Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas.
Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas.
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DOI：
10.7555/jbr.30.20160033
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发表时间：
2016-11
影响因子：
2.3
通讯作者：
Miao Y
中科院分区：
文献类型：
--
作者：
Zhao L;Cai B;Lu Z;Tian L;Guo S;Wu P;Qian D;Xu Q;Jiang K;Miao Y
胰腺星状细胞（PSC）的原代培养仍然是体外研究的重要基础。然而，目前缺乏分离大量PSC的有效方法。我们报告了一种从正常大鼠胰腺和人胰腺导管腺癌（PDAC）组织中分离PSC的新方法。在大鼠麻醉和剖腹手术后，将钝插管通过十二指肠的反肠系膜侧插入胰管，并将胰腺缓慢输注酶溶液，直到所有小叶完全分散。然后将胰腺预孵育，精细切碎并孵育以获得细胞悬液。将细胞悬液过滤、洗涤并用Nycodenz溶液进行梯度离心后获得PSC。用锋利的刀片将新鲜的人PDAC组织切成1×1×1 mm 3的立方体。将组织块置于培养板底部，新鲜血浆（来自同一患者的EDTA抗凝血浆，与CaCl 2混合）洒在样本周围。在适当条件下培养5-10天后，收获活化的PSC。与多次注射技术相比，导管内灌注酶溶液简化了大鼠PSC分离的过程，并且改进的生长方法显着缩短了活化细胞的生长时间。我们对PSC分离方法的改进显著提高了分离效率，缩短了培养周期，从而为PSC相关研究提供了便利。
Primary cultures of pancreatic stellate cells (PSCs) remain an important basis for in vitro study. However, effective methods for isolating abundant PSCs are currently lacking. We report on a novel approach to isolating PSCs from normal rat pancreases and human pancreatic ductal adenocarcinoma (PDAC) tissue. After anaesthesia and laparotomy of the rat, a blunt cannula was inserted into the pancreatic duct through the anti-mesentery side of the duodenum, and the pancreas was slowly infused with an enzyme solution until all lobules were fully dispersed. The pancreas was then pre-incubated, finely minced and incubated to procure a cell suspension. PSCs were obtained after the cell suspension was filtered, washed and subject to gradient centrifugation with Nycodenz solution. Fresh human PDAC tissue was finely minced into 1×1×1 mm3 cubes with sharp blades. Tissue blocks were placed at the bottom of a culture plate with fresh plasma (EDTA-anti-coagulated plasma from the same patient, mixed with CaCl2) sprinkled around the sample. After culture for 5–10 days under appropriate conditions, activated PSCs were harvested. An intraductal perfusion of an enzyme solution simplified the procedure of isolation of rat PSCs, as compared with the multiple injections technique, and a modified outgrowth method significantly shortened the outgrowth time of the activated cells. Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period, thus facilitating future PSC-related research.