Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet Isolation

Authors

  • H. Noguchi,

    Corresponding author
    1. Diabetes Research Institute Kyoto
    2. Research Fellow of the Japan Society for the Promotion of Science (SPD)
    3. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • M. Ueda,

    1. Diabetes Research Institute Kyoto
    2. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • Y. Nakai,

    1. Diabetes Research Institute Kyoto
    2. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • Y. Iwanaga,

    1. Diabetes Research Institute Kyoto
    2. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • T. Okitsu,

    1. Diabetes Research Institute Kyoto
    2. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • H. Nagata,

    1. Diabetes Research Institute Kyoto
    2. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • Y. Yonekawa,

    1. Diabetes Research Institute Kyoto
    2. Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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  • N. Kobayashi,

    1. Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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  • T. Nakamura,

    1. Department of Experimental Therapeutics, Transplantational Research Center, Kyoto University Hospital
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  • H. Wada,

    1. Department of Thoracic Surgery, Kyoto University Graduate School of Medicine
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  • S. Matsumoto

    1. Diabetes Research Institute Kyoto
    2. Transplantation Unit, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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Abstract

Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.

Introduction

Islet allotransplantation can achieve insulin independence in patients with type I diabetes (1,2). Since the Edmonton protocol was announced, more than 500 type I diabetes in more than 50 institutions have undergone islet transplantation to cure their disease. However, current isolation techniques usually recover fewer than half the islets from a given pancreas, necessitating islet transplantation from two or more donors to achieve euglycemia (1).

Donor pancreata are usually preserved with University of Wisconsin (UW) solution. Recent reports have shown that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW to preserve not only the whole pancreas but also in islet transplantation (3–7). The TLM is especially important for preserving pancreata before islet isolation because it helps to preserve the organ, which UW preservation results in the deterioration of both islet isolation efficacy and post-transplant islet function (3,4,7). Recently, successful islet transplantation from single donors in some diabetic patients has also been reported by using TLM in clinical setting (8). With TLM, the pancreas is directly oxygenated by PFC during pancreas preservation and maintains a high level of adenosine triphosphate (ATP) in tissues, which maintains cellular integrity and retains parenchymal and nonparenchymal viability (9,10).

However, UW solution has several disadvantages: it is chemically unstable, it must be cold stored until use and its short shelf life makes it expensive. It is also highly viscous, which may complicate the initial organ flush (11). Recently, our university developed a new preservation solution, ET-Kyoto solution, and its effectiveness in cold lung storage has been demonstrated in clinical lung transplantation (12,13). It also is effective for skin flap storage and its clinical application is beginning in this field (14). ET-Kyoto solution contains trehalose and gluconate as distinct components. Trehalose has a cytoprotective effect against stress, and gluconate acts as an extracellular oncotic agent, which prevents cells from swelling (15). As such effective components and other ingredients are chemically stable, ET-Kyoto solution, but not UW solution, can be stored at room temperature for a long period.

ET-Kyoto solution also has a high sodium-low potassium composition with comparatively low viscosity. In clinical islet transplantation, preserving solution is used not only for storage but also for initial flushing when the pancreas is obtained. High potassium in UW solution causes insulin release from pancreatic β cells (16) and the high viscosity of UW solution may prevent sufficient flushing. Moreover, UW solution inhibits the activity of Liberase, an enzyme blend for pancreatic digestion (17,18). In this study, we investigated the features of modified ET-Kyoto solution (M-Kyoto solution) compared with UW solution for islet isolation.

Materials and Methods

Preservation solution

M-Kyoto solution was prepared specifically for this experiment. We used M-Kyoto solution, UW solution (ViaSpan, DuPont Pharmaceuticals, Wilmington, DE), and PFC. The components and other features of the solutions are shown in Table 1.

Table 1. Composition and other features of each preserving solution
 EffectUWM-Kyoto
  1. HES = hydroxyethyl starch; ROS = reactive oxygen species.

