Center for Biomedical Engineering, School of Engineering, Brown University, Providence, Rhode Island.
PerkinElmer, 940 Winter St, Waltham, Massachusetts.
Cytometry A. 2019 Aug;95(8):885-897. doi: 10.1002/cyto.a.23736. Epub 2019 Mar 9.
Isolation of circulating tumor cells (CTCs) has generated clinical and academic interest due to the important role that CTCs play in cancer metastasis and diagnosis. Here, we present a PDMS and glass prototype of a microfluidic device for the immunomagnetic, immiscible phase filtration based capture, and isolation of MCF-7 breast cancer cells, from various sample matrices including PBS-based buffer, blood plasma, and unprocessed whole blood. Following optimization of surface energy of an oil-water interface, microfluidic geometry, and bead-binding kinematics, our microfluidic device achieved 95 ± 4% recovery of target cells from PBS-based buffer with 95% purity, 90 ± 3% recovery of target cells from blood plasma and recovery of ~70 ± 5% from unprocessed whole blood with purity >99% with 1 ml blood samples with 1,000 spiked target cells. From quantitative studies to assess the nonspecific carryover of contaminants from whole blood, we found that our system accomplishes a >175 fold depletion in platelets, >900 fold depletion in erythrocytes, and >1,700 fold depletion in leukocytes with respect to unprocessed whole blood, enabling us to avoid sample pre-processing. In addition, we found that ~95% of the isolated target cells were viable, making them suitable for subsequent molecular and cellular studies. We quantify and propose mechanisms for the carryover of platelet, erythrocyte, and leukocyte contamination in purified samples, rather than relying on sample pre-processing. These results validate the continued study of our platform for extraction of CTCs from patient samples and other rare cell isolation applications. © 2019 International Society for Advancement of Cytometry.
由于循环肿瘤细胞（CTCs）在癌症转移和诊断中发挥着重要作用，因此对其进行分离引起了临床和学术界的兴趣。在这里，我们展示了一种基于 PDMS 和玻璃的微流控器件原型，用于基于免疫磁珠的不混溶相过滤，从各种样品基质中捕获和分离 MCF-7 乳腺癌细胞，包括基于 PBS 的缓冲液、血浆和未经处理的全血。在优化油水界面的表面能、微流控几何形状和磁珠结合运动学之后，我们的微流控器件从基于 PBS 的缓冲液中以 95%的纯度实现了 95%±4%的目标细胞回收，从血浆中实现了 90%±3%的目标细胞回收，从未经处理的全血中以纯度>99%的方式回收了约 70%±5%的目标细胞，而 1 ml 血液样本中含有 1000 个掺入的目标细胞。从定量研究评估全血中污染物的非特异性携带情况，我们发现我们的系统在血小板方面实现了>175 倍的耗尽，在红细胞方面实现了>900 倍的耗尽，在白细胞方面实现了>1700 倍的耗尽，相对于未经处理的全血，从而能够避免样品预处理。此外，我们发现约 95%的分离出的目标细胞是存活的，这使它们适合于随后的分子和细胞研究。我们量化并提出了在纯化样品中血小板、红细胞和白细胞污染的携带机制，而不是依赖于样品预处理。这些结果验证了我们的平台继续用于从患者样本中提取 CTCs 和其他稀有细胞分离应用的研究。 © 2019 国际细胞分析学会。
