isolation of synovial fibroblasts

Desvelando la artritis inflamatoria: explorando las funciones de las células sinoviales

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.

Autor destacado: Aislamiento y cultivo de macrófagos sinoviales primarios y fibroblastos a partir de tejido de artritis murina  

Aislamiento y cultivo de macrófagos sinoviales primarios y fibroblastos a partir de tejido murino de artritis

Watch this Scientific Journal Video about Isolation of Synovial Fibroblasts at JoVE.com

Isolation of Synovial Fibroblasts  

Aislamiento de fibroblastos sinoviales

Utilice las suspensiones celulares obtenidas después de la digestión de las articulaciones de la rodilla del ratón y siembre la suspensión en el plato recubierto de colágeno. Incubar la placa durante una hora a 37 grados centígrados en una atmósfera humidificada con un 5% de dióxido de carbono. Después de la incubación, recoja las células no adherentes con una pipeta.Lave el plato recubierto de colágeno con medio de cultivo y recoja el medio. Luego, agregue medio fresco a la placa para cultivar las células adherentes que exhiben una morfología fibroblastoide. Trate las células con tripsina al 0,05% en la solución salina equilibrada de Hank y haga pasar las células subconfluentes.Si se requieren células similares a fibroblastos de alta pureza, realice pases repetidos. Asegúrese de utilizar celdas con menos de cinco pasajes. Se aislaron células similares a fibroblastos del tejido inflamatorio de la artritis, inducidas en ratones hembra C57BL/6 de siete a ocho semanas de edad.La pureza de las células aisladas se evaluó mediante RTQPCR La expresión de ARNm de marcadores de fibroblastos sinoviales, como Vcam1, Cdh11, Col6a1 y Csf1 en las células aisladas, sugirió que las fracciones ricas en fibroblastos se aislaron del tejido de la sinovitis.

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JoVE Journal

Biology

Isolation of Synovial Fibroblasts

679 vistas. Utilice las suspensiones celulares obtenidas después de la digestión de las articulaciones de la rodilla del ratón y siembre la suspensión en el plato recubierto de colágeno. Incubar la placa durante una hora a 37 grados centígrados en una atmósfera humidificada con un 5% de dióxido de carbono. Después de la incubación, recoja las células no adherentes con una pipeta.Lave el plato recubierto de colágeno con medio de cultivo y recoja el medio. Luego, agregue medio fresco a l...

Video: Isolation of Synovial Fibroblasts  

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 ...