不同器官保存液对成人胰岛分离、纯化及功能的影响

目的探讨不同器官保存液保存胰腺对胰岛分离、纯化及功能的影响。方法以成人胰腺为研究对象,分别以高渗枸橼酸钾溶液(HCA液)与威斯康新大学器官保存液(UW液)灌洗、保存胰腺,进行胰岛分离及纯化,比较两组分离、纯化后收获的胰岛数及胰岛当量数(IEQ),并在体外评价其功能。结果两组在胰腺重量、热缺血时间、冷缺血时间没有明显差异的情况下,胰腺分离、纯化后所获得的胰岛数及胰岛当量数的差异无显著性(P>0.05),纯化后的胰岛细胞在体外低糖与高糖刺激下,其胰岛素的分泌量及C肽的释放量的差异也无显著性(P>0.05);两个组胰岛细胞的活率均在85%以上。结论在胰岛移植中,可以使用HCA液作为胰腺保存液。     

机械分离成人尸体胰腺提高胰岛数量和质量

目的通过机械分离胰腺的方法,提高成人胰岛数量和质量。方法切取8个成人尸体胰腺,并保存于4℃UW液中。首先经主胰管插管灌注复合胶原酶溶液,使胰腺膨胀,然后将胰腺剪切成5~8块,放入Ricordi室中,于37℃下机械震荡消化。收集的胰岛溶于100mlUW液中,放置30min。先将Ficoll液泵入COBE2991细胞分离仪中,然后泵入UW/胰岛制剂,最后加入M199液冲洗。收集纯化的胰岛,计算胰岛当量(IEQ)和纯度,检测胰岛的活性。结果热缺血时间为0~1min,冷缺血时间0.5~4h。纯化前收集的胰岛数量平均为(376.7±50.2)×103IEQ,纯化后为(378.6±56.7)×103IEQ;胰岛收获率平均为(86.8±9.4)%,镶嵌的胰岛平均占(11.0±2.7)%;收获的胰岛纯度平均为(88.3±5.1)%;经AO-PI染色后检测胰岛的活度为(91.3±7.8)%。胰岛素分泌指数平均为5.87±0.81。结论通过机械分离胰腺的方法,可以获得更多的胰岛数量和更高的胰岛活性,适合用于临床移植。     

乌司他丁对犬胰岛的保护作用

目的判定在机械分离纯化犬胰岛过程中,胰腺原位灌洗时应用乌司他丁(UTI)的保护作用,观察胰岛的获取产量。方法将20只犬随机分为两组,每组10只。HC-A组:胰腺经腹主动脉原位灌洗时用冷HC-A液2500ml;HC-A UTI组:胰腺原位灌洗时,HC-A液中加用UTI10000U/kg。两组均经主胰管用4℃胶原酶V控压灌注,在RicordiChamber系统中消化,用Ficoll连续梯度离心纯化。记录消化时间、胰岛外分泌腺包裹率、胰岛纯度、纯化后胰岛在体外用低糖与高糖刺激下胰岛素分泌量、C-肽的释放量及收获的胰岛当量(IEQ),并对纯化后的胰岛进行光镜及电镜观察。结果HC-A UTI组与HC-A组在胰腺消化时间、胰岛外分泌腺包裹率、胰岛纯度、纯化后的胰岛在体外经低糖与高糖刺激下胰岛素分泌量、C-肽的释放量以及胰岛的形态和结构上比较,差异均无统计学意义(P>0.05)。HC-A组收获胰岛[(3.42±1.47)×104]IEQ,HC-A UTI组收获胰岛[(6.17±2.86)×104]IEQ,两组差异有统计学意义(P<0.05)。结论在犬胰岛分离和纯化过程中,胰腺获取原位灌洗液中加用UTI能保护胰腺组织,增加胰岛产量。     