Na (mmol/L)Cation29100
K (mmol/L)Cation12543.5
Mg (mmol/L)Cation5
Gluconate (mmol/L)Anion100
Phosphate (mmol/L)Buffer2525
Sulfate (mmol/L)Buffer5
Lactobionate (mmol/L)Buffer100
Raffinose (mmol/L)Sugar30
Trehalose (mmol/L)Sugar120
Adenosine (mmol/L)Energy source5
Alloprinol (mmol/L)ROS scavenger1
Glutathione (mmol/L)ROS scavenger3
HES (g/L)Colloid5030
Ulinastatin (×103 U/L)Trypsin inhibitor100
Osmorality (mOsm) 320366
ViscosityAt 4.5°C (cPs)5.504.09
 At 37°C (cPs)2.561.70

Porcine islet isolation

Porcine pancreata were obtained at a local slaughterhouse. About 10 min after the cessation of heart beating, the operation was started. After removing the pancreas, it started cooling but was not flushed by any solutions. We immediately inserted a cannula into the main pancreatic duct and placed the pancreas into a UW/PFC two-layer preservation container or a M-Kyoto/PFC two-layer preservation container. Operation time was defined as the time elapsed between the start of operation and removal of the pancreas. Warm ischemic time (WIT) was defined as the time elapsed between cessation of heart beating and placement of the pancreas into the preservation solution. Cold ischemic time (CIT) was defined as the time elapsed between placement of the pancreas into the preservation solution and the start of islet isolation.

Islet isolation was conducted in accordance with the method described in the Edmonton protocol (1,7,19–21). In brief, after decontamination of the pancreas, the ducts were perfused in a controlled fashion with a cold enzyme blend of Liberase HI (1.4 mg/mL; Roche Molecular Biochemicals, Indianapolis, IN). The distended pancreas was then cut into nine pieces, placed in a sterilized Ricordi chamber and shaken gently. While the pancreas was being digested by recirculating the enzyme solution through the Ricordi chamber at 37°C, we monitored the extent of digestion with dithizone staining by taking small samples from the system. Once digestion was confirmed as completed, RPMI 1640 medium (Invitrogen Japan KK, Tokyo) was introduced into the system, and then the system was cooled to stop further digestive activity. The digested tissue was collected 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. Islets were purified with a continuous density gradient of high-osmolality Ficoll based on the Edmonton islet isolation protocol (1) in an apheresis system (COBE 2991 cell processor, Gambro Laboratories, Denver, CO).

Islet evaluation

The crude number of islets in each diameter class was determined by counting islets after dithizone staining (3 mg/mL final concentration) (Sigma Chemical Co., St. Louis, MO) using an optical graticule. The crude number of islets was then converted to the standard number of islet equivalents (IE; diameter standardizing to 150 μm) (22). Gross morphology was qualitatively assessed by two independent investigators scoring the islets for shape (flat vs. spherical), border (irregular vs. well-rounded), integrity (fragmented vs. solid/compact), uniformity of stain (not uniform vs. perfectly uniform) and diameter (all <100 μm vs. >10% >200 μm) (7,22). Each parameter was graded from 0–2 with 0 equaling the worst and 2 the best score, so that the worst islet preparations were given a score of 0 and the best a score of 10. Spherical, well-rounded, solid/compact, uniformly stained and large islets were characterized as the best islets.

Islet viability after purification was assessed using acridine orange (10 μmol/L) and propidium iodide (15 μmol/L) (AO/PI) staining to visualize living and dead islet cells simultaneously (7,22,23). Fifty islets were inspected and their individual viability was determined visually, followed by calculation of their average viability (7).

Assessment of islet function

Islet function was assessed by monitoring the insulin secretory response of the purified islets during glucose stimulation according to a procedure described by Shapiro and colleagues (1). Briefly, 1200 IE were incubated with either 2.8 mM or 25 mM glucose in RPMI 1640 for 2 h at 37°C and 5% CO2. The supernatant was collected and insulin levels were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (ALPCO Insulin ELISA kit; ALPO Diagnostics, Windham, NH). The stimulation index was calculated by determining the ratio of insulin released from islets in high glucose concentration to the insulin released in a low concentration. The data were normalized by total proteins from the cell lysate. All assessments were made in triplicate and the data (mean ± SE) were expressed as a percentage of the control values in each experiment to eliminate variables caused by differences among donor pancreata.

In vivo assessment

Mice with severe combined immunodeficiency disease (SCID; CLEA Japan, Inc., Meguro, Tokyo) were used for the experiments. The recipients were rendered diabetic by a single injection of streptozotocin (STZ) at a dose of 220 mg/kg. Hyperglycemia was defined as a glucose level of >350 mg/dL detected twice consecutively after STZ injection. The 2000 IE pig islets obtained from each groups were transplanted into the renal subcapsular space of the left kidney of a diabetic SCID mouse. During the 30-day post-transplantation 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 pre-transplantation blood glucose levels or pre-transplantation body weight were observed among the four groups of mice. Mouse studies were approved by the review committee of Kyoto University Graduate School of Medicine.

