Carroll, P

Demetris, AJ

Ricordi, C

Sever, CE

Starzl, TE

Tzakis, A

Zeng, Y

Transplantation Proceedings

English

PubMed

SEVER, C. E

DEMETRIS, A. J

ZENG, Y

CARROLL, P

TZAKIS, A

STARZL, T. E

RICORDI, C

University of Miami: Scholarship Miami

Cellular composition of islet cell suspensions for transplantation

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i I Cellular Composition of Islet Cell Suspensions for Transplantation C.E. Sever. A.J. Demetris, Y. Zeng, P. Carroll. A. Tzakis, T.E. Starzl, and C. Ricordi A SIGNIFICANT number of nonislet cells still remains in cell suspensions from human islet cell prepara-tions obtained by our previously described method. I •2 Nonislet components such as acinar cells or immunogenic hematolymphoid cells could have adverse effects on initial graft taking as well as long-term graft survival through mechanisms of enzymatic injury and initiation of rejec-tion.J In this study, we have examined in detail the cellular I composition of islet cell suspensions using paraffin-embed-ded cell pellets of the isolates immediately after purifica-tion and after 1 week in culture medium. MATERIALS AND METHODS Human Islets obtained from SIX different multiorgan donors were separated and purified by the automated method and Euro-Collins-Ficoll gradient separauon using the COBE 2991 cell pro-cessor.1.1 A pOnlon of the suspension was fixed in buffered formaldehyde. and cell pellets were obtained by centrifugation at 800 f( for 3 minutes. Additional pellets were prepared after the remaining suspensions were maintained in CMRL-l066 culture medium at 30"C in 5% CO2 for I week. All formalin-fixed pel-lets were paraffin embedded. secuoned. and stained with the avi-din-biotin-complex (ABC) immunoperoxidase method. as de-scribed by Hsu.· A panel of antibodies was used with antibodies directed against insulin. glucagon. chromogranin. actin (all Bio-genex. San Ramon. CAl, AE-I. AE-3 (Boehringer Mannheim. Indianapolis. IN). vimentin (Sigma. St Louis. Mo). factor VIIIR-Ag. Sioo. LCNCD45. MAC-387.lysozyme. UCHL-1. L261CD20 (all DAKO. Santa Barbara. CaiiO. LN3/HLA-DR (Biotest Diag-nosucs. Denville. NJ). and Leu-221CD43 (Becton-Dickinson. Mountam View. CaiiO. Small fractions from two preparations were fixed in 2% glutaraldehyde. postfixed in 1% osmium tetrox-ide. embedded in Epon-Araldite resin. stained with 4% uranyl acetate followed by lead citrate. and examined with a Phillips EM 300 transmission electron microscope. RESULTS AND DISCUSSION Immediately after separation. the isolates contained 30% to 80% islet cells; however, all other cell types found in normal pancreas were also present. This included HLA-DR and SI00 positive dendritic cells. which are known as potent stimulators of immune reactions. includ-ing rejection. S The difference in purity of the various TranspIantIJIion Proceed'ngs. Vol 24. No 3 (Junel. 1992: p 979 preparations was mainly due to a different content of islet and acinar cells; whereas ductal. soft tissue. and hema-tolymphoid cells comprised approximately 10% to 20% in all preparations. One week of culture had different effects on moderately and highly purified preparations: In less purified isolates (30% to 40% islets). the islet cell content dropped to as low as 5%. In addition. remaining islets cells weakly expressed HLA-DR. Remaining cells were almost exclusively acinar cells; however. scattered endothelial cells and a few lymphoid aggregates as well as very rare HLA-DR positive macrophages could still be detected. In contrast. highly purified isolates (70% to 80% islets I mainly showed decreased insulin content without significant loss of islet cells or new HLA-DR expression. Epithelial, soft tissue. and hematolymphoid elements decreased only min-imally. It appears that reduction of immunogenic cells in islet cell suspensions cannot be achieved solely by primar-ily mechanical purification procedures but requires addi-tional measures. Short-term culture only reduces but does not eliminate immunogenic cells and is deleterious to islet cells if the initial content of acinar cells is high. REFERENCES I. Ricordi C. Lacy PEJ. Finke H. et a1: Diabetes 37:4\3. 1988 2. Ricordi C. Alejandro R. Zeng Y. et a1: Transplant Proc 23:783. 1991 3. Setum CM. Hegrc 00. Serie JR. et a1: Transplantation 49: 1175. 1990 4. Hsu SM. Raine L. Fanger H: J Histochem Cytochem 29:5n. 1981 5. FausUnan DL. Steinman RM. Gebel HM. et al: Proc Nat! Acad Sci USA:3864. 1984 From the Departments of Pathology and Surgery. Pittsburgh Transplant Institute. University of Pittsburgh. Pittsburgh. Pennsyl-vania. Supported in pan by Juvenile Diabetes Foundation Research Grant no. 1911421. Address reprint requests to Camillo Ricordi. MD. University 01 Pittsburgh, School 01 Medicine. Department of Surgery. Division of Transplantation. 3601 Fifth Avenue. Pittsburgh. PA 15213. © 1992 by Appleton & Lange 0041-13451921$3.001 +0 979 

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Cellular composition of islet cell suspensions for transplantation

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