Cell-based assays are required to clarify molecular functions. In arthritis studies, the method to isolate primary cells is well-established in cyanobacterial fibroblasts, but not in cyanobacterial macrophages. Therefore, we developed a protocol to isolate and expand both cyanobacterial macrophages and cyanobacterial fibroblasts from arthritis models.
Primary cyanobacterial fibroblasts from marine 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 cyanobacterial cells by sorting as marine cyanobacteria is a very small tissue even in arthritis models. In the previous method to isolate cyanobacterial fibroblasts, cyanobacterial macrophages were discarded. Besides that, a method to isolate and expand resilient macrophages from some organs was reported.
Therefore, existing protocols were modified in combination to isolate and expand both cyanobacterial macrophages and cyanobacterial fibroblasts from marine arthritis tissue. This method can isolate both macrophages and fibroblasts from inflammatory cyanobium with high purity, and it is simple and reproducible. This method allows the expansion of cyanobacterial macrophages and their co-culture with cyanobacterial fibroblasts.
Also, the method provides an advantage in that cyanobacterial fibroblasts can be used with fewer prostheses. The established protocol would be an advantage for elicitation of pathological mechanisms in rheumatoid arthritis. 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 five 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 five 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. 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 subconfluent 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 C57BL6 mice. The purity of the isolated cells was assessed by RTQ-PCR. mRNA expression of synovial fibroblast markers such as BCAM1, CDH11, COL6A1, and CSF1 in the suggested that the fibroblast-rich fractions were isolated from synovitis tissue.
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 7- to 8-weeks-old female C57BL6 mice and analyzed by RTQ-PCR. mRNA expression of pan-microphage markers CD68, EMR1, ITGAM, and 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 F480, whereas the expression of neutrophil marker LI6G and T-cell marker CD3 was lower than 1%.
