Name: Mouse Leptomeninges Dissociation and Enzymatic Digestion for Efficient Culturing of Leptomeningeal Cells
Uploaded: 2023-09-08T14:55:00
Description: Watch this Scientific Journal Video about Mouse Leptomeninges Dissociation and Enzymatic Digestion for Efficient Culturing of Leptomeningeal Cells at JoVE.com

mouse leptomeninges dissociation and enzymatic digestion for efficient culturing of leptomeningeal cells

Estabelecimento de culturas LLEC in vitro usando seleção de células magnéticas

The newly discovered leptomeningeal lymphatic endothelial cells (LLECs) form a meshwork of individual cells within the leptomeninges, exhibiting a distinct distribution pattern when compared to peripheral lymphatic endothelial cells1,2. The cellular functions and clinical implications associated with LLECs remain largely uncharted territory. In order to pave the way for functional research on LLECs, it is imperative to establish an in vitro model for their study. Therefore, this study has devised a comprehensive protocol for the isolation and primary culture of LLECs.
Mice are the preferred animal model due to their suitability for genetic manipulation in disease research. Previous studies have successfully isolated lymphatic endothelial cells from various mouse tissues, including lymph nodes3, mesenteric tissue4, dermal tissue5, collecting lymphatics6, and lung tissue7. These isolation procedures have primarily relied on techniques such as magnetic-activated cell sorting (MACS) and flow cytometry sorting8,9,10. Additionally, research efforts have led to the establishment of rat arachnoid cell lines and rat lymphatic capillary cell lines11,12. Despite the existence of explant culture techniques for leptomeninges13, there exists an urgent need for a standardized protocol for the isolation and culture of LLECs. Consequently, this study has successfully harvested and cultured LLECs by meticulously dissociating leptomeninges under the guidance of a microscope and promoting LLECs expansion through the use of vascular endothelial growth factor-C (VEGF-C). The distinctive marker for lymphatic endothelial cells is lymphatic vessel hyaluronic receptor-1 (LYVE-1)14. This multi-step protocol selectively isolates LYVE-1-positive LLECs using MACS and subsequently verifies their purity through flow cytometric analysis and immunofluorescent staining.
The primary steps of this multi-step protocol can be summarized as follows: flask coating, dissociation of leptomeninges, enzymatic digestion of leptomeninges, cell expansion, magnetic cell selection, and subsequent culture of LLECs. Finally, the purity of the isolated LLECs is confirmed through flow cytometric analysis and immunofluorescent staining. The overarching aim of this study is to present a reproducible, multi-step protocol for the isolation of LLECs from mouse leptomeninges and their subsequent in vitro culture. This protocol is poised to greatly facilitate investigations into the cellular functions and clinical implications of LLECs.

Autor em destaque: Revelando funções celulares e potenciais implicações clínicas das células endoteliais linfáticas leptomeníngeas 

Colheita e Cultura Primária de Células Endoteliais Linfáticas Leptomeníngeas

This protocol is designed for areas of by other researchers only interested where established the multi procedure to and cultural primary areas Our protocol ultimately led to the establishment of our primary culture of area CS with a purity level exceeding 95%There is currently no existing protocol for harvesting and culturing Our results paved the way for further investigating the similar function of LLECS in virtual. At RUC, our recent discovery intracranial cellular population, the biological significance of RUC We will conduct further research on the cellular function and review its clinical implication.

Watch this Scientific Journal Video about Mouse Leptomeninges Dissociation and Enzymatic Digestion for Efficient Culturing of Leptomeningeal Cells at JoVE.com

Mouse Leptomeninges Dissociation and Enzymatic Digestion for Efficient Culturing of Leptomeningeal Cells  

Dissociação de leptomeninges de camundongos e digestão enzimática para cultivo eficiente de células leptomeníngeas

Para começar, incite cuidadosamente a pele do rato sacrificado, começando pela abertura do crânio e estendendo-se em direção à área frontal. Usando tesouras, levante e remova delicadamente o crânio sem danificar as leptomeninges, coletando todo o cérebro. Submergir todo o cérebro no tampão de lavagem e lavá-lo suavemente para remover o sangue da superfície.Transfira o cérebro para uma placa de Petri estéril sem cortar. Em seguida, sob um microscópio, use pinças de ponta fina para extrair as leptomeninges da superfície do cérebro. Usando microtesouras estéreis, corte o tecido das leptomeninges em fragmentos.Reparar a mistura de enzimas digestivas usando os componentes fornecidos. Adicione 10 mililitros de mistura aos fragmentos e incube a 37 graus Celsius por 15 minutos. Agite suavemente para separar os fragmentos do fundo do tubo.Em seguida, adicione 10 mililitros de tampão de parada. Centrifugar a suspensão a 300 g durante cinco minutos a quatro graus Celsius. E usando uma pipeta, remova cuidadosamente o sobrenadante.Adicione 10 mililitros de PBS frio e filtre quaisquer aglomerados através de um filtro de 70 mícrons em um tubo estéril de 50 mililitros. Placa de 10 a quinta células por centímetro quadrado em um frasco T25 revestido com fibronectina com cinco mililitros de meio de cultura e incubar a 37 graus Celsius com 5% de dióxido de carbono por 24 horas. Após a incubação, substitua o meio por um meio fresco para eliminar as células não aderidas.

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Neuroscience

Mouse Leptomeninges Dissociation and Enzymatic Digestion for Efficient Culturing of Leptomeningeal Cells

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...