使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响
June 1st, 2019
我们采用硅橡胶 (PDMS) 制造高通量牵引力测定。这种新颖的测定适用于研究各种生物和生物医学过程和疾病中细胞收缩性的物理变化。通过测量上皮到中位过渡期间收缩性增加的TGF-α依赖性增加,我们证明了这种方法的效用。
我的实验室对机械生物学感兴趣，它研究细胞如何发挥和受到机械力的影响。我们可以量化机械生物学的一个方法就是测量细胞收缩性。在粘附细胞中，收缩表现在对细胞基板施加的牵引力中。我们使用通常称为牵引力显微镜或 TFM 的方法量化这些力。TFM在各种各样的不同方法中实现，但一个常见的方面是，它是一种相当连续和缓慢的实验室技术，通常在像这里这样的单道菜中完成。这限制了我们一次评估一个人口或条件。在整个细胞培养改善前一段时间，但创建多井菜，我们认为，这不是伟大的，我们可以TFM与相同的增加吞吐量和相同的时间和试剂效率？这是我们的解决方案。因此，我们创造了一个96井牵引力盘。它看起来非常类似于传统的96井组织培养皿，然而，与这道菜，我们不仅可以检查96种不同的细胞培养条件，但也量化其细胞收缩性。这提供了一个比单盘方法更高的吞吐量解决方案，使我们能够检查不同的细胞条件和收缩反应。在癌症转移期间，癌细胞从初级部位传播到二级部位，在我们的体内传播疾病。癌细胞的这种转移行为占癌症相关死亡的90%。上皮癌细胞获得更迁移和侵入性表型的拟议机制之一是上皮-乳质过渡，EMT。在EMT期间，他们变得更加迁移和侵入，改变他们的身体行为。为了更好地了解这些物理行为的变化，我们利用软硅基质测量通过TGF诱导的小鼠乳腺细胞收缩的变化，在这项研究中，我们调查了TGF-影响细胞物理行为的不同浓度和孵化时间。聚二甲基硅氧烷是一种非常合规的橡胶，最低 Young 的模量约为 0.6 千帕。这种材料的刚度也可以使用不同浓度的交联剂（包括 SYLGARD 184.To测量我们使用的流变仪的PDMS的机械性能。混合 PDMS 的 A 部分和 B 部分后，以一对一的重量比加上指定重量百分比的固化剂，将准备的批次装载到流变表板上。确保装载足够的样品以完全覆盖主轴区域。确保主轴完全接触样品，边缘没有空位，这一点很重要。任何额外的样品应仔细修剪，并擦掉板。在0.5%Newsha的振荡剪切应变下进行时间和频率扫描，测量每个样品粘弹性的最终存储模量和时间依赖性。
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概述
本研究介绍了一种由硅胶（PDMS）制造的高通量牵引力测定法，旨在研究各种生物过程中细胞收缩性的物理变化。该方法的实用性通过测量上皮-间充质转化过程中TGF-β依赖性收缩性增加得到了验证。
关键研究组成部分
科学领域
- 细胞生物学
- 生物力学
- 生物医学工程
背景
- 细胞收缩性在各种生物过程中至关重要。
- 了解收缩性的变化可以为疾病提供洞见。
- 传统方法可能缺乏通量和灵敏度。
- PDMS是一种用于创建测定法的多功能材料。
研究目的
- 开发一种高通量测定法来测量细胞收缩性。
- 评估TGF-β对上皮-间充质转化过程中收缩性的影响。
- 提供一种研究细胞在各种条件下行为的可靠方法。
使用的方法
- 使用PDMS制造牵引力测定法。
- 测量细胞对TGF-β的收缩性反应。
- 高通量分析以评估多个样本。
- 评估上皮-间充质转化动力学。
主要结果
- 成功测量了细胞收缩性变化。
- 证明了TGF-β依赖性收缩性增加。
- 验证了基于PDMS的测定法的实用性。
- 提供了细胞转化过程力学的洞见。
结论
- PDMS牵引力测定法对于研究细胞收缩性是有效的。
- 确认了上皮-间充质转化过程中收缩性增加。
- 这种方法可应用于各种生物和生物医学研究。
常见问题
Executive Industry Relevance
This high-throughput traction force microscopy platform enables quantitative assessment of cellular contractility in a scalable format, supporting mechanistic de-risking in early discovery. By linking TGF-β-induced epithelial-to-mesenchymal transition to measurable biophysical changes, the method provides predictive value for target validation in fibrosis and metastasis research. The PDMS-based system offers reproducibility and compatibility with existing multi-well workflows, enhancing enterprise reuse across discovery biology and assay development pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of TGF-β as a modulator of cellular contractility during EMT, supporting target hypothesis testing.
- Operational Value: Provides dose-dependent and time-resolved contractility data to prioritize mechanistic targets in fibrosis and cancer pathways.
- Predictive Value: Quantifies biophysical phenotypes linked to EMT, aiding in preclinical target selection and pathway de-risking.
Screening & Assay Development
- Scientific Value: Delivers quantitative, substrate-independent contractility measurements for assay standardization in mechanobiology screening.
- Operational Value: Compatible with 96-well formats, enabling parallel screening of compound libraries or cytokine doses.
- Assay Readiness: Uses stable, inert PDMS substrates with tunable modulus (0.4–100 kPa) and indefinite shelf life, reducing batch variability.
Translational & Preclinical Research
- Translational Continuity: Links TGF-β dose and duration to contractile output, supporting biomarker-aligned preclinical models of EMT.
- Mechanistic De-risking: Enables evaluation of contractility changes as a functional readout in disease-relevant systems prior to in vivo validation.
- Risk-Adjusted Advancement: Supports go/no-go decisions by correlating molecular intervention with biophysical phenotype modulation.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation through lead identification, providing biophysical readouts that inform early-phase decision-making.