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

炎症性関節炎の解明:滑膜細胞の機能の探索

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.

著者スポットライト:マウス関節炎組織からの一次滑膜マクロファージおよび線維芽細胞の単離と培養  

マウス関節炎組織からの初代滑膜マクロファージおよび線維芽細胞の単離と培養

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  

滑膜炎組織の消化

解剖したマウスの膝関節を培養皿に入れることから始めます。培養液を吸引し、新鮮な培地を加える。洗濯を3〜4回繰り返します。次に、実体顕微鏡下で、微細な点ピンセットを使用して培養液内のすべての関節を引っ張って脱臼させます。骨を折らないように注意しながら、脛骨とできるだけ多くの血管、腱、靭帯を取り除きます。サンプルごとに2つの15ミリリットルチューブを準備します。ピンセットを使用して、軟部組織で脱臼した骨を最初のチューブに移します。両方の後足から得られた各サンプルについて、4ミリリットルの消化培地をチューブに加える。残留細胞および組織断片を収集するために、培養液を解剖皿から第2の15ミリリットルチューブに移す。培地を500 Gで室温で5分間遠心分離します。上清を除去した後、ペレットを1ミリリットルの消化培地で再懸濁する。そして、この溶液を最初の15ミリリットルのチューブに移して、ほとんどすべての組織、合計5ミリリットルの消化培地が含まれるようにします。次に、ハイブリダイゼーションオーブンで振とうしながら、摂氏37度で60〜120分間サンプルを消化します。インキュベーション後、ピペッティングでサンプルを混合し、40ミクロンのセルストレーナーを通して細胞溶液を50ミリリットルのチューブにろ過します。10ミリリットルの培養液をセルストレーナーを通してチューブに加えます。室温で5分間、300 Gで遠心分離します。上清を除去し、細胞ペレットを10ミリリットルの培地で再懸濁する。遠心分離を繰り返し、上清を捨て、ペレットを2ミリリットルの培養液で再懸濁します。

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 ...