Objective To develop a methodology for direct isolation of intact infiltrating lymphocytes from fresh renal cancer tissue,in the attempt to evaluate their immunological functions.Methods Freshly collected tissues were mechanically smashed and homogenized into a single cell suspension.The,tumor infiltrating lymphocytes were isolated through Ficoll density gradient centrifugation.Meanwhile,CD3-positive T cells were sorted using EasySep? human CD3 positive selection Kit.Results The simple method of rapid isolation of tumor infiltrating lymphocytes from fresh renal cancer tissue was successfully established.Seven tissues were successfully separated.The results of flow cytometry showed that,CD3 + T cells accounted for (24.10 ± 17.60) ％,CD4 + and CD8 + T cells account for (37.69 ± 9.83) ％and (37.86 ± 10.47)％ respectively.Through magnetic bead purificaiton,the purity of CD3 + T cells was 91.6％,which was suitable for subsequent immunological and molecular analysis.Conclusions Compared with enzymatic digestion,this approach can protect the surface receptors of cell membrane,and indicate the infiltration of immunocytes in the tumor.The new method is beneficial to provide complete tumor immune microenvironment information.In addition,homogenates prepared using the gentle dissociator show time-saving and increased viability.%目的 建立一种快速简便的不经过消化酶处理而直接分离、纯化新鲜肾癌组织浸润淋巴细胞的新方法.方法 手术切除的新鲜肾癌组织经过机械剪碎、温和匀浆处理制备成单细胞悬液,再通过Ficoll密度梯度离心分离肿瘤浸润淋巴细胞,密度梯度分离后经CD3磁珠分选纯化.结果 成功建立了快速从新鲜肾癌组织分离肿瘤浸润淋巴细胞的新方法,成功完成7例组织分离.分析结果显示CD3+T细胞占(24.10±17.60)％,CD4+和CD8+细胞分别占(37.69 ±9.83)％、(37.86±10.47)％;进一步经磁珠纯化后CD3+T细胞纯度达91.6％,适用于后续分析.结论 本法优势在于:①与酶消化法相比,有利于保护细胞膜表面受体,最大程度全面反映肿瘤组织中淋巴细胞亚群的浸润情况;②短暂、快速、温和匀浆处理,操作时间短,有利于维持细胞的高活力.
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