Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation.
Islet preparation purity is overestimated, and less pure fractions have lower post-culture viability before clinical allotransplantation.
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DOI：
10.1016/j.transproceed.2014.06.011
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发表时间：
2014-07
影响因子：
0.9
通讯作者：
Papas KK
中科院分区：
文献类型：
--
作者：
Kitzmann JP;Karatzas T;Mueller KR;Avgoustiniatos ES;Gruessner AC;Balamurugan AN;Bellin MD;Hering BJ;Papas KK
使用离体胰岛同种异体移植（IT）替代β细胞是治疗1型糖尿病伴低血糖无意识的新兴疗法。目前的标准方案要求在IT之前进行36-72小时的培养。我们检查了13个具有≥2个纯度级分的临床胰岛制备物，以确定培养物对活力的影响。在标准胰岛分离和纯化后，将纯胰岛级分在37°C和5%C02下放置12-24小时，随后移至22°C，而较不纯的级分在22°C下培养整个持续时间。培养物密度目标为100-200胰岛当量（IEQ）/cm 2的范围，针对纯度进行调整。评估胰岛的纯度（双硫腙染色）、数量（沉淀体积和DNA）和活力（标准化为DNA含量[OCR/DNA]和膜完整性的耗氧率）。结果表明，纯度被高估，特别是在纯度较低的馏分。观察到的颗粒尺寸明显大于预期，并且使用dsDNA测定（如果可用）定量组织量，证明了这一点。与纯的相比，较不纯的级分显示出显著较低的OCR/DNA和膜完整性。2种纯度组分之间的活力差异可能是由于多种原因，包括缺氧、营养缺乏、毒性代谢产物蓄积和/或腺泡组织杂质释放的蛋白水解酶（未被人血清白蛋白中和）。目前的临床胰岛培养方案应进一步检查，特别是对于纯度较低的部分，以确保移植前的活力维持。即使相对较小，存活率的差异也是重要的，因为引入受体的死亡或垂死组织的量可能会显著增加，特别是对于纯度较低的制剂。
Replacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36–72-hour culture period beforeIT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. After standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C,whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IEQ)/cm2 adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content[OCR/DNA] and membrane integrity). Results indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin. Current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability beforetransplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.