Mammalian cell culture media Human Fibroblast-Like Synoviocytes-Osteoarthritis: HFLS-OA

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

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EuroClone Online Store

Protocol tips
The synovial tissues were minced and treated for 4 h with 2.5 mg/mL of type I collagenase (Sigma Aldrich, Saint Louis, MO, USA) in Dulbecco's modified Eagle's medium (DMEM) (Euroclone, Italy) at 37°C in 5% CO2. Dissociated cells were then centrifuged at 1000 ×g, resuspended in DMEM supplemented with 10% fetal calf serum (FCS) (Fetalclone1 Hyclone Logan, UT, USA), 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin (Euroclone, Italy), and plated in 75 cm2 flasks (Primo Cell Culture Flask, Euroclone, Italy). After overnight culture, nonadherent cells were removed, and adherent cells were cultivated in DMEM supplemented with 10% FCS. The cultures were kept at 37°C in 5% CO2, and the medium was replaced every 3 days.

The purity of the cells was tested by flow-cytometric analysis using phycoerythrin-conjugated anti-CD14 (PharMingen, San Diego, CA, USA) and fluorescein isothiocyanate phycoerythrin-conjugated anti-CD3, anti-CD19, anti-CD14, or anti-Thy-1 (CD90) monoclonal antibodies (R&D Systems Minneapolis, MN). A FACS Calibur flow cytometer (488Ex/620Em) (Becton Dickinson, San José, CA, USA) was used for the analysis. At passage 3, the cells were morphologically homogeneous and exhibited the appearance of FLS, with typical bipolar configuration under inverse microscopy. Most cells (>98%) expressed the surface markers for fibroblasts (Thy-1) and were negative for the expression of CD3, CD19, and CD14. Synoviocytes from passages 3–8 were used in each experiment.
Human Synoviocyte Growth Medium

Cell Applications Inc

Protocol tips
Primary human fibroblast-like synoviocytes (HFLS) and HFLS-rheumatoid arthritis (HFLS-RA) were purchased from the Culture Collections, Public Health England (PHE, Salisbury, UK). The cells were cultured in Synoviocyte Growth Medium (Culture Collections, PHE) and maintained in a humidified incubator at 37°C with 5% CO2 and filtered air.
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