Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.
Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.
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.
The existing protocol for harvesting and culturing LLECs in vitro has not been previously reported. This study introduces a reproducible, multi-procedural protocol for harvesting and culturing LLECs from mouse leptomeninges.
While this multi-procedural protocol is reproducible, there are several key considerations. For example, fibronectin-coated T25 flasks promote the adhesion of LLECs and function by eliminating non-adherent cells, thereby ensuring a more homogenous cellular populat…
The authors have nothing to disclose.
The study was supported by grants from the National Natural Science Foundation of China (81960226, 81760223), the Natural Science Foundation of Yunnan Province (202001AS070045, 202301AY070001-011), and the Scientific Research Foundation of Yunnan Province Department of Education (2023Y0784).
Block buffer | Beyotime | P0102 | Store aliquots at –4 °C |
Collagenase I | Solarbio | C8140 | Store aliquots at –20 °C |
DAPI | Beyotime | P0131 | Store aliquots at –20 °C |
DMEM | Solarbio | 11995 | Store aliquots at –4 °C |
D-PBS | Solarbio | D1041 | Store aliquots at –4 °C |
EGM-2 MV Bullet Kit | Lonza | C-3202 | Store aliquots at –4 °C |
FBS | Solarbio | S9010 | Store aliquots at –20 °C |
Fibronectin | Solarbio | F8180 | Store aliquots at –20 °C |
FlowJo Software | BD Biosciences | V10.8.1 | |
LYVE-1 antibody | eBioscience | 12-0443-82 | Store aliquots at –4 °C |
Magnetic separator | Miltenyi | 130-042-302 | Sterile before use |
Magnetic separator stand | Miltenyi | 130-042-303 | Sterile before use |
Microbeads | Miltenyi | 130-048-801 | Store aliquots at –4 °C |
P/S | Solarbio | P1400 | Store aliquots at –20 °C |
Papain | Solarbio | G8430-25g | Store aliquots at –20 °C |
PBS | Solarbio | D1040 | Store aliquots at –4 °C |
PDPN antibody | Santa | sc-53533 | Store aliquots at –4 °C |
PFA | Solarbio | P1110 | Store aliquots at –4 °C |
PROX1 antibody | Santa | sc-81983 | Store aliquots at –4 °C |
Selection column | Miltenyi | 130-042-401 | Sterile before use |
Trypsin | Gibco | 25200072 | Store aliquots at –20 °C |
VEGF-C | Abcam | ab51947 | Store aliquots at –20 °C |
VEGFR-3 antibody | Santa | sc-514825 | Store aliquots at –4 °C |