Determination of ATP production

Tissue samples were taken from the pancreas after 2 h preservation in UW/PFC or M-Kyoto/PFC. To measure ATP production, tissue samples were washed twice with ice-cold PBS and solubilized. The amount of ATP was then measured using an ATP assay system (Toyo Inki, Tokyo, Japan), according to the manufacturer's instructions. Briefly, after allowing the reagents to equilibrate to room temperature, 10 μL of cell extracts were added to 100 μL of the reagents. The samples were measured by luminometer.

Measurement of trypsin inhibition ability of solutions and trypsin activity during isolation

In order to assess the trypsin inhibition of UW solution, M-Kyoto solution and phosphate-buffered saline (PBS), 3 mL of 0.3 mM N-benzoyl-L-arginine ethylester reagent (BAEE; sigma, Tokyo, Japan) were incubated for 5 min at 25°C and then 5 μL of 0.25% trypsin, 30 μL of PBS, and 15 μL of UW solution, M-Kyoto solution, or PBS (control) were added. To measure trypsin activity at the end of digestion, snap-frozen 1 mL aliquots of supernatant were taken from the Ricordi system. Trypsin activity was measured by absorption spectrophotometry (λ253 nm) using BAEE for the trypsin substrate, according to a previous report (3). Absorbance was measured at 1, 2, 3, 4, 5 and 6 min. A BAEE unit was defined as the Δoptical density of 0.001 per min.

Inhibition of collagenase digestion

The inhibition rate of collagenase digestion was measured in accordance with the modified method as previously described (18). In brief, pancreatic blocks measuring approximately 0.5 cm3 were placed into 2 mL of each solution in a 10 mL test tube and pre-incubated for 5 min at 37°C. Collagenase was dissolved in each solution at a concentration of 4 mg/mL and 2 mL of this was added to each tube, giving a final enzyme concentration of 2 mg/mL. The test tubes were placed in a water-bath at 37°C and every 5 min they were manually shaken for 10 s and examined. The digestion process was monitored by the appearance of fragmented tissue in the supernatant and scored between + and +++ depending upon the amount of tissue seen (18). A single individual performed all assessments. The time at which ++ of fragmented tissue appeared was noted for each tube, and this time was used to compare the rate of digestion.

Search for inhibitory components of collagenase digestion

To examine whether trehalose and ulinastatin inhibit the collagenase digestion phase, four solutions, based on M-Kyoto solution, were used as shown in Table 2. The inhibition rate of collagenase digestion was measured in accordance with the modified method as previously described (18).

Table 2. Composition and other features of each preserving solution
SolutionM-Kyoto12 (ET-Kyoto)3
  1. HES = hydroxyethyl starch.

Na (mmol/L)100100100100
K (mmol/L)43.543.543.543.5
Gluconate (mmol/L)100100100100
Phosphate (mmol/L)25252525
HES (g/L)30303030
Trehalose (mmol/L)120120
Ulinastatin (×103 U/L)100100

Rat islet isolation

Islets were isolated from inbred male Lewis rats (Charles River Laboratories, Wilmington, MA), weighing 300–380 g. Rat studies were approved by the review committee of Kyoto University Graduate School of Medicine. The common bile duct was cannulated using a 24-gauge catheter (Baxter, Deerfield, IL) and fixed. The distal common bile duct was then clamped. The pancreas, spleen and duodenum were removed en bloc and preserved in each solution. Pancreata were preserved at 4°C for 2 or 6 h. Islets were isolated using the modified method of Sawada (24). After preservation, each pancreas was distended using 2 mg/mL of collagenase solution (24 mg of Serva collagenase; Serva, Heidelberg, Germany) in 12 mL of Hanks’ balanced salt solution (HBSS). The spleen and the duodenum were removed, and the pancreas was incubated in a 50 mL conical tube, without shaking, at 37°C for 22 min. The digested pancreas was washed with UW solution three times by centrifugation (150 g, 3 min, 8°C). It was then purified with a discontinuous density gradient (1.030, 1.095, 1.105, 1.125 g/cm3 in a modified M-Kyoto/Optiprep solution (Nycomed Pharma AS, Oslo, Norway). Islet preparations were evaluated for yield by means of dithizone staining (22).

