[A method for primary culture of pulmonary microvascular endothelial cells].

OBJECTIVE

To explore a simple and practical method of primarily culturing rat pulmonary microvascular endothelial cells (PMECs) in vitro, and observe the cell growth status and identify the PMECs.

METHODS

Wistar rats (n=40, aged 4-5 weeks) were sacrificed to take the lung tissue. After removal of pleura, the peripheral lung tissues were cut into pieces (1 mm(3)) in aseptic condition. The endothelial cells were cultured in the DMEM medium containing heparin sodium and in the RPMI1640 medium supplemented with special additives or not, respectively. Cell growth and morphology was observed under an inverted microscope. The expression of CD31 in cells was detected by immunofluorescence staining.

RESULTS

After incubation for 24 hours, PMECs in the medium containing special additives were the most in number and purity compared with the other two culture systems. At 24 hours, endothelial cells migrated from the lung tissue, and at 14 days, the cells aggregated and grew obviously, exhibiting a polygon shape, being tightly arranged and paving the base of Petri dish. After sub-culturing, the cells spread much more and most cells became spindle shaped, which showed a tendency of endothelial cell angiogenesis in vitro. CD31 was positive in immunofluorescence staining.

CONCLUSION

The adherent culture method of tissue explants in the medium added by the special additives was proved to a good method to obtain a high-purity rat PMECs in vitro.