One of the significant challenges in the isolation of human islets for transplantation is the effect of cold ischemia (1, 2). University of Wisconsin (UW) solution has been used for over a decade as the primary flush and preservation media for pancreata intended for islet isolation (3). Recently, some centers have started using Histidine-Tryptophan Ketoglutarate (HTK) as a lower-cost alternative to UW (HTK=$150/liter, UW=$282/liter) for the flush and transport of human organs intended for transplantation.
HTK contains histidine as buffer, tryptophan as membrane stabilizer, ketoglutarate as metabolism substrate and contains less potassium than UW solution. Comparable results between UW and HTK preservations have been described in outcomes of liver, kidney, and pancreas transplants (4–6). Each solution has proposed benefits: UW is effective in preservation of abdominal organs for a longer period than HTK. Kidneys can be preserved up to 72 hr with UW before suffering irreversible preservation injury versus 48 hr with HTK (7). Livers may be stored for 24 hr with UW versus 15 hr with HTK (8). HTK is not indicated for use in preservation of the pancreas for whole organ transplant according to the product information (Custodiol manufacturer product information), although some studies have shown it is effective for this purpose (5, 6). Kidneys preserved with UW have also been shown to have better outcomes than those preserved with HTK (9), in particular when kidney storage exceeds 24 hr (10). HTK has lower viscosity than UW and diffuses more readily into the extracellular space (11).
The purpose of our study was to compare the islet isolation results from pancreata flushed and transported with HTK versus those flushed and transported with UW solution.
Potential donors were screened at the time of offer. Donors with positive serology results (other than cytomegalovirus), diabetic or suspected diabetic donors, and donors with sepsis, cancer, or methanol toxicity were ruled out. Characteristics for both groups are outlined in Table 1.
Isolations were performed at the University of Illinois at Chicago (UIC) on pancreata received from three different organ procurement organizations (OPO). Michigan and Indiana OPOs routinely used HTK, and the local OPO (Illinois) exclusively used UW. Between March 2004 and October 2005, a series of 86 human islet isolations were compared based upon use of HTK or UW as an organ flush/preservation media (41 for HTK vs. 45 for UW solutions). At the time of organ recovery the pancreata were flushed with 4100±255 ml of HTK or 3488±191 ml of UW solutions. After procurement, the pancreas was placed in 500 to 1000 ml liters of the same preservation solution as used to flush the donor. The pancreata were digested using a modified automated method (12). After collection and wash, the tissue was incubated in UW solution (DuPont Pharma, Bad Homburg, Germany) for 30 min, islets were purified by a continuous Ficoll gradient (Biocoll, Cedarlane, ON, Canada) on a cell separator (Cobe 2991, Cobe, Lakewood, CO). Islets were cultured in Culture Media (Mediatech, Herndon, VA) at 37°C and 5% CO2 for up to 72 hr.
Islet isolation outcome was assessed by quantification of islet mass after dithizone staining, expressed in equivalent islet numbers (EIN) (13). Islet viability and percent live cells was determined by fluorescent staining with Syto-Green/Ethidium Bromide (14, 15). In vitro islet function was performed by static glucose incubation and expressed as stimulation index (SI) (16). In vivo islets function was assessed in a subgroup of marginal donors (which were suboptimal and they had been deemed unacceptable by more than one center) through transplantation of islets from seven preparations of the HTK pancreata and four of the UW pancreata into diabetic nude mice (15 mice were allocated to HTK and 11 mice to UW group). Mice were rendered diabetic by a single intraperitoneal injection of streptozotocin (Sigma, 220 mg/kg body wt). Islet grafts of 1,000 EIN from each group were transplanted under kidney capsule (17). Successful transplantation was defined by reduction of glycemia levels to ≤200 mg/dl on two separate days following transplantation. Five to seven weeks after transplantation, normoglycemic recipients underwent nephrectomy in order to remove the islet graft. Return to hyperglycemia was interpreted as proof of islet graft functional dependence. Graft function was also assessed by the lag period to achieve normoglycemia. Statistical analysis was carried out by Student’s t test and Pearson chi-square test where applicable. P values of <0.05 were regarded as statistically significant.
