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

Establecimiento de cultivos in vitro de LLEC mediante selección magnética de células

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 destacado: Desvelando las funciones celulares y las posibles implicaciones clínicas de las células endoteliales linfáticas leptomeníngeas 

Recolección y cultivo primario de células endoteliales 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  

Disociación de leptomeninges de ratón y digestión enzimática para el cultivo eficiente de células leptomeníngeas

Para comenzar, incide con cuidado la piel del ratón sacrificado, comenzando desde la abertura del cráneo y extendiéndose hacia el área frontal. Con unas tijeras, levante y retire delicadamente el cráneo sin dañar las leptomeninges, recogiendo todo el cerebro. Sumerge todo el cerebro en el tampón de lavado y enjuágalo suavemente para eliminar la sangre de la superficie.Transfiera el cerebro a una placa de Petri estéril sin picar. Luego, bajo un microscopio, use pinzas de punta fina para extraer las leptomeninges de la superficie del cerebro. Con unas tijeras estériles, corte el tejido de las leptomeninges en fragmentos.Repare la mezcla de enzimas digestivas utilizando los componentes dados. Agregue 10 mililitros de mezcla a los fragmentos e incube a 37 grados centígrados durante 15 minutos. Agite suavemente para separar los fragmentos del fondo del tubo.Luego, agregue 10 mililitros de tampón de parada. Centrifugar la suspensión a 300 g durante cinco minutos a cuatro grados centígrados. Y con una pipeta, retire con cuidado el sobrenadante.Agregue 10 mililitros de PBS frío y filtre los grumos a través de un colador de 70 micrones en un tubo estéril de 50 mililitros. Colocar 10 a la quinta celda por centímetro cuadrado en un matraz T25 recubierto de fibronectina con cinco mililitros de medio de cultivo, e incubar a 37 grados centígrados con dióxido de carbono al 5% durante 24 horas. Después de la incubación, reemplace el medio con un medio nuevo para eliminar las células no adheridas.

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JoVE Journal

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 ...