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0996. Improventment of method primary cultivation and identification of rat pulmonary microvascular endothelial cells
Intensive Care Medicine Experimental volume 2, Article number: P81 (2014)
Introduction
Pulmonary microvascular endothelial cell(PMVEC) is a very important tool to study acute respiratory distress syndrome. Several methods for in vitro cultivation of PMVEC have been established, but all the methods damage PMVECs either by use of proteolytic emzymes or by mechanical trauma. This study was to improve the method for primary cultivation of rat pulmonary microvascular endothelial cells and identify the primary cultivated cells.
Objectives
To improve the method for primary cultivation of rat pulmonary microvascular endothelial cells and identify the primary cultivated cells
Methods
Pulmonary microvascular endothelial cells were derived from peripheral lung tissue of Sprague-Dawley rats. Primary cultivation method was improved from the procedures of animal selection, lung tissue perfusion, tissue piece pasting, culture medium selection and so on. Inverted microscope was used to observe morphological characteristics of pulmonary microvascular endothelial cells. Immunohistochemical staining for expression of VIII-related antigen and CD31, and observed by fluorescence microscope for binding with lectin from BSI (FITC-BSI binding away ) to identified pulmonary microvascular endothelial cells. The cell purity was detected with flow cytometry.
Results
The PMVECs were exhibited as polygon and presented typical cobblestone-like morphology after fusion to monolayer with contact inhibition. PMVECs turned to be fusiform and presented a swirling or aggregate growth pattern after transfer of culture. Immunohistochemical staining revealed that the expression of CD31 and factor VIII-related antigen was positive. Besides, there were positive findings for FITC-BSI assay. The vitality and growth rate of PMVECs were in good condition. The cell purity was 93.2% with the improvement method of primary cultivation.
Conclusions
The improved method for primary cultivation is easy to handle, with favorable repeatability and success rate. PMVECs obtained with this method will display lower contamination, higher purity, faster growth and better status.
References
Magee JC, Stone AE, Oldham KT, et al.: Isolation, culture, and characterization of rat lung microvascular endothelial cells. Am J Physiol 1994, 267: L433–41.
Chen SF, Fei X, Li SH: A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries. Microvasc Res 1995, 50: 119–28. 10.1006/mvre.1995.1044
Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells Am J Physiol Heart Circ Physiol 2003, 284: H215–24.
Grant acknowledgment
This study was supported by grant from the National Natural Science Foundation of china (81071536) and Youth Fund of the National Natural Science Foundation of china (81201452).
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Yong-Jun, L., Jie, M., Ping-Ping, W. et al. 0996. Improventment of method primary cultivation and identification of rat pulmonary microvascular endothelial cells. ICMx 2 (Suppl 1), P81 (2014). https://doi.org/10.1186/2197-425X-2-S1-P81
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DOI: https://doi.org/10.1186/2197-425X-2-S1-P81