A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

Abstract

Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84–100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.

Unraveling the chromosome 17 patterns of FISH in interphase nuclei: an in-depth analysis of the HER2amplicon and chromosome 17 centromere by karyotyping, FISH and M-FISH in breast cancer cells

“…The triple negative phenotype was confirmed by immunohistochemistry (IHC) for the estrogen receptor (ER) (Clone SP1, 1:50 diluted, Cell Marque, Rocklin, California), progesterone receptor (PR) (Clone 1A6, 1:50 diluted, Leica Biosystems, Newcastle Upon Tyne, United Kingdom) and by FISH for the HER2 gene on a cell block obtained after centrifugation of an aliquot of the effusion. The remaining part was used to set up a short-term primary culture according to a protocol recently described [ 15 ]. The epithelial origin of the cells was confirmed by the positive expression of cytokeratins (clones AE1/AE3 and PCK26, pre-diluted, Ventana-Diapath, Tucson, AZ, USA) and by the absence of the mesothelial marker calretinin (polyclonal; 1:100 diluted, Invitrogen) using an immunohistochemical procedure on cells grown directly on sterilized slides [ 15 ].…”

Immunohistochemical and molecular profiling of histologically defined apocrine carcinomas of the breast

“…A local ethical committee approved the study. Informed consents were available and signed by all patients whose tumor samples were sequenced; as at our institution, written informed consent was obtained from patients to use both residual fresh neoplastic and archival tissues [12]. All cases were reviewed, and the apocrine cytomorphology [1] was confirmed (S.V., Z.G., and A.S.).…”

TOP2A gene copy gain predicts response of epithelial ovarian cancers to pegylated liposomal doxorubicin

“…Immuno-histochemical studies were performed as described [18] to detect topoisomerase II alpha using a monoclonal rabbit antibody (D10G9, Cell Signalling Technology), in paraffin-embedded tumor material. Ten images of each sample were then acquired by optical microscope (20x) connected with CCD camera.…”