Related Experiment Video
Updated: Jul 22, 2025
Isolation and Purification of Murine Cardiac Pericytes
Published on: August 16, 2019
A method to isolate human dermal microvascular pericytes without the use of magnetic beads sorting in vitro
Yinhua Zhao1, Jiaxing Ma1, Yue Cui1
1Plastic and Reconstructive Surgery, Beijing Anzhen Hospital, Capital Medical University, 2 Anzhen road, Chaoyang district, Beijing 100029, China.
Abstract:
Human dermal microvascular pericytes (HDMPCs) are a critical component of the skin flap microvasculature and play a role in regulating flap blood flow and integrity. Pericytes were isolated mostly via magnetic bead sorting in the published literature. In this study, we discuss in detail how to separate and concentrate pericytes from human facial flaps using enzyme digestion and differential adherence instead of magnetic bead sorting. Cultured HDMPCs were seen to have well-spread irregular edges, with most cells having two longitudinal pericytic processes. The phalloidin staining revealed that HDMPCs had prominent stress fibers, and the nucleus deviated to the side that interacted with the neighboring pericytic processes. Flow cytometry analysis showed that the positive rates of NG2 in the first and second passages were 91.2% ± 0.7% and 98.2% ± 0.1% separately. And the immunofluorescence and western blot results demonstrated a positive expression of α smooth muscle actin (αSMA), platelet-derived growth factor receptor β (PDGFRβ), and NG-2, while the endothelial cell marker CD31 was negatively expressed. In summary, we established a straightforward methodology for selectively isolating and identifying HDMPCs as well as generating high-purity cell cultures in vitro without the use of magnetic bead sorting.
Insights
This study presents a new method for isolating human dermal microvascular pericytes (HDMPCs) from facial skin flaps using enzyme digestion and differential adherence. This technique avoids magnetic bead sorting, yielding high-purity pericyte cultures.
Area of Science:
- Cell Biology
- Vascular Biology
- Tissue Engineering
Background:
- Human dermal microvascular pericytes (HDMPCs) are crucial for skin flap microvasculature, regulating blood flow and integrity.
- Existing isolation methods often rely on magnetic bead sorting, which can be limiting.
Purpose of the Study:
- To develop and detail a novel, efficient method for isolating and culturing HDMPCs from human facial flaps.
- To establish a high-purity HDMPC cell culture without using magnetic bead sorting.
Main Methods:
- Utilized enzyme digestion and differential adherence for pericyte separation from human facial flaps.
- Characterized cultured HDMPCs using phalloidin staining, flow cytometry, immunofluorescence, and western blot analysis.
Main Results:
- Cultured HDMPCs exhibited characteristic morphology with prominent stress fibers.
- Flow cytometry confirmed high purity (91.2%–98.2%) of NG2-positive pericytes across passages.
- Immunofluorescence and western blot showed positive expression of αSMA, PDGFRβ, and NG-2, with negative CD31 expression.
Conclusions:
- A straightforward and effective methodology for isolating and identifying HDMPCs has been established.
- This non-magnetic bead sorting approach generates high-purity pericyte cultures in vitro.
- The method offers a valuable alternative for pericyte isolation in research and potential therapeutic applications.
