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 digestion medium as follows: supplement the culture medium with 1 mg/mL collagenase type IV. Adjust the collagenase concentration just before use.</li>
	<li>Dilute type I-C collagen to a concentration of 0.15 mg/mL with 1 mM HCl solution. Flood culture dishes (diameter of 40 or 60 mm) with the diluted collagen solution. After 6-12 h at room temperature, remove the collagen solution from the dishes and dry at room temperature. The collagen-coated dishes can be kept at room temperature for at least 1 week. Wash the pre-coated dishes with phosphate buffered saline (PBS) or medium before use.</li>
	<li>Prepare sterile surgical instruments, such as scissors, tweezers with serrated tips, and fine-point tweezers. Soak in 70% ethanol before use.</li>
</ol>
2. Preparation of synovitis tissue in mice ( Figure 1A)
<ol>
	<li>Prepare a mouse with inflammatory arthritis in the hind paws. 
	NOTE: Female C57BL/6 mice (18-20 g), 7-8 weeks postnatal, with collagen antibody-induced arthritis (CAIA) or K/BxN serum transfer arthritis (STA) were used for this protocol8. Isolating SMs from non-swollen (i.e., healthy) tissue might be difficult, as the number of cells is insufficient.</li>
	<li>Anesthetize the mice by an intraperitoneal injection of 80 mg/kg ketamine and 16 mg/kg xylazine. Clean the mice with 70% ethanol.</li>
	<li>Cut open the pectoral region with scissors to expose the heart. Cut the right auricle of the heart with scissors, and then stick a 23 G butterfly needle into the left ventricle via apex of the heart, followed by a reflux of 15-20 mL of sterile PBS using a syringe to manually remove peripheral blood (approximately 1 mL/2 s).</li>
	<li>Decorticate the hind paws by cutting the skin using scissors and pulling skin using tweezers with serrated tips. 
	NOTE: After this step, the usage of tweezers is recommended for the handling of samples. Don&#39;t touch decorticated tissue with fingers, to avoid the attachment of murine hair.</li>
	<li>Dislocate the metatarsophalangeal joints by pulling, followed by cutting the ligaments of the joints using scissors to remove the toes.</li>
	<li>Cut the tendons of the lower leg muscles near the ankle using scissors. Grasp the tendon with tweezers and peel the muscles proximally in the lower leg to expose the tibia. Remove the fibula.</li>
	<li>Dislocate the knee joint by pulling, followed by cutting the ligaments of the joints using scissors to detach the tibia with the swollen hind paw. Keep the samples in ice-cold culture medium (0.3 mL/cm2) until processing to step 3.1.</li>
</ol>
3. Digestion of synovitis tissue&#160;( Figure 1B)
<ol>
	<li>Aspirate the culture medium, avoiding the sample, and then add fresh culture medium (0.3 mL/cm2). Repeat the washing process three or four times. 
	NOTE: From this step, the handling of samples should be performed aseptically in a clean bench or safety cabinet.</li>
	<li>Dislocate all the joints of the samples by pulling using fine-point tweezers in the culture medium under a stereomicroscope (at 10x-20x magnification). Fine-point tweezers are convenient in this step. Remove the tibia and as many vessels, tendons, and ligaments as possible. Be careful not to break the bones when dislocating.</li>
	<li>Prepare two 15 mL tubes per sample. Transfer the dislocated bones with soft tissues to the first 15 mL tube using tweezers. Add 4 mL of digestion medium per sample, obtained from both hind paws, into the tube.</li>
	<li>To collect residual cells and tissue fragments, transfer the medium in which the sample was contained to the second 15 mL tube. Centrifuge the medium at 500 x g for 5 min at room temperature. After removing the supernatant, resuspend the pellet with 1 mL of digestion medium and transfer the cell and tissue fragment solution to the first 15 mL tube containing almost all the tissue (total of 5 mL of digestion medium/hind paws).</li>
	<li>Digest the sample for 60-120 min at 37 &#176;C with shaking in a hybridization oven. 
	&#8203;NOTE: The optimal time to digest the samples should be decided. The time is dependent on the degree of swelling in the ankles and collagenases. In most cases, 60-120 min is sufficient. After 60 min of incubation, collect a part of the digested sample by pipetting, and observe under a microscope. If the digestion is insufficient, the incubation should continue, and the digestion checked every 30 min.</li>
	<li>Pipet the solution well. Filter the cell solution through a cell strainer (40 &#181;m pore size) to a 50 mL tube.</li>
	<li>Add 10 mL of culture medium to the 50 mL tube through the cell strainer. Centrifuge at 300 x g for 5 min at room temperature.</li>
	<li>After removing the supernatant, resuspend with 10 mL of culture medium. Repeat the centrifugation. After removing the supernatant, resuspend with 2 mL of culture medium.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65196/65196fig01.jpg" /> 
Figure 1: Procedure of sampling of murine arthritis tissue and collagenase digestion. (A) (i) Murine hind paw with inflammatory arthritis. (ii) Removal of the skin on the hind paw. (iii) Dislocation of the metatarsophalangeal joints and removal of the toes. (iv) Cutting of the tendons in the ankle. (v) Removal of the muscles in the lower legs. (vi) Dislocation of the knee joint. (B) Left; excised legs in culture medium. Right; dislocated tarsus and metatarsus in culture medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65196/65196fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Isolation of synovial fibroblasts ( Figure 2A)
<ol>
	<li>Seed all cell suspensions obtained from both ankles on the collagen-coated dish. 
	NOTE: If using ankles with weak or moderate swelling to obtain the cells, a 40 mm diameter dish is applied. The collagen-coated dish size can be changed to a 60 mm diameter dish if both ankles have severe swelling.</li>
	<li>Add culture medium (approximately 222 &#181;L/cm2). Incubate for 1 h at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>Collect non-adherent cells using a pipet (use in step 5.1). Wash the collagen-coated dish with culture medium and collect the medium. Culture the adherent cells in fresh medium (Figure 2B,i). Most of the cells that quickly adhere to the collagen-coated dish exhibit a fibroblastoid (spindle-shaped) morphology.</li>
	<li>Passage sub-confluent cells by treatment with 0.05% trypsin in Hanks&#39; balanced salt solution (HBSS). In this method, the contamination of other cells is limited, even if in the initial expansion. If more purified fibroblast-like cells are required, perform repeated passaging to allow enhancement of the purity; however, the expansion of the cells&#39; cytoplasm in adhesion is also observed (Figure 2B,ii). Since excessive passages affect the loss of na&#239;ve characteristics in the cells, use cells with fewer than 5 passages.</li>
</ol>
5. Isolation of synovial macrophages ( Figure 2A)
<ol>
	<li>Seed all non-adherent cells from step 4.4 on dishes (diameter of 40 or 60 mm) that have not been coated with collagen. 
	NOTE: Non-adherent cells include macrophages, other lymphocytes, and residual fibroblasts from synovitis tissue.</li>
	<li>Culture the bulk cells for 1 day at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>To remove non-adherent lymphocytes, aspirate the cultured medium, and then add fresh culture medium. Repeat this process two or three times (Figure 2B,iii).</li>
	<li>Culture the adherent bulk cells for 1-2 weeks in culture medium, with medium changes every 2 days while maintaining confluence (Figure 2B,iv). 
	NOTE: The number of SMs slowly increases under co-culture conditions with SFs. Thus, the co-culture period should be adjusted as needed.</li>
	<li>Wash with PBS or HBSS two times. Treat with 0.05% trypsin in HBSS (approximately 55 &#181;L/cm2) for 3 min at 37 &#176;C in a humidified atmosphere with 5% CO2. Fibroblasts easily detach from the culture dish by trypsin treatment, and macrophages exhibit resistance to trypsin treatment. Use this property for the selection of synovial macrophages.</li>
	<li>Add culture medium (approximately 222 &#181;L/cm2) gently to 0.05% trypsin in HBSS. After this step, do not pour the medium directly onto the cells.</li>
	<li>To remove detached cells, aspirate the cultured medium, and then add fresh culture medium gently. Repeat this process two or three times. Maintain the remaining cells on the dish in fresh culture medium until use (Figure 2B,v). 
	NOTE: Following trypsin treatment, adherent cells exhibit macrophage-like morphological characteristics.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65196/65196fig02.jpg" /> 
Figure 2: Separation of macrophage-rich and fibroblast-rich fractions from inflammatory arthritis tissue. (A) Schema of the procedure to separate macrophage-rich and fibroblast-rich cells from arthritis tissue. (B) Representative phase contrast images of stages of the procedure, (i) to (v) in Figure 2A. Scale bar represents 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65196/65196fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

