Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Summary

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.

Abstract

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.

Introduction

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 cells^1,2. 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 sorting^8,9,10. Additionally, research efforts have led to the establishment of rat arachnoid cell lines and rat lymphatic capillary cell lines^11,12. 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.

Protocol

This research received approval from the Animal Experiment Ethics Committee of Kunming Medical University (kmmu20220945). All experiments adhered to established animal care guidelines. Leptomeningeal lymphatic endothelial cells (LLECs) were harvested from male C57Bl/6J mice aged 6-8 weeks and weighing between 20-25 g. These mice were procured from Kunming Medical University in Kunming, China. The entire experimental procedure was conducted under strict sterile conditions. All the centrifugation steps are performed at room temperature unless otherwise specified.

1. Preparation of reagents and instruments

NOTE: All steps involving solutions must be conducted within a class II biohazard cabinet.

1. Prepare the washing buffer by mixing phosphate-buffered saline (PBS) with calcium and magnesium, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (P/S) (see Table of Materials). Keep this buffer cold at 4 °C. It is essential to prepare it fresh on the day of use and degas the buffer.
2. Prepare the digestive enzymes by combining 10 mL of PBS (without calcium and magnesium) with 2 mL of 0.25% trypsin, 1 mL of 20 mg/mL papain, and 200 µL of 1 mg/mL collagenase I (see Table of Materials).
   NOTE: Ensure the use of aliquots of appropriate volumes to prevent repeated freeze-thaw cycles. Store these aliquots at -20 °C.
3. Prepare the culture medium by utilizing the commercially available endothelial cell growth medium kit, which includes VEGF-C (100 ng/mL) and 1% P/S (see Table of Materials).
4. Prepare the stopping buffer by supplementing Dulbecco's modified Eagle's medium (DMEM) with 10% FBS to stop the digestion process.
5. Prepare sterile surgical instruments: these instruments should include scissors, serrated-tip tweezers, and fine-point tweezers.

2. Flask coating

1. Coat the T25 flask with a 100 µg/mL fibronectin solution (see Table of Materials) and incubate overnight at 37 °C. Prior to use, remove the coating solution using a pipette.
2. Wash the flask three times with PBS, subsequently aspirate the PBS, and allow the T25 flask to air dry.

3. Leptomeninges dissociation

NOTE: Always use pre-cooled buffers and solutions at 4 °C.

1. Anesthetize the mice with excess inhalation of 4% isoflurane and then sacrifice them by swift decapitation using scissors that have been previously cleaned with 70% ethanol.
2. Carefully incise the skin along the midline, starting from the opening at the back of the skull and extending toward the frontal area.
3. Delicately remove the skull, lifting it gently with the scissors to avoid damaging the leptomeninges, ensuring the entire brain, including the leptomeninges, is obtained.
4. Submerge the whole brain in a washing buffer and gently flush it to remove surface blood.
5. Transfer the brain into a sterile Petri dish without chopping, and extract the leptomeninges from the brain's surface using fine-point tweezers under a microscope.
6. Cut the leptomeninges tissue into fragments using sterile micro-scissors.

4. Leptomeninges enzymatic digestion

1. Add 10 mL of the enzyme mix (step 1.2) to the fragments and incubate at 37 °C for 15 min. Gently agitate to ensure the fragments detach from the bottom of the tube. Afterward, add 10 mL of stopping buffer (step 1.4) to halt the digestion.
2. Centrifuge at 300 x g for 5 min at 4 °C and carefully remove the supernatant using a pipette.
3. Add 10 mL of cold PBS, and pass the mixture through a 70 µm strainer into a sterile 50 mL tube to filter out any clumps.

5. Cell expansion

1. Plate 1 x 10⁵ cells per cm² into a fibronectin-coated T25 flask (step 2) with 5 mL of culture medium.
2. Incubate the cells at 37 °C with 5% CO₂ for 24 h. Afterward, remove the medium to eliminate non-attached cells.
3. Sustain the culture by replacing 50% of the medium every two days. Repeat this process two or three times.

6. Magnetic cell selection

NOTE: Work swiftly, maintain cell coldness, and utilize pre-cooled solutions to prevent non-specific cell labeling.

