Name: Digestion of Synovitis Tissue
Uploaded: 2023-02-24T13:30:00
Description: Watch this Scientific Journal Video about Digestion of Synovitis Tissue at JoVE.com

digestion of synovitis tissue

염증성 관절염 밝혀내기: 활막 세포의 기능 탐구

Rheumatoid arthritis (RA) is an autoimmune disease characterized by hyperplasia of the synovium, leading to joint destruction1,2. Tissue-resident macrophages and fibroblasts are present in healthy synovium to maintain joint homeostasis. In RA patients, synovial fibroblasts (SFs) proliferate, and immune cells, including monocytes, infiltrate into the synovium and joint fluid, processes associated with inflammation1,3,4. Synovial macrophages (SMs), which include resident macrophages and peripheral blood monocyte-derived macrophages, and SFs are aberrantly activated and have important roles in RA pathogenesis. Recent studies have suggested that cell-cell interactions between SMs and SFs contributes to both the exacerbation and remission of RA5,6. 
To understand RA pathogenesis, several rodent models of inflammatory arthritis have been used, including K/BxN serum transfer arthritis, collagen-induced arthritis, and collagen antibody-induced arthritis. Cell-based assays are generally required to clarify molecular functions in arthritis. Therefore, primary cells from animal models of arthritis have been isolated. The method to isolate SFs from murine arthritis tissue is well established, and these cells have contributed to the elucidation of molecular mechanisms in arthritis pathogenesis7,8. On the other hand, bone marrow-derived macrophages, blood monocyte-derived macrophages, and macrophage cell lines have often been used as macrophage resources for arthritis studies9,10. Since macrophages can acquire functions associated with their microenvironment, general sources of macrophages may lack responses specific to arthritis tissue. In addition, it is difficult to obtain enough synovial cells by sorting, as murine synovium is a very small tissue even in arthritis models. The lack of usage of synovial macrophages for in vitro studies has been a limitation in arthritis studies. The establishment of a protocol to isolate and expand synovial macrophages would be an advantage for the elucidation of pathological mechanisms in RA.
In the previous method to isolate SFs, SMs were discarded7. Besides that, a method to isolate and expand resident macrophages from some organs was reported11. Therefore, existing protocols were modified in combination. The modification aims to achieve the primary culture of both SMs and SFs with high purity. The overall goal of this method is to isolate and expand both SMs and SFs from murine arthritis tissue.

저자 스포트라이트: Isolation and Culture of primary Synovial macrophages and Fibroblasts from murine Arthritis Tissue (쥐 관절염 조직에서 원발성 활막 대식세포 및 섬유아세포의 분리 및 배양)  

쥐 관절염 조직에서 Primary Synovial Macrophages and Fibroblasts의 분리 및 배양

Cell-based assays are required to clarify molecular functions. In arthritis studies, the method to isolate primary cells is well established in synovial fibroblasts, but not in synovial macrophages. Therefore, we developed a protocol to isolate and expand both synovial macrophages and synovial fibroblasts from arthritis models.Primary synovial fibroblasts from murine arthritis tissue have contributed to the elucidation of molecular mechanisms in arthritis pathogenesis. On the other hand, bone marrow-derived macrophages, blood monocyte-derived macrophages, and macrophage cell lines have often been used as macrophage resources for arthritis studies. Since macrophages can acquire functions associated with their microenvironment, general sources of macrophages may lack responses specific to arthritis tissue.In addition, it is difficult to obtain enough synovial cells by sorting, as murine synovium is a very small tissue even in arthritis models. In the previous method to isolate synovial fibroblasts, synovial macrophages were discarded. Besides that, a method to isolate and expand resident macrophages from some organs was reported.Therefore, existing protocols were modified in combination to isolate and expand both synovial macrophages and synovial fibroblasts from murine arthritis tissue. This method can isolate both macrophages and fibroblasts from inflammatory synovium with high purity, and it is simple and reproducible. This method allows the expansion of synovial macrophages under co-culture with synovial fibroblasts.Also, the method provides an advantage, in that synovial fibroblasts can be used with fewer passages. The established protocol would be an advantage for elucidation of pathological mechanisms in rheumatoid arthritis.

Watch this Scientific Journal Video about Digestion of Synovitis Tissue at JoVE.com

