A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells
A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells
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DOI：
10.1039/c2lc40072c
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发表时间：
2012-01-01
期刊：
影响因子：
6.1
通讯作者：
Ingber, Donald E.
中科院分区：
文献类型：
--
作者：
Kang, Joo H.;Krause, Silva;Ingber, Donald E.
在这里，我们描述了一种组合的微流体-微磁细胞分离装置，该装置已被开发用于从全血中分离、检测和培养循环肿瘤细胞（CTC），并使用来自乳腺癌荷瘤小鼠的血液证明其实用性。该装置由聚二甲基硅氧烷制成，包含一个微流体结构，该微流体结构具有主通道和冗余的“双收集”通道，该通道由两排死端侧室排列，用于肿瘤细胞收集。使用计算模拟来优化微器件设计，以确定使用上皮细胞粘附分子（EpCAM）抗体包被的磁性微珠（直径2.8 μ m）进行细胞分离的尺寸、磁力和流速。使用该装置，分离效率以线性方式增加，当仅将2至80个乳腺癌细胞掺入从野生型小鼠采集的小体积（1.0 mL）血液中时，分离效率接近90%。该设备的高灵敏度可视化能力还允许检测其死端侧室内的单个细胞。当在肿瘤进展的不同阶段从携带FVB C3（1）-SV 40 T抗原乳腺肿瘤的转基因小鼠中取出血液时，使用抗EpCAM珠在装置中分离并在死端侧室内磁性收集的细胞也对泛细胞角蛋白-FITC和DAPI染色呈阳性，对CD 45-PerCP呈阴性，并表达SV 40大T抗原，从而确认其为CTC。使用这种分离方法，我们检测到雌性转基因小鼠血液中CTC数量的时间依赖性增加，20周后血液中出现的转移性肿瘤细胞数量急剧增加，此时肿瘤转变为浸润性癌，并在该模型中表现出转移生长增加。重要的是，与先前描述的CTC分离方法相反，从携带乳腺肿瘤的动物取出的少量血液中收集的乳腺肿瘤细胞保持活力，并且它们可以容易地从这些装置中取出并在培养物中扩增以用于额外的分析研究或潜在的药物敏感性测试。
Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 mu m diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.