CULTURE OF HUMAN KIDNEY PROXIMAL TUBULAR CELLS - THE EFFECT OF VARIOUS DETACHMENT PROCEDURES ON VIABILITY AND DEGREE OF CELL DETACHMENT
CULTURE OF HUMAN KIDNEY PROXIMAL TUBULAR CELLS - THE EFFECT OF VARIOUS DETACHMENT PROCEDURES ON VIABILITY AND DEGREE OF CELL DETACHMENT
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DOI：
10.1159/000154771
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发表时间：
1995-09-01
影响因子：
--
通讯作者：
HENKE, W
中科院分区：
文献类型：
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作者：
JUNG, K;HAMPEL, G;HENKE, W
肾近端小管细胞培养已成为研究中广泛使用的工具。由于管状上皮细胞培养形成单层，在实验前必须从培养瓶中分离出来，本研究的目的是评估在将单层转化为单细胞悬液的过程中细胞可能受到的损伤。研究了各种分离方法（刮取、乙二胺四乙酸（EDTA）孵育、胰蛋白酶/EDTA孵育、单独使用胶原酶以及胶原酶和EDTA共同孵育，以及使用各种胶原酶制剂），以获得有活力、未受损的人类细胞。乳酸脱氢酶活性的测定被用作评估细胞活力/损伤的指标。将各种分离方法对人类细胞乳酸脱氢酶释放的影响与对肾细胞系MDCK和LLC - PK1的影响进行了比较。与人类细胞相比，连续细胞系在分离后释放的乳酸脱氢酶活性显著较低。刮取人类细胞造成的损伤程度最高，显示约90%的总乳酸脱氢酶释放，而胶原酶的乳酸脱氢酶释放量在8% - 25%之间，取决于所使用的胶原酶制剂。建议在培养的人肾近端小管细胞分离中使用胶原酶。来自宝灵曼（Boehringer Mannheim）的胶原酶P制剂（200毫克/100毫升；在37℃下孵育10分钟）似乎特别合适。
Culture of renal proximal tubular cells has become a widely used tool in research. As tubular epithelial cell cultures form monolayers which have to be detached from the culture flasks prior to experiments, it was the objective of this study to evaluate possible damage suffered by the cells during the procedure of transforming monolayers into single-cell suspensions. Various detachment procedures (scraping, EDTA incubation, trypsin/EDTA incubation, incubation with collagenase alone and collagenase and EDTA together, and the use of various collagenase preparations) were studied in order to obtain viable, non-damaged human cells. The measurement of lactate dehydrogenase activity was used as an indicator in the assessment of cell viability/damage. The effect of the various methods of detachment on the release of lactate dehydrogenase from human cells was compared with that from the renal cell lines, MDCK and LLC-PK1. The released lactate dehydrogenase activity following the detachment was significantly lower in the continuous cell lines as compared to the human cells. Scraping of the human cells caused the highest degree of damage, showing a release of about 90% of the total lactate dehydrogenase, whereas collagenases showed a lactate dehydrogenase release between 8 and 25% depending on the collagenase preparation used. The use of collagenase in the detachment of cultured human kidney proximal tubular cells is recommended. The collagenase P preparation from Boehringer Mannheim (200 mg/100 ml; incubation at 37 degrees C for 10 min) appears to be particularly suitable.