A method of isolation and culture of microvascular endothelial cells from mouse skin.
OBJECTIVE
The study of isolated microvascular endothelial cells from mice has long been impeded due to the many difficulties encountered in isolating and culturing these cells. We focused on developing a method to isolate microvascular endothelial cells from the skin fragments of newborn mice. We also aimed at establishing optimal culture conditions to sustain the growth of these cells.


RESULTS
Isolation of murine dermal microvascular endothelial cells (mDMEC) from P3 newborn mice was based first on enzymatic separation of the skin epidermal layer from the dermis using dispase and then on disaggregating dermal cellular elements using collagenase. The cells obtained from the dermis were subjected to a continuous density gradient centrifugation. Cells situated between densities 1.033 and 1.047 were then cultured on collagen IV-coated culture flasks using optimized growth culture conditions. Cells were characterized by endothelial appearance and by the presence and genetic expression of endothelial markers like CD31, NOS3, VEGFR-2 and Tie-2. Uptake of acetylated low-density lipoprotein (Ac-LDL) was used as a functional assay.


CONCLUSIONS
The methodology described herein for isolation and culture of murine microvascular endothelium offers a distinctive advantage for those using mouse models to study endothelial cell biology.
Cha
Sung Tae
ST
Department of Surgery, Division of Plastic and Reconstructive Surgery, Engineered Wound Repair Laboratory at Davis Building, Cedars Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA.
Talavera
Dodanim
D
Demir
Erhan
E
Nath
Anjali K
AK
Sierra-Honigmann
M Rocio
MR
D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000096:biosynthesis; D015820:Cadherins / Q000096:biosynthesis; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D008077:Lipoproteins, LDL / Q000378:metabolism; D051379:Mice; D008833:Microcirculation; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008954:Models, Biological; D010641:Phenotype; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism* / Q000473:pathology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D018952:Antigens, CD34; D015820:Cadherins; D008077:Lipoproteins, LDL; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D012333:RNA, Messenger; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099152:Cadherin 5