Results

Porcine islet isolation characteristics

The characteristics of porcine islet isolation protocols are shown in Table 3. There were no significant differences in pancreas size or operation time between the two groups. Phase I was significantly longer for the UW/PFC group but phase II was similar for the two groups.

Table 3. Pig islet isolation characteristics
 UW/PFCM-Kyoto/PFCp-value
  1. Data are expressed as mean ± SE.

Pancreas size (g)103.4 ± 13.298.0 ± 8.6NS
Operation time (min)9.2 ± 0.86.8 ± 1.3NS
Warm ischemic time (min)18.2 ± 0.620.8 ± 1.2NS
Cold ischemic time (min)148.4 ± 9.7133.6 ± 9.2NS
Phase I period (min)15.6 ± 1.68.0 ± 0.6<0.01
Phase II period (min)27.2 ± 1.430.2 ± 2.4NS
Viability (%)93.3 ± 3.095.4 ± 1.2NS
Score7.8 ± 0.87.8 ± 0.7NS
Purity (%)91.0 ± 2.986.0 ± 2.9NS
Post-purification recovery rate (%)63.9 ± 11.355.8 ± 5.0NS
Islet size pre-purification (μm)175.3 ± 36.0184.1 ± 34.8NS
Islet size post-purification (μm)138.6 ± 35.3142.4 ± 50.8NS
Stimulation index1.69 ± 0.301.65 ± 0.32NS

Islet yield both before and after purification was significantly higher in the M-Kyoto/PFC group (n = 5) compared with the UW/PFC group (n = 5) (before purification: M-Kyoto/PFC; 6946 ± 583 IE/g, UW/PFC; 4395 ± 387 IE/g, after purification: M-Kyoto/PFC; 3936 ± 566 IE/g, UW/PFC; 2885 ± 290 IE/g) (Figure 1). Other porcine islet characteristics are shown in Table 3. There were no other significantly different characteristics between the two groups.

Figure 1.

Islet yield before purification (A) and after purification (B). M-Kyoto/PFC group (n = 5) had significantly improved islet yield before and after purification compared with the UW/PFC group (n = 5). Data are expressed as the mean ± SE.

Transplant experiments

To assess the islet graft function of each group in vivo, 2000 IE islets of each group were then transplanted below the kidney capsule of STZ-induced diabetic SCID mice. The blood glucose levels of four of the five mice (80.0%) receiving islets from the UW group decreased gradually and reached normoglycemia. The blood glucose levels remained stable thereafter and returned to pre-transplantation levels after kidney-bearing islets were removed 30 days after transplantation. The blood glucose levels of four of the five mice (80.0%) receiving islets from the M-Kyoto group decreased gradually and reached normoglycemia (Figure 2A). The attainability of post-transplantation normoglycemia for the M-Kyoto group was the same as for the UW group. Morphologic studies showed the presence of islets under the kidney capsule of all SCID mice 30 days after transplantation. The islet grafts of each group in the normoglycemic mice showed intense insulin staining (data not shown).

Figure 2.

Transplant experiment and cellular ATP content. (A) Normoglycemic rate of STZ-induced diabetic SCID mice after islet transplantation. Immediately after isolation, 2000 IE islets were transplanted below the kidney capsule of diabetic SCID mice. Normoglycemia was defined as two consecutive post-transplant blood glucose levels showing less than 200 mg/dL. (UW group n = 5; M-Kyoto group n = 5) (B) The ATP concentration of the cell lysate after preservation was measured using an ATP assay system. There was no significant difference between the UW/PFC (n = 5) and M-Kyoto/PFC (n = 5) groups. Data are expressed as the mean ± SE.

Adenine nucleotides

There was no significant difference in ATP content after preservation in the study pancreas between the UW/PFC (n = 5) and M-Kyoto/PFC (n = 5) groups (Figure 2B). These data suggest that different islet isolation effects between the two preservation solutions are not due to their differences as energy sources.

Inhibition of trypsin activity by M-Kyoto solution

Previous reports show that trypsin inhibition by Pefabloc with TLM preservation improves islet yields (3). M-Kyoto solution includes ulinastatin, and it was therefore examined whether M-Kyoto solution inhibited trypsin activity. Compared with UW solution or PBS (control), M-Kyoto solution significantly inhibited trypsin activity (Figure 3A). We then measured trypsin activity in the supernatant after digestion. At the end of digestion, trypsin activity was reduced in the M-Kyoto/PFC group compared with the UW/PFC group (Figure 3B). These observations showed that M-Kyoto solution reduced trypsin activity in the digestion step during pancreas preservation.

