Mv2 basal medium

Ethical approval was granted by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number: P1000002); informed consent obtained from healthy donors before collection of peripheral blood (80 mL). The peripheral blood mononuclear cells (PBMCs) were fractionated from other blood components by centrifuge on a Ficoll-Paque Plus (Amersham Biosciences, Uppala, Sweden), as per manufacturer's instructions. CD34-positive progenitor cells were obtained from the isolated PBMCs using a CD34 MicroBead kit and a MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). Maintenance and characterization of CD34-positive EPCs were performed as described previously [45 (link), 46 (link)]. Briefly, human CD34-positive EPCs were maintained and propagated in MV2 complete medium consisting of MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany), with 20% chemically defined FBS (HyClone, Logan, UT). Cells were seeded onto 1% gelatin-coated plastic ware and cultured in humidified air containing 5% CO₂ at 37°C for further treatment.

Human synovial fibroblasts were isolated by collagenase treatment of synovial tissue samples obtained from patients with OA during knee replacement surgery and samples of non-arthritic synovial tissues obtained at arthroscopy after trauma/joint derangement at China Medical University Hospital. Protocol for study was approved by the Institutional Review Board at China Medical University Hospital and informed consent was obtained from each donor. OASFs were isolated, cultured, and characterized as previously described.^{49 (link)} Human endothelial progenitor cells (EPCs) were approved by the Institutional Review Board of Mackay Medical College at New Taipei City, Taiwan (reference number: P1000002). All subjects gave written consent before enrollment. Briefly, CD34-positive EPCs were maintained and propagated in MV2 complete medium consisting of MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany) of 20% FBS (HyClone, Logan, UT, USA). Cultures seeded onto 1% gelatin-coated plasticware were maintained at 37 °C in a humidified atmosphere of 5% CO₂.^{50 (link)}

Protocol for EPC culture was approved by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number P1000002). All subjects gave informed written consent before enrolling in our study. Peripheral blood (80 mL) was obtained from healthy donors, and mononuclear cells were isolated using the Ficoll-Paque PLUS centrifuge (Amersham Biosciences, Uppsala, Sweden), according to manufacturer's instructions. CD34-positive progenitor cells were isolated from mononuclear cell fraction by CD34 MicroBead kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). EPCs were maintained and characterized as described previously [45 (link)]. Briefly, human CD34-positive EPCs were cultured in MV2 complete medium that contained MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany) and was also supplemented with 20% defined FBS (HyClone, Logan, UT, USA). Cultures were seeded on 1% gelatin-coated plasticware and maintained at 37°C in a humidified 5% CO₂ atmosphere.

The study protocol for isolation of human EPCs was approved by the Institutional Review Board of Mackay Memorial Hospital, Taipei, Taiwan (reference number: 13MMHIS062). Peripheral blood mononuclear cells (PBMCs) were centrifuged from healthy donors using Ficoll-Paque PLUS solution (Amersham Biosciences, Uppala, Sweden), according to the manufacturer’s protocol. CD34⁺ progenitor cells were isolated from PBMCs using the CD34 MicroBead kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany), then cultured in MV2 complete medium containing MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany) supplemented with 20% defined fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and maintained at 37°C in a humidified atmosphere of 5% CO₂ [23 (link), 24 (link)].