Alejandro, R

Casavilla, A

Jaffe, R

Mintz, DH

Ricordi, C

Starzl, TE

Tzakis, A

Zeng, Y

Transplantation Proceedings

English

PubMed

Name not available

Human islet isolation and purification from pediatric-age donors

Ricordi, C

Alejandro, R

Zeng, Y

Tzakis, A

Casavilla, A

Jaffe, R

Mintz, D H

Starzl, T E

University of Miami: Scholarship@Miami

D-Scholarship@Pitt

Human Islet Isolation and Purification From Pediatric-Age Donors C. Ricordi, R. Alejandro, Y. Zeng, A. Tzakis, A. Casavilla, R. Jaffe, D.H. Mintz, and T.E. Starzl THE separation and purification of human islets from the pancreas of pediatric-age donors has been con-sidered difficult if not impossible because of easy fragmen-tation of the islets and/or their incomplete separation from the enclosing acinar tissue (embedding). I.:! The purpose of this study was to develop a method to separate and purify large numbers of viable human islets from pediatric do-nors. After preliminary studies on swine islet isolation3 which resemble pediatric human isolation in many ways, a modification of the automated method for adult human islet isolation was developed. MATERIALS AND METHODS Six pancreata obtained from pediatric donors 'age 7 to 18) were distended by intraductal injection of 200 to ~<;<g mL of Hanks' solution with I mglmL collagenase (Boehringer-Mannheim, Type P) and loaded into a digestion chamber (volume 450 mLl contain-ing a stainless steel screen (400-J.LI11 pore size). and 7 solid glass marbles. I After a first phase of recirculation of the collagenase solution in a closed system (12 to 18') in .. ruch intrachamber temperature was progressively raised from ~ to 37°C (ftow rate: 50 mUmin). the islets were collected during a dilution phase of 15 to 30' (flow rate: 300 mUmin). Any mechanical trauma to the islets during separation was carefully avoided. ~ Final purification of the islets was obtained by discontinuous density gradient separation with Euro-Collins solution used as vehicle for the FicoH powder (Sigma. DL-4(0). Three layers (densities = 1108, 1096. and 1037) have been used.· After centrifugation on a COBE 2991 cell processor.s.1> the islets were retrieved from both 1.0371 1.096 (purity 80% to 95%) and from 1.0961\.108 (purity 40% to 50%) interfaces. Morphologic and functional integrity of the islet preparation have been assessed following ~ criteria recently proposed for islet isolation assessment. 7 The functional integrity of the final preparation was tested by transplantation of an aliquot of the final preparation into diabetic nude mice- and by aHotrans-plantation in patients who underwent total paocreatectomy as part of an upper abdominal exenteration9 for malignancy. The main modifications from the procedure for adult human islet isolation I consisted in the absence of heating circuit bypass, the absence of a cooling circuit, the absence of shaking during the recirculation phase. the increased volume of the digestion cham-ber. and the increased pore size of the stainless steel screen. RESULTS AND DISCUSSION The results of 6 consecutive pediatric islet isolations are summarized in Table I. An average of over 430,000 islets were obtained after the purification process, representing over 9500 islets per gram of pancreatic tissue. The purity of the final islet preparation ranged between 40% and 90% islets. Histologic studies showed that the islets obtained by this procedure were weU preserved. Significant basal and stimulated C-peptide production have been shown in all patients who received pediatric islet allografts. The results of clinical islet transplantation. described in detail elsewhere,lo demonstrated that this procedure resulted in numbers of viable pancreatic islets from a single pediatric donor sufficient to prevent pancreatectomy-induced diabe-tes mellitus following intraportal islet allotransplantation. From the DepartmenlS of Surgery and Pathology, University of Pittsburgh, Pittsburgh, PA; Diabetes Research Institute, University of Miami School of Medicine, Miami, FL; cnj Department of Surgery. Institute H. San Ralaele University 01 Milan, Milan. Italy. Supported by research grants from the Veterans Administration, Project Grant Nos. OK 29961 and OK 25802 from the National InsliMes of Health (Bethesda. Md) and the Diabetes Research Institute Foundation (Mami. Fla). Address reprint requests 10 CamillO Ricordi. MD. Department of Surgery. 3601 Fifth Avenue, Pittsburgh. PA 15213. ~ 1991 by Appleton & Lange 0041-13451911$3.001+0 Table 1. Donor Pancreas Data and Postpurtflcatlon Islet Isolation Results Donor Pancreas PosIpurifocallOn Islet Assess I oent Weight err Number' v'*- Purity No. Age (g) (h) (x 1(00) (lit.) 1SIeI;9 ('II. IsIeIS) 18 48.2 8 47" 0.839 9834 60 2 7 23.2 8 562 0.993 24.224 50 3 18 108.0 7 105 0.186 972 70 4 18 100.0 6 285 0.504 2850 40 5 17 40.0 10 459 0.811 11.475 90 6 16 90.0 6 717 1.2ffl 7967 70 Mean 433.7 0.767 9553 63.3 SO 195.4 0.345 7528 16.0 'CIT = cold ischemia lime. tEstimated number of islets with an average diameter 01150 p.m. Transplantation Proceedings, Vol 23. No 1 (February), 1991: pp 783-784 783 784 The combination of the automated method for islet sepa-ration together with a purification process that involves the COBE cell separator represents a further step toward complete automation of cadaveric pancreatic islet isolation and purification. REFERENCES I. Ricordi C, Lacy PE, Finke H. et aJ: Diabetes 37:413, 1988 2. Anderson 0, Kneteman NM. Scharp OW: Transplant Proc 19:916. 1987 3. Ricordi C, Socci C, DavaJli AM. et al: Surgery 107:688. 1990 RICORDI, ALEJANDRO. ZENG ET AL 4. Sharp OW, Lacy PE. Santiago lV. et al: Diabetes 39:515, 1990 5. Lake SP. Basset D. Larkins A, et al: Diabetes 38:143. 1989 (suppl I) 6. Alejandro A, Strasser S, Zucker PF, et al: Transplantation 50:207,1990 7. Ricordi C. Gray D\\-'R, Hering Bl. et al: Acta Diabetologica Latina (in press) 8. Ricordi C, Scharp OW, Lacy PE: Transplantation 45:994, 1988 9. Tzakis A. Todo S. Stanl TE: Transplant Proc 22:273, 1990 10. Tzakis A, Ricordi C, Alejandro R, et aJ: Lancet PPS:4M~I 1990 ". , 

Human islet isolation and purification from pediatric-age donors.

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Human islet isolation and purification from pediatric-age donors.

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