BruCells has been actively involved in the discussion of the EMEA guideline on Cell Based Medicinal Products (CBMP). Catherine De Greef was present at the DC-THERA Cluster 4 Meeting in Bamberg (July 2007) where among other topics these guidelines were discussed and where the basis for a written statement has been worked out. During the finalisation of the statement, BruCells has mainly put forward the notion that the guidelines are not suitable to CBMP for the authorization of investigational trials, but are applicable in the procedure of obtaining market approval.

(Basic manufacturing and QC SOPs (Generation of Peptide loaded DCs, Generation of cytokines and media components, Generation of autologous plasma and serum, Staining of cells for FACS Analysis, Cell Counting by Trypanblue Staining) have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

(Basic manufacturing and QC SOPs (Generation of Peptide loaded DCs, Generation of cytokines and media components, Generation of autologous plasma and serum, Staining of cells for FACS Analysis, Cell Counting by Trypanblue Staining) have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

A standard operating procedure for the identification of preclinical tests for cellular vaccines and release criteria for DC vaccines has been generated in our lab.

Draft templates for IMP have been generated.

Draft templates for CTP have been generated.

We possess an SOP for the assessment of Th subsets via flow cytometry.

Sample pre-processing and biotin labeling were performed using the Affymetrix GeneChipH cDNA Synthesis Kit and IVT Labeling Kit (Affymetrix) according to the manufacturer’s protocols. Microarrays were then hybridized on Affymetrix GeneChipH HG-U133A 2.0 microarrays, and scanned according to the manufacturer’s instructions on a GeneChipH Scanner 3000 (Affymetrix). Extraction, hybridization and scanning were performed by the Genopolis consortium (University of Milano-Bicocca, Italy).

We have a clinical protocol for immunotherapy of melanoma patients in preparation, however many details are not yet finalized. Everything is carried out under GMP conditions according to SOPs. In brief, patients with stage III or stage IV melanoma will undergo surgical resection of a melanotic lesion and leukapheresis will be performed on the following day. DCs will be generated from monocytes after elutriation of the leukapheresis product. On day 5 of culture immature DC will be pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed le...

We have a current clinical protocol for active immunotherapy in advanced melanoma patients (GMP SOPs covering the protocols are also available). The vaccine is composed by autologous DCs pulsed with allogenic melanoma peptides (SK-Mel 24, HLA-A1 and HLA-A2 positive) plus KLH as immunological tracer and it is produced under GMP condition at MolMed S.p.A. (DIBIT, San Raffaele, Milan, Italy). HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma undergo leukapheresis. DCs are generated from adherent peripheral blood monocytes, at day 6 DC are pulsed with ...

Array construction: The yeast oligonucleotide array was constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene. Probe preparation and hybridization: We used the indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3- aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) was incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesi...

The cells were collected and the pellet were 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) were added and the falcon were mixed by vortex. After 10’ of incubation in 65 °C water bath, the samples were incubated on ice and then centrifugated at 5000 rpm in an tabletop centrifuge. The supernatants were transferred to a new tube and added 2 ml of acid phenol to repeat the extraction. After the centrifugation (10’ at 5000 rpm) the supernata...

Different strains of Saccharomyces cerevisiae were 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) Monocyte-derived DCs were added at a final concentration of 5x105 cells/ml into 96-well plates. Serial diluition of yeast cells and preparations were added to the MoDCs. Cytokine accumulation was evaluated in the supernatants at 24h by ELISA, according to a standard protocol and ...

PBMC were isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM). Monocytes were isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch-Gladbach,Germany). Cells were 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). Differentiation of monocytes into dendriti...

Lentiviral infection of target cells: Day 1: seed cells Day 2: remove the medium from the cells. Mix the medium containing the virus gently by pipetting and add to the cells (+ 6 ?g/ml of polybrene). Incubate the cells at 37°C overnight. Day 3: change medium and start selection for stably transduced cells

Production of lentiviruses: Producing lentivirus in 293 FT Cells: Day 1: 4 x 106 293 FT in 10 ml DMEM (+10 % FCS (Tet free), +P/S) in a 10cm plate Day 2: transfection Day 3: change of medium Day 4: harvesting

a) Incubate cells in Fc-block (1:50 in PBS + 2 % FCS) 30 min on ice b) Staining of the cells in PEalphaDC80 in PBS + 2 % FCS + c) Fc-block for 30 min on ice d) FACS analysis

Monocyte source: leukapheresis product (rarely: buffy coats of healthy blood donors, research use only) => gives high yield of monocytes (15-25 x 10e8) Monocyte isolation: affinity purification (CliniMacs) or counter-flow centrifugation (Elutra) => good recovery, high purity, high viability Culture: in culture bags (Teflon Bags, Cell Genix) in serum free medium (CellGro from Cell Genix) => no contact-mediated activation, no interference by serum proteins For research use (sometimes) generation of adherent DC by plating monocytes on plastic. Cytokines: clinical gra...

Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma Staining of GM-DC/Raw with anti-CD11c antibodies Coculture of DCs and cells to be taken up (1(-2)x105 + 1(-2)x105 cells in 12-well) for 4 h to 24 h at 37°C Take supernatant and freeze at -80°C for analysis of DC-stimulation Trypsinize cells have and fix in 4 % PFA (or formalin) FACS analysis