An improved method for primary culture of renal epithelial cells
- Feng, Zhou
- Xian-jin, Du
- Chang-yuan, Dong
- Jie, Zhang
Abstract
BACKGROUND:
Cell strain of epithelial cells is focus of domestic studies. However, long period of ex vivo may contribute to cell variation. Primary culture is similar to in vivo status, and is hopeful to provide sources for renal epithelial cell in renal tissue engineering.
OBJECTIVE:
To explore the condition and approach for the culture of primary renal epithelial cell, so as to establish an effective method for renal epithelial cell culture.
DESIGN, TIME AND SETTING:
Single sample observation was performed at Laboratory of Molecular Virology and Cancer, State Key Laboratory of Virology, Wuhan University between December 2007 and March 2008.
MATERIALS:
Twelve neonatal SPF male SD rats at 2-4 days old were used.
METHODS:
Bilateral kidney tissues were harvested from SD rats, cut into pieces of 1 mm×1 mm×1 mm, digested with 2.5 g/L pancreatic enzyme and 0.4 g/L EDTA at 37 °C for 30 minutes. The cells were filtered by 200-mesh steel net to remove non-digested tissues and cell groups. The cell suspension was centrifuged at 1 000 r/min for 5 minutes, and collected cells were cultured in DMEM (pH=7.2) to prepare cell suspension, seeded in culture flask at the density of 4 × 108 /L, and incubated in CO2 at 37 °C. In addition, cell suspension was seeded in a culture flask and cultured for 30 minutes. Cell attachment was observed. Cell culture solution and unattached cells were placed to another flask for continuous culture. This process was repeated to purify renal epithelial cells.
MAIN OUTCOME MEASURES:
The survival rate of renal epithelial cell was detected by 4 g/L trypan-blue staining. Those without staining were survival cells. The survival rate, morphological changes of renal epithelial cell and the rate of monolayer adherence were observed.
RESULTS:
The survival rate of the renal epithelial cell was 95.20%, and the adherence rate was about 60% after 4 hours and 85% after 12 hours. After 24 hours, cells had pseudopodia, and exhibited fusiform or polygon-shaped with one or two nuclei. Cells rapidly grew, and formed cell cluster within 3-4 days, and lamellar finally.
CONCLUSION:
This method is an effective one to cultivate renal epithelial cell with high survival rate.
Figure
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