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

Magnetic Cell Selection을 사용한 In Vitro LLEC 배양 확립

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.

저자 스포트라이트: 연수막 림프 내피 세포의 세포 기능 및 잠재적 임상적 영향 밝혀내기 

연수막 림프 내피 세포의 수확 및 1차 배양

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  

마우스 렙토메닝 분해 및 효소 분해를 위한 연수막 세포의 효율적인 배양

시작하려면 희생된 쥐의 피부를 두개골 입구에서 시작하여 정면 부위로 뻗어 조심스럽게 절개합니다. 가위를 사용하여 연수막을 손상시키지 않고 두개골을 섬세하게 들어 올려 제거하고 뇌 전체를 수집합니다. 뇌 전체를 세척 버퍼에 담그고 부드럽게 씻어내어 표면 혈액을 제거합니다.뇌를 자르지 않고 멸균된 페트리 접시에 옮깁니다. 그런 다음 현미경으로 미세한 핀셋을 사용하여 뇌 표면에서 렙토메닝을 추출합니다. 멸균 마이크로 가위를 사용하여 렙토메닝 조직을 조각으로 자릅니다.주어진 성분을 사용하여 소화 효소 혼합물을 복구하십시오. 혼합물 10mL를 조각에 넣고 섭씨 37도에서 15분 동안 배양합니다. 부드럽게 저어 튜브 바닥에서 조각을 분리합니다.그런 다음 10밀리리터의 중지 버퍼를 추가합니다. 서스펜션을 섭씨 4도에서 5분 동안 300g으로 원심분리합니다. 그리고 피펫을 사용하여 상층액을 조심스럽게 제거합니다.10밀리리터의 콜드 PBS를 추가하고 70미크론 스트레이너를 통해 덩어리를 여과하여 멸균된 50밀리리터 튜브에 넣습니다. 평방 센티미터당 10개에서 5번째 세포를 5밀리리터의 배양 배지가 있는 피브로넥틴 코팅된 T25 플라스크에 넣고 섭씨 37도에서 5% 이산화탄소로 24시간 동안 배양합니다. 배양 후 배지를 새 배지로 교체하여 부착되지 않은 세포를 제거합니다.

mouse-leptomeninges-dissociation-enzymatic-digestion-for-efficient

Research

JoVE Journal

Neuroscience

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

304 조회수.  The First Affiliated Hospital of Kunming Medical University. 시작하려면 희생된 쥐의 피부를 두개골 입구에서 시작하여 정면 부위로 뻗어 조심스럽게 절개합니다. 가위를 사용하여 연수막을 손상시키지 않고 두개골을 섬세하게 들어 올려 제거하고 뇌 전체를 수집합니다. 뇌 전체를 세척 버퍼에 담그고 부드럽게 씻어내어 표면 혈액을 제거합니다.뇌를 자르지 않고 멸균된 페트리 접시에 옮깁니다. 그런 다음 현미경으로 미세한 핀...

비디오: 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 ...

연구

신경과학

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.