Collagenase b solution

Grinding the organs through 200 μm mesh sieves yielded single‐cell suspensions from spleens and inguinal lymph nodes. The excised hearts were digested in a water bath at 37°C with 0.1% collagenase B solution (Roche Diagnostics), and then passed through 70 μm cell strainers.²⁰ Mononuclear cells and granulocytes were isolated and collected from peripheral blood, bone marrow, spleen, and heart using Lymphocyte Separation Medium (1.0770–1.0800 g/mL; MP Biomedicals) or Histopaque®‐1119 (1.1190 g/mL; Sigma‐Aldrich) and used in subsequent experiments.

Human bone marrow stromal cells (HBMSCs) were isolated and cultured from patients undergoing elective hip replacement surgery with ethical approval of the Southampton and South West Hampshire Local Research Ethics Committee (194/99/1). Cells were cultured at 37°C, 5% CO₂/balanced air until confluent in basal minimal essential medium, Eagle α-modification (α-MEM) containing 10% FCS and 1% penicillin/streptomycin (P/S) (23 (link)). HBMSC cultures were expanded no higher than passage 3 in tissue culture flasks for all studies.
Human umbilical cords were obtained from consenting, healthy mothers at the Princess Anne Hospital, Southampton, United Kingdom, under ethical approval from Southampton and South West Hampshire Local Research Ethics Committee (LREC 05/Q1702/102). HUVECs were isolated and cultured as previously described (24 (link)) with minor modifications. Cells were dissociated from the vessel wall by infusion with 5 mg/ml (w/v) of Collagenase B solution (Roche Diagnostics, Burgess Hill, United Kingdom) and cultured at 37°C, 5% CO₂/balanced air in endothelial cell culture medium consisting of medium 199 supplemented with 1% P/S, 10% FCS, ECGS/Heparin (ECGS/H) 0.4% (v/v) (Promocell).

Human immortalized articular chondrocyte C28/I2 cells were purchased from Merck Millipore and cultured in DMEM/F12 (Gibco) containing 10% FBS (Gibco), 1% (vol/vol) antibiotic/antimycotic (Gibco), and 1% L-glutamine (Gibco) in a humidified atmosphere at 37°C and 5 % CO₂. For the isolation of primary hACs, cartilage was first dissected from the joint surface, rinsed with PBS, and cut into small pieces. The cartilage pieces were incubated with 2 mg/ml pronase solution (Roche) for 90 minutes at 37°C and digested overnight at 37°C in 1.5 mg/ml collagenase B solution (Roche). Then, the preparation was filtered through a 70 μM strainer and cells were plated in culture flasks and cultured in a humidified atmosphere at 37°C and 5% CO₂. Culture medium consisted of DMEM/F12 (Gibco), 10% FBS (Gibco), 1% (vol/vol) antibiotic/antimycotic (Gibco), and 1% L-glutamine (Gibco).