这里展示的模型的发展得到了DOD赠款W81XWH-12-1-0163、NIH赠款R21 CA179981和R21CA196202以及佐藤转移研究基金的支持。

1. 细胞制备
<ol><li>当 70-90% 汇流时，通过遵循所用细胞系的建议指南从组织培养皿中释放细胞。<ol>
<li>例如，为 PC-3 细胞吸气生长介质，用 5 mL 的无钙和无镁磷酸盐缓冲盐水 （PBS） 清洗 10 厘米的细胞盘。</li>
		<li>在使用制造商协议添加 1 mL 的 0.25% 试用素之前，先吸气 PBS。</li>
		<li>在倒置显微镜下观察细胞分离后，加入含有10%胎儿牛血清的5 mL DMEM：F12介质，以抑制试金石。</li>
	</ol>
</li>
	<li>将?...

<ol>
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本文演示了FSS在悬架中使用注射器和针头对癌细胞的应用。使用这个模型，癌细胞已经证明对高水平FSS的短暂脉冲比非转化上皮细胞15，22，24更耐药。此外，使用此模型接触FSS可导致细胞僵硬度迅速增加，RhoA激活，皮质F-actin和肌素II基收缩性增加24，27。快速机械适应（CTC的能力，?...

转移，或癌症的扩散超过最初的肿瘤部位，是导致癌症死亡率的主要因素1。在转移过程中，癌细胞利用循环系统作为高速公路传播到全身2、3的遥远部位。在前往这些部位的途中，循环肿瘤细胞（CTCs）存在于动态流体微环境内，不像其原始原发肿瘤3、4、5。有人提出，这种流体微环境是转移4的众多障碍之一。转移效率低下的概念大体一致，即大多数进入循环的CTC要么灭亡，要么不形成生产性转移殖民地6，7，8。然而，为什么从单个反恐委员会的角度来看，转移效率低下，这一点不太确定，而且仍然是一个活跃的调查领域。CTC与细胞外基质分离，剥夺了原发性肿瘤中可能存在的可溶性生长和生存因子，并且以与原发性肿瘤4大不相同的方式暴露在免疫系统和血液动力中。这些因素都可能导致CTC的存活率低，但其相对贡献尚不清楚。本文讨论了血液动力如何影响CTC的问题。
研究血液动力对CTC的影响是相当具有挑战性的。目前，没有工程体外系统可以复制整个空间动力学（心到毛细血管）和人类血管系统的流变特性。此外，CTC如何体验循环系统还不完全清楚。实验证据表明，大多数癌细胞不会像血细胞那样持续循环。相反，由于它们的尺寸相对较大（直径为10-20μm），大多数CTC被卡在毛细血管床（直径为6-8μm）的可变时间长度（s至天），在那里他们可能会死亡，奢侈，或被转移到下一个毛细血管床8，9，10，11。然而，有一些证据表明，CTC的大小可能更异质的体内，较小的CTC是可检测到的12。因此，根据距离和血流速度，CTC只能在这些诱捕期之间自由循环几秒钟，尽管缺乏对这种行为的定量描述。
此外，根据CTC进入循环的位置，它们可能通过肺部和其他外周部位的多个毛细发床，在到达最终目的地之前通过左右心脏。一路上，CTC 暴露于各种血液动力学应力，包括液切变应力 （FSS）、在微循环中诱捕过程中的压缩力，以及在可能表现出类似白细胞的沿着血管壁滚动的情况下的牵引力。因此，对流通进行建模的能力和对要建模的CTC行为的理解都是有限的。由于这种不确定性， 体外 模型系统的任何发现都应在实验脊椎动物体内以及最终在癌症患者中得到验证。
有了上述警告，本文演示了一个相对简单的模型，将FSS应用于悬浮细胞，以探讨FSS对CTC的影响首次描述在2012年15。FSS 是血流摩擦血管壁的结果，在较大血管的层压流条件下产生抛物线速度梯度。细胞在血管壁附近经历更高水平的FSS，在血管中心附近经历较低的FSS水平。流体粘度、流速和流经管道的尺寸会影响 FSS，如哈根-波塞耶方程所述。这适用于血流作为牛顿流体的行为，但不适用于微循环。生理FSS的范围超过几个数量级，淋巴（<1 dyn/cm2）中最低，心脏瓣膜和动脉粥样硬化斑块周围区域最高（>500 dyn/cm2）5。动脉中的平均壁切应力为10-70 dyn/cm2和静脉1-6 dyn/cm2，静脉16，17。
在心脏，细胞可能暴露在阀门传单周围的湍流，其中非常高水平，但非常短的FSS可能会经历18，19。虽然生物处理领域长期以来一直研究FSS对悬浮哺乳动物细胞的影响，但这种信息对于理解FSS对CTC的影响可能价值有限，因为它通常侧重于长期应用的低得多的FSS水平。如下所述，使用注射器和针头，可以将相对较高的（数万至数千丁/厘米2）FSS用于相对较短的（毫秒）的细胞悬架。自最初描述这个模型15以来，其他人已经利用它来研究FSS对癌细胞21，22，23的影响。FSS 的多个"脉冲"可在短时间内应用于细胞悬浮，以促进下游实验分析。例如，该模型可用于测量细胞通过 FSS 抵抗机械破坏的能力，测量细胞的可行性作为应用脉冲数量的函数。或者，可以通过收集细胞进行各种下游分析来探索FSS暴露对癌细胞生物学的影响。重要的是，部分单元格悬架保留为静态控制，以比较 FSS 与可能与单元格分离和悬架中保持的时间相关的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL round bottom tubes</td>
			<td>Falcon - Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G 1/2&#34; Needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black bottom plate</td>
			<td>Costar - Corning</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioluminescence detector</td>
			<td>AMI</td>
			<td>AMI HTX</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, Fraction V</td>
			<td>Sigma</td>
			<td>10735086001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Titer Blue</td>
			<td>Promega</td>
			<td>G8081</td>
			<td></td>
		</tr>
		<tr>
			<td>crystal violet</td>
			<td>Sigma</td>
			<td>C0775</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>GoldBio</td>
			<td>D-LUCK</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide (NaN3)</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>70-3005</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling the effects of hemodynamic stress on circulating tumor cells using a syringe and needle

