Heuff G, Steenbergen J J, Van de Loosdrecht A A, Sirovich I, Dijkstra C D, Meyer S, Beelen R H
Department of Surgical Oncology, University Hospital, Free University, Amsterdam, Netherlands.
J Immunol Methods. 1993 Feb 26;159(1-2):115-23. doi: 10.1016/0022-1759(93)90148-z.
Kupffer cell (KC)-mediated cytotoxicity against tumor cells is of interest, since the liver is a major site of metastatic growth of primary colorectal cancer. KC isolation methods from rat livers, to study the tumoricidal properties of these cells, are based on perfusion of the liver and are therefore not suitable for human KC isolation from liver biopsies. In view of application to isolate KC from small wedge human liver biopsies, we have developed an isolation procedure for rat KC that does not require perfusion techniques. Liver tissue fragments were incubated with pronase with continuous pH registration and neutralization. KC were subsequently separated from other non-parenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. KC and other non-parenchymal cells were identified by immunophenotyping with a cytoplasmic monoclonal antibody ED1 and by ultrastructural analysis. About 3 x 10(6) KC per gram liver were isolated with a final purity of > 95% without loss of viability. To ensure that functionally competent KC were isolated, we assayed cytotoxicity against CC531 tumor cells in a recent developed cell-mediated MTT assay. Maximum cytotoxicity of KC was approximately 40% at an effector to target ratio of 10. In conclusion our approach seems to be a useful and simple method to isolate KC with good functional properties from rat livers, without the need for perfusion techniques.
由于肝脏是原发性结直肠癌转移生长的主要部位，库普弗细胞（KC）介导的对肿瘤细胞的细胞毒性备受关注。从大鼠肝脏中分离KC以研究这些细胞的杀肿瘤特性的方法基于肝脏灌注，因此不适用于从人肝活检组织中分离人KC。鉴于要从人肝小楔形活检组织中分离KC的应用需求，我们开发了一种无需灌注技术的大鼠KC分离程序。将肝组织碎片与链霉蛋白酶一起孵育，并持续记录pH值及进行中和。随后通过 Nycodenz 梯度离心将KC与其他非实质细胞分离，并通过逆流离心淘析进行纯化。通过用细胞质单克隆抗体ED1进行免疫表型分析和超微结构分析来鉴定KC和其他非实质细胞。每克肝脏可分离出约3×10⁶个KC，最终纯度>95%，且不失活力。为确保分离出功能正常的KC，我们在最近开发的细胞介导的MTT试验中检测了对CC531肿瘤细胞的细胞毒性。在效应细胞与靶细胞比例为10时，KC的最大细胞毒性约为40%。总之，我们的方法似乎是一种有用且简单的方法，无需灌注技术即可从大鼠肝脏中分离出具有良好功能特性的KC。
