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As to the lymph nodes, these were cut into small pieces and cells were flushed out with PBS.
Microsatellite analysis was performed on various samples related to the establishment of Capi3 : DNA from donor patient peripheral blood mononuclear cells, initial tumor biopsy, xenografted tumor pieces and cells grown in vitro.
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Total RNA was extracted from tumour pieces and cell lines as described (Ichimura et al, 1996).
For immunocytochemistry, small tissue pieces and cell cultures were fixed with 4% paraformaldehyde in the cold overnight or for 30 minutes, respectively.
In brief, the mucosa from surgical patients was cut into 1-mm pieces and epithelial cells were removed in Hank's Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (PAA, Cölbe, Germany) with 2mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, Taufkirchen, Germany).
The ventricle was cut in pieces, and individual cells were released by gently agitating the muscle pieces.
The pieces and outgrowing cells were cultivated in DMEM high glucose medium with 10% fetal calf serum.
Tumor tissue from solid tumor samples was minced into small pieces and tumor cells were isolated by collagenase dispersion followed by Percoll density gradient centrifugation [ 21].
The tissue was cut into 5 mm pieces and epithelial cells released by two cycles of treatment with 1 mM EDTA and 1 mM dithiothreitol (DTT) in HEPES-buffered Hank's balanced salt solution containing 2% of fetal calf serum at 37°C for 15 min with slow stirring (Lundgren et al., 2005).
Briefly, the scaffolds were cut in pieces, trypsinized, shaken and cells were collected via centrifugation.
After cells reached confluence, tissue pieces were removed and cells were trypsinized.
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