Cell Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA.
Cell Transplant. 2010;19(12):1537-46. doi: 10.3727/096368910X516600. Epub 2010 Aug 17.
Islet isolation and purification using a continuous density gradient may reduce the volume of tissue necessary for implantation into patients, therefore minimizing the risks associated with intraportal infusion in islet transplantation. On the other hand, the purification procedure might result in a decreased number of islets recovered due to various stresses such as exposure to cytokine/chemokine. While a Ficoll-based density gradient has been widely used in purification for clinical trials, purification with iodixanol (OptiPrep) has been recently reported in islet transplant series with successful clinical outcomes. The aim of the current study was to compare the effects of the purification method using OptiPrep-based and Ficoll-based density gradients. Human islet isolations were performed using a modified automated method. After the digestion phase, pre-purification digests were divided into two groups and purified using a semiautomated cell processor with either a continuous Ficoll- or OptiPrep-based density gradient. The quantity, purity, viability, and cellular composition of islet preparations from each group were assessed. Cytokine/chemokine and tissue factor production from islet preparations after 48-h culture were also measured. Although islet purity, post-purification IEQ, islet recovery rate, FDA/PI, and fractional β-cell viability were comparable, β-cell mass after 48-h culture significantly improved in the OptiPrep group when compared to the Ficoll group. The production of cytokine/chemokine including IL-1β, TNF-α, IFN-γ, IL-6, IL-8, MIP-1β, MCP-1, and RANTES but not tissue factor from the OptiPrep group was significantly lower during 48-h culture after isolation. Each preparation contained the similar number of ductal cells and macrophages. Endotoxin level in both gradient medium was also comparable. The purification method using OptiPrep gradient media significantly reduced cytokine/chemokine production but not tissue factor from human islet preparations and improved β-cell survival during pretransplant culture. Our results suggest that the purification method using OptiPrep gradient media may be of assistance in increasing successful islet transplantation.
使用连续密度梯度进行胰岛分离和纯化可以减少植入患者所需的组织量，从而最大限度地降低胰岛移植中门静脉内输注相关的风险。另一方面，由于暴露于细胞因子/趋化因子等各种应激，纯化过程可能导致回收的胰岛数量减少。虽然基于菲可的密度梯度已广泛用于临床试验中的纯化，但最近在胰岛移植系列中报道了使用碘克沙醇（OptiPrep）进行纯化，取得了成功的临床结果。本研究的目的是比较使用基于 OptiPrep 和基于菲可的密度梯度的纯化方法的效果。使用改良的自动化方法进行人胰岛分离。消化阶段后，将预纯化消化物分为两组，并使用半自动细胞处理器，使用连续的基于菲可或 OptiPrep 的密度梯度进行纯化。评估每组胰岛制剂的数量、纯度、活力和细胞组成。还测量了胰岛制剂在 48 小时培养后产生细胞因子/趋化因子和组织因子的情况。尽管胰岛纯度、纯化后 IEQ、胰岛回收率、FDA/PI 和β细胞活力分数相似，但与菲可组相比，OptiPrep 组在 48 小时培养后的β细胞质量显著提高。与菲可组相比，OptiPrep 组在分离后 48 小时培养过程中产生的细胞因子/趋化因子（包括 IL-1β、TNF-α、IFN-γ、IL-6、IL-8、MIP-1β、MCP-1 和 RANTES）但不包括组织因子的产量显著降低。每个制剂都含有相似数量的导管细胞和巨噬细胞。两种梯度培养基中的内毒素水平也相当。OptiPrep 梯度培养基的纯化方法可显著降低人胰岛制剂中细胞因子/趋化因子的产生，但不降低组织因子，同时在移植前培养过程中提高β细胞的存活率。我们的结果表明，OptiPrep 梯度培养基的纯化方法可能有助于增加成功的胰岛移植。