1. Preparation of instruments and reagents: attach a magnetic separator to a magnetic separator stand (see Table of Materials). Connect a selection column to the magnetic separator, and position a 70 µm cell strainer atop the selection column. Place a 50 mL tube beneath the selection column to collect the flow.
2. Enzymatic digestion: once the cells reach 80% confluence, aspirate the medium and rinse the cells with PBS. Add 0.25% trypsin to detach adherent cells and incubate at 37 °C for 5 min. Stop the digestion by adding the stopping buffer. Centrifuge the cells at 300 x g for 5 min at room temperature and remove the supernatant.
3. Antibody incubation: resuspend 1 x 10⁷ total cells in 100 µL of PBS and add 10 µL of LYVE-1 antibody (see Table of Materials). Mix thoroughly and incubate for 30 min in the dark at 4 °C.
   1. Spin the cells at 300 x g for 5 min and discard the supernatant. Rinse the cells by introducing 1 mL of PBS and centrifuging at 300 x g for 5 min. Completely remove the supernatant.
4. Microbeads labeling: resuspend the cells in 100 µL of PBS and add 20 µL of Microbeads (see Table of Materials). Mix well and incubate for 30 min in the dark at 4 °C.
   1. Spin the cells at 300 x g for 5 min and decant the supernatant. Subsequently, cleanse the cells with 1 mL of PBS through centrifugation at 300 x g for 5 min. Finally, completely remove the supernatant.
5. Magnetic negative exclusion: resuspend the cells in 4 mL of PBS. Pass the cells through a 70 µm cell strainer to eliminate clumps. Prepare the selection column (see Table of Materials) by rinsing it with 3 mL of PBS, then apply the cell suspension into the selection column.
   1. Perform washing steps with 3 mL of PBS and collect LYVE-1-negative cells into a 50 mL tube positioned below the selection column to allow them to pass through.
6. Magnetic positive selection: pipette 6 mL of PBS into the selection column. Instantly flush out the magnetically labeled cells by firmly pushing the plunger into the selection column to obtain LYVE-1-positive LLECs.
   ​NOTE: Always wait until the selection column reservoir is empty before proceeding to the next step.

7. LLECs culture

1. Centrifuge the LYVE-1-positive LLECs at 300 x g for 5 min, and then carefully remove the supernatant.
2. Plate 1 x 10⁵ cells per cm² into a fibronectin-coated T25 flask with 5 mL of culture medium.
3. Maintain the culture by replacing 50% of the medium every other day. When the cells reach 80% confluence, detach the cells with 0.25% trypsin and perform cell passage. Utilize the cells for subsequent experiments after 2-3 passages.

8. Flow cytometric analysis

NOTE: The Flow cytometric analysis was conducted following the previously described procedure¹⁵.

1. Add 0.25% trypsin to detach adherent cells and incubate at 37 °C for 5 min. Then, add the stopping buffer to halt the digestion.
2. Centrifuge the cells at 300 x g for 5 min and completely aspirate the supernatant. Resuspend 1 x 10⁶ cells in 100 µL of PBS.
3. Add 1 µL of LYVE-1 antibody. Mix thoroughly and incubate for 10 min in the dark at room temperature.
4. Resuspend the cells in an appropriate amount of PBS buffer for analysis using flow cytometry and FlowJo software (see Table of Materials).

9. Immunofluorescent staining

NOTE: The immunofluorescent staining was conducted following the procedure described previously¹⁶.

1. Wash the cells with PBS three times. Fix the cells with 4% paraformaldehyde (PFA) for 10 min at room temperature. Discard the PFA and wash the cells with PBS three times.
2. Apply the block buffer for 1 h at room temperature. Remove the block buffer from the coverslips.
3. Add the LYVE-1 antibody in the staining buffer to the cells and incubate in the dark. Wash the cells three times with PBS.
4. Add an appropriate secondary antibody (see Table of Materials) in the staining buffer to the cells and incubate in the dark. Wash the cells three times with PBS.
5. Counterstain with 4',6-diamidino-2-phenylindole (DAPI). The cells are now ready for immunofluorescence microscopy.

Results

This study presents a reproducible, multi-step protocol for harvesting lymphatic endothelial cells (LLECs) from mice and subsequently establishing their primary culture in vitro. The key steps involve flask preparation and fibronectin coating, dissociation of leptomeninges, obtaining a single-cell suspension through enzymatic digestion, and inducing LLECs expansion with VEGF-C. LYVE-1-positive LLECs are then selectively isolated using magnetic-activated cell sorting (MACS). Finally, immunofluorescence staining a...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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