Digestion of Synovitis Tissue  

활액막염 조직의 소화

해부된 마우스 무릎 관절을 배양 접시에 넣는 것으로 시작합니다. 배양 배지를 흡인하고 신선한 배양 배지를 추가합니다. 세탁을 서너 번 반복하십시오.그런 다음 실체 현미경으로 미세한 핀셋을 사용하여 잡아당겨 배양 배지의 모든 관절을 탈구시킵니다. 경골과 가능한 한 많은 혈관, 힘줄 및 인대를 제거하고 뼈가 부러지지 않도록주의하십시오. 샘플 당 두 개의 15 밀리리터 튜브를 준비하십시오.핀셋을 사용하여 연조직이있는 탈구 된 뼈를 첫 번째 튜브로 옮깁니다. 양쪽 뒷발에서 얻은 각 샘플에 대해 4 밀리리터의 소화 배지를 튜브에 첨가하십시오. 잔류 세포 및 조직 단편을 수집하기 위해, 배양 배지를 해부 접시로부터 제2 15 밀리리터 튜브로 옮긴다.배지를 500G에서 실온에서 5분 동안 원심분리합니다. 상청액을 제거한 후, 1 밀리리터의 소화 매체로 펠렛을 재현 탁시킨다. 그리고이 용액을 처음 15 밀리리터 튜브로 옮겨 거의 모든 조직, 총 5 밀리리터의 소화 매체를 포함합니다.그런 다음 섭씨 37도에서 60 내지 120분 동안 샘플을 하이브리드화 오븐에서 진탕하여 분해한다. 인큐베이션 후, 샘플을 피펫팅으로 혼합하고, 40 미크론 셀 스트레이너를 통해 세포 용액을 50 밀리리터 튜브로 여과한다. 세포 여과기를 통해 10 밀리리터의 배양 배지를 튜브에 첨가한다.실온에서 5분 동안 300G에서 원심분리합니다. 상청액을 제거하고 세포 펠릿을 10 밀리리터의 배양 배지로 재현탁한다. 원심분리를 반복하고, 상층액을 버리고, 2밀리리터의 배양 배지로 펠릿을 재현탁한다.

Research

JoVE Journal

Biology

Digestion of Synovitis Tissue

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...

연구

생물학

Begin by placing the dissected mouse knee joints in a culture dish. Aspirate the culture medium and add fresh culture medium. Repeat the washing three or four times.Then under a stereo microscope, dislocate all the joints in the culture medium by pulling them using fine point tweezers. Remove the tibia and as many vessels, tendons, and ligaments as possible, being careful not to break the bones. Prepare two 15 milliliter tubes per sample.Using tweezers, transfer the dislocated bones with soft tissues to the first tube. For each sample obtained from both hind paws add four milliliters of digestion medium to the tube. To collect residual cells and tissue fragments transfer the culture medium from the dissection dish to the second 15 milliliter tube.Centrifuge the medium at 500 G for 5 minutes at room temperature. After removing the supernatant, resuspend the pellet with one milliliter of digestion medium. And transfer this solution to the first 15 milliliter tube so that it contains almost all the tissue, in total five milliliters of digestion medium.Then digest the sample for 60 to 120 minutes at 37 degrees Celsius with shaking in a hybridization oven. After incubation, mix the sample by pipetting and filter the cell solution through a 40 micron cell strainer into a 50 milliliter tube. Add 10 milliliters of culture medium to the tube through the cell strainer.Centrifuge at 300 G for 5 minutes at room temperature. Remove the supernatant and resuspend the cell pellet with 10 milliliters of culture medium. Repeat the centrifugation, discard the supernatant, and resuspend the pellet with two milliliters of culture medium.

Isolation of Synovial Fibroblasts

Use the cell suspensions obtained after digestion of mouse knee joints, and seed the suspension on the collagen-coated dish. Incubate the dish for one hour at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide. After incubation, collect non-adherent cells using a pipette.Wash the collagen coated dish with culture medium, and collect the medium. Then, add fresh medium to the dish to culture the adherent cells that exhibit a fibroblastoid morphology. Treat the cells with 0.05%trypsin in Hank's balanced salt solution, and passage the sub confluent cells.If highly pure fibroblast-like cells are required, perform repeated passaging. Ensure to use cells with less than five passages. Fibroblast-like cells were isolated from inflammatory arthritis tissue, induced in seven to eight weeks old female C57BL/6 mice.The purity of the isolated cells was assessed by RTQPCR mRNA expression of synovial fibroblast markers, such as Vcam1, Cdh11, Col6a1, and Csf1 in the isolated cells suggested that the fibroblast-rich fractions were isolated from synovitis tissue.

활막 섬유아세포의 분리

Isolation of Synovial Macrophages

Before beginning, perform synovial fibroblast isolation and use the non-adherent cells collected from the collagen-coated dish in this procedure. Seed the non-adherent cells on 40 to 60 millimeter dishes that have not been coated with collagen. Culture the bulk cells for one day at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide.To remove non-adherent lymphocytes, aspirate the cultured medium and then add fresh culture medium. Culture the adherent bulk cells for one to two weeks with medium changes every two days, while maintaining confluence. Then wash two times with PBS or HBSS.Select for synovial macrophages by treating with 0.05%trypsin in HBSS and incubate for three minutes at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide. Then add culture medium gently to 0.05%trypsin in HBSS. After this step, do not pour the medium directly onto the cells.To remove detached cells, aspirate the cultured medium and then add fresh culture medium gently. Repeat two or three times and maintain the cells on the dish in fresh culture medium until use. Macrophage-like cells were isolated from seven to eight weeks old female C57BL/6 mice and analyzed by RT-qPCR.mRNA expression of pan-macrophage markers CD68, EMR1, ITGAM, CSF1R showed macrophage rich isolation from the synovitis tissue. To establish the purity of macrophages, surface protein markers for macrophages and other cell types were analyzed by flow cytometry. More than 90%of the cells expressed macrophage markers CD45, CD11b and F4/80, whereas the expression of neutrophil marker Ly6G and T-cell marker CD3 was lower than 1%