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

Summary

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

Abstract

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.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by hyperplasia of the synovium, leading to joint destruction^1,2. 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 inflammation^1,3,4. Synovial macrophages (SMs), which include resident macrophages and peripheral blood monocyte-derived....

Protocol

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 digestion medium as follows: supplement the culture medium with 1 mg/mL collagenase type IV. Adjust the collagenase concentration just before use.
3. Dil....

Results

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.......

Discussion

This method developed here improves upon previous techniques for isolating both SFs from murine arthritis and resident macrophages from a number of organs^7,11. 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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5.0 g/L Trypsin/5.3 mmol/L EDTA solution	nacalai tesque	35556-44	Diluted with HBSS
Antibiotic–antimycotic (anti/anti)	Gibco	15240-062
Butterfly needle	TERUMO	SV-23DLK	23G
Cell strainer	Falcon	352340	40 µm pore, Nylon
Cellmatrix Type I-C	Nitta gelatin	637-00773	Type I-C collagen
Centriguge tube 15	TPP	91014	15 mL tube
Centriguge tube 50	TPP	91050	50 mL tube
Collagenase from C. Histolyticum	Sigma	C5138	Type IV collagenase
Dulbecco’s Modified Eagle Medium GlutaMax (DMEM)	Gibco	10569-010
Fetal bovine serum (FBS)	SIGAM	173012	Heat inactivation was performed
Hanks' balanced salt solution (HBSS)	Wako	085-09355
Scissors	Bio Research Center	PRI28-1525A
Tissue culture dish 40	TPP	93040	For cell culture
Tissue culture dish 60	TPP	92006	For cell culture
Tweezers	KFI	1-9749-31	Fine-point
Tweezers	Bio Research Center	PRI28-1522	Serrated tip
ZEISS Stemi 305	ZEISS	STEMI305-EDU	Stereomicroscope