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A method for the massive separation of highly purified, adult porcine islets of langerhans

A method for the massive separation of highly purified, adult porcine islets of langerhans
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  A Method for the Massive Separation of Highly Purified Adult Porcine Islets of Langerhans zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Riccardo Calafiore, Filippo Calcinaro, Giuseppe Basta, Massimo Pietropaolo, Albert0 Falorni, Maurizio Piermattei, and Paolo Brunetti zyxwvutsrqponmlkjihgfedcbaZYXWVU A method for the massive and reproducible isolation of highly purified, adult porcine islets of Langerhans is described. The successful combination of donor animal-strain selection with srcinal procedures for pancreas retrieval and enzymatic digestion permitted us to separate uniquely massive concentrations of pure porcine islets with no need for mechanical disruption of the pancreatic tissue. Following our procedure, porcine islets, which fully retain viability and function, can be harvested easily and rapidly. Xenotransplantation of such islets, immunoprotected within algin/polyaminoacidic microcap- sules, was associated with complete reversal of hyperglycemia in rodents with either spontaneous or streptozotocin- induced diabetes mellitus. 0 1930 by W B Saunders Company zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA T HE RESTRICTED availability of cadaveric human donor pancreata continues to hamper progress of islet cell transplantation into clinical trials. An attractive alterna- tive would be to employ non-human pancreatic tissue as a resource for donor islets. We have developed a method for the large-scale isolation of highly purified islets of Langerhans from the pancreas of adult pigs. These islets could be ideal candidates as donor pancreatic tissue, provided that their immunologic patterns were altered, because of both the molecular affinity between pork and human insulin and the large availability of pigs. In comparison with procedures previously described,’ our method provides incomparably higher and uniquely massive concen- trations of pure porcine islets. We report that such islets have been proven to retain full viability as well as functional competence, either in vitro or in vivo, after xenograft in diabetic rodents. MATERIALS AND METHODS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Organ Procurement A local slaughterhouse provided us with female adult pigs (breed- ing strain: Large White), 8 months old, weighing an average 100 kg., that received no treatment prior to pancreas retrieval. The porcine pancreas has a duodenal and splenic lobe. Since the latter has been shown to contain larger concentrations of islets, we processed only this portion of the organ. The pancreas was retrieved, under fairly sterile conditions, imme- diately after the animals were shot through the head but prior to cardiac arrest, with care being taken to avoid significant bleeding. These maneuvers were performed to reduce the overall ischemia time and to protect islet viability. Once isolated the pancreas was placed into a sterile bag containing Hank’s balanced salt solution (HBSS) (Flow, Rickmansworth, UK), supplemented with 10 bovine serum (Flow) and antibiotics (Flow) at 4OC. The organ was immediately transported to the laboratory and handled within 45 minutes of retrieval. Islet Isolation A multienzymatic solution (Table 1) whose final volume, in milliliters, was equal to twice the pancreas weight, in grams, was prepared. After removing all mesenteric leaflets and the tail fibrous cap, the organ was considered clean and cannulated, via the main duct, with an indwelling, suture-secured, 16-gauge polyethylene catheter (Ab- bocath T-16, Abbott Laboratories LTD. Queensborough, UK). A volume of the enzymatic solution in milliliters, equal to the pancreas zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Metabolism, Vol39, No 2 (February). 