Name: Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue
Uploaded: 2023-02-24T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue at JoVE.com

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 Grant, The Sumitomo Foundation, SENSHIN Medical Research Foundation, The Mochida Memorial Foundation (to NS); and a Takeda Science Foundation Medical Research grant, UCB Japan (UCBJ) project grant, and the JSBMR&#160;Frontier Scientist grant 2019 (to YI).

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
<ol>
	<li>Prepare the culture medium as follows: supplement Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (anti-anti).</li>
	<li>Prepare the digest...

Unveiling Inflammatory Arthritis&amp;#58; Exploring Functions of Synovial Cells

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolically+engineered+stem+cell-derived+exosomes+to+regulate+macrophage+heterogeneity+in+rheumatoid+arthritis.">Metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis.</a> Science Advances. 7 (23), 0083 (2021).</li><li>Ogawa, K., Tsurutani, M., Hashimoto, A., Soeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+propagation+method+for+resident+macrophages+by+co-culture+and+subculture,+and+their+isolation+from+various+organs.">Simple propagation method for resident macrophages by co-culture and subculture, and their isolation from various organs.</a> BMC Immunology. 20 (1), 34 (2019).</li><li>Andr&#228;, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+T-cell+functionality+after+flow+cytometry+sorting+revealed+p38+MAPK+activation.">An evaluation of T-cell functionality after flow cytometry sorting revealed p38 MAPK activation.</a> Cytometry Part A. 97 (2), 171-183 (2020).</li><li>Ryan, K., Rose, R. 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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 ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5.0 g/L Trypsin/5.3 mmol/L EDTA solution</td>
			<td>nacalai tesque</td>
			<td>35556-44</td>
			<td>Diluted with HBSS</td>
		</tr>
		<tr>
			<td>Antibiotic&#8211;antimycotic (anti/anti)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly needle</td>
			<td>TERUMO</td>
			<td>SV-23DLK</td>
			<td>23G</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td>40 &#181;m pore, Nylon</td>
		</tr>
		<tr>
			<td>Cellmatrix Type I-C</td>
			<td>Nitta gelatin</td>
			<td>637-00773</td>
			<td>Type I-C collagen</td>
		</tr>
		<tr>
			<td>Centriguge tube 15</td>
			<td>TPP</td>
			<td>91014</td>
			<td>15 mL tube</td>
		</tr>
		<tr>
			<td>Centriguge tube 50</td>
			<td>TPP</td>
			<td>91050</td>
			<td>50 mL tube</td>
		</tr>
		<tr>
			<td>Collagenase from C. Histolyticum</td>
			<td>Sigma</td>
			<td>C5138</td>
			<td>Type IV collagenase</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium GlutaMax (DMEM)</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>SIGAM</td>
			<td>173012</td>
			<td>Heat inactivation was performed</td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution (HBSS)</td>
			<td>Wako</td>
			<td>085-09355</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Bio Research Center</td>
			<td>PRI28-1525A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish 40</td>
			<td>TPP</td>
			<td>93040</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Tissue culture dish 60</td>
			<td>TPP</td>
			<td>92006</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>KFI</td>
			<td>1-9749-31</td>
			<td>Fine-point</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Bio Research Center</td>
			<td>PRI28-1522</td>
			<td>Serrated tip</td>
		</tr>
		<tr>
			<td>ZEISS Stemi 305</td>
			<td>ZEISS</td>
			<td>STEMI305-EDU</td>
			<td>Stereomicroscope</td>
		</tr>
	</tbody>
</table>

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.

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

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

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.

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...

Watch this Scientific Journal Video about Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue at JoVE.com

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 ...

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Biology

Digestion of Synovitis Tissue

Begin by placing the dissected mouse knee joints in a culture dish. Aspirate the culture medium and add fresh culture medium. Repeat the washing three or four times.Then under a stereo microscope, dislocate all the joints in the culture medium by pulling them using fine point tweezers. Remove the tibia and as many vessels, tendons, and ligaments as possible, being careful not to break the bones. Prepare two 15 milliliter tubes per sample.Using tweezers, transfer the dislocated bones with soft tissues to the first tube. For each sample obtained from both hind paws add four milliliters of digestion medium to the tube. To collect residual cells and tissue fragments transfer the culture medium from the dissection dish to the second 15 milliliter tube.Centrifuge the medium at 500 G for 5 minutes at room temperature. After removing the supernatant, resuspend the pellet with one milliliter of digestion medium. And transfer this solution to the first 15 milliliter tube so that it contains almost all the tissue, in total five milliliters of digestion medium.Then digest the sample for 60 to 120 minutes at 37 degrees Celsius with shaking in a hybridization oven. After incubation, mix the sample by pipetting and filter the cell solution through a 40 micron cell strainer into a 50 milliliter tube. Add 10 milliliters of culture medium to the tube through the cell strainer.Centrifuge at 300 G for 5 minutes at room temperature. Remove the supernatant and resuspend the cell pellet with 10 milliliters of culture medium. Repeat the centrifugation, discard the supernatant, and resuspend the pellet with two milliliters of culture medium.

Isolation of Synovial Fibroblasts

Use the cell suspensions obtained after digestion of mouse knee joints, and seed the suspension on the collagen-coated dish. Incubate the dish for one hour at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide. After incubation, collect non-adherent cells using a pipette.Wash the collagen coated dish with culture medium, and collect the medium. Then, add fresh medium to the dish to culture the adherent cells that exhibit a fibroblastoid morphology. Treat the cells with 0.05%trypsin in Hank's balanced salt solution, and passage the sub confluent cells.If highly pure fibroblast-like cells are required, perform repeated passaging. Ensure to use cells with less than five passages. Fibroblast-like cells were isolated from inflammatory arthritis tissue, induced in seven to eight weeks old female C57BL/6 mice.The purity of the isolated cells was assessed by RTQPCR mRNA expression of synovial fibroblast markers, such as Vcam1, Cdh11, Col6a1, and Csf1 in the isolated cells suggested that the fibroblast-rich fractions were isolated from synovitis tissue.

Isolation of Synovial Macrophages

Before beginning, perform synovial fibroblast isolation and use the non-adherent cells collected from the collagen-coated dish in this procedure. Seed the non-adherent cells on 40 to 60 millimeter dishes that have not been coated with collagen. Culture the bulk cells for one day at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide.To remove non-adherent lymphocytes, aspirate the cultured medium and then add fresh culture medium. Culture the adherent bulk cells for one to two weeks with medium changes every two days, while maintaining confluence. Then wash two times with PBS or HBSS.Select for synovial macrophages by treating with 0.05%trypsin in HBSS and incubate for three minutes at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide. Then add culture medium gently to 0.05%trypsin in HBSS. After this step, do not pour the medium directly onto the cells.To remove detached cells, aspirate the cultured medium and then add fresh culture medium gently. Repeat two or three times and maintain the cells on the dish in fresh culture medium until use. Macrophage-like cells were isolated from seven to eight weeks old female C57BL/6 mice and analyzed by RT-qPCR.mRNA expression of pan-macrophage markers CD68, EMR1, ITGAM, CSF1R showed macrophage rich isolation from the synovitis tissue. To establish the purity of macrophages, surface protein markers for macrophages and other cell types were analyzed by flow cytometry. More than 90%of the cells expressed macrophage markers CD45, CD11b and F4/80, whereas the expression of neutrophil marker Ly6G and T-cell marker CD3 was lower than 1%