Trypsin edta cell dissociation reagent

Synovial membrane (SM) was collected from the same horses immediately following bone marrow aspiration. All synovial membrane was collected aseptically from dorsal aspect of the antebrachiocarpal and middle carpal joint of normal carpi. Following harvest, synovial membrane was rinsed in phosphate buffered saline (saline) with penicillin (100 U/mL) and streptomycin (100 μg/mL). Synovial membrane (~400 mg) was then debrided with a sterile syringe plunger and incubated at 37°C in 200 μL FBS for 20 min. Samples were re-suspended in DMEM with 4.5 g/L D-glucose, 2 mM L-Glutamine, and 1 mM sodium pyruvate (ThermoFisher Scientific, Hampton, NH), penicillin (100 U/mL)-streptomycin (100 μg/mL) solution, and 10% FBS. Medium was changed every 48 h. Cells were passaged when they reached ~ 80% confluency using Trypsin-EDTA Cell Dissociation Reagent (Gibco™, ThermoFisher Scientific, Waltham, MA). Passage 2 cells were used for differentiation assays. Cell number and viability was determined using the Cellometer™ Auto 2000 Cell Viability Counter and ViaStain™ AOPI staining solution.

Bone marrow was collected aseptically from the sternebrae of horses being euthanized for unrelated reasons immediately following euthanasia. Using an 11-gauge Jamshidi bone marrow biopsy needle (VWR Scientific, Bridgeport, NJ) and 60 mL syringe containing 30,000 U of heparin, 30 mL of bone marrow was aspirated. Bone marrow samples were processed via density centrifugation with Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA) prior to seeding into flasks containing medium consisting of Dulbecco's Modified Eagle Medium (DMEM) with 1 g/L of D-glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (ThermoFisher Scientific, Hampton, NH), penicillin (100 U/mL)-streptomycin (100 μg/mL) solution (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (VWR Life Science Seradigm, VWR, Radnor, PA), and basic fibroblastic growth factor (bFGF, 1 ng/mL) (Invitrogen, Carlsbad, CA). Medium was changed every 48 h. Cells were passaged when they reached ~80% confluency using Trypsin-EDTA Cell Dissociation Reagent (ThermoFisher Scientific, Waltham, MA). Passage 2 (P2) cells were used for differentiation assays. Cell number and viability was determined using the Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA) and ViaStain™ AOPI staining solution (Nexcelom Bioscience LLC, Lawrence, MA).