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Neuroscience
Colheita e Cultura Primária de Células Endoteliais Linfáticas Leptomeníngeas
Colheita e Cultura Primária de Células Endoteliais Linfáticas Leptomeníngeas
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Colheita e Cultura Primária de Células Endoteliais Linfáticas Leptomeníngeas

Full Text
2,302 Views
06:44 min
September 8, 2023

DOI: 10.3791/65872-v

Hong-Ji Deng1, Kun Wu2, Han-Fu Yu1, Yong-Jin Zhang1,3, Yun-Cong Li1, Chong Li1, Fei Wang1

1Department of Neurosurgery,The First Affiliated Hospital of Kunming Medical University, 2Department of Clinical Laboratory,The First Affiliated Hospital of Kunming Medical University, 3Clinical Medical Research Center,The First Affiliated Hospital of Kunming Medical University

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Overview

This study presents a protocol for harvesting and culturing leptomeningeal lymphatic endothelial cells (LLECs) from mice, an intracranial cell type with largely unexplored functions. The established in vitro primary cultures of LLECs can facilitate research into their cellular functions and potential clinical implications.

Key Study Components

Area of Science

  • Neuroscience
  • Cell Biology
  • In Vitro Cultures

Background

  • Leptomeningeal lymphatic endothelial cells (LLECs) are a recently identified cell type in the skull.
  • Their functions and characteristics remain poorly understood in the field of neuroscience.
  • Existing protocols for LLEC cultivation are lacking, necessitating this reproducible method.
  • The study aims to enable further investigation into the potential roles and clinical relevance of LLECs.

Purpose of Study

  • To establish a reliable method for harvesting LLECs from mice.
  • To cultivate these cells in vitro for future research.
  • To investigate the biological significance of LLECs in the nervous system context.

Methods Used

  • The study involved cell culture techniques for LLECs derived from mouse brains.
  • Primary LLEC cultures were established with a specified protocol, achieving over 95% purity.
  • Key steps included tissue digestion, centrifugation, and separation via magnetic selection.
  • Cell behavior was observed and verified through immunofluorescence staining.

Main Results

  • The developed protocol successfully yielded LLECs with high purity, revealing their distinct morphology and characteristics.
  • Immunofluorescent staining confirmed LLECs' identity and differentiation from other cell types.
  • Cultured LLECs exhibited typical endothelial-like features over time.

Conclusions

  • This study provides a foundational protocol for LLEC study, facilitating exploration of their roles in health and disease.
  • The in vitro system opens avenues for understanding LLEC functions in the central nervous system.
  • Future investigations may clarify LLECs’ clinical implications and biological significance in neuroscience.

Frequently Asked Questions

What are the advantages of using the established protocol?
The protocol provides a reproducible method for isolating and cultivating LLECs with high purity, enabling reliable experimentation and understanding of these cells.
How are LLECs harvested from mouse brains?
LLECs are harvested by carefully removing leptomeninges from the brain surface, followed by enzymatic digestion and cell culture techniques.
What type of data or outcomes can researchers expect?
Researchers can obtain insights into the cellular characteristics of LLECs, including their morphology and specific marker expressions.
How can the method be adapted for other research purposes?
Variations of the protocol can be applied to study other brain-derived cell types or explore different biochemical environments by modifying digestion enzymes and media conditions.
What are the limitations of the described method?
The method primarily focuses on LLECs from mice, which may not fully represent LLECs in other species, limiting broader applicability without further validation.

As células endoteliais linfáticas leptomeníngeas (LLECs), um tipo de célula intracraniana recentemente identificado, têm funções pouco compreendidas. Este estudo apresenta um protocolo reprodutível para a colheita de LLECs de camundongos e estabelecimento de culturas primárias in vitro . Este protocolo é projetado para permitir que os pesquisadores se aprofundem nas funções celulares e potenciais implicações clínicas de LLECs.

Este protocolo é projetado para áreas de por outros pesquisadores apenas interessados onde estabelecido o procedimento múltiplo para e áreas primárias culturais Nosso protocolo finalmente levou ao estabelecimento de nossa cultura primária de área CS com um nível de pureza superior a 95%Não há atualmente nenhum protocolo existente para colheita e cultivo Nossos resultados abriram o caminho para investigar mais a função similar de LLECS em virtual. Na RUC, nossa recente descoberta da população celular intracraniana, o significado biológico da RUC Realizaremos mais pesquisas sobre a função celular e revisaremos sua implicação clínica.

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Células Endoteliais Linfáticas Leptomeníngeas LLECs Colheita Cultura Primária População Celular Intracraniana Células Endoteliais Linfáticas Periféricas Pesquisa Funcional In Vitro Protocolo Revestimento de Fibronectina Dissecção de Leptomeninges Digestão Enzimática Suspensão de Célula Única Fator de Crescimento Endotelial Vascular-C (VEGF-C) Receptor Hialurônico de Vaso Linfático-1 (LYVE-1) Classificação de Células Ativadas por Magnésio (MACS) Estabelecimento de Cultura Primária Confirmação de Pureza Imunofluorescência Coloração Análise por Citometria de Fluxo

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