Expansion of CSF-Derived Mononuclear Cells

CSF-derived mononuclear cells were expanded as previously reported.23 (link),24 (link) Briefly, 2000 cells per well were seeded in 96-well U-botton microtiter plates together with 2 × 10⁵ nonautologous, irradiated PBMC (45 Gy), 1 μg/mL of PHA-L (Sigma, St Louis, MO), and IL-2 supernatant (2000× diluted) (kindly provided by Dr. Sallusto, Institute for Research in Biomedicine, Bellinzona, Switzerland). Medium consisted of IMDM (PAA) containing 100 U/mL penicillin/streptomycin (PAA), 50 μg/mL gentamicin (BioWhittaker, Cambrex, East Rutherford, NJ, USA), 2 mmol/L l-glutamine (GIBCO, Invitrogen, Waltham, MA, USA) and 5% heat-decomplemented human serum (PAA). Additional IL2 was added every 3–4 days. After 2 weeks cells were pooled and analyzed, cryopreserved, or restimulated again with 1 μg/mL PHA, IL-2 and allogeneic irradiated PBMC. We know from our previous experience that this expansion protocol preserves the TCR repertoire.24 (link)