<ol>
	<li>Smolen, J. S., Aletaha, D., McInnes, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+arthritis.">Rheumatoid arthritis.</a> Lancet. 388 (10055), 2023-2038 (2016).</li><li>McInnes, I. B., Schett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+rheumatoid+arthritis.">The pathogenesis of rheumatoid arthritis.</a> The New England Journal of Medicine. 365 (23), 2205-2219 (2011).</li><li>Kurowska-Stolarska, M., Alivernini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+tissue+macrophages:+friend+or+foe.">Synovial tissue macrophages: friend or foe.</a> RMD Open. 3 (2), (2017).</li><li>Hannemann, N., Apparailly, F., Courties, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+macrophages:+from+ordinary+eaters+to+extraordinary+multitaskers.">Synovial macrophages: from ordinary eaters to extraordinary multitaskers.</a> Trends in Immunology. 42 (5), 368-371 (2021).</li><li>Alivernini, S., 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=Distinct+synovial+tissue+macrophage+subsets+regulate+inflammation+and+remission+in+rheumatoid+arthritis.">Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.</a> Nature Medicine. 26 (8), 1295-1306 (2020).</li><li>Saeki, N., Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+synovial+macrophage+metabolism+by+synovial+fibroblasts+under+inflammatory+conditions.">Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions.</a> Cell Communication and Signaling. 18 (1), 188 (2020).</li><li>Armaka, M., Gkretsi, V., Kontoyiannis, D., Kollias, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+the+isolation+and+culture+of+normal+and+arthritogenic+murine+synovial+fibroblasts.">A standardized protocol for the isolation and culture of normal and arthritogenic murine synovial fibroblasts.</a> Protocol Exchange. , (2009).</li><li>Saeki, N., 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=Epigenetic+regulator+UHRF1+orchestrates+proinflammatory+gene+expression+in+rheumatoid+arthritis+in+a+suppressive+manner.">Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner.</a> The Journal of Clinical Investigation. 132 (11), (2022).</li><li>Midwood, K., 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=Tenascin-C+is+an+endogenous+activator+of+Toll-like+receptor+4+that+is+essential+for+maintaining+inflammation+in+arthritic+joint+disease.">Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease.</a> Nature Medicine. 15 (7), 774-780 (2009).</li><li>You, D. 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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The authors declare that they have no competing interests.

