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

PLOS ONE, Dec 2019

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.

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

et al. (2013) A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study. PLoS ONE 8(9): e75193. doi:10.1371/journal.pone.0075193 A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study Laura Annaratone 0 Caterina Marchio` 0 Rosalia Russo 0 Luigi Ciardo 0 Sandra Milena Rondon-Lagos 0 Margherita Goia 0 Maria Stella Scalzo 0 Stefania Bolla 0 Isabella Castellano 0 Ludovica Verdun di Cantogno 0 Gianni Bussolati 0 Anna Sapino 0 Jorge Sans Burns, University Hospital of Modena and Reggio Emilia, Italy 0 1 Department of Medical Sciences, University of Turin , Turin , Italy , 2 Department of Laboratory Medicine, Azienda Ospedaliera Citta` della Salute e della Scienza di Torino , Turin , Italy , 3 Institut Victor Babes , Bucharest , Romania 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 4uC 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 4uC. 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. - Funding: This work was supported by Ricerca Sanitaria Finalizzata RF-2010-2310674 and AIRC Grants (IG 10787 to AS and MFAG 13310 to CM). GB is funded in part by Persother (SMIS-CSRN:549/12.024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Primary cultures represent an invaluable tool to set up functional experimental conditions that are instrumental to demonstrate biological mechanisms directly on human-derived tumor cells, however, creation of tissue cultures from solid tumors is troublesome and often unproductive particularly when dealing with primary cultures of carcinomas. Major biological issues are related to a relatively slow doubling time of epithelial cancer cells and to a rapid overgrowth with fibroblasts [1], depending also on the type of source lesions. In addition, technical aspects may affect the success rate of primary cultures in general. Such technical issues may stem from pre-analytical procedures routinely employed in surgical theaters and pathology laboratories. Indeed, two simple rules are mandatory for obtaining proper samples for cell cultures: (i) to acquire fresh specimens as soon as possible after completing the surgical procedure; (ii) to avoid both bacterial and fungal contamination of the specimens. The first rule matches with the need to reduce the ischemia process following the surgical procedure that stops with proper specimen fixation, since it allows activation of tissue enzymes, autolysis and degradation of proteins and nucleic acids [2,3]. However, logistics management of specimens from the surgical theater to the pathology lab has to be considered with the priority that involves material handling, packaging and transportation. Depending on the hospital structure the transport of surgical specimens from the surgical theater to the pathology lab may prolong the ischemia time. In addition, transfer may be performed using the most variable types of boxes, transport media and at different temperatu (...truncated)


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Laura Annaratone, Caterina Marchiò, Rosalia Russo, Luigi Ciardo, Sandra Milena Rondon-Lagos, Margherita Goia, Maria Stella Scalzo, Stefania Bolla, Isabella Castellano, Ludovica Verdun di Cantogno, Gianni Bussolati, Anna Sapino. A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study, PLOS ONE, 2013, Volume 8, Issue 9, DOI: 10.1371/journal.pone.0075193