摘要： 目的·改进乳腺癌类器官培养传代方法，建立富集肿瘤相关成纤维细胞（cancer associated fibroblasts，CAFs）的共培养体系。方法·采用不同类型胶原酶（Ⅰ型、Ⅲ型、Ⅳ型）处理5例乳腺癌新鲜肿瘤组织，通过细胞计数法计数组织消化后的细胞数量，用流式细胞仪分析细胞活性；采用含有不同含量成纤维细胞生长因子7（fibroblast growth factor 7，FGF7）、FGF10以及表皮生长因子（epidermal growth factor，EGF）的培养基对乳腺癌原发灶单细胞进行三维培养，对比观察不同细胞因子含量对类器官培养成功率和状态的影响；采用不同离心速度，对比类器官传代方法优劣，简化传代步骤。建立原代CAFs与类器官共培养体系，CCK8实验研究CAFs对类器官生长的影响，光学显微镜下观察类器官的形态变化。结果·相比于Ⅰ型胶原酶和Ⅲ型胶原酶，Ⅳ型胶原酶消化乳腺癌组织可得到较高的细胞回收效率（
P=0.045，
P=0.017），并保持较高的细胞活性（
P=0.005，
P=0.048）；优化类器官培养基成分（不添加FGF7和FGF10，添加低浓度EGF），并提高离心转速至900×
g，可得到更经济、有效、快速的类器官培养传代方法。相比单独培养的类器官，CAFs与类器官共培养，进一步提高类器官体外生长速度（
P<0.05），增加类器官形态的异质性。结论·优化后的类器官培养体系可显著增加乳腺癌类器官培养成功率，简化培养步骤并降低培养成本。原代CAFs和类器官共培养体系的建立为研究乳腺癌异质性和肿瘤微环境提供了良好的体外模型。
关键词: 乳腺癌, 类器官, 改进培养, 肿瘤相关成纤维细胞, 共培养 Abstract: Objective·To improve the cultivating and passaging method of breast cancer organoids, and establish a co-culture system enriching cancer associated fibroblasts (CAFs).
Methods·Different types of collagenases (type Ⅰ, type Ⅲ and type Ⅳ) were used to digest fresh tissues from 5 breast cancer patients. The number of cells after tissue digestion was counted by cell counting method, and cell viability was analyzed by cell flow cytometry. Three-dimensional culture of primary breast cancer single cells was carried out by using culture system containing different contents of fibroblast growth factor 7 (FGF7), FGF10 and epidermal growth factor (EGF). The success rate of cell culture and the growing status of organoids were observed and compared. Different centrifugation speeds were used to compare the advantages and disadvantages of passaging methods and simplify the passaging steps. CCK8 assay was used to study the effect of CAFs on the growth of organoids in the co-culture system of primary CAFs and organoids, and the morphological changes of organoids were observed under optical microscope.
Results·Compared with type Ⅰ and type Ⅲ collagenase, type Ⅳ collagenase got the highest cell yields (P=0.045, P=0.017), and maintained the highest cell viability (P=0.005, P=0.048). By optimizing the composition of organoid medium (omitting FGF7 and FGF10, reducing EGF concentration) and passaging process (improving centrifugal velocity to 900×g), a more economical, effective and rapid method of organoid culture was obtained. Compared with organoids cultured alone, the growth rate (P<0.05) and heterogeneity of organoids increased when organoids were co-cultured with CAFs.
Conclusion·The optimized culture system can significantly increase the success rate of organoids, simplify the culture steps and reduce the culture cost. The establishment of primary CAFs and organoids co-culture system provides a good in vitro model for the study of breast cancer heterogeneity and tumor microenvironment.
Key words: breast cancer, organoid, improved culture condition, cancer-associated fibroblast (CAF), co-culture PDF全文下载地址:
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