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 with other methods, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) which may induce damage and stress in cells due to treatment with antibodies and the physical effects associated with sorting12,13. The resultant cells can be used for investigation with relatively few unnecessary external stimuli. Previously, in vitro analysis of tissue-resident macrophages from the synovium has been difficult, as the number of cells isolated is small, even under arthritic conditions in mice. This method allows the expansion of SMs under co-culture with SFs, as well as the previous method for macrophages from the liver, spleen, lung, and brain11. In addition, the qPCR result suggests that only one passage of SFs allows a high enough level of purity (Figure 3B), although a previous method to isolate SFs required at least three passages7. The lack of a purity check of SFs by flow cytometric analyses is a limitation of this study. However, the method also provides an advantage in that SFs can be used with fewer passages.
The critical step to isolate synovial cells with high purity is to avoid the contamination of bacteria and/or bone marrow-derived cells. Disinfected mice should be managed with sterile tools. In particular, isolated ankle samples should contain as little hair as possible. If a lot of hair is present in the samples, increased washing (step 3.1) should be performed. Also, bones obtained from arthritis are often brittle14; dislocation of the metatarsophalangeal joints requires great care to avoid fracture, and contamination of bone marrow-derived cells by a fracture would interrupt the quality of synovial cells. If the bone is fractured, the broken bone should be removed immediately.
A recent single-cell RNA sequence analysis revealed that several sub-populations of SMs and SFs are present in the synovium5,15. In this method, bulk cells, including heterogeneous sub-populations, may be cultured. In general, the heterogeneity of the cells is lost under in vitro conditions16. Indeed, a previous RNA-sequence analysis indicated that cells obtained by this method have more characteristics, including some sub-populations in terms of a gene expression pattern, than a particular characteristic of a sub-population6. This suggests that cells obtained by this method may have some characteristics of the microenvironment of synovial tissue. Therefore, this technique anticipates being useful as a new approach to studying cell populations in inflammatory arthritis.
Since the development of swelling after experimental arthritis induction is generally monitored in ankles8,10,15, the method of synovial cell isolation from only ankle tissues was provided here. The cells obtained by the methods developed here could be used with other joints such as knee and/or other arthritis models. However, the purity, cell number, and proliferative capacity might be different. This method also could be used for the isolation of macrophages from human synovium; however, verification is required.

