Journal of neuroscience methods · 2025
Isolation, culture, and characterization of primary endothelial cells and pericytes from mouse sciatic nerve.
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Huang Yan, Jin Hai-Rong, Liu Fang-Yuan, Fridayana Fitri Rahma, Vo Minh Nhat, Ryu Ji-Kan, Yin Guo Nan
Аннотация
BACKGROUND: The recovery of injured peripheral nerves relies on angiogenesis, where newly formed blood vessels act as pathways guiding Schwann cells across the wound to support axon regeneration. While some research has examined this process, the specific mechanisms of angiogenesis in peripheral nerve healing remain unclear. In vitro models are vital tools to investigate these mechanisms; however, no current in vitro culture methods exist for isolating vascular cells, such as endothelial cells (ECs) and pericytes, specifically from sciatic nerves.
NEW METHOD: We developed a straightforward and reliable technique for isolating ECs and pericytes from injured sciatic nerves, optimized for use in in vitro studies. Cell types were characterized using specific markers and phenotypic assessments, with flow cytometry confirming cell identity and determining cell purity.
RESULTS: Our method successfully isolated high-purity ECs and pericytes from injured sciatic nerves. Immunofluorescence analysis showed that primary cultured ECs exhibited strong positive staining for CD31, while pericytes stained strongly for NG2 and PDGFRβ. Flow cytometric analysis confirmed that ECs achieved a purity of 90.22 %, and pericytes reached a purity of 92.01 %. Both cell types were capable of forming organized capillary-like structures, and in co-culture systems, pericytes effectively wrapped around ECs.
COMPARISON WITH EXISTING METHODS: Current isolation methods for ECs and pericytes from sciatic nerves are limited. Although techniques exist for isolating these cells from other tissues, they often rely on enzymatic digestion, which can damage cell surface proteins and reduce cell viability. Our method allows for the efficient isolation of intact ECs and pericytes from sciatic nerve tissue without such drawbacks, providing a robust platform for in vitro studies.
CONCLUSIONS: This newly developed method offers an effective approach to isolate ECs and pericytes from the sciatic nerve, contributing a valuable tool for investigating the function and pathology of angiogenesis in the context of sciatic nerve injury recovery.
NEW METHOD: We developed a straightforward and reliable technique for isolating ECs and pericytes from injured sciatic nerves, optimized for use in in vitro studies. Cell types were characterized using specific markers and phenotypic assessments, with flow cytometry confirming cell identity and determining cell purity.
RESULTS: Our method successfully isolated high-purity ECs and pericytes from injured sciatic nerves. Immunofluorescence analysis showed that primary cultured ECs exhibited strong positive staining for CD31, while pericytes stained strongly for NG2 and PDGFRβ. Flow cytometric analysis confirmed that ECs achieved a purity of 90.22 %, and pericytes reached a purity of 92.01 %. Both cell types were capable of forming organized capillary-like structures, and in co-culture systems, pericytes effectively wrapped around ECs.
COMPARISON WITH EXISTING METHODS: Current isolation methods for ECs and pericytes from sciatic nerves are limited. Although techniques exist for isolating these cells from other tissues, they often rely on enzymatic digestion, which can damage cell surface proteins and reduce cell viability. Our method allows for the efficient isolation of intact ECs and pericytes from sciatic nerve tissue without such drawbacks, providing a robust platform for in vitro studies.
CONCLUSIONS: This newly developed method offers an effective approach to isolate ECs and pericytes from the sciatic nerve, contributing a valuable tool for investigating the function and pathology of angiogenesis in the context of sciatic nerve injury recovery.