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stimulation step
DCs are stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT.
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establishing cell culture step
Different strains of Saccharomyces cerevisiae are cultured and collected in different conditions:
different growth phase (exponential and stationary phase)
different growth media (standard and promoting pseudohyphal growth)
different cell form (spheroplast, spore and whole cell)
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cell coculturing step
The T cell transfer will consist of T cells expanded by co-culture with tumor lysate loaded DC.
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cell coculturing step
Coculture of DCs and cells to be taken up (1(-2)x10^5 + 1(-2)x10^5 cells in 12-well) for 4 h to 24 h at 37°C
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cell coculturing step
Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs
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cell coculturing step
Purified CD8 T cells using MACS cell separation are co-cultured with electroporated DC.
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centrifugation step
After the spin (10’ at 3,000 rpm), the supernatants are precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Centrifugation (30’ at 12000 rpm).
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centrifugation step
The samples are centrifugated at 5000 rpm in an tabletop centrifuge.
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establishing cell culture step
Day 1: seed cells
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maintaining cell culture step
Day 3: change of medium
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exposure of material to environment step
A portion of pulsed and unpulsed mature dendritic cells will be frozen and used to monitor immunological responses elicited by the DC vaccine and transferred T cells during the clinical trial.
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establishing cell culture step
Cells are cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).
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adding substance to cell culture step
Autologous DC are pulsed with the apoptotic allogenic prostate carcinoma cell line LNCap to produce the vaccine for patients with advanced prostate carcinoma..
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adding substance to cell culture step
Pulsing of autologous DCs with apoptotic autologous ovarian carcinoma cells.
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stimulation step
Serotype A C. albicans strain SC5314 is cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth. The culture is treated as for yeast cells. After microscopical inspection of the purity of hyphal culture, this is treated as for yeast cells. [It should be noted that in all the experiments performed in this study live microrganisms were used.]
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establishing cell culture step
2 x 200 x 10e6 monocytes are transferred into culture bags by sterile welding and cultured for 5 days at a density of 2.5-5 x 10e6 cells/mL in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix). After 2 days one volume fresh medium containing 2x concentrated cytokines is added.
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adding substance to cell culture step
The cells are collected and the pellets are resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) are added and the falcon is mixed by vortex.
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adding substance to cell culture step
Serial diluition of yeast cells and preparations are added to the MoDCs.
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manufacturing step
The yeast oligonucleotide array is constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene.
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evaluation step
The polarization is assessed by flow cytofluorimetry and cytokine production.