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Osteoarthritis (OA) is a disease caused by loss of articular cartilage and cartilage degeneration. Surgery is often done when symptoms cannot be controlled and the disease is getting worse, yet this operation does not guarantee the disappearance of the disease. Synovial Membrane Mesenchymal Stem Cells (SM-MSCs) enables the treatment of osteoarthritis (OA) because of their high regeneration ability, have the ability to differentiate into chondrocyte derivatives, and have been known to have high potential for articular cartilage repair technology. This research was conducted to optimize seeding density of MSCs on MSCs population doubling time using 3D Mini Tide Bioreactor. SM-MSC was cultured temporarily in flask culture and placed in a 2D rocker for optimization of seeding density (30.000cells/carrier and 50.000cells/carrier) in 3D culture with fed system. Macro-carrier used is BioNOCII which is made from 100% PET material (thermostat polymer). Morphological of MSCs attachment in BioNOCII macro-carrier were stained using acridine orange staining kit and visualized using inverted microscope. The results showed that SMMSCs cells could be isolated from SM tissues using explant methods in xeno-free medium. The isolated SM-MSCs showed high expression of CD90, CD44, CD105, CD73 (>99%) and did not express any negative markers of MSCs. Isolated cells have high proliferation ability in 2D culture with a PDT value of ± 1.5 days. Cells that have been isolated have the ability to differentiate into adipocyte, chondrocyte, and osteocyte cells. Percent attachment of MSCs on macro-carrier were >90%. The lowest population doubling time of MSCs on macro-carrier is approximately 2.8 days in the 50.000 cells/carrier seeding density.