在转移过程中，来自固体组织的癌细胞，包括绰号，获得淋巴和血源循环，在那里他们暴露在机械应激由于血液同源流动。其中一个压力是循环肿瘤细胞（CTCs）的经验是液体剪切应力（FSS）。虽然癌细胞可能由于间歇性流动而在肿瘤内经历低水平的FSS，但CTC在没有细胞外基质附着的情况下暴露在更大的FSS水平上。从生理学上讲，FSS的震级范围超过3-4级，淋巴（<1分/厘米2）中含量较低，当细胞穿过心脏和心脏瓣膜时，最高水平短暂存在（>500dynes/cm2）。有几个 体外 模型设计为在不同的时间范围内模拟不同范围的生理剪切应力。本文描述了一个模型，利用简单的注射器和针头系统来研究高水平FSS的短暂（毫秒）脉冲对癌细胞生物学的影响。

先前已证明，对FSS诱发的机械破坏的耐药性升高是多种癌细胞系的保存表型，而与未转化的上皮细胞比较器15、24相比，从肿瘤中新分离出来的癌细胞是一种保存的表型。在这里，从各种组织起源（表2）的其他癌细胞系测试，以证明这些细胞的大多数显示生存能力≥20%后，10脉冲的FSS在250μL/s。一个例外是MiaPaCa2细胞，它对FSS的机械破坏相对?...

