Primary Cell Preparation of Human Renal Tubular Cells for Transcriptome Analysis

Abstract

We initiated a toxicogenomics project using Affymetrix GeneChip^® HG-U133A and HG-U133B arrays harboring 45,000 probe sets representing more than 39,000 transcripts to analyze gene expression in primary cultures of human cells after exposure to chemicals that cause tissue toxicity. In order to assess the quality of the samples studied, we prepared primary human renal cortical cell cultures from surgically resected human kidney and evaluated the origin of the cells and the effects of cryopreservation. We analyzed the primary cultures using GeneChip and compared their expression patterns with those in the Novartis Research Foundation (GNF) Gene Expression Database. The comparison with the GNF database revealed that the gene expression pattern of the cultured cells was compatible with kidney cells, indicating that we had purified human renal cortical cells. Due to the purification procedure, the primary cultured cells could be a mixture of renal components; however, we identified the major population as renal proximal tubule cells by assessing gamma-GTP activity and Glut2 antigen expression. We compared gene expression in the cells before and after cryopreservation. The expression of 567 selected housekeeping genes was unchanged by cryopreservation (Pearson's correlation coefficient r = 0.980; p < 0.0001). The analysis of more than 39,000 transcripts after normalization revealed no significant changes in expression. These results indicate that our method is satisfactory for obtaining adequate primary cell cultures of renal origin and that gene expression was not significantly changed by cryopreservation.

Keywords :

• Cryopreservation
• GeneChip
• Genomics
• Primary Culture
• Renal Tubular Cell
• Transcriptome

This work was supported by a Research on Advanced Medical Technology grant from the Ministry of Health, Labor, and Welfare of Japan (No. 14190301). The human tissue experiments in this research were reviewed by the Bioethics Committee of Jichi Medical School and the Bioethics Committee for Human Gene Analysis and were carried out under the control of the Guidelines of the Government of Japan (http://www2.ncc.go.jp/elsi/).