Synoviocyte growth medium

Human FLS (as control), RA patient FLS and synoviocyte growth medium were purchased from Cell Applications (Santiago, California, USA) [31 (link)]. FLS were cultured at 37 °C in 5% CO₂ in synoviocyte growth medium containing 100 μg/mL streptomycin and 100 U/mL penicillin. FLS in the exponential phase of growth were seeded into 10-cm dishes (1 × 10⁶ cells/dish).
After FLS had spread across the dishes, they were fixed for 15 min in 2% paraformaldehyde, blocked for 1 h with rabbit serum (Sigma), then incubated for 1 h with antibody against the FLS marker vimentin (1:50; Abcam, Cambridge, MA, USA). The dishes were washed three times with 0.01% saponin in phosphate-buffered saline (PBS) for 15 min each, then incubated for 1 h with secondary antibody conjugated with fluorescein (Jackson Immuno Research, West Grove, PA, USA). Dishes were washed in PBS, the nuclear stain DAPI was added, the coverslips were washed three times with 0.01% saponin in PBS for 15 min each, and then they were washed twice in PBS for 10 min each. The dishes were mounted on slides using anti-fade mounting medium and analyzed under an IX2-ILL100 fluorescence microscope (Olympus, Tokyo, Japan).

Primary human synovial fibroblasts isolated from one normal donor and one RA subject were obtained from Cell Applications Inc (San Diego, CA, USA). Synovial fibroblasts were cultured with Synoviocyte Growth Medium (Cell Applications Inc. San Diego, CA, USA) at 37 °C and 5% CO₂. When the cells had grown to confluence, the normal synovial fibroblasts and RA synovial fibroblasts were harvested for total RNA extraction and transcriptome profiling. First passage synovial fibroblasts were used for this study to avoid the influence of repeated passages and accumulation of chromosomal aberrations, which may influence gene expression. The study was exempted from the institutional review board requirement because the research utilized de-identified human cells obtained from commercial entities.

Human fibroblast-like synoviocytes isolated from adult normal (HFLS) and osteoarthritic synovial tissue (HFLS-OA) were obtained from Cell Applications, Inc. (San Diego, CA, USA). The isolated cells were cryopreserved at the first passage. The cryopreserved vials of HFLS and HFLS-OA were thawed and cultured in Synoviocyte Growth Medium (Cell Applications, Inc. San Diego, CA, USA) and incubated in a 37°C, 5% CO₂ humidified incubator until confluence. The cells were then harvested for total RNA extraction using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The quality of extracted RNAs were confirmed using ND-1000 spectrophotometer (Nanodrop Technology, Wilmington, DE, USA) for detection of OD₂₆₀/OD₂₈₀ absorbance ratio and Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA, USA) for RNA integrity number (RIN) with RNA 6000 labchip kit (Agilent Technology, Santa Clara, CA, USA). The OD₂₆₀/OD₂₈₀ absorbance ratio was 1.95 for HFLS and 1.94 for HFLS-OA, while the RINs were 9.9 and 10 for HFLS and HFLS-OA, respectively, indicating good quality of the extracted RNA.

Fibroblast-like synoviocytes from healthy and RA donors and the corresponding Synoviocyte Growth Medium were commercially purchased from Cell Applications, Inc. Glucose, oligomycin, HIF inhibitor V and 2-DeoxyGlucose were from Sigma-Aldrich. Recombinant human TNF was from R&D systems, (5Z)-7-oxozeanol from Tocris. We used a highly potent Glut1 inhibitor from the 1H-pyrazolo[3,4-d]pyrimidine class.

Primary human fibroblast-like synoviocytes (FLS) from the knee (Cell Applications) were allowed to attach to the underside of 5µm pore-sized transwell inserts (Corning) in synoviocyte growth medium (Cell Applications) before the inserts were turned and placed in a 24-well plate (Corning) and cultured for 3-5 days. HMEC endothelial cells (ATCC) were subsequently added to the inside of the inserts and cultured in MCDB 131 medium (Gibco) supplemented with 10% foetal bovine serum, 10ng/ml hEGF, non-essential amino acids, sodium pyruvate and PenStrep for 72hrs before use.
The prepared inserts were placed in a new 24-well plate (Corning) with 400µl MCDB-131 medium supplemented with 20% synovial fluid (instead of 10% FBS). 0.2x10⁶ recently isolated monocytes from healthy donors (described above) were added to the inserts, and monocytes were allowed to migrate for 3hrs at 37°C, 5% CO₂ followed by removal of the inserts. As a control, monocytes were instead added directly to wells containing the same medium and synovial fluids, but without inserts. The cells were incubated overnight at 37°C, 5% CO₂. Next day, monocytes were detached, counted, and used for surface marker analysis (CD14, MerTK, CD86, HLA and CD16 (clone 3G8, APC-H7, BD) all diluted 1:200 and T-cell proliferation assays as described above.

RA-FLS and NH-FLS were purchased from Cell Applications (San Diego, CA, USA) and were maintained in synoviocyte growth medium (Cell Applications). HDMECs were purchased from ScienCell Research Laboratories (6076 Corte Del Cedro, Carlsbad, CA, USA) and were maintained in Endothelial cell Growth Medium (EGM)-2 from the same company. All cells were maintained in a humidified atmosphere of 5% CO₂ at 37^°C and cells of passages from three to five were used for the experiment.