1990: pp 175-l 8 1 weight in grams, was forced through the cannulated duct, through- out overall four to five minutes, by a 60-mL plastic syringe (Becton Dickinson, Dun Laoghaire Co LTD, Dublin, Ireland), which re- sulted in visible distention of the organ. The distended pancreas was then immersed in an apparatus, driven by a peristaltic pump (Minipuls 2, Gilson Medical Electronics, Inc, Middleton, WI) that replaced the syringe, in a bath at 37 containing the balance of the enzymatic solution and continuously perfused at a Row rate of 7 mL/min (Fig 1). Pressures generated in the pancreas were 15 to 20 and 3 bars, during manual distention and perfusion, respectively. The solution seeped through the organ and exited into the aqueous bath where it was, once again, drawn up by the pump and recirculated through the pancreas for overall 30 minutes. The organ and aqueous bath were thereafter shaken at 110 cycles/min at 37OC, for an additional 10 minutes, when the pancreatic tissue came finely apart. Digestion time was established after processing 70 pancreata that were retrieved from Large White pigs of comparable age, sex, and body weight. The preparation was, at this point, chilled with 200 mL of cold HBSS supplemented with 10 heat-inactivated newborn calf serum (Flow). Occasional undigested tissue chunks were discarded, since they were not proven to affect the final yield in viable islet cells. This process disengaged massive concentrations of islets, making them float away from the remainder of the pancreatic tissue and collect in a wide portion of the supernatant. Therefore a pancreatic tissue suspension that was already enriched in free islets was easily and rapidly harvested simply by skimming it from surface layers of the aqueous bath, under gentle beaker shaking, by a stainless, 14-gauge cannula (American Scientific Products, Miami, FL) mounted on a 60-mL plastic syringe (Fig 2). This process continued, by diluting tissue suspension with cold HBSS, several times until islets were shown in the supernatant. Hence following this method there was no need for mechanical disruption of the digest through choppers, macerators, or other devices, since appropriate enzymatic digestion alone was associated with massive liberation of islets. After several washes in HBSS at 450 g at 4OC for 5 minutes each, islet tissue, whose packed cell volume never exceeded 20 mL. was then resus- From the Istituto di Patologia Speciale Medica e Metodologia Clinica, University of Perugia, School of Medicine, 06100 Perugia and Consorzio interuniversitario per il Trapianto d’Organo (DPR 31/l O/1 988). Italy. Supported by Grant No. 8700201.56, given by the Italian Na- tional Councilfor Research (CNR). Address reprint requests to Riccardo CalaJiore. MD, Istituto di Patologia Speciale Medica, Via E. Dal Pozzo, 06100 Perugia, Italy. o 1990 by W.B. Saunders Company. 0026-0495/90/3902-0011 3.00/O 175  176 CALAFIORE ET AL Table zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA . Multienzymatic Solution for the Digestion of Porcine Pancreas zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Islet Culture Reagent Collagenase ype V Elastase Deoxyribonu- cleasa Calcium chloride (anydrous) Specific Activity 450 Ujmg solid 45 U/mg solid 600 Kunitz U/ mg protein Source Sigma Sigma Sigma Sigma Concentration 2 mg/mL 0.5 mg/mL 0.3 mg/mL 1 mg/mL Tissue aliquots, comprised of 100,000 islets, were placed in 100 x 15 mm Petri dishes (Becton Dickinson Labware, Lincoln Park, NJ) containing 10 mL of RPM1 1640 supplemented with heat- inactivated 10 fetal calf serum (Flow) and antibiotics and thereaf- ter incubated at 37OC n humid atmosphere of 95 air/CO,. Culture media were replaced every 48 hours. Morphological Studies pended at room temperature in a solution of Ficoll400 DL (Sigma Chemical Co, St. Louis, MO) in HBSS to a final density of 1,110. Discontinous gradients were prepared, with slight modifications of procedures previously described? by layering 15 mL of Ficoll/ HBSS, at densities of 1,090, 1,080 and 1,040 each on 80-mL 1,100 density tissue/Ficoll aliquots in round-bottom 150-mL glass bottles. The gradients were centrifuged at 400 g for 35 minutes at 4°C. Massive concentrations of pure islets were collected from the top of the 1,040 Ficoll layer, whereas no islets but only nonendocrine pancreatic cell components were found in the other layers and in the tissue pellet. The final, packed, islet-cell volume, assessed after harvest and wash in HBSS, at 450 g and 4OC ranged from 0.4 to 0.6 ml, with