Culturing Pancreatic Islets in Microfluidic Flow Enhances Morphology of the Associated Endothelial Cells
Culturing Pancreatic Islets in Microfluidic Flow Enhances Morphology of the Associated Endothelial Cells
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DOI：
10.1371/journal.pone.0024904
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发表时间：
2011-09-22
期刊：
影响因子：
3.7
通讯作者：
Rocheleau, Jonathan V.
中科院分区：
文献类型：
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作者：
Sankar, Krishana S.;Green, Brenda J.;Rocheleau, Jonathan V.
胰岛在体内高度血管化，每种胰岛素分泌的β细胞与至少一个内皮细胞（EC）相关。这种结构在隔离后立即维持；然而，在培养过程中，EC 会慢慢退化，失去密度和分支形态。我们假设这种恶化发生在由于组织内介质扩散有限而没有血流的情况下。为了改善组织内的介质交换，我们创建了一种微流体装置来以一定流速培养胰岛。与传统培养的胰岛相比，在该装置中培养来自 C57BL6 小鼠的胰岛，培养基流量在 1 至 7 ml/24 小时之间，导致 EC 密度和连接长度增加一倍。含有荧光右旋糖酐的培养基以依赖于流速的方式到达装置中的胰岛中心，这与改善的渗透一致。我们还使用无血清培养基观察到 EC 形态的恶化，通过添加牛血清白蛋白（一种已知的在组织中扩散有限的抗凋亡信号）来挽救 EC 形态的恶化。我们进一步检查了流动对β细胞的影响，显示出胰岛周围流体剪切应力最大的细胞对葡萄糖刺激的Ca(2+)反应减弱。然而，我们观察到正常的双光子 NAD(P) H 反应和胰岛其余部分的胰岛素分泌。这些数据揭示胰岛 EC 形态的恶化部分是由于组织内血清白蛋白的扩散受限。这些数据进一步揭示了微流体装置作为独特的平台，通过引入细胞间流动来克服培养基成分的有限扩散来优化胰岛培养。
Pancreatic islets are heavily vascularized in vivo with each insulin secreting beta-cell associated with at least one endothelial cell (EC). This structure is maintained immediately post-isolation; however, in culture the ECs slowly deteriorate, losing density and branched morphology. We postulate that this deterioration occurs in the absence of blood flow due to limited diffusion of media inside the tissue. To improve exchange of media inside the tissue, we created a microfluidic device to culture islets in a range of flow-rates. Culturing the islets from C57BL6 mice in this device with media flowing between 1 and 7 ml/24 hr resulted in twice the EC-density and - connected length compared to classically cultured islets. Media containing fluorescent dextran reached the center of islets in the device in a flow-rate-dependant manner consistent with improved penetration. We also observed deterioration of EC morphology using serum free media that was rescued by addition of bovine serum albumin, a known anti-apoptotic signal with limited diffusion in tissue. We further examined the effect of flow on beta-cells showing dampened glucose-stimulated Ca(2+)-response from cells at the periphery of the islet where fluid shear-stress is greatest. However, we observed normal two-photon NAD(P) H response and insulin secretion from the remainder of the islet. These data reveal the deterioration of islet EC-morphology is in part due to restricted diffusion of serum albumin within the tissue. These data further reveal microfluidic devices as unique platforms to optimize islet culture by introducing intercellular flow to overcome the restricted diffusion of media components.