Islet isolation outcomes were evaluated and found to be statistically similar between both groups (Table 2). Digestion time as well as digestion rate was similar between the groups. The final islet counts expressed in EIN were statistically similar (HTK: 383,085±33,606 vs. UW: 328,514±26,523, P=0.14). In the HTK group, 63.4% (26/41) of isolations resulted in a yield of over 300,000, where as in the UW group this was achieved in 46.7% (21/45; P=0.12). The number of islet preparations transplanted to human type I diabetic patients was higher in HTK than UW group (10/41 vs. 4/45, P=0.05). The final viability results postisolation were similar between both groups (HTK: 82.9±1.3% vs. UW: 82.7±1.6%, P=0.93). Static glucose challenge demonstrated no significant difference between two groups (HTK: 5.28±0.63 vs. UW: 4.91±0.45, P=0.62). In terms of in vivo function in diabetic nude mice transplant model, the groups studied were not significantly different; however, there was a trend for better in vivo function for islets isolated from HTK preserved pancreata. In all, 73% (11/15) of the mice transplanted with HTK islets reached normoglycemia compared to 36% (4/11) of mice transplanted with UW islets, P=0.06. The lag time in days for mice to reach normoglycemia was also similar between both groups (1.0±0.09 vs. 2.8±0.66 days) for HTK and UW respectively (P=0.06).
Our study demonstrates that both HTK and UW provide similar preservation of the pancreas for the purpose of islet isolation. With growing demand for sufficient amount of islets for transplantation, it is crucial that optimal pancreas procurement and preservation is achieved. Ensuring good preservation of the pancreas is necessary to protect the organ from the effects of cold ischemia (18); necessary to maintain an appropriate amount of viable isolated islets for transplant (1, 2). In the past, both HTK and UW have shown to be effective as flush and transport solutions for the recovery of the pancreas for whole organ transplant (5, 6), and now our study suggests the same for a pancreas intended for islet isolation. There appeared to be no significant difference in digestion, islet yield, purity, viability and stimulation index between HTK and UW groups.
HTK is less expensive than UW because it substitutes mannitol for the starch in UW. Mannitol in HTK maintains the same osmolarity for a lower price. The proposed longer storage time benefit of UW does not apply to the pancreas for islet isolation, since we will only accept pancreata with up to 12 hr of cold ischemia. However, in this study, mean cold ischemia time was even higher in HTK preserved donors. Moreover, the donor quality was rather lower in HTK group as the percentage of other organs suitable for transplantation was lower than UW group. Our study suggests that a comparable amount of HTK may be used to flush the pancreas to the amount of UW used. The higher volumes of HTK previously described were based upon a 10 min flush. This time appears to be longer than needed; and a smaller volume may be used at a slower rate. In addition, the viscosity of HTK solution is similar to water and the organ could be more readily flushed and cooled down. Our transplant model of marginal pancreas donors suggests that the HTK islets performed equally or even slightly better than the UW islets. A higher number of animals reached normoglycemia and with a shorter lag period than UW islets, although this difference was not statistically significant. In terms of isolation success and actual patient transplantation, higher percentages were observed with HTK-treated donors.
Two-layer method (TLM) for preservation of the pancreas has been shown to improve islet isolation and transplantation outcomes in organs with prolonged cold ischemia time (19). TLM was originally developed with perfluorocarbon and Euro-Collins solution (EC), which was later replaced by UW solution. As an aqueous solution, biochemical characteristics of HTK are similar to UW or EC solutions and it could also be used for TLM, though not been investigated in this study. Diliz-Perez et al. showed that cold preservation of hepatocytes is as effective in HTK/PFC as UW/PFC solutions (20). Future studies should specifically explore the suitability of HTK in TLM for extended pancreas preservation.
In conclusion, both UW and HTK solutions are acceptable for the in situ perfusion and transport of human pancreata for islet isolation. The use of HTK or UW demonstrated to be equally effective in preserving islet yield, viability and function. HTK may be the preservation solution of choice for a pancreas intended for islet isolation due to its lower cost and comparable performance to UW solution.
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