the estimated purity of the islets, as assessed by light microscopy (see below), overcoming 95 . Isolated islets were examined either at the end of the isolation or after 48- to 72-hour incubation periods. Islet aliquots and specimens of the native pancreas were fixed in 10 buffered formaldehyde, dehydrated with ethanol, and finally embedded in paraffin. Micro- tome sections, 5 p thick, were mounted on glass slides and stained with aldheyde fucsin. Additional islet aliquots were examined by electron microscopy after processing preparations in agreement with procedures previously described.4 Viability of fresh islets was also assessed by staining with ethidium bromide and fluorescein diacetate’ under fluorescence microscope (Leitz Orthomat IV, Ernst Leitz Wetzlar GmbH, Wetzlar, West Germany). Size distribution of either isolated or native pancreatic islets was assessed by morphometric evaluations of sections stained with aldheyde fucsin, with a light microscope equipped with a 240-mm scaled screen (Reichert Univar, Vienna, Austria). In Vitro Studies ssessment of Islet Yield The final islet yield was assessed under inverted phase microscope (Jenoptik Jena GmbH, Jena, DDR) equipped with a grid in the eyepiece, by counting random aliquots of the islet suspension. Prior to counting, islets were incubated with an alcoholic solution of 0.03 Diphenylthiocarbazone (DTZ, Sigma Chemical Co), which stains specifically in red insulin-containing tissue,3 thus facilitating quanti- tative and qualitative assessment of the final islet preparation. Bach islet count was performed as follows: After diluting the final islet suspension with 15 mL RPM1 1640 (Flow) under gentle agitation, a 0.2-mL random aliquot was transferred to a 50-mm Petri dish for count. Average 10,000 islets were examined each time. Only islets whose diameter resulted higher than 60 pm (see below) were quoted for the assessment of the final islet yield. Insulin content was measured by acid-ethanol extraction in aliquots of the final islet preparation and expressed as insulin recovery/g of pancreatic tissue. Porcine islet insulin-secretory function was assessed in either static incubation or perifusion studies. Batches of 25 islets were either incubated for 2 hours at 37OC with HBSS containing glucose at different concentrations (5.5 mmol/L to 16.5 mmol/L), with or without 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX) and 10 mmol/L arginine or continuously perifused for overall 2 hours with the same secretagogues according to methods previously described.6 At the end of each study media were collected for insulin assay. In Vivo Studies After 24 hours culture, islets were enveloped within algin polyami- noacidic microcapsules according to methods previously described.6 Polymer/islet mixture was adjusted so as to make each capsule contain an average of four to six islets. Microencapsulated porcine islets, which were associated with full retention of physiologic insulin-secretory kinetics in vitro were xenoengrafted, after an peristaltic pump \ C C enzymatic solution - bath pancreas Figl. Schematic represen- tations of the apparatus for enzymatic digestion of the por- cine pancreas.  MASSIVE SEPARATION OF PORCINE ISLETS OF LANGERHANS 177 r====l Fig 2. Schematic represen- tation of porcine pancreatic di- gest at the end of the perfu- TISSUE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ELLET sion. additional 24 hours of culture, into six NOD mice with spontaneous and six Lewis rats with streptozotocin (65 mg/kg)-induced diabetes mellitus. In either rodent strain overt diabetes was diagnosed for at least 3 weeks prior to transplantation (fasting blood glucose, FBG > 400 mg/dL). Mice received 5,000 (capsules = 833 to 1,250) and rats 10,000 (capsules = 1,666 to 2,500) microencapsulated islets, intraperitoneally (IP), with the animals under general anesthe- sia. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB ssays Insulin collected from media or acid ethanol extracts was deter- mined by radioimmunoassay (RIA).’ Blood glucose of transplanted animals was measured by a glucose oxidase method (Beckman glucose analyzer 2, Beckman Instruments, Fullerton, CA). RESULTS Islet Yield and Morphology Table 2 shows overall islet and insulin recoveries from 12 separate porcine pancreata, achieved by our method. We isolated an average of 11,000 islets/g of pancreas, which represents a uniquely massive yield in comparison with results previously reported. Islet-size distribution, assessed 1 rJ ,Y : l zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Ll 4 I I L ‘. - I I L-, , 1 L, ,‘,‘,‘,~/‘,1,/,11’,‘,‘,~ ‘,‘,“‘,‘I’,‘, 10 20 30 .lo 50 60 70 80 90 100 110 120 130 140 160 16 170 180 190 200 r by morphometric examination of native pancreas versus isolated islets is represented by Fig 3. Retrieved tissue was comprised of over 95 pure islets, as determined by dissect- ing microscope, after DTZ staining (Fig 4). These islets were shown to be viable by microscopy examination after either ethidium bromide plus fluorescein diacetate or aldheyde fucsin (Fig 5) staining. Islet morphological integrity was finally established by electron microscopy examination that was associated with the presence of well-differentiated islet cells (Fig 6). Islet In Vitro Function Insulin release above baseline, in response to 16.5 mmol/L glucose with or without 0.1 mmol/L IBMX and to 5.5 mmol/L glucose plus 10 mmol/L arginine, was substantial and significant, as compared to baseline, and was sustained throughout 72 hours of culture maintenance, after either static incubation (Fig 7) or perifusion (Fig 8), showing that isolated porcine islets were associated with physiologic func- tional competence. 1 ;’ I L. ADIPOSE TISSUE FREE ISLETS n H isolated k -- - i native pancreas Fig 3. Schematic repreren- tation of isolated versus native pancreatic porcine-islet-size distribution after morphomet- ric assessment. IAMETER OF ISLETS (pm 1  178 Table 2. Islet zyxwvutsrqpon nd Insulin Recoveries From Porcine P8ncre8t8 Pancreas weight (g) 80 f 8 Islet recovery (islets/g pancreas) 11,166 f 901 Insulin recovery* (U/g pancreas) 1.5 f 0.2 NOTE. Mean values f SEM are shown for 12 separate pancreata. *Insulin was extracted by acid ethanol from aliquots of pure islets at the end of the isolation procedure. zyxwvutsrqponm Islet In Vivo Function All animals receiving intraperitoneal implants of microen- capsulated porcine islets showed complete remission of hyperglycemia (Fig 9) by day 3 of treatment, which was sustained in 100 of recipients for at least 15 or 50 days in NOD mice or Lewis rats, respectively. DISCUSSION Several methods have been reported for the isolation of islets of Langerhans from the rodent,* canine,9 porcine,’ bovine,‘Ov” and human” pancreas to validate the potential introduction of islet-cell transplantation in the therapy of insulin-dependent diabetes mellitus. So far this objective has been successfully achieved in rodentsI and in dogs,14 grant- ing that sufficient islet-cell mass and either immunoalter- ation of islets or immunosuppression of recipients were provided. On the other hand, no success has been achieved yet in humans in terms of complete remission of diabetes, neither could insulin be withdrawn following islet transplan- CALAFIORE ET AL Fig 5. Photomicrcgrclph of freshly isol8ted Porcine islets of L8ngerh8nc after staining with aldheyde fucsin under light micrc- scope (srcinal msgnific8tion x400). Fig 4. Photomicrogr8ph of freshly isol8ted porcine islets of Langerh8ns 8fter st8ining with diphenylthicc8rbazone. under stereomicro- *cope (srcinal m8gnification X40).  MASSIVE SEPARATION OF PORCINE ISLETS OF IANGERHANS zyxwvuts 79 Fig 6. Electron photomicrc- graph of freshly isolated por- cine islets of Lengerhanr show- ing well-differentiated islet-cell endocrine granuli. 2 1 zyxwvutsrqpo 0. L I I I fresh 72”h GLUCOSE : GLUCOSE 5.5 mM GLUCOSE 18.5 mM GLUCOSE 16.5 mM + ISMX 0.1 mM GLUCOSE 5.5 mM + ARGININE 10 mM Q p 4 0.02 @a,0 peo.01 Fig 7. Isolation release from isolated porcine islets in ra- sponse to insulin secretegogues after 2 hours static incubation. 120- 5 ?i loo g $j 80- a z * SW 60- $ $j 40- - p dI 20- zyxwvutsrqpon 0 J 5.5 mM 16.5 mM+lBMX 0.1 mM 5.5 mM 5.5 mM + ARGMINE 10 mM I II I I I O+ Fresh Fig 6. Insulation secretory kinetics of isolated porcine is- lets during perifusion with insu- lin secretagogues. I I I I 1 I , 1 , I 1 I I a I -20 0 10 20 30 40 50 60 70 80 90 loo 110 120 130 TIME (minutes)
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