An effective method of isolating microvascular endothelial cells from the human dermis

Abstract

Dermal microvascular endothelial cells (DMECs) play central roles in inflammation and angiogenesis and have become important cell models for studying various skin diseases. However, primary DMECs are difficult to culture and often contaminated by mesenchymal stem cells, fibroblasts, and other stromal cells. Surgically removed superfluous foreskin was first cut into pieces, digested with two types of enzymes, and dispersed into single cells. Cells obtained from the dermis were then subjected to Percoll density gradient centrifugation and cells located between densities 1.033 and 1.047 g/ml were further purified with endothelial growth medium containing decreasing concentrations of puromycin. Obtained HDMECs were identified by microscopy, flow cytometry, quantitative reverse‐transcription polymerase chain reaction, western blot analysis, and immunofluorescent staining. The expression of CD31 (PECAM‐1), CD34, VEGFR2, VWF (Von Willebrand Factor), VE‐Cadherin (CD144), and NOS was positive. HDMECs were found to have abilities of angiogenesis and uptake of acetylated low‐density lipoprotein. Growth curves and cell viability were analyzed, and a growth pattern consisting of the “latency phase‐logarithmic growth phase‐stagnation phase” was determined. In this study, a simple, rapid, effective, and low‐cost method is established to isolate HDMECs from the foreskin with a purity of over 91% and high viability. The method showed good repeatability and allowed a stable passage. This study provides technical support and theoretical guidance for studying the physiological characteristics of HDMECs, the pathogenesis of the skin associated, and other microvascular diseases.

Increased angiogenesis and migration of dermal microvascular endothelial cells from patients with psoriasis

“…Due to technical difficulty in isolation and culture of human dermal microvascular endothelial cells (HDMECs), 19‐22 currently immunohistochemistry in skin tissue 23,24 and human umbilical vein endothelial cells 25,26 are major approaches in the study of pathogenesis of psoriasis. In the present study, we isolated HDMECs from the human dermis and compared the functions, including angiogenesis, migration and metabolism, of HDMECs from psoriatic lesions vs. that from the skin of healthy subjects.…”

Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

“…To systematically study the regulatory role of CTHRC1 in dermal microvascular development at the cellular level, we first isolated the DMECs from dermal tissues of WT and Cthrc1 −/− mice using a modified protocol ( Figure 3a ) [ 25 ]. At the initial isolation stage, both DMECs exhibited a typical ‘cobblestone’ appearance.…”

“…Primary DMECs were isolated from the 1-day-old mouse skin using a modified protocol [ 25 ]. Briefly, freshly harvested mouse skin was digested with 2.5 mg/ml dispase II at 4°C for 12 h. The dermal tissue was separated from the epidermal layer by mechanical dissociation, minced, and digested with 4 mg/ml type I collagenase at 37°C for 2 h. Following filtration and centrifugation, the cell pellet was resuspended in EGM-2 (MV Microvascular Endothelial Cell Growth Medium-2 BulletKit; CC-3162, Lonza, Switzerland) containing 1 μg/ml puromycin (Solarbio, China) and clutured for 50 h at 37°C and 5% CO 2 .…”

Promotion effect of angelica sinensis extract on angiogenesis of chicken preovulatory follicles in vitro

“…The extract method is similar to separation of luteal microvascular endothelial cells ( Sakurai et al, 2011 ), which is widely used for the separation of microvascular endothelial cells from heart, lung and brain. CD31 and vWF are recognized as specific markers of microvascular endothelial cells ( Hou et al, 2020 ). The microvascular endothelial cells isolated and cultured in this study showed positive results of vWF and CD31 through immunofluorescence analysis.…”

“…After centrifuging for 1 min at 400 g in a vertical centrifuge, the solution was divided into 3 layers. Cells in middle layer (a density of 1.033–1.047g/ Ml; Hou et al, 2020 ) were carefully absorbed into a 15 mL centrifuge tube, and centrifuged for 5 min at 250 g . The supernatant was discarded.…”