使用交流介电泳分离非转移性（MCF-7）和非肿瘤（MCF-10A）乳腺癌细胞的微流体装置
Summary
与非肿瘤乳腺上皮细胞相比，乳腺癌细胞表现出不同的介电特性。据推测，基于介电特性的这种差异，可以出于免疫治疗目的将两个群体分开。为了支持这一点，我们模拟了一种微流体装置来分选MCF-7和MCF-10A细胞。
Abstract
介电泳装置能够利用施加外部电场使样品体积中的癌细胞极化的原理，以无标记、经济高效、稳健和准确的方式检测和操纵癌细胞。本文演示了如何使用细胞混合物中的流体动力介电泳（HDEP）利用微流体平台对非转移性乳腺癌细胞（MCF-7）和非肿瘤乳腺上皮细胞（MCF-10A）进行高通量连续分选。通过在HDEP微流控芯片中并排放置的两个电极之间产生电场，它们之间有微米大小的间隙，非肿瘤乳腺上皮细胞（MCF-10A）可以被推开，在主通道内表现出负DEP，而非转移性乳腺癌细胞在悬浮在细胞培养基中时遵循其过程，由于电导率高于膜电导率。为了证明这一概念，对不同值的介质电导率进行了模拟，并研究了细胞的分选。进行了参数研究，发现合适的细胞混合物电导率为0.4 S/m。通过保持介质电导率固定，建立了足够的0.8 MHz交流频率，通过改变电场频率提供最大的分选效率。使用所演示的方法，在选择合适的细胞混合物悬浮介质电导率和施加的AC频率后，可以实现最大的分选效率。
Introduction
在乳腺组织内和乳腺组织周围发展的恶性肿瘤是全球女性乳腺癌的常见原因，导致严重的健康问题1。如果在早期发现，转移前的乳腺肿瘤可以通过手术治疗，但如果忽视，它们会扩散到他们的肺、脑和骨骼，对患者的生活产生严重影响。后期提供的治疗，如放射和化学疗法，具有严重的副作用2。最近的研究报告称，乳腺癌的早期诊断可将死亡率降低 60%3。因此，必须努力实现个性化的早期检测方法。为此，从事不同科学和技术领域的研究人员利用微流体技术开发了用于乳腺癌早期诊断的设备4。这些方法包括细胞亲和显微色谱、磁性激活微细胞分选仪、基于大小的癌细胞捕获和分离以及片上介电泳（DEP）5，6。文献中报道的这些微流体技术能够对定义明确的样品进行精确的细胞操作、实时监测和分类，这是许多诊断和治疗应用中的中间步骤5。这些分选机制与微流体的集成提供了对靶细胞7，8，9，10的灵活可靠的操作。这种集成的主要优点之一是能够处理纳升到微升体积的流体样品，并且还能够操纵样品流体的电特性。通过调节微流体装置内悬浮液的电导率，可以根据生物细胞的大小和介电特性的差异对生物细胞进行分类11，12。
在这些技术中，片上DEP通常是首选，因为它是一种利用生物样品电特性的无标记细胞分选技术。据报道，DEP 可以操纵生物样品，例如 DNA 13、RNA 14、蛋白质 15、细菌16、血细胞 17、循环肿瘤细胞 （CTC） 18 和干细胞 19。采用DEP分选生物样品的微流体装置已在文献20中广泛报道。据报道，基于储库的DEP微流体（rDEP）装置用于分选活菌和非活酵母细胞，可保护细胞免受电化学反应的不利影响21，22。Piacentini等人报道了一种卡斯特尔化微流体细胞分选机，该分选机以97%23的效率从血小板中分离红细胞。据报道，具有不对称孔和嵌入式电极的片上DEP器件也可以对活细胞和非活细胞进行分类24。Valero和Demierre等人通过在通道25，26的两侧引入两个微电极阵列来修改卡斯特尔式微流体细胞分选仪。这有助于将细胞聚焦在通道的中心。Zeynep等人提出了一种基于DEP的微流体装置，用于从白细胞27中分离和浓缩MCF7乳腺癌细胞。他们报告说，从白细胞中提取MCF7细胞的效率在74%-98%之间，频率为1 MHz，施加的电压范围为10-12 Vpp。补充表1表示基于DEP的微流体分选设备之间基于其设计，电极配置和操作参数（施加频率和电压）的定性和定量比较。
最近，研究人员试图测量微流控芯片28，29内乳腺上皮细胞（MCF-10A）和非转移性乳腺癌细胞（MCF-7）介电行为的差异。Jithin等人还使用频率在200 MHz和13.6 GHz30之间的开放式同轴探针技术表征了不同癌细胞系的介电响应。MCF-7和MCF-10A细胞系介电响应的这些差异可用于在运行时将它们分离，并可能导致个性化早期诊断设备的开发。
在本文中，我们使用交流介电泳模拟了非转移性乳腺癌细胞（MCF-7）和非肿瘤乳腺上皮细胞（MCF-10A）的受控分选。电场的变化区域会影响微流控芯片内部的分选。所提出的技术易于实现，并允许将分选技术集成到各种微流控芯片布局中。通过计算流体动力学（CFD）模拟，通过改变悬浮细胞的流体培养基的电导率，研究非转移性乳腺癌细胞和非肿瘤乳腺上皮细胞的分离。在这些模拟中，表明通过保持电导率恒定并改变施加的频率，可以控制癌细胞和健康细胞的分离。
Protocol
注意：此处的实验方案使用 COMSOL（一种多物理场仿真软件）来模拟使用交流电泳对非转移性乳腺癌细胞 （MCF-7） 和非肿瘤乳腺上皮细胞 （MCF-10A） 进行受控分选。 1.芯片设计及参数选择 打开多物理场软件并选择 空白模型。右键单击 全局定义 ，然后选择 参数。将 表 1 中给出的参数作为文本文件导入全?…
Representative Results
研究基于DEP对非转移性乳腺癌（MCF-7）和非肿瘤乳腺上皮（MCF-10A）细胞进行有效分选的最佳操作参数为了在进行介电泳时成功分离具有不同介电特性的非转移性乳腺癌（MCF-7）和非肿瘤乳腺上皮（MCF-10A）细胞，它们的K因子应通过保持应用频率固定37，38来区分。使用公式4实现了非转移性乳腺癌细胞和非肿瘤乳腺上皮细胞在外加电场下的?…
Discussion
以前已经报道过用于细胞培养，捕获和分选的微流体装置47，52，53。在洁净室中制造这些设备是一个昂贵的过程，必须通过CFD仿真来量化所提出的微流体设备的输出和效率。本研究介绍了基于非转移性乳腺癌细胞（MCF-7）和非肿瘤乳腺上皮细胞（MCF-10A）介电特性的AC介电泳微流控装置的设计和模拟23。
<p cla…Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了巴基斯坦高等教育委员会的支持。
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ur Rehman, A., Zabibah, R. S., Kharratian, S., Mustafa, A. Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis. J. Vis. Exp. (186), e63850, doi:10.3791/63850 (2022).