Cha Sung Tae, Talavera Dodanim, Demir Erhan, Nath Anjali K, Sierra-Honigmann M Rocio
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.
Microvasc Res. 2005 Nov;70(3):198-204. doi: 10.1016/j.mvr.2005.08.002. Epub 2005 Sep 26.
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.
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.
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.
长期以来，从小鼠中分离微血管内皮细胞的研究因在分离和培养这些细胞过程中遇到的诸多困难而受阻。我们专注于开发一种从新生小鼠皮肤碎片中分离微血管内皮细胞的方法。我们还旨在建立最佳培养条件以维持这些细胞的生长。
从出生3天的新生小鼠中分离鼠真皮微血管内皮细胞（mDMEC），首先使用dispase酶将皮肤表皮层与真皮分离，然后使用胶原酶分解真皮细胞成分。从真皮获得的细胞进行连续密度梯度离心。然后将密度在1.033至1.047之间的细胞接种到包被有IV型胶原的培养瓶中，使用优化的生长培养条件进行培养。通过内皮细胞形态以及内皮标志物如CD31、NOS3、VEGFR - 2和Tie - 2的存在及基因表达对细胞进行鉴定。使用乙酰化低密度脂蛋白（Ac - LDL）摄取作为功能检测。
本文所述的分离和培养鼠微血管内皮的方法，为那些使用小鼠模型研究内皮细胞生物学的人提供了独特优势。