Figure 3.

Impact of M-Kyoto solution on trypsin activity during pancreas preservation. (A) Measurement of trypsin inhibition. BAEE were incubated for 5 min at 25°C and then trypsin and UW solution (n = 5), M-Kyoto solution (n = 5), or PBS (control; n = 5) was added. Trypsin activity was measured by absorption spectrophotometry (λ253 nm) using BAEE reagent. (B) Measurement of trypsin activity in supernatant after digestion. We collected and snap-froze 1-mL aliquots of supernatant at the completion of digestion (UW solution; n = 5, M-Kyoto solution; n = 5). Trypsin activity was measured by absorption spectrophotometry using BAEE reagent. Absorbance was measured at 1, 2, 3, 4, 5 and 6 min. A BAEE unit was defined as the Δoptical density of 0.001 per min. Data are expressed as the mean ± SE.

Inhibition of collagenase digestion

Table 3 shows that phase I was significantly longer for the UW/PFC group, which may have been due to the collagenase inhibition of UW solution. To assess the inhibitory effect of collagenase by UW solution and M-Kyoto solution, the inhibition rates were measured. The median digestion time was 58.8 ± 2.4 min for Hank's (control), 103.8 ± 9.0 min for UW, and 75.0 ± 4.1 min for M-Kyoto solution (Table 4). These data suggest that M-Kyoto solution inhibited collagenase digestion less than UW solution, resulting in better islet yields.

Table 4. Inhibition of collagenase digestion
SolutionDigestion timeMedian ratiop-value
  1. Data are expressed as mean ± SE.

  2. M-UW = UW solution with ulinastatin.

Hank's (n = 4)58.8 ± 2.41.0 
UW solution (n = 4)103.8 ± 9.01.8<0.01
M-UW solution (n = 4)105.0 ± 6.51.8<0.01
M-Kyoto solution (n = 4)75.9 ± 4.11.3<0.05
Solution 1 (n = 4)60.0 ± 2.91.0NS
Solution 2 (ET-Kyoto; n = 4)71.3 ± 2.41.2<0.05
Solution 3 (n = 4)63.8 ± 2.41.1NS

We further examined whether trehalose and ulinastatin inhibit the collagenase digestion phase. We used four solutions based on M-Kyoto solution (Table 2). The median digestion time was 60.0 ± 2.9 min for solution 1, 71.3 ± 2.4 min for solution 2, 63.8 ± 2.4 min for solution 3, and 75.0 ± 4.1 min for M-Kyoto solution. Solution 2 and M-Kyoto solution slightly inhibited collagenase activity compared with solutions 1 and 3 (Table 4). These data suggest that trehalose slightly inhibits collagenase activity and ulinastatin does not inhibit collagenase digestion.

Detailed assessment of M-Kyoto solution

To assess the effect of M-Kyoto solution in more detail, four solutions (UW, M-Kyoto, ET-Kyoto, and UW with ulinastatin (M-UW) solution) were used. Initially, the trypsin inhibitory effect of four solutions was examined. Compared with UW solution, ET-Kyoto solution, or PBS (control), M-Kyoto solution significantly inhibited trypsin activity (Figure 4A). Although M-UW solution significantly inhibited trypsin activity compared with PBS, the inhibitory effect was weaker than M-Kyoto solution (Figure 4A). Some components in UW solution may inhibit ulinastatin activity. We then measured trypsin activity in the supernatant after digestion. At the end of digestion, trypsin activity was reduced in the M-Kyoto/PFC and M-UW/PFC groups compared with the UW/PFC and ET-Kyoto/PFC groups (Figure 4B). These observations showed that the solutions with ulinastatin reduced trypsin activity in the digestion step during pancreas preservation.

Figure 4.

