Elsevier

Surgery

Volume 126, Issue 2, August 1999, Pages 299-304
Surgery

Society of University Surgeons
Anoikis, extracellular matrix, and apoptosis factors in isolated cell transplantation,☆☆

Presented at the 60th Annual Meeting of the Society of University Surgeons, New Orleans, La, Feb 11-13, 1999.
https://doi.org/10.1016/S0039-6060(99)70169-8Get rights and content

Abstract

Background: Isolated cell transplantation (ICT) of pancreatic islets (PI), nerve tissue, hepatocytes, and other cells is an exciting new concept of transplantation. PI transplantation can be successful in reversing diabetes but, unlike whole pancreas, grafts have a unique and unexplained high failure rate, with over 60% loss by 3 to 6 months. We established that PI of rhesus monkeys have a high rate of death within 48 hours of isolation as a result of apoptosis, as measured with the Annexin V assay (Pharmingen, San Diego, Calif). In contrast, PI incompletely separated from the extracellular matrix (ECM) remained viable for prolonged periods in culture and performed superiorly in perifusion assays (insulin secretion of 4.6 ± 0.8 times basal secretion). Methods: We studied the ability of the anti-AP Bcl-2 molecule, known to block anoikis (a mechanism of AP due to cell-ECM separation), to prevent apoptosis of isolated PI. Results: PI transduced with an adenovirus-Bcl-2 gene complex showed a high viability and a low AP rate in culture versus control rhesus PI. Conclusion: In summary, PI protected from AP by a surrounding ECM mantle or by Adenovirol Vector (AdV) transduction of the Bcl-2 gene showed superior viability without AP in vitro and in vivo evidence of a preserved insulin secretion response to glucose. (Surgery 1999;126:299-304)

Section snippets

Rhesus islet preparation

The rhesus macaque islets were isolated from pancreata removed under clinical conditions of procurement from a heart-beating donor animal. Optimal procurement techniques were used, including retrograde perfusion of the coeliac, superior mesenteric, and renal arteries with University of Wisconsin (UW) preservation fluid and removal and duct cannulation of the donor pancreas. The pancreas was kept at 4°C in iced UW solution. Pancreas digestion was performed by means of a modified Ricordi

Results

Fig 1 shows the initial observation demonstrating a high rate of death in free isolated islets cultured 24 to 48 hours.

. Graph demonstrates high rate of viability of freshly isolated free and highly purified rhesus islets. The viability by EB/AO testing was >95% in 4 preparations. In sharp contrast, cells cultured for 24 to 48 hours showed a very low rate of viability, of less than 20% (P < .01).

After culture, the mean viability of the freely isolated islets (EB/AO stain) was strikingly reduced

Discussion

Islet replacement by whole-organ pancreas graft or isolated islet transplantation has been demonstrated to be the therapy of choice for maintaining a constant euglycemic state.19 Unlike whole-organ pancreas grafts done for insulin-dependent diabetes mellitus, isolated islet transplants have a high rate of early (up to 3 months) failure of more than 60%.10 Thus, early donor islet failure is a phenomenon not seen in other whole-organ transplants.

The traumatic nature of islet isolation and the

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Supported by National Institutes of Health grants #R01 Al 122293, Al 39793, and Juvenile Diabetes Foundation International grant #198242.

☆☆

Reprint requests: Francis T. Thomas, MD, Professor of Surgery, The University of Alabama at Birmingham, Suite 557-A, 1808 7th Avenue, South, Birmingham, AL 35294-0012.

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