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).

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.
<ol>
	<li>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 &#176;C. It is essential to prepare it fresh on the day of use and degas the buffer.</li>
	<li>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 &#181;L 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 &#176;C.</li>
	<li>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).</li>
	<li>Prepare the stopping buffer by supplementing Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% FBS to stop the digestion process.</li>
	<li>Prepare sterile surgical instruments: these instruments should include scissors, serrated-tip tweezers, and fine-point tweezers.</li>
</ol>
2. Flask coating
<ol>
	<li>Coat the T25 flask with a 100 &#181;g/mL fibronectin solution (see Table of Materials) and incubate overnight at 37 &#176;C. Prior to use, remove the coating solution using a pipette.</li>
	<li>Wash the flask three times with PBS, subsequently aspirate the PBS, and allow the T25 flask to air dry.</li>
</ol>
3. Leptomeninges dissociation
NOTE: Always use pre-cooled buffers and solutions at 4 &#176;C.
<ol>
	<li>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.</li>
	<li>Carefully incise the skin along the midline, starting from the opening at the back of the skull and extending toward the frontal area.</li>
	<li>Delicately remove the skull, lifting it gently with the scissors to avoid damaging the leptomeninges, ensuring the entire brain, including the leptomeninges, is obtained.</li>
	<li>Submerge the whole brain in a washing buffer and gently flush it to remove surface blood.</li>
	<li>Transfer the brain into a sterile Petri dish without chopping, and extract the leptomeninges from the brain&#39;s surface using fine-point tweezers under a microscope.</li>
	<li>Cut the leptomeninges tissue into fragments using sterile micro-scissors.</li>
</ol>
4. Leptomeninges enzymatic digestion
<ol>
	<li>Add 10 mL of the enzyme mix (step 1.2) to the fragments and incubate at 37 &#176;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.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C and carefully remove the supernatant using a pipette.</li>
	<li>Add 10 mL of cold PBS, and pass the mixture through a 70 &#181;m strainer into a sterile 50 mL tube to filter out any clumps.</li>
</ol>
5. Cell expansion
<ol>
	<li>Plate 1 x 105 cells per cm2 into a fibronectin-coated T25 flask (step 2) with 5 mL of culture medium.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 24 h. Afterward, remove the medium to eliminate non-attached cells.</li>
	<li>Sustain the culture by replacing 50% of the medium every two days. Repeat this process two or three times.</li>
</ol>
6. Magnetic cell selection
NOTE: Work swiftly, maintain cell coldness, and utilize pre-cooled solutions to prevent non-specific cell labeling.
<ol>
	<li>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 &#181;m cell strainer atop the selection column. Place a 50 mL tube beneath the selection column to collect the flow.</li>
	<li>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 &#176;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.</li>
	<li>Antibody incubation: resuspend 1 x 107 total cells in 100 &#181;L of PBS and add 10 &#181;L of LYVE-1 antibody (see Table of Materials). Mix thoroughly and incubate for 30 min in the dark at 4 &#176;C.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Microbeads labeling: resuspend the cells in 100 &#181;L of PBS and add 20 &#181;L of Microbeads (see Table of Materials). Mix well and incubate for 30 min in the dark at 4 &#176;C.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Magnetic negative exclusion: resuspend the cells in 4 mL of PBS. Pass the cells through a 70 &#181;m 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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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. 
	&#8203;NOTE: Always wait until the selection column reservoir is empty before proceeding to the next step.</li>
</ol>
7. LLECs culture
<ol>
	<li>Centrifuge the LYVE-1-positive LLECs at 300 x g for 5 min, and then carefully remove the supernatant.</li>
	<li>Plate 1 x 105 cells per cm2 into a fibronectin-coated T25 flask with 5 mL of culture medium.</li>
	<li>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.</li>
</ol>
8. Flow cytometric analysis
NOTE: The Flow cytometric analysis was conducted following the previously described procedure15.
<ol>
	<li>Add 0.25% trypsin to detach adherent cells and incubate at 37 &#176;C for 5 min. Then, add the stopping buffer to halt the digestion.</li>
	<li>Centrifuge the cells at 300 x g for 5 min and completely aspirate the supernatant. Resuspend 1 x 106 cells in 100 &#181;L of PBS.</li>
	<li>Add 1 &#181;L of LYVE-1 antibody. Mix thoroughly and incubate for 10 min in the dark at room temperature.</li>
	<li>Resuspend the cells in an appropriate amount of PBS buffer for analysis using flow cytometry and FlowJo software (see Table of Materials).</li>
</ol>
9. Immunofluorescent staining
NOTE: The immunofluorescent staining was conducted following the procedure described previously16.
<ol>
	<li>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.</li>
	<li>Apply the block buffer for 1 h at room temperature. Remove the block buffer from the coverslips.</li>
	<li>Add the LYVE-1 antibody in the staining buffer to the cells and incubate in the dark. Wash the cells three times with PBS.</li>
	<li>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.</li>
	<li>Counterstain with 4&#39;,6-diamidino-2-phenylindole (DAPI). The cells are now ready for immunofluorescence microscopy.</li>
</ol>

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

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The authors declare that they have no conflict of interest to disclose.

