Isolation and in vitro culture of rare cancer stem cells from patient-derived xenografts of pancreatic ductal adenocarcinoma.
Isolation and in vitro culture of rare cancer stem cells from patient-derived xenografts of pancreatic ductal adenocarcinoma.
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DOI：
10.1021/ac401165s
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发表时间：
2013-08-06
影响因子：
7.4
通讯作者：
Allbritton, Nancy L.
中科院分区：
文献类型：
--
作者：
Gach, Philip C.;Attayek, Peter J.;Herrera, Gabriela;Yeh, Jen Jen;Allbritton, Nancy L.
描述了大量可释放微结构（微托盘）的构建以及筛选和分离方案，用于从异质细胞群中分选罕见的，大约1万分之一的癌症干细胞（CSCs）。在大型玻璃基板上制备了10.1 × 7.1 cm的微托盘阵列（50 × 50 × 75 μm结构和25 μm微托盘间隙），提供了大约130万个可释放的微结构阵列。图像分析算法的发展，以允许阵列筛选鉴定荧光标记的细胞，在不到15分钟内使用一个具有计算机控制的翻译台的广视场显微镜。将转染绿色荧光蛋白（green fluorescent protein， GFP）的HeLa细胞与野生型HeLa细胞按1:104 ~ 1:106的比例混合在阵列上培养，筛选鉴定荧光细胞，测试装置的运行情况。然后，通过聚焦激光脉冲选择性释放含有感兴趣细胞的微托盘，并将其收集在具有高活力的编号聚二甲基硅氧烷（PDMS）底物上。该技术与荧光激活细胞分选（FACS）的直接比较表明，微托盘阵列提供了更高的分选后纯度（100%），产量（100%）和罕见细胞分离的活力（94 - 100%）。为了证明该技术的价值，对来自Panc-1细胞系和患者来源的异种移植物的胰腺肿瘤细胞进行了CD24、CD44和CD326的筛选；胰腺csc的表面标记物。细胞分离培养后，63±23%的Panc-1细胞和35%的分选人异种移植物细胞形成了具有高表达水平的CD24、CD44和CD326的肿瘤球体。从相对较小的样本量中分离稀有细胞的能力将促进我们对细胞生物学的理解和新治疗策略的发展。
Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10,000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.1 cm array of micropallets (50 × 50 × 75 μm structures and 25 μm micropallet gap) was fabricated on a large glass substrate, providing an array of approximately 1.3 million releasable microstructures. Image analysis algorithms were developed to permit array screening for identification of fluorescently labeled cells in less than 15 minutes using an epifluorescent wide-field microscope with a computer controlled translational stage. Device operation was tested by culturing HeLa cells transfected with green fluorescent protein (GFP) admixed with wild-type HeLa cells at ratios of 1:104 to 1:106 on the array followed by screening to identify flourescent cells. Micropallets containing cells of interest were then selectively released by a focused laser pulse and collected on a numbered poly(dimethylsiloxane (PDMS) substrate with high viability. A direct comparison of this technology with fluorescence-activated cell sorting (FACS) demonstrated that micropallet arrays offered enhanced post sorting purity (100%), yield (100%) and viability (94 – 100%) for rare cell isolation. As a demonstration of the technology’s value, pancreatic tumor cells from Panc-1 cell lines and patient-derived xenografts were screened for the presence of CD24, CD44 and CD326; surface markers of pancreatic CSCs. Following cell isolation and culture, 63 ± 23% of the isolated Panc-1 cells and 35% of sorted human xenograft cells formed tumor spheroids retaining high expression levels of CD24, CD44 and CD326. The ability to isolate rare cells from relatively small sample sizes will facilitate our understanding of cell biology and the development of new therapeutic strategies.
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影响因子：
158.5
作者：
Cristofanilli, M;Budd, GT;Hayes, DF
通讯作者：
Hayes, DF
影响因子：
14
作者：
Gach, Philip C.;Sims, Christopher E.;Allbritton, Nancy L.
通讯作者：
Allbritton, Nancy L.
DOI：
10.1073/pnas.0404036101
发表时间：
2004-07-20
影响因子：
11.1
作者：
Krivacic, RT;Ladanyi, A;Bruce, RH
通讯作者：
Bruce, RH
影响因子：
6.1
作者：
Kang, Joo H.;Krause, Silva;Ingber, Donald E.
通讯作者：
Ingber, Donald E.
影响因子：
11.2
作者：
Li, Chenwei;Heidt, David G.;Simeone, Diane M.
通讯作者：
Simeone, Diane M.