Watch this Scientific Journal Video about Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle at JoVE.com

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

在这里，我们演示了一种方法，将液剪应力应用于悬浮中的癌细胞，以模拟血液动力学应激对循环肿瘤细胞的影响。

使用注射器和针头模拟血液学应激对循环肿瘤细胞的影响

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...

该协议将悬浮中的癌细胞暴露在液切变应力的短暂脉冲中，以模拟转移性癌细胞在循环系统中如何暴露于血液动力的某些方面。应用高水平流体剪切应力的短暂脉冲是一种相对简单的技术。在尝试此协议时，小心未封顶的针头，不要弯曲它们，因为这将改变施加力剪切应力。此程序可能会产生气溶胶，因此请使用适当的安全措施。首先，通过吸气生长介质，用5毫升无钙和无镁PBS清洗10厘米的盘，从组织培养皿中释放70%至90%的聚合PC3细胞。然后，吸气PBS，并添加一毫升0.25%的试用素。尝试后，观察倒置显微镜下细胞的分离。要抑制试丁香，添加5毫升DMEM/F12介质含有10%FBS。接下来，将细胞悬浮放入圆锥管中，确定细胞浓度和细胞总数。将细胞悬浮离心在 300 x g 下三分钟，然后吸气超纳特，并在无血清组织培养介质中重新吸气颗粒。在 7 毫升线处切开 14 毫升聚苯乙烯管的底部，并将混合细胞悬架放入切割管中。单独收集细胞静态控制样本，然后再进行液切应力暴露，用于执行检测。要将剩余的细胞悬浮样品暴露在液切变应力下，请将细胞悬浮液拉入 5 毫升注射器中，并连接 30 米半英寸针头。将带未封盖针头的注射器放置并固定在注射器泵上，并设置流速，以达到所需的液切变应力水平。运行注射器泵，以大约 45 度角在切管中收集剪切样品，以减少发泡。小心地从注射器泵中取出注射器，并用钳子取出针头，注意不要触摸针头。重复此过程，直到细胞悬浮暴露在所需的液切变应激脉冲数量。在使细胞暴露在液切变应力之前，对静态样品进行可行性分析。对于酶检测，将 100 微升从重复中的静态样品中转移到 96 井板中。然后，在液体剪切应力暴露后收集 100 微升样品，并将其放置在 96 井板中。在每口样品井和仅含有100微升中等微升的油井中加入20微升每毫升0.15毫克的再沙祖林溶液。在37摄氏度的组织培养孵化器中孵育井板两小时，然后使用板读器测量荧光和吸收，并通过将每个流体剪应力暴露样品的平均信号与静态控制样本进行比较，获得活细胞的百分比。同样，从静态样本中收集阿利报价，以执行文本手稿中描述的流细胞学和克隆性测定。在具有代表性的分析中，在使细胞暴露在一些液切变应激脉冲中后，使用再沙祖林转化来评估合成活检乳腺上皮癌细胞的可行性。尽管每个细胞系都显示不同的电阻型材，但在10次液切变应激暴露脉冲后，其生存能力没有显著差异。来自各种组织起源的其他癌细胞系表明，在10脉冲的液切变应激之后，其生存能力超过20%，但MIA PaCa-2细胞除外，由于对液切变应激的机械破坏敏感性，其生存能力低于10%。在应用流体剪切应力之前收集静态样品时，确保细胞悬浮是同质混合的。小心地用钳子取下针头，不要弯曲或触摸针头。除了测量细胞的生存能力外，人们还可以评估基因表达、细胞信号、增殖和迁移的变化。以此作为接触液切变应激的模型，可以研究癌细胞如何抵抗液切变应激的破坏，并评估液切变应激对癌细胞生物学的影响。本技术已被我们的实验室和其他人用于探索液切变应力对循环肿瘤细胞的影响。这些研究表明，液切变应力迅速改变癌细胞的机械特性，这有助于癌细胞抵抗液切变应力破坏的能力。