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Biology

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

Published: February 24th, 2023

DOI:

10.3791/65196

1Division of Medical Research Support, Advanced Research Support Center, Ehime University, 2Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, 3Department of Pathophysiology, Graduate School of Medicine, Ehime University

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 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.

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....

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Experiments involving animals were approved by the Animal Experiment Committee of Ehime University and were performed in accordance with Ehime University Guidelines for Animal Experiments (37A1-1*16).

1. Preparation of instruments, reagents, and culture medium

  1. Prepare the culture medium as follows: supplement Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (anti-anti).
  2. Prepare the digest.......

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Female C57BL/6 mice at 7-8 weeks of age underwent collagen antibody-induced arthritis. Macrophage-like cells and fibroblast-like cells were independently isolated from inflammatory arthritis tissue according to the procedure described above (Figure 2A,B). Macrophage-like cells were immediately used after step 5.7. Fibroblast-like cells were initially cultured to be sub-confluent after step 4.4, and then passaged to a new culture dish followed by usage. To evaluate whether SM.......

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This method developed here improves upon previous techniques for isolating both SFs from murine arthritis and resident macrophages from a number of organs7,11. The modified method can isolate both macrophages and fibroblasts from inflammatory synovium with high purity, and it is simple and reproducible. Since the method does not require complex instruments such as a cell sorter, anyone can conduct it. In addition, the present technique avoids concerns associated .......

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The authors thank the staff at the Division of Medical Research Support, the Advanced Research Support Center (ADRES), and the members of the Division of Integrative Pathophysiology, Proteo-Science Center (PROS), Ehime University, for their technical assistance and helpful support. This study was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants JP17K17929, JP19K16015, JP21K05974 (to NS) and JP23689066, JP15H04961, JP15K15552, JP17K19728, JP19H03786 (to YI); grants from the Osaka Medical Research Foundation for Intractable Diseases, The Nakatomi Foundation, The Japanese Society for Bone and Mineral Research (JSBMR) Rising Stars ....

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NameCompanyCatalog NumberComments
5.0 g/L Trypsin/5.3 mmol/L EDTA solutionnacalai tesque35556-44Diluted with HBSS
Antibiotic–antimycotic (anti/anti)Gibco15240-062
Butterfly needleTERUMOSV-23DLK23G
Cell strainerFalcon35234040 µm pore, Nylon
Cellmatrix Type I-CNitta gelatin637-00773Type I-C collagen
Centriguge tube 15TPP9101415 mL tube
Centriguge tube 50TPP9105050 mL tube
Collagenase from C. HistolyticumSigmaC5138Type IV collagenase
Dulbecco’s Modified Eagle Medium GlutaMax (DMEM)Gibco10569-010
Fetal bovine serum (FBS)SIGAM173012Heat inactivation was performed
Hanks' balanced salt solution (HBSS)Wako085-09355
ScissorsBio Research CenterPRI28-1525A
Tissue culture dish 40TPP93040For cell culture
Tissue culture dish 60TPP92006For cell culture
TweezersKFI1-9749-31Fine-point
TweezersBio Research CenterPRI28-1522Serrated tip
ZEISS Stemi 305ZEISSSTEMI305-EDUStereomicroscope

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