Detailed assessment of M-Kyoto solution. (A) Measurement of trypsin inhibition. BAEE were incubated for 5 min at 25°C and then trypsin and UW solution (n = 5), M-UW solution (n = 5), ET-Kyoto solution (n = 5), M-Kyoto solution (n = 5), or PBS (control; n = 5) was added. Trypsin activity was measured by absorption spectrophotometry (λ253 nm) using BAEE reagent. (B) Measurement of trypsin activity in supernatant after digestion of porcine pancreas. We collected and snap-froze 1-mL aliquots of supernatant at the completion of digestion (UW solution; n = 5, M-UW solution; n = 5, ET-Kyoto solution; n = 5, M-Kyoto solution; n = 5). Trypsin activity was measured by absorption spectrophotometry using BAEE reagent. Absorbance was measured at 1, 2, 3, 4, 5 and 6 min. A BAEE unit was defined as the Δoptical density of 0.001 per min. (C) Cellular ATP content. The ATP concentration of the cell lysate after preservation of porcine pancreas (UW; n = 3, M-UW; n = 3, ET-Kyoto; n = 3, M-Kyoto; n = 3, UW/PFC; n = 3, M-UW/PFC; n = 3, ET-Kyoto/PFC; n = 3, M-Kyoto/PFC; n = 3) was measured using an ATP assay system. (D) Rat islet yield after purification. The M-Kyoto/PFC group (n = 4) had significantly improved islet yield after purification compared with the UW/PFC (n = 4), M-UW/PFC (n = 4), or the ET-Kyoto/PFC (n = 4) group. Data are expressed as the mean ± SE.

We next examined ATP content after preservation. The pancreas tissues were preserved for eight conditions [simple preservation using UW (n = 3), M-UW (n = 3), ET-Kyoto (n = 3) or M-Kyoto (n = 3) solution and TLM preservation using UW/PFC (n = 3), M-UW/PFC (n = 3), ET-Kyoto/PFC (n = 3) or M-Kyoto/PFC (n = 3)]. There was no significant difference in ATP content after preservation of the study pancreas between simple preservation groups (Figure 4C). ATP levels were 7–9 times higher in TLM groups than simple preservation groups. In TLM groups, there was no significant difference in ATP content (Figure 4C).

We further examined whether these solutions inhibit the collagenase digestion phase. As shown in Table 4, UW solution and M-UW solution inhibited collagenase digestion compared with Hank’s, ET-Kyoto, and M-Kyoto solution.

To examine the effect of the solutions on islet isolation, a rat model was used. Islet yield after purification was significantly higher in the M-Kyoto/PFC group (n = 4) compared with the UW/PFC (n = 4), M-UW/PFC (n = 4), and ET-Kyoto/PFC (n = 4) groups (UW/PFC; 622 ± 133 IE, M-UW/PFC; 730 ± 114 IE, ET-Kyoto/PFC; 700 ± 116 IE, M-Kyoto/PFC; 1207 ± 76 IE) for 6-h preservation (Figure 4D). Islet yield after purification was also significantly higher in the M-Kyoto/PFC group (n = 4) compared with the UW/PFC (n = 4), M-UW/PFC (n = 4), and ET-Kyoto/PFC (n = 4) groups for 2 h preservation (data not shown). These data suggest that M-Kyoto/PFC preservation is the best in these groups.

Discussion

The cadaveric pancreas is injured due to brain death (25,26), hypotension, and vasopressor therapy, and from warm ischemia after donor cross-clamping and cold ischemic storage in UW solution (27). There is a clear relationship between these injuries and the reduced success of subsequent islet isolation (28,29). Particularly in Japan, pancreatic islets can only be isolated from nonheart-beating donors (NHBDs) for clinical islet transplantation due to competition with pancreatic organ transplantation for procuring donor pancreata after brain death. We, therefore, need to develop an efficient isolation technique for NHBD pancreata.

We have recently demonstrated that islet isolation and transplantation with NHBDs using the modified Ricordi method (Kyoto Islet Isolation Method) effectively cures type I diabetes. The transplantation rate (transplantation number/isolation number) is more than 80%, higher than recent published data using brain-death heart-beating donors (30). The Kyoto isolation method includes ductal injection, the modified TLM (M-Kyoto/PFC), and iodixanol-based purification. It is not clear whether ductal injection or the preservation or purification method might be important in the Kyoto islet isolation method. In this study, we compared M-Kyoto/PFC preservation with the regular two-layer (UW/PFC) preservation method using a porcine model. The data in this article showed that M-Kyoto/PFC preservation significantly improved islet yields compared with UW/PFC preservation.

