Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa
Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa
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DOI：
10.1016/j.jim.2012.12.006
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发表时间：
2013-03-01
影响因子：
2.2
通讯作者：
Esteve, Maria
中科院分区：
文献类型：
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作者：
Carrasco, Anna;Mane, Josep;Esteve, Maria
理想的免疫细胞分离方法应该提供最大的细胞产量而不干扰功能特性。与手术样本相比，肠道内窥镜活检允许研究所有疾病阶段，但具有可用组织量最小的缺点，使得方案优化是强制性的。我们比较了第一次两种方法的分离结肠上皮和五种方法的固有层细胞分离结肠活检标本（机械，酶和器官培养协议）。流式细胞术检测淋巴细胞数量、活性和表型（CD 45+、CD 103+、CD 3+、CD 4+、CD 8+、CD 19+、CD 16 -56 +）。两种上皮分离方案均未实现适当的上皮分离，但高强度离子螯合法更准确。无表型修饰的固有层淋巴细胞的最大细胞产率通过过夜平滑酶消化获得。与低酶法相比，高剂量胶原酶孵育导致固有层CD 4+淋巴细胞显著减少（p = 0.004）。机械培养和活检培养分别因细胞产量低、表型改变和污染率高而不可取。(C)2013爱思唯尔有限公司版权所有。
An ideal method of immune cell isolation should provide maximum cell yield without disturbing functional properties. Intestinal endoscopic biopsies, in contrast to surgical samples, allow the study of all disease stages but have the drawback of a minimum amount of tissue available, making protocol optimization mandatory. We compared for the first time two methods of separation of colonic epithelium and five methods of lamina propria cell isolation for colonic biopsy specimens (mechanical, enzymatic and organ culture protocols). Lymphocyte number, viability and phenotype (CD45 +, CD103 +, CD3 +, CD4 +, CD8 +, CD19 +, CD16-56 +) were analyzed by flow cytometiy. Neither of the two epithelial detachment protocols achieved proper epithelial separation, though the high intensity ion chelation method was more accurate. Maximum cell yield of lamina propria lymphocytes without phenotypic modification was obtained with overnight smooth enzymatic digestion. High dose collagenase incubation caused a marked decrease in CD4 + lymphocytes of the lamina propria as compared to low enzymatic method (p = 0.004). Mechanical and biopsy culture are not advisable methods because of the low cell yield, and phenotypic alterations and high contamination rate, respectively. (C) 2013 Elsevier B.V. All rights reserved.