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

磁気細胞選択法を用いたIn vitro RHEC培養法の樹立

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.

著者スポットライト:軟髄膜リンパ内皮細胞の細胞機能と潜在的な臨床的意味を明らかにする 

軟髄膜リンパ管内皮細胞の採取と初代培養

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  

マウス軟髄膜細胞の効率的な培養のための軟髄膜解離と酵素消化

まず、犠牲にしたマウスの皮膚を、頭蓋骨の開口部から前頭部に向かって慎重に切開します。はさみを使用して、軟髄膜を傷つけることなく頭蓋骨を繊細に持ち上げて取り外し、脳全体を収集します。脳全体を洗浄バッファーに浸し、静かに洗い流して表面の血液を取り除きます。みじん切りにせずに脳を滅菌シャーレに移します。次に、顕微鏡下で、細かいピンセットを使用して、脳の表面から軟髄膜を抽出します。滅菌マイクロハサミを使用して、軟髄膜組織を断片に切断します。与えられた成分を使用して消化酵素ミックスを修復します。フラグメントに10ミリリットルの混合物を加え、摂氏37度で15分間インキュベートします。静かに攪拌して、チューブの底から破片を取り外します。次に、10ミリリットルの停止バッファーを追加します。懸濁液を300gで摂氏4度で5分間遠心分離します。そして、ピペットを使用して、上清を慎重に取り除きます。10ミリリットルの冷たいPBSを加え、凝集塊を70ミクロンのストレーナーでろ過し、50ミリリットルの滅菌チューブに入れます。フィブロネクチンでコーティングされたT25フラスコに10〜5個の細胞を5ミリリットルの培地を入れてプレートし、5%二酸化炭素と摂氏37度で24時間インキュベートします。インキュベーション後、培地を新しい培地と交換して、付着していない細胞を排除します。

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Research

JoVE Journal

Neuroscience

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

311 ビュー.  The First Affiliated Hospital of Kunming Medical University. まず、犠牲にしたマウスの皮膚を、頭蓋骨の開口部から前頭部に向かって慎重に切開します。はさみを使用して、軟髄膜を傷つけることなく頭蓋骨を繊細に持ち上げて取り外し、脳全体を収集します。脳全体を洗浄バッファーに浸し、静かに洗い流して表面の血液を取り除きます。みじん切りにせずに脳を滅菌シャーレに移します。次に、顕微鏡下で、細かいピン?...

ビデオ: 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 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.

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 ...

神経科学

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.