实验大鼠胰岛分离移植技术方法的比较分析

目的 探索高效的大鼠胰岛分离移植技术方法.方法 应用Wistai-Furth大鼠,于体内或体外胶原酶经胰管灌注膨化胰腺,联合不同密度Ficoll液或Histopaque液纯化胰岛细胞,评估胰岛的数量、纯度、胰岛当量以及肾被膜下胰岛移植的有效性.结果 体外经胰管灌注膨化胰腺结合Histopaque液纯化提取胰岛的数量、纯度和胰岛当量值均显著高于体内灌注组各数值(P＜0.01),其提取时间无显著差别.1000个胰岛细胞移植进入左肾被膜下,有效的逆转了糖尿病大鼠高血糖,其远期糖耐受结果优于500和800个胰岛细胞移植组.结论 体外灌注膨化消化胰腺结合Histopaque液纯化胰岛的分离方法是一种满意的分离技术.1000个胰岛细胞是保证肾被膜下胰岛移植成功的最低有效数量.     

胰岛分离、纯化制备的改进

目的 探讨大型哺乳动物胰岛机械化大量分离、纯化的方法,为人类胰岛移植物的大量制备摸索创造条件.方法 应用改进的机械化胰岛分离、纯化系统,用HCA和UW液顺序原位灌洗犬胰腺,主副胰管插管,4℃胶原酶-V(1.5 g/L)+胰酶抑制剂pefabloc(0.4 mmol/L)灌注后,Ricordi-Chamber消化罐消化,4℃COBE2991连续密度梯度离心纯化,测定胰岛当量(IEQ)、胰岛纯度及存活率、胰岛素及C-肽的释放量、培养24 h后光镜及电镜观察.结果 胰腺消化时间为(25.0±6.0)min,胰岛外分泌腺包裹率为(9.4±2.4)%,消化后胰岛收获量为(17.2±3.6)×104IEQ/每个胰腺,纯化后胰岛收获量为(8.3±2.0)×104IEQ/每个胰腺,胰岛纯度为(89.7±3.5)%.纯化胰岛体外低糖与高糖刺激下胰岛索分泌量及C-肽的释放量良好,培养24 h后形态结构及功能正常.结论 本实验室改进的胰岛机械分离方法及各设备运行可靠,获得的胰岛形态功能良好,可望用于临床人类胰岛的大量制备.     

成年猪胰岛的分离与纯化

目的　研究成年猪胰岛的分离与纯化方法。方法　胰岛的分离采用导管内连续灌注胶原酶消化法 ,纯化采用Nycodenz连续密度梯度离心。 结果　消化后每个胰腺平均可获得 (3970 0 0±185 96 3)个胰岛或 (986 0± 3 0 43)个胰岛 /克胰腺组织 ,纯化后为 (1480 0 0± 890 0 0 )个胰岛 /胰腺或(3 75 0± 493)个胰岛 /克胰腺组织 ,纯度 >95 %。体外培养示胰岛对糖刺激反应良好 ;免疫组织化学检查证实胰岛细胞存活。结论　经本法获得的胰岛具有较高的纯度及良好的功能 ,可为临床提供可靠的移植物来源。     

大鼠胰岛分离、纯化技术改良

目的:探讨SD大鼠胰岛分离、纯化的较优方案,并评价依该法分离所得胰岛的生物学特性。方法:以浓度为0.75mg/ml的Ⅺ型胶原酶经胆管充分灌注大鼠胰腺;完整分离后以37℃水浴振荡消化17min,Dextron70不连续密度梯度(1.091、1.081及1.037)离心法分离、纯化胰岛;以DTZ染色鉴定胰岛并计算得率,AO-PI染色检测胰岛活性,糖刺激试验判定胰岛功能,胰岛"细胞免疫细胞化学鉴定"细胞,光镜观察体外培养胰岛的形态学变化。结果:纯化后,每只大鼠胰岛收获量为(743.60±43.07)个,胰岛纯度>90%,胰岛细胞活率>90%。在45min内每10个胰岛细胞在低糖和高糖刺激下分泌的胰岛素浓度分别为(25.35±7.00)μIU/ml和(86.48±12.75)μIU/ml。结论:依改良后的分离纯化方案分离大鼠胰岛可获得高纯度高活率的胰岛细胞,且细胞形态正常、功能良好。     