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><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&amp;ndash;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 &amp;micro;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&amp;rsquo;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&#039; 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.

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 SMs and SFs were successfully isolated, the following experiments were performed.
To assess the purity of the isolated cells, mRNA expression of various cell markers was analyzed by RT-qPCR. Cd68, Emr1, Itgam, and Csf1r were used as pan-macrophage markers, and Cdh11, Col6a1, Csf1, and Vcam1 were used as SF markers. Rplp0 was used as reference gene6,8. All marker genes were normalized by Rplp0 expression. Both cell-type markers were analyzed in the macrophage-like cells, and fibroblast-like cells were obtained from arthritis tissue. SF markers were expressed in fibroblast-like cells and pan-macrophage markers were expressed in macrophage-like cells, suggesting that macrophage-rich and fibroblast-rich fractions were isolated from synovitis tissue, respectively (Figure 3A,B).
To establish the purity of macrophages, surface protein markers for macrophages and other cell types were analyzed by flow cytometry. F4/80 and CD11b were used for macrophage markers, Ly6G for a neutrophil marker, and CD3 for a T cell marker6. The gating strategy was used as previously shown6. More than 90% of the cells expressed CD45, CD11b, and F4/80, whereas the expression of Ly6G and CD3 was lower than 1% (Figure 4).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65196/65196fig03.jpg" /> 
Figure 3: mRNA expression of cell-type specific markers. (A) mRNA expression of pan-macrophage markers (Cd68, Emr1, Itgam, Csf1r) in synovial macrophages (SMs) and synovial fibroblasts (SFs) by RT-qPCR (n = 4). (B) Expression levels of SF markers (Cdh11, Col6a1, Csf1, Vcam1) in SMs and SFs (n = 4). Data are presented as averages &#177; SD. ** indicates p &#60; 0.01 by an unpaired t-test. Data was obtained from four independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65196/65196fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65196/65196fig04.jpg" /> 
Figure 4: Cell-type specific cell surface protein expression. Representative histograms obtained in flow cytometric analyses of leukocyte surface markers (CD45, CD11b, F4/80, Ly6g, CD3) in synovial macrophages (SMs). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65196/65196fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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JoVE Journal

Biology

7.2K Views.  Ehime University. Celgebaseerde assays zijn nodig om moleculaire functies te verduidelijken. In artritisstudies is de methode om primaire cellen te isoleren goed ingeburgerd in synoviale fibroblasten, maar niet in synoviale macrofagen. Daarom hebben we een protocol ontwikkeld om zowel synoviale macrofagen als synoviale fibroblasten uit artritismodellen te isoleren en uit te breiden.Primaire synoviale fibroblasten uit muizenartritisweefsel hebben bijgedragen aan de opheldering van moleculaire mechanismen...

Video: Auteur in de schijnwerpers: isolatie en cultuur van primaire synoviale macrofagen en fibroblasten uit muizenartritisweefsel  

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Developmental Biology

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Medicine

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Immunology and Infection

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

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

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