Isolation and cultivation of human, ovine, equine, and canine BMSCs
Human BMSCs were obtained from the Translational Biomedical Research Group, Center for Regenerative Therapies, Dresden. Ovine bone marrow aspirates obtained from the chest of a merino sheep were provided by the Department of Orthopaedics, University Clinic Carl Gustav Carus, Dresden. Equine and canine BMSCs were isolated by the Department of Veterinary Anatomy, University of Giessen. Density gradient centrifugation by Ficoll (Biochrom, Berlin, Germany) was performed to enrich human, ovine, equine, and canine bone marrow stromal cells (BMSCs). Thereafter, they were cultured in T-175 flasks (Greiner Bio-One, Frickenhausen, Germany) in alpha medium (Biochrom) containing 10% fetal calf serum (FCS) (Sigma), 1% L-glutamine (PAA, Pasching, Austria) and 1% penicillin/streptomycin (PAA) in a humidified atmosphere with 20% O2, 5% CO2 at 37°C (Thermo Scientific BBD 6220 CO2 Incubator, Omnilab, Bremen, Germany). After 5 days the culture medium was exchanged and thereafter every 3-4 days until the culture reached 80-90% confluence.
Cultivation of HUVECs
Human umbilical vein endothelial cells (HUVECs) were purchased from Promocell, Heidelberg, Germany and cultured in Endothelial Cell Growth Medium (ready-to-use, Promocell) without further supplements.
Characterization of human, ovine, equine, and canine BMSCs
CD105 and CD271 MicroBead kits (Miltenyi Biotec) were used for magnetic labelling and positive selection of the BMSCs according to the manufacturer’s instructions.
Generation of HCM
Human, ovine, equine, and canine BMSCs were cultured in T-175 flasks in alpha medium containing 10% fetal calf serum,1% L-glutamine and 1% penicillin/streptomycin (PAA) in a normoxic chamber until the cultures reached 80-90% confluence. After the passage, cells were cultured overnight in alpha medium containing 5% fetal calf serum,1% L-glutamine and 1% penicillin/streptomycin (PAA). On the next day, cultures were transferred into another incubator and cultured in a humidified atmosphere with 1% O2, 5% CO2 at 37°C for 48 h (Thermo Scientific HERAcell 150i, Waltham, MA, USA). Thereafter, the supernatants (HCM) were aliquoted into 2 ml tubes (Eppendorf) and stored at -80°C to be used in migration assays. Control media were generated in the same way but at normoxic conditions.
Human, ovine, equine and canine VEGF and HMGB1 ELISA of HCM
In order to quantify the content of VEGF and HMGB1 in human, ovine, equine and canine HCM, an enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s instruction. VEGF kits for human and canine ELISA were from R&D (Abingdon, England), for ovine from TSZ Biotang (Waltham, USA) and for equine from Genorise (Philadelphia, USA). HMGB1 kit was from IBL (Hamburg, Germany).
Migration assay
Cell migration assays were performed in Corning Transwell®-96 permeable support chambers with porous polyester membranes with a pore size of 8.0 μm (Corning Incorporated Life Sciences, Munich, Germany). Human BMSCs (P2-11), ovine BMSCs (P5-P12), equine BMSCs (P4-7), and canine BMSCs (P5-8) were resuspended at 5 × 104 cells/ml in Alpha Medium supplemented with 1% L-glutamine, 1% penicillin/streptomycin, without BSA and seeded into the upper chamber. HUVECs (P6) were resuspended in endothelial cell growth medium without supplements. 200 ng/ml human, ovine, equine and canine VEGF-A (all from Biomol, Hamburg, Germany) or HCM from corresponding MSCs were used as chemoattractive agents in the lower compartment. The 96-well plate was incubated overnight-night at 37°C in a normoxic incubator. The cells on the upper membrane compartment were removed manually. Then the migrated cells adhering to the underside of the membrane and from the lower chamber were dislodged by incubating the inserts in 0.25% trypsin for 4 min followed by treatment with trypsin neutralisation solution (Promocell). AlamarBlue® (Invitrogen, Darmstadt, Germany) was used to stain the cells overnight. Cell number was calculated at excitation 560 nm/emission 590 nm using Tecan microplate reader (Männedorf, Switzerland).
Results are described as the mean percentage of migrated cells over control cells, the latter show basal migration without chemotactic signal. Each condition was tested in four wells, experiments for human BMSCs and ovine BMSCs were repeated six times, for HUVEC, equine BMSCs and canine BMSCs three experiments were performed.
Statistical analysis
T test was performed using the program available at http://www.daten-consult.de/forms/ttestunabh.html website.