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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Block buffer</td>
			<td>Beyotime</td>
			<td>P0102</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Solarbio</td>
			<td>C8140</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>P0131</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Solarbio</td>
			<td>11995</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Solarbio</td>
			<td>D1041</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>C-3202</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Solarbio</td>
			<td>S9010</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Solarbio</td>
			<td>F8180&#160;</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td>V10.8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE-1 antibody</td>
			<td>eBioscience</td>
			<td>12-0443-82</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>Magnetic separator</td>
			<td>Miltenyi</td>
			<td>130-042-302</td>
			<td>Sterile before use</td>
		</tr>
		<tr>
			<td>Magnetic separator stand</td>
			<td>Miltenyi</td>
			<td>130-042-303</td>
			<td>Sterile before use</td>
		</tr>
		<tr>
			<td>Microbeads</td>
			<td>Miltenyi</td>
			<td>130-048-801</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>P/S</td>
			<td>Solarbio</td>
			<td>P1400</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Solarbio</td>
			<td>G8430-25g</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>D1040</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>PDPN antibody</td>
			<td>Santa</td>
			<td>sc-53533</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>PROX1 antibody</td>
			<td>Santa</td>
			<td>sc-81983</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
		<tr>
			<td>Selection column&#160;</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>Sterile before use</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>VEGF-C&#160;</td>
			<td>Abcam</td>
			<td>ab51947</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
		</tr>
		<tr>
			<td>VEGFR-3 antibody</td>
			<td>Santa</td>
			<td>sc-514825</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
		</tr>
	</tbody>
</table>

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 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.

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

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.

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...

Watch this Scientific Journal Video about Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells at JoVE.com

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 ...

harvest-primary-culture-leptomeningeal-lymphatic-endothelial

Research

JoVE Journal

Neuroscience

868 Views.  The First Affiliated Hospital of Kunming Medical University. 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 biologi...

Video: Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

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

To begin, carefully incise the skin of the sacrificed mouse, starting from the skull's opening and extending towards the frontal area. Using scissors, delicately lift and remove the skull without damaging the leptomeninges, collecting the entire brain. Submerge the whole brain in the washing buffer, and gently flush it to remove surface blood.Transfer the brain into a sterile Petri dish without chopping. Then, under a microscope, use fine-point tweezers to extract the leptomeninges from the brain's surface. Using sterile microscissors, cut the leptomeninges tissue into fragments.Repair the digestive enzyme mix using the given components. Add 10 milliliters of mix to the fragments and incubate at 37 degrees Celsius for 15 minutes. Gently agitate to detach the fragments from the bottom of the tube.Then, add 10 milliliters of stopping buffer. Centrifuge the suspension at 300 g for five minutes at four degrees Celsius. And using a pipette, carefully remove the supernatant.Add 10 milliliters of cold PBS, and filter any clumps through a 70 micron strainer into a sterile 50 milliliter tube. Plate 10 to the fifth cells per square centimeter into a fibronectin-coated T25 flask with five milliliters of culture medium, and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours. After incubation, replace the medium with a fresh medium to eliminate non-attached cells.

Establishing Primary In Vitro Culture of Leptomeningeal Lymphatic Endothelial Cells (LLECs)

Begin by attaching a magnetic separator to its stand. Connect a selection column to the magnetic separator and place a 70 micron cell strainer on top of the selection column. Place a 50 milliliter tube beneath the selection column to collect the flow.Once the mouse brain cells reach 80%confluency, aspirate the medium and rinse the cells with PBS. Add 0.25%trypsin to detach the adherence cells and incubate at 37 degrees Celsius for five minutes. Then add a stopping buffer.Centrifuge the cell suspension at 300 G for five minutes, and remove the supernatant. For antibody staining, resuspend 10 to the seven cells in 100 microliters of PBS. Then add 10 microliters of LYVE-1 antibody solution and mix thoroughly.Incubate the mixture for 30 minutes in the dark at four degrees Celsius. After incubation, centrifuge the cells and discard the supernatant. Then rinse the cells by adding one milliliter of PBS and centrifuging at 300 G for five minutes.For microbead labeling, resuspend the pellet in 100 microliters of PBS and 20 microliters of microbeads. Make swell and incubate for 30 minutes in the dark at four degrees Celsius. Then centrifuge the suspension and wash the pellet with one milliliter of PBS.Again, centrifuge and resuspend the pellet in four milliliters of PBS for magnetic negative exclusion. Pass the cell suspension through a 70 micron strainer to eliminate clumps. Prepare the selection column by rinsing it with three milliliters of PBS.Then add cell suspension into the selection column. Wash the column with three milliliters of PBS and collect LYVE-1 negative cells into a 50 milliliter tube. For magnetic positive selection, pipette six milliliters of PBS into the selection column.Then flush the magnetically labeled cells by firmly pushing the plunger into the selection column to obtain LYVE-1 positive LLECs. Next, centrifuge the positive cell suspension and remove the supernatant. Plate 10 to the fifth cells per square centimeter into a T25 flask with five milliliters of culture medium.Maintain the culture by replacing 50%of the medium every alternate day. When the cells reach 80%confluency, detach them as demonstrated previously before performing cell passage. Flow cytometry analysis revealed that the percentage of LYVE-1 positive cells in passage two was not significantly different from passage three after MACS, indicating a purity greater than 95%Immunofluorescent staining confirmed co-staining of LYVE-1 with PDPN, VEGFR-3 and PROX1, establishing the identity of LLECs.LYVE-1 positive cells did not express F48 and platelet derived growth factor beta, effectively distinguishing LLECs from macrophages and fibroblasts. Leptomeningeal cells before MACS displayed heterogeneous morphology ranging from round spheres to fused fiber shapes. However, post MACS, LLECs exhibited typical endothelial-like features such as spindle and cobblestone shapes.