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High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways.

Nistri S, Mazzetti L, Failli P, Bani D - Biol Proced Online (2002)

Bottom Line: We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase.This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation.The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Italy. V. le G. Pieraccini, 6, I-50139 Florence. Italy.; Department of Preclinical and Clinical Pharmacology, University of Florence, Italy. V. le G. Pieraccini, 6, I-50139 Florence. Italy. daniele.bani@unifi.it

ABSTRACT
We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.

No MeSH data available.


Related in: MedlinePlus

Phase-contrast microscopy of putative RCE cells adherent to the floor of a 25 cm2 flask 4 h after seeding. Most of them appear as small-sized, round-shaped bodies. A few cells already show a more flattened shape (arrow). Bar = 100 μm
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Figure 2: Phase-contrast microscopy of putative RCE cells adherent to the floor of a 25 cm2 flask 4 h after seeding. Most of them appear as small-sized, round-shaped bodies. A few cells already show a more flattened shape (arrow). Bar = 100 μm

Mentions: Many viable cardiomyocytes, characterized by typical rod-like shape and rhythmic contractions, can be usually seen. Upon trypsin digestion of the supernatant and subsequent seeding in growth medium into 25 cm2 flasks for 4 hours, most RCE cells appear as small-sized, round-shaped bodies adherent to the flask floor (Fig. 2).


High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways.

Nistri S, Mazzetti L, Failli P, Bani D - Biol Proced Online (2002)

Phase-contrast microscopy of putative RCE cells adherent to the floor of a 25 cm2 flask 4 h after seeding. Most of them appear as small-sized, round-shaped bodies. A few cells already show a more flattened shape (arrow). Bar = 100 μm
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC145554&req=5

Figure 2: Phase-contrast microscopy of putative RCE cells adherent to the floor of a 25 cm2 flask 4 h after seeding. Most of them appear as small-sized, round-shaped bodies. A few cells already show a more flattened shape (arrow). Bar = 100 μm
Mentions: Many viable cardiomyocytes, characterized by typical rod-like shape and rhythmic contractions, can be usually seen. Upon trypsin digestion of the supernatant and subsequent seeding in growth medium into 25 cm2 flasks for 4 hours, most RCE cells appear as small-sized, round-shaped bodies adherent to the flask floor (Fig. 2).

Bottom Line: We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase.This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation.The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Italy. V. le G. Pieraccini, 6, I-50139 Florence. Italy.; Department of Preclinical and Clinical Pharmacology, University of Florence, Italy. V. le G. Pieraccini, 6, I-50139 Florence. Italy. daniele.bani@unifi.it

ABSTRACT
We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.

No MeSH data available.


Related in: MedlinePlus