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 ...