热缺血时间对大鼠胰岛结构和功能的影响

目的 探讨胰腺热缺血时间对胰岛形态和功能的影响。方法 以Lewis大鼠的胰腺为研究对象，分别在大鼠被处死后立即、25min后及30min后以胶原配灌注，然后分离、纯化胰岛，观察各组所得胰岛的形态、活性、胰岛当量数(IEQ)与功能。结果 各组均可获得形态完好的胰岛，胰岛细胞活率在85％以上；各组IEQ的差异元显著性；热缺血时间25min组与立即灌注组相比，纯化后的胰岛细胞在低糖与高糖刺激下，胰岛素分泌量的差异元显著性，而热缺血时间30min组与立即灌注组相比，胰岛素的分泌能力显著降低(P＜0．01)。结论 热缺血时间不超过25min时，分离、纯化后得到的胰岛在结构与功能方面的改变不显著，若热缺血时间延长到30min时，胰岛功能出现减退。     

小鼠胰岛的分离纯化及体外功能检测的实验研究

目的：探讨获得高质量小鼠胰岛的分离纯化方法，评价其功能。方法：采用胆总管内胶原酶灌注膨胀消化胰腺的方法分离小鼠胰岛，不连续密度梯度离心法纯化胰岛，用双硫腙（Dithizone，DTZ）对胰岛进行特异性染色计算胰岛产量及纯度，以葡萄糖和茶碱刺激胰岛素释放检测胰岛功能。结果：胰岛的产量和活性主要与胰腺均匀膨胀和胶原酶的消化时间有关。平均每个小鼠胰腺能得到150～250个高质量胰岛，活性〉95％，纯度〉90％。葡萄糖及茶碱（carbachol，Cch）刺激后胰岛素释放量明显增加。结论：改良的胆总管内胶原酶灌注膨胀消化小鼠胰腺及不连续密度梯度Ficoll-400纯化胰岛的方法，可获得产量较高、纯度及功能较好的胰岛。     

器官联合切取时供胰切取和修整方法的探讨

目的 探讨胰肾联合移植中供胰的切取和修整方法.方法 采用腹部器官联合切取法.取腹部“十”字切口,原位经腹主动脉和门静脉插管,分别以0～4℃高渗枸橼酸盐嘌呤溶液(HC-A液)2000 ml+ UW液1000ml灌洗,灌洗压力约为100 cm H2O(9.8 kPa);以0～4℃生理盐水1000 ml+甲硝唑200ml灌洗肠道,联合整块切取肝、肾、胰、脾和十二指肠,然后在工作台上分离、修整胰腺和肾脏.采用此法共完成40例胰肾联合移植.结果 整块器官的热缺血时间平均为3.2 min(2～5 min),器官灌注良好,无血管损伤.胰腺冷缺血时间为10.6 h(8～15 h),肾脏冷缺血时间为8.5h(4～16 h).术后停用胰岛素的时间平均为9.5 d(4～17 d),空腹血糖为6.7μmol/L(4.4～10.7μmol/L),糖化血红蛋白为4.4％(4.1％～4.7％).术后1个月肌酐87.2(56～121) μmol/L.术后发生移植肾功能恢复延迟2例,血肌酐均于1个月内恢复正常.结论 正确的供胰切取、保存及修整方法是保证移植成功的关键因素.     

Influence of collagenase loading on long-term preservation of pig pancreas by the two-layer method for subsequent islet isolation

BACKGROUND: The introduction of the two-layer method (TLM) for long-term human pancreas preservation revealed the enormous potential of TLM to improve graft function of isolated islets. It is still unclear whether pig islets can be successfully isolated from pancreases after prolonged cold ischemia. To clarify this question, pig pancreases were subjected to 7-hour preservation by University of Wisconsin solution (UWS) storage or TLM. Another aim was to verify whether TLM can be synergistically combined with intraductal collagenase injection before cold storage. METHODS: After intraductal flush with UWS, organs were distended with 4.4 PZ-U/g of UWS-dissolved collagenase NB-8 and neutral protease adjusted to respectively 1.1, 0.2, 0.5, or 0.8 DMC-U/g for pancreases freshly procured (n=6) or distended with enzymes before (TLM preloaded, n=7) or after cold storage (UWS storage, n=4; TLM postloaded, n=10). RESULTS: Purified islet yield decreased from 429,200+/-86,700 islet equivalents (IEQ) in unstored pancreases to respectively 37,670+/-19620, 210,400+/-22900 and 238,000+/-26600 IEQ in UWS-stored (P<0.01), TLM-preloaded, or postloaded organs (P<0.05). Purity (>90%), viability (>95%), and insulin content were not different between groups. Islets from UWS-stored pancreases fragmented extensively, preventing further assessment of in vivo function. Compared with other experimental groups, islets from TLM-preloaded organs were characterized by enhanced basal and stimulated insulin release. Sustained normoglycemia was observed in diabetic nude mice transplanted with islets from TLM-postloaded or unstored pancreases in contrast with transient function in TLM-preloaded islets. CONCLUSIONS: This study demonstrates that significant amounts of intact pig islets can be isolated after prolonged pancreas preservation by TLM. Enzyme administration before TLM preservation decreases islet graft function.     

