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

Manyetik Hücre Seçimi Kullanılarak İn Vitro LLEC Kültürlerinin Oluşturulması

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.

Yazar Gündemi: Leptomeningeal Lenfatik Endotel Hücrelerinin Hücresel Fonksiyonlarının ve Potansiyel Klinik Etkilerinin Ortaya Çıkarılması 

Leptomeningeal Lenfatik Endotel Hücrelerinin Hasadı ve Primer Kültürü

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  

Leptomeningeal Hücrelerin Verimli Kültürü için Fare Leptomeninges Dissosiyasyonu ve Enzimatik Sindirim

Başlamak için, kurban edilen farenin derisini, kafatasının açıklığından başlayarak ve ön bölgeye doğru uzanarak dikkatlice kesin. Makas kullanarak, leptomeninglere zarar vermeden kafatasını nazikçe kaldırın ve çıkarın, tüm beyni toplayın. Tüm beyni yıkama tamponuna batırın ve yüzeydeki kanı çıkarmak için hafifçe yıkayın.Beyni doğramadan steril bir Petri kabına aktarın. Daha sonra, mikroskop altında, leptomeningleri beynin yüzeyinden çıkarmak için ince uçlu cımbız kullanın. Steril mikromakas kullanarak, leptomeninges dokusunu parçalara ayırın.Verilen bileşenleri kullanarak sindirim enzimi karışımını onarın. Parçalara 10 mililitre karışım ekleyin ve 37 santigrat derecede 15 dakika inkübe edin. Parçaları tüpün dibinden ayırmak için hafifçe çalkalayın.Ardından, 10 mililitre durdurma tamponu ekleyin. Süspansiyonu 300 g'da dört santigrat derecede beş dakika santrifüjleyin. Ve bir pipet kullanarak, süpernatanı dikkatlice çıkarın.10 mililitre soğuk PBS ekleyin ve kümeleri 70 mikronluk bir süzgeçten geçirerek steril 50 mililitrelik bir tüpe süzün. Beş mililitre kültür ortamı içeren fibronektin kaplı bir T25 şişesine santimetre kare başına 10 ila beşinci hücre yerleştirin ve 24 saat boyunca% 5 karbondioksit ile 37 santigrat derecede inkübe edin. İnkübasyondan sonra, bağlı olmayan hücreleri ortadan kaldırmak için ortamı yeni bir ortamla değiştirin.

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Research

JoVE Journal

Neuroscience

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

311 Görüntüleme.  The First Affiliated Hospital of Kunming Medical University. Başlamak için, kurban edilen farenin derisini, kafatasının açıklığından başlayarak ve ön bölgeye doğru uzanarak dikkatlice kesin. Makas kullanarak, leptomeninglere zarar vermeden kafatasını nazikçe kaldırın ve çıkarın, tüm beyni toplayın. Tüm beyni yıkama tamponuna batırın ve yüzeydeki kanı çıkarmak için hafifçe yıkayın.Beyni doğramadan steril bir Petri kabına aktarın. Daha sonra, mikroskop altında, leptomeningleri beynin yüzeyinden çıkarmak i?...

Video: 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 ...

Nörobilim

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.