) than the University of Wisconsin solution (UW) group (n = 5; p < 0.05). Data are expressed as the mean ± SE.

Table 2.

Porcine Islet Characteristics

	UW (n = 5)	HN-1 (n = 5)
Undigested tissue (g)	8.0 ± 2.0	10.4 ± 4.7
Embedded islets (%)	30.6 ± 6.5	20.4 ± 6.2
Viability (%)	98.1 ± 0.5	98.5 ± 0.2
Purity (%)	66.5 ± 5.9	68.6 ± 5.6
Postpurification recovery rate (%)	79.9 ± 4.9	87.4 ± 4.4
Stimulation index	1.84 ± 0.23	1.93 ± 0.08

Data are expressed as mean ± SE.

Assessment of Islet Function In Vitro

To assess the islet quality in the two groups in vitro, the ATP contents of isolated islets were measured. The ATP contents were significantly higher in the HN-1 group than in the UW group (UW: 0.54 ± 0.03 pmol/IEQ, HN-1: 0.81 ± 0.05 pmol/IEQ, p < 0.01) (Fig. 2). These data suggest that the quality of islets may be superior in the HN-1 group than in the UW group, although there were no significant differences in stimulation index or viability between the two solutions (Table 2).

Figure 2.

ATP content of porcine islets. The adenosine triphosphate (ATP) concentration of the cell lysate after islet purification was measured using an ATP assay system. ATP was normalized to islet equivalents (pmol/IEQ). The HN-1 group (n = 5) had significantly higher ATP content compared with the UW group (n = 5) (p < 0.01). Data are expressed as the mean ± SE.

In Vivo Assessment

To assess the islet graft function of each group in vivo, 1,500 IEQs of each group were transplanted below the kidney capsule of STZ-induced diabetic nude mice. The blood glucose levels of 3 of the 10 mice (30%) receiving islets from the UW group decreased gradually and reached normoglycemia. The blood glucose levels of 8 of the 10 mice (80%) receiving islets from the HN-1 group reached normoglycemia. The blood glucose levels remained stable thereafter and returned to pretransplantation levels after islet-bearing kidneys were removed (30 days after transplantation) (Fig. 3). The attainability of posttransplantation normoglycemia was significantly higher in the HN-1 group than in the UW group (p < 0.05). These data suggest that HN-1 preservation is superior to UW preservation.

Figure 3.

Evaluation of purified islet quality of each group in vivo. Normoglycemic percentage of STZ-induced diabetic nude mice after islet transplantation. A total of 1,500 IEQs were transplanted below the kidney capsule of diabetic nude mice. Normoglycemia was defined as two consecutive posttransplant blood glucose levels of less than 200 mg/dl. n = 10 for both groups.

DISCUSSION

In this study, we showed that HN-1 solution was superior to UW solution in pancreas preservation. UW solution was initially developed for pancreas preservation. It is widely used for the preservation of all types of organs and is currently the gold standard for abdominal organ preservation in general and the pancreas in particular. However, it has been reported that UW inhibits the collagenase digestion phase of islet isolation, resulting in poor yields and islets of poor viability (2,19). It has been reported that the components in UW solution found to be inhibitory for collagenase activities were magnesium, low Na⁺/high K⁺, hydroxyethyl starch (HES), and adenosine. In addition, allopurinol in combination with either lactobionate or glutathione was also markedly inhibitory. The most inhibitory solution tested was a combination of three components, raffinose, glutathione, and lactobionate (2). HN-1 solution has high Na⁺/low K⁺, and the concentration of magnesium, HES, adenosine, allopurinol, raffinose, and glutathione in HN-1 solution is lower than that in UW solution (unpublished data). These findings show that HN-1 solution is a more effective cold storage solution in pancreas preservation for islet isolation than UW solution.

We and other groups recently reported the comparison of several solutions for pancreas preservation for islet transplantation. Hubert et al. reported that islet yields were inferior in the Celsior group than the UW group because Celsior solution induced cell swelling and pancreatic edema after only 4 h of cold storage (3). On the other hand, Niclauss et al. assessed the impact of UW, Celsior, and Institute Georges Lopez-1 (IGL-1) solutions on human islet isolation and transplant outcome. Islet yield, success rates, transplant rates, β-cell secretory function, and viability were similar for all three groups (4). It was reported that UW and histidine–tryptophan–ketoglutarate (HTK) solution demonstrated equal effectiveness in the preservation of human pancreata intended for islet isolation (1,15). We reported that pancreas preservation with modified extracellular-type trehalose-containing (ET)-Kyoto (MK) solution significantly improved islet yields compared with UW preservation. The advantages of MK solution are characterized by trypsin inhibition with minimal interference to collagenase activities (14). We also compared MK solution with modified HTK solution or modified Celsior solution. MK solution is better for pancreas preservation before islet isolation than modified HTK solution (12) or modified Celsior solution (9). Thus, several new insights about preservation solutions have been reported, but MK solution may be the best solution for pancreas preservation at present. We will compare MK solution with HN-1 solution in preservation solution for islet isolation in a future study.

In conclusion, we showed that HN-1 solution is better for pancreas preservation than UW solution, almost invariably a gold standard perfusion solution. Although further studies are required in order to evaluate the precise impact for pancreas preservation, there is a good possibility that HN-1 preservation makes it feasible to use marginal donors for efficient islet transplantation into type 1 diabetic patients.