Repurification: Rescue Rather Than Routine Remedy (2005)

Reversal of type 1 diabetes has become a consistent result in recipients of a critical islet mass immunosuppressed with a steroid-free regimen (1Shapiro AM Lakey JR Ryan EA et al.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen (see comments)..N Engl J Med. 2000; 343: 230-238Crossref PubMed Scopus (4378) Google Scholar,2Shapiro AM Ricordi C Hering B Edmonton's islet success has indeed been replicated elsewhere..Lancet. 2003; 362: 1242Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). However, results of isolation and purification of islets from a deceased human donor pancreas have remained much less consistent. On average, islet yields from only 1 of 2-3 donor pancreases exceed 5000 islet equivalents (IE) per kg recipient body weight and thus meet islet batch product release criteria for transplant. In addition, typically 2 islet preparations with a total yield of 10 000 IE/kg are deemed necessary to achieve insulin-independence post-transplant in a type 1 diabetic recipient. Our ability to improve the efficiency of human islet processing will therefore be a critical determinant of the availability of islet transplants for the treatment of diabetes. Factors contributing to the inconsistency in human islet preparation include donor and donor pancreas characteristics, pancreas procurement and preservation protocols, islet isolation procedures, islet purification practices and islet culture techniques. The article by Ichii et al. in this issue of the American Journal of Transplantation (3Ichii H Pileggi A Molano RD et al.Rescue purification maximizes the use of human islet preparations for transplantation..Am J Transplant. 2004; 5: 21-30Abstract Full Text Full Text PDF Scopus (97) Google Scholar) describes a novel method for recovering islets from predominantly exocrine tissue-containing, less pure islet fractions obtained after continuous density gradient purification of the pancreatic digest. The novel approach, referred to as rescue gradient purification (RGP), increased the islet mass available for transplant by 27.9 ± 19.9%, and inclusion of rescued islets in the final islet preparation allowed obtaining the minimal islet number required for release for transplant in 12 of 25 preparations. Ichii et al. also present data suggesting that RGP does not compromise the viability and potency of human islets and demonstrate sustained C-peptide production, HbA1c normalization and reduced insulin requirements in recipients of islet transplants containing islets subjected to RGP. How important will RGP be as a new islet processing method and what are the implications of the study? The average number of ‘rescued’ islets reported by Ichii etal. is impressive and document that RGP is a straightforward, remarkably effective approach to increasing the proportion of islet preparations that meet the minimal islet mass requirements for transplant. Besides addressing the efficacy in recovering islets, consideration must be given to the impact of RGD on islet function. Previous studies have demonstrated that ficoll exposure negatively impacts islet function (4Scharp DW Kemp CB Knight MJ Ballinger WF Lacy PE The use of ficoll in the preparation of viable islets of Langerhans from the rat pancreas..Transplantation. 1973; 16: 686-689Crossref PubMed Scopus (206) Google Scholar,5Salvalaggio PR Deng S Ariyan CE et al.Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function..Transplantation. 2002; 74: 877-879Crossref PubMed Scopus (103) Google Scholar). The effects on in vivo function may be minimal with a large dose islet transplant but may be critical with a marginal dose human islet transplant. Ichii et al. have performed a series of in vitro and in vivo studies to address this question. In vitro islet testing suggests that RGP has no unfavorable effect on islet cell viability and on islet insulin secretory capacity. However, it is generally agreed that the sensitivity of these assays in terms of unraveling islet damage is poor. This limitation makes the results of in vivo islet testing particularly important. Unfortunately, the in vivo evidence provided that RGP is not detrimental to islet function is, at best, preliminary. In the diabetic nude mouse bioassay, 3 of 25 islet preparations were analyzed for their ability to reverse diabetes. A detrimental effect of RGD was not apparent, but only large-dose islet grafts (i.e. 2000 IE per mouse) were analyzed in this bioassay, allowing only serious islet damage to be detected. Most investigators monitor islet function in this bioassay for about 4 weeks. Gotoh et al. (6Gotoh M Kanai T Dono K Porter J Maki T Monaco AP Gammairradiation as a tool to reduce immunogenicity of islet allo- and xenografts..Horm Metab Res Suppl. 1990; 25: 89-96Google Scholar) demonstrated late loss of function of 200 syngeneic mouse islets between 40 and 100 days post-transplant with increasing doses of pre-transplant γ-irradiation (24, 40 and 80 Gy). It may therefore prove useful to extend the monitoring period of human-to-nude mouse islet grafts whenever new islet processing techniques are evaluated. The clinical data presented by Ichii et al. on RGD islet function, albeit incomplete, perhaps provide the most compelling rationale for adopting RGP. Additional studies are encouraged and should include a longitudinal comparison of the magnitude of first phase insulin secretion in response to intravenous glucose in recipients of islet grafts that do and do not contain a substantial proportion of RGD islets. ‘Rescue’ implies “freeing from imminent danger by prompt or vigorous action” and should therefore not be considered as routine remedy. Low islet purities generally result from acinar tissue swelling (7London NJM Robertson GSM Chadwick DR James RFL Bell PRF Adult islet purification..in: Ricordi C Methods in Cell Transplantation. RG Landes, Austin, TX1995: 439-454Google Scholar), leading to migration of acinar tissue from layers of high density to layers of lower density. Acinar swelling most likely reflects poor acinar tissue viability caused by ischemia, proteolytic enzymes and other insults encountered. Acinar swelling may also point to compromised islet viability that is not identified by commonly practiced in vitro testing. We should therefore pursue a more proactive approach and seek to maintain the substantial differences in density between intact islet (1.070 g/cm3) and acinar tissue (1.100 g/cm3) by applying cytoprotective strategies throughout donor management, pancreas retrieval, pancreas preservation and pancreas dissociation so that effective separation of islet and acinar tissue can be accomplished on a consistent basis with only one density gradient separation. More innovative approaches, such as magnetic retraction and positive selection of liberated islets (8Pinkse GG Steenvoorde E Hogendoorn S et al.Stable transplantation results of magnetically retracted islets: a novel method..Diabetologia. 2004; 47: 55-61Crossref PubMed Scopus (17) Google Scholar), would speed up islet processing considerably and obviate the need for any density gradient separation. As long as these techniques are not developed for human islet processing, RGP represents our best remedy for recoverable islets.