UW solution is used extensively as a cold-storage solution during procurement and transport of the pancreas prior to islet isolation; however, it has been observed that UW inhibits the collagenase digestion phase of islet isolation, resulting in poor islet yields and islets of poor viability (17,18). It has been reported that the components in UW solution found to be most inhibitory were magnesium, low Na+/high K+, hydroxyethyl starch (HES), and adenosine, that allopurinol in combination with either lactobionate or glutathione was markedly inhibitory, and that the most inhibitory solution tested was a combination of three components, raffinose, glutathione and lactobionate (18). M-Kyoto solution has high Na+/low K+ and, of the UW components, it includes only HES at a lower concentration. It has also been shown that the Na+/K+ ratio, adenosine, allopurinol and glutathione are not essential for the cold storage of pancreatic digest prior to islet purification (31). Moreover, trehalose and ulinastatin inhibit collagenase digestion less than UW solution, as shown in Table 4. These findings show that M-Kyoto is a more effective cold-storage solution in pancreas preservation for islet isolation than UW solution.

In the TLM, the pancreas is directly oxygenated through oxygenated PFC and produces ATP. ATP is utilized to drive a sodium pump, maintain cell integrity and repair warm ischemic injury (9,10,32,33,34). After significant warm ischemic injury, adenosine in UW solution is used as a substrate of ATP, however, without significant warm ischemia, ATP is generated from adenosine diphosphate (ADP) or adenosine monophosphate (AMP) located in the cells. In this study, there was no difference in ATP levels between the M-Kyoto/PFC and UW/PFC groups. Since the WIT was less than 30 min in this study, adenosine is not important for ATP generation, and ATP levels after preservation in M-Kyoto/PFC were similar to UW/PFC preservation. It seems that in terms of ATP generation, M-Kyoto/PFC is similar to UW/PFC.

Trypsin from pancreatic acinar cells destroys islets. Previous studies have shown that trypsin inhibition by Pefabloc during human pancreas digestion improves islet yield and reduces the fraction of embedded islets (3,35), suggesting that trypsin may degrade the ductules, reducing the delivery of collagenase solution to the immediate neighborhood of the islets. In this article, we have demonstrated that modified TLM preservation, including ulinastatin, eliminated trypsin activity during pancreas preservation, and ET-Kyoto/PFC preservation without ulinastatin resulted in lower islet yields in our rat (Figure 4) and porcine model (data not shown).

Although the differences are not significant, the islet recovery rate in the M-Kyoto group (55.8%) was lower than in the UW group (63.9%). The purity in the M-Kyoto solution group (86.0%) was also lower than in the UW group (91.0%). These differences may be due to the density of exocrine tissue and islets. Hypothermic storage of both the pancreas and pancreatic digestion alters tissue density, thereby affecting islet purification (36). Since UW solution has a higher effect on cell shrinkage than M-Kyoto solution, UW storage may produce high islet purity compared with M-Kyoto storage; indeed, the density of exocrine tissue before purification in the UW group (1.115 ± 0.004 g/cm3) is heavier than in the M-Kyoto group (1.109 ± 0.005 g/cm3). Therefore, when M-Kyoto solution is used in pancreas preservation, the pre-incubation time before purification using UW solution should be extended.

After the Edmonton protocol was reported, islet transplantation advanced significantly, including the utilization of nonheart-beating donors (30,37), single donor islet transplantation (8,38), and living-donor islet transplantation (19). These advances were based on advanced pancreas transport systems (3–10), revised immunosuppressant protocols (39), improved islet isolation methods (3) and enhanced islet engraftment (40). Although experiments of β-cell regeneration from stem cells have proceeded (41–44), there is still no reliable method to make β cells. Until a new method to generate β cells is developed, improving the efficacy of islet transplantation seems the most realistic and prudent method to cure diabetes.

In conclusion, we succeeded in efficiently isolating large numbers of islets using a new preservation solution. The advantage of M-Kyoto solution is trypsin inhibition by ulinastatin and less collagenase inhibition without decreasing the energy level of the preserved pancreas. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata. M-Kyoto/PFC preservation makes it feasible to use NHBDs for efficient islet transplantation into type I diabetes (30). The improved viable islet yield resulting from these innovations means that transplantable islets can be recovered from marginal pancreata, and patients can be cured with islets from just one donor.

Acknowledgment

The authors wish to thank Dr. Toshiya Sawada (Akita University) for technical advice and Ms. Akemi Ishii for assistance.

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