Fetal calf serum (fcs)

At 3 or 10 wk post-immunization, animals were euthanized, and spleens were isolated by gross dissection. Tissues were processed into single-cell suspensions in complete medium (CM) consisting of RPMI 1640 supplemented with 20 mM L-glutamine, 10 mM HEPES, 50 ug/mL streptomycin, 50 U/mL penicillin, 50 mM 2-mercaptoethanol (Sigma–Aldrich, St. Louis, Missouri) and 10% FCS (Hyclone, Thermo Fisher Scientific, Waltham, Massachusetts). All reagents were purchased from Gibco unless otherwise specified. Red blood cell lysis was performed using RBC Lysing Buffer (Sigma–Aldrich). Lung tissues were also harvested, minced in sterile 25ml beakers, and incubated in complete digestion medium (CDM) in a 37° C water bath prior to processing into single-cell suspensions as described above. CDM consisted of CM without FCS, and with addition of 1mg/ml collagenase I and 30ug/ml DNase I (Worthington Biochemical Corp, Lakewood, NJ).

The post-mortem usage of biological material for biomedical research taken from the food chain requires no ethical permit according to the Swiss and European law. Porcine notochordal cells (NC) were isolated from the nucleus pulposus (NP) tissue of porcine tails obtained from 4- to 5-month-old pigs collected from the local abattoir. Tails with a high percentage of NC (~80%) were selected for the experiment. The percentage of NCs was assessed and counted using a haemocytometer and brightfield microscopy to confirmed the NC phenotype as large in cell size and presence of vacuoles. Bovine nucleus pulposus cells (NPC) were harvested from the NP tissue of ~1-year-old bovine tails obtained from the local slaughter house. Porcine and bovine NP tissues were digested by 0.19% pronase (Roche, Basel, Switzerland) for 1 hour and subsequent collagenase type 2 at a concentration of 32 U/mL in 25 mL of HG-DMEM supplemented with 10% FCS (Worthington, London, UK) overnight (~14 hours) at 37°C and standard culture conditions (5% CO2 and 100% humidity)[14 (link), 23 (link)].