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/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/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. E., Jones, D. R., Lopez, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sheath+fluid+impacts+the+depletion+of+cellular+metabolites+in+cells+afflicted+by+sorting+induced+cellular+stress+(SICS).">Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS).</a> Cytometry Part A. 99 (9), 921-929 (2021).</li><li>Llorente, I., Garc&#237;a-Casta&#241;eda, N., Valero, C., Gonz&#225;lez-&#193;lvaro, I., Casta&#241;eda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoporosis+in+rheumatoid+arthritis:+dangerous+liaisons.">Osteoporosis in rheumatoid arthritis: dangerous liaisons.</a> Frontiers in Medicine. 7, 601618 (2020).</li><li>Croft, A. P., 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+fibroblast+subsets+drive+inflammation+and+damage+in+arthritis.">Distinct fibroblast subsets drive inflammation and damage in arthritis.</a> Nature. 570 (7760), 246-251 (2019).</li><li>Wei, 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=Notch+signalling+drives+synovial+fibroblast+identity+and+arthritis+pathology.">Notch signalling drives synovial fibroblast identity and arthritis pathology.</a> Nature. 582 (7811), 259-264 (2020).</li></ol>

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 ...

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.

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

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 ...

isolation-culture-primary-synovial-macrophages-fibroblasts-from

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6.8K Views.  Ehime University. The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue. 

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

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...