Serine-protease inhibition during islet isolation increases islet yield from human pancreases with prolonged ischemia.

BACKGROUND: Islet isolation from the pancreatic tissue matrix remains highly variable. Recent evidence suggests that intrinsic human pancreatic proteases, including trypsin, may inhibit effective collagenase enzymatic activity during islet isolation, thereby impairing the isolation success. In this study we have hypothesized that serine protease inhibition applied during pancreatic digestion, could improve yield and/or functional viability of islets isolated from human pancreases. METHODS: Twelve organ donor pancreases with 12.9+/-0.6 hr cold storage (mean+/-SEM) were perfused via their ducts with Liberase-HI enzyme in the presence (n=6) or absence (n=6) of 0.4 mM Pefabloc. All were then gently dissociated and their purified islets separated with Ficoll density gradient centrifugation. RESULTS: Donor-related factors (age, gender, cold storage time, body mass index, and pancreas weight) did not differ significantly between the two experimental groups. Pefabloc supplementation did not affect the digestion time, islets remaining trapped in exocrine tissue, or final islet purity. Islet recovery was increased in the Pefabloc-treated group (mean+/-SEM yield 323.8+/-80.8 x 10(3) islet equivalents vs. 130.8+/-13.6 x 10(3) islet equivalents, P<0.05). Cellular composition, DNA and insulin content, and insulin secretory activity of the isolated islets was similar. CONCLUSIONS: Inhibition of intrinsic protease activity within pancreases after prolonged cold storage improves isolation of viable islets.     

Successful pancreas preservation by a perfluorocarbon-based one-layer method for subsequent pig islet isolation

BACKGROUND: The oxygenation of human pancreas by the two-layer method (TLM) during cold storage was recently established for clinical islet transplantation. Simplification of TLM would facilitate the application of perfluorocarbon (PFC) as a regularly used preservation solution for subsequent islet transplantation. The present study examined whether PFC can be used in a one-layer method (OLM) for long-term pancreas preservation before isolation of adult pig islets. METHODS: Resected pancreases were intraductally flushed with cold University of Wisconsin solution and immediately processed (n=6) or subjected to 7-hour storage by OLM (n=8) or TLM (n=10). Subsequently, pancreases were intraductally distended with collagenase NB-8 supplemented with neutral protease. Isolation and purification were performed as previously described. RESULTS: Compared with unstored pancreases (3,670+/-740 islet equivalents [IEQ]) purified islet yield in TLM-stored organs (2,080+/-290 IEQ, P<0.05) was significantly decreased in contrast with OLM-preserved pancreases (3,110+/-520 IEQ, NS). No differences were observed between groups regarding purity (>90%), trypan-blue exclusion (>95%), adenosine triphosphate content, and mitochondrial viability of islets. Stimulation index during static glucose incubation (20 vs. 2.8 mm) was decreased after storage by TLM (1.81+/-0.20, P<0.05) but not by OLM (2.27+/-0.57) if compared with unstored pancreases (2.47+/-0.36). However, transplantation into diabetic nude mice resulted in sustained normoglycemia of recipients of either group until nephrectomy of graft-bearing kidneys was performed. CONCLUSIONS: This study demonstrates that PFC alone can be used in a one-layer procedure for successful pig-pancreas preservation. This simplification can facilitate the broad application of PFC as pancreas preservation solution without reducing its benefits demonstrated by TLM.     

Improvement in islet yield from a cold-preserved pancreas by pancreatic ductal collagenase distention at the time of harvesting.

This study tried to improve the number of viable islets isolated from a pancreas because a sufficient number cannot be obtained when the organ is preserved in the manner used for pancreas transplantation. The mechanism involved in the decrease in islet yield during preservation was studied to try to develop a better method for islet preparation. First, the integrity of the ductal system was compared between fresh and 6-hr simply preserved (in Hanks' balanced salt solution) rat pancreases. The ductal pressure after ductal injection of HBSS reached a plateau earlier and was significantly lower for the preserved pancreases (0.073 +/- 0.026 min, 410 +/- 17 mmHg, n = 5) than for the fresh ones (0.176 +/- 0.086 min, 561 +/- 103 mmHg, n = 7, P less than 0.05). Second, the extent of pancreatic distention was examined following ductal injection of barium gelatin solution. Solution leakage occurred earlier and distention was less in the preserved pancreas. In addition, the gelatin was found in the capillaries within some islets of the preserved pancreas. These results indicated that the preservation led to a rapid loss of integrity of the ductal system before collagenase injection. We therefore tested the efficacy of ductal collagenase injection at the time of harvesting: 15 ml of 1.0 mg/ml collagenase HBSS was intraductally injected and the pancreas was preserved at 4 degrees C for 2, 4, 6, and 24 hr. The isolation procedure was similar to that used for the fresh pancreas. The yield was significantly better than that of the simply preserved pancreas at 4 hr (241 +/- 22, n = 3, vs. 140 +/- 58, n = 3, P less than 0.05) and at 6 hr (171 +/- 58, n = 14, vs. 32 +/- 33, n = 6, P less than 0.01). These isolated islets were spherical-oval and their viability was confirmed by the ability to reverse STZ-induced diabetes in mice. These results indicated that the integrity of the ductal system, which is necessary for distention of the whole pancreas, was lost during preservation. To solve this problem, ductal collagenase injection should be done at the time of pancreas harvesting and then followed by simple preservation. This method is recommended to obtain viable islets from a preserved pancreas.     

Portal Venous Oxygen Persufflation of the Donation after Cardiac Death pancreas in a rat model is superior to static cold storage and hypothermic machine perfusion

Success of clinical pancreatic islet transplantation depends on the mass of viable islets transplanted and the proportion of transplanted islets that survive early ischaemia reperfusion injury. Novel pancreas preservation techniques to improve islet preservation and viability can increase the utilization of donation after cardiac death donor pancreases for islet transplantation. Rat pancreases were retrieved after 30 min of warm ischaemia and preserved by static cold storage, hypothermic machine perfusion or retrograde portal venous oxygen persufflation for 6 h. They underwent collagenase digestion and density gradient separation to isolate islets. The yield, viability, morphology were compared. In vitro function of isolated islets was compared using glucose stimulated insulin secretion test. Portal venous oxygen persufflation improved the islet yield, viability and morphology as compared to static cold storage. The percentage of pancreases with good in vitro function (stimulation index > 1.0) was also higher after oxygen persufflation as compared to static cold storage. Retrograde portal venous oxygen persufflation of donation after cardiac death donor rat pancreases has the potential to improve islet yield.     

Comparison of automated and manual methods for islet isolation

The authors used the principal features of a collagenase perfusion technique and an automated dissociation technique to determine if islets could be isolated from the large mammal pancreas and to compare the effects of the two methods on isolated islets. The pancreases of 16 dogs were cannulated and perfused with collagenase at 4 degrees C, then warmed to 37 degrees C. Group 1 (eight) pancreases were perfused at 37 degrees C until digested, then dissociated manually by teasing and trituration. Group 2 (eight) pancreases were transferred to a closed chamber for continued collagenase digestion and dissociation at 37 degrees C. Islets were purified using identical Ficoll gradients. Aliquots were stained with dithizone and evaluated for number, size and purity. Total islet volume was calculated. Group 2 pancreases were thoroughly digested leaving only a few residual ducts, but undigested fragments persisted in group 1 pancreases. Islet size was similar in both groups. There was a greater islet volume before and after Ficoll purification in group 2, but the difference was not significant. Purity was greater than 90% in both groups. Perifusion with 28 mM glucose elicited a biphasic insulin release from islets in both groups. The data show that the combined protocol enables mass isolation of purified islets from the canine pancreas. Compared with the manual technique, the automated protocol for pancreas dissociation tends to improve the yield of islets without compromising islet size and viability. It provides the advantages of a closed system with increased control over the extent of collagenase digestion.     

Studies of the isolation and viability of human islets of Langerhans

Pancreas obtained from 34 adult human cadaver organ donors was divided into proximal and distal segments, and the duct to each segment was cannulated. Collagenase was injected into the proximal duct of 7 glands and into the distal duct of 7 others; the duct of the opposite segment was perfused with collagenase. The pancreas was then dispersed by teasing, trituration, and passage through filters. Perfused proximal and distal segments released 1461 +/- 287 and 2728 +/- 797 islets/g (+/- SEM) versus 710 +/- 149 (P less than 0.05) and 1950 +/- 636 after injection. Twenty other pancreases were perfused with collagenase warmed rapidly to 39 degrees C (n = 4) or warmed slowly to 37 degrees C (n = 6) or 39 degrees C (n = 10): the yield was 1625 +/- 632, 1320 +/- 116, and 2009 +/- 277 islets/g respectively. Total yields from the latter were 76 X 10(3) large (greater than 100 microns) and 85 X 10(3) small (less than 100 microns) islets with recoveries of 61% and 42%, respectively, after Ficoll density gradient purification. Histology showed highly purified islets. Perifusion with glucose elicited a biphasic release of insulin with the mean response (microU/islet/min) rising to a first peak of 0.5 and constant second phase secretion of 0.25, followed by a return to baseline. Reduced response was observed for islets from pancreas stored greater than 6 hr and tissue obtained from multiple centers. Less insulin was produced by freshly isolated islets, islets less than 100 microns, and after Ficoll separation. Secretion was similar for islets derived from proximal or distal segments. Perfusion of collagenase via the ducts of human pancreas improves islet isolation and Ficoll gradient separation yields highly purified islets. Important factors influencing insulin secretion are the source of donor tissue, cold storage of pancreas, Ficoll purification, islet size, and tissue culture.     

Experimental pancreatic preservation prior to islet isolation and transplantation

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amylase levels in preservation solutions as a marker of exocrine tissue injury and as a prognostic factor for pancreatic islet isolation

Occurrence of primary graft nonfunction of pancreatic islets demands research for new methods of organ preservation during cold ischemia conditions. Digestive enzymes released during preservation injure the islets for subsequent rewarming and islet isolation processes. The aim of our study was to assess the amylase level in preservation solution as a marker of exocrine tissue injury, allowing the prognosis of islet yield and viability. The experiments undertaken on rats used three commercially available preservation solutions: ViaSpan (UW); Custodiol (HTK); and Euro-Collins (EC). After 180 minutes of cold ischemia, the highest islet recovery was observed among pancreata stored in UW solution (508 +/- 139 vs HTK 344 +/- 103; P <.05 vs EC 322 +/- 113; P <.05). These islets also revealed the highest insulin stimulation index in glucose static tests (1.19 +/- 0.30 vs HTK, 0.87 +/- 0.43; P <.01, vs EC.25 +/-.06; P <.001). The highest amylase level in the preservation solution was associated with a decreased yield of islets during the isolation process and lowest insulin stimulation index (increasing 139 +/- 18% for EC, 108 +/- 12% for HTK; P <.05 vs 87 +/- 10% for UW; P <.05). Our data strongly suggest, that the dynamic of amylase release during pancreas preservation at 4 degrees C correlates with a reduced number and viability of isolated islets. These results suggest that measurement of amylase levels after pancreas preservation may have potential clinical application as a marker to evaluate pancreatic tissue injury.     

Lessons from in vitro perifusion of pancreatic islets isolated from 80 human pancreases

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve delta-AUC alpha)], and the correlation among them was determined. SI positively correlated with delta-AUC alpha (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = -0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01).