Failli Alessandra, Consolini Rita, Legitimo Annalisa, Spisni Roberto, Castagna Maura, Romanini Antonella, Crimaldi Gaetano, Miccoli Paolo
Department of Medicina della Procreazione e dell'Età Evolutiva, Laboratory of Immunology, University of Pisa, Pisa, Italy.
Tumori. 2009 May-Jun;95(3):343-7. doi: 10.1177/030089160909500312.
Because colorectal cancer is a significant cause of morbidity and mortality in the Western population, knowledge of the molecular and biological alterations associated with its development is important. Since primary human colon cancer cultures from fresh tumor tissue are technically difficult to obtain, experiments in most laboratories are performed on colon epithelial cell lines, but these represent just one stage of tumor progression. Only primary cultures of neoplastic colonocytes may reflect the actual responsiveness of tumors at certain developmental stages to antitumor agents.
This paper analyzes several critical points concerning primary cultures, ranging from cell isolation to culture conditions, and compares different methodological approaches to isolate and cultivate a pure fraction of viable tumor cells. Samples of resected colorectal cancers were collected from 20 patients (stage T3 or T4). We compared in vitro several approaches of tissue disaggregation including mechanical disaggregation and enzymatic dissociation with trypsin or collagenase. Isolated cells were maintained in a short-term serum-free culture system. Evaluation of the purity and tumoral nature of isolated cells was performed by immunochemistry.
We established the antibiotic concentration necessary during transport and washing of the specimens to prevent microbial overgrowth. We demonstrated that the number of viable cells was dependent on the dissociation method used. Mechanical disaggregation is not a valid dissociation method because of the high mortality of cells and might be used only in samples for molecular analysis. Comparison of the enzymatic digestion procedures showed that digestion with trypsin allowed the highest recovery of viable cells.
In this paper we analyzed several critical aspects of cell culture procedures and designed a methodological approach suitable for functional studies of colorectal cancer.
由于结直肠癌是西方人群发病和死亡的重要原因，了解与其发生相关的分子和生物学改变至关重要。由于从新鲜肿瘤组织获取原发性人类结肠癌培养物在技术上存在困难，大多数实验室的实验是在结肠上皮细胞系上进行的，但这些仅代表肿瘤进展的一个阶段。只有肿瘤性结肠细胞的原代培养物可能反映肿瘤在某些发育阶段对抗肿瘤药物的实际反应性。
本文分析了从细胞分离到培养条件等原代培养的几个关键点，并比较了分离和培养纯的存活肿瘤细胞组分的不同方法。从20例患者（T3或T4期）收集切除的结直肠癌样本。我们在体外比较了几种组织解离方法，包括机械解离以及用胰蛋白酶或胶原酶进行酶解。分离的细胞维持在短期无血清培养系统中。通过免疫化学对分离细胞的纯度和肿瘤性质进行评估。
我们确定了标本运输和洗涤过程中防止微生物过度生长所需的抗生素浓度。我们证明存活细胞的数量取决于所使用的解离方法。机械解离不是一种有效的解离方法，因为细胞死亡率高，仅可用于分子分析的样本。酶消化程序的比较表明，用胰蛋白酶消化可使存活细胞回收率最高。
本文分析了细胞培养程序的几个关键方面，并设计了一种适合结直肠癌功能研究的方法。
