Department of Medical Sciences, University of Turin, Turin, Italy.
PLoS One. 2013 Sep 30;8(9):e75193. doi: 10.1371/journal.pone.0075193. eCollection 2013.
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.
原代培养物是建立功能实验条件的宝贵工具；然而，从实体瘤中创建组织培养物是麻烦且常常没有成效的。几个因素会影响原代培养物的成功率，包括手术 theater 和病理实验室中使用的分析前程序的技术问题。我们最近引入了一种新的外科标本采集、转移和保存方法，要求在手术 theater 立即对切除的标本进行真空密封，然后在 4°C 下按时间控制转移到病理实验室。在这里，我们研究了从真空包装和冷却（VPAC）保存的组织中获得的短期原代细胞培养物的可行性和性能。从大于 2 厘米的肿瘤的 52 个外科标本中采集组织碎片，记录手术和 VPAC 时间（后者对应于冷缺血时间）。通过台盼蓝染料排除试验测定细胞活力，并进行苏木精和伊红以及免疫组织化学染色，以评估培养细胞的形态和免疫表型特征。在 52 个 VPAC 保存组织中，有 44 个（85%）的细胞活力范围为 84-100%。手术和 VPAC 时间的长短均影响细胞活力：关键的手术时间约为 1 小时 30 分钟，而当在 4°C 下真空保持约 24 小时时，细胞仍保持良好的活力。细胞可在培养中至少传代 3 次。免疫细胞化学证实了不同细胞群体的表型，即上皮样细胞中细胞角蛋白的表达和梭形细胞中波形蛋白的表达。我们的结果表明，VPAC 保存的组织可能是创建原代细胞培养物的可靠来源，并且仔细监测手术和冷缺血时间有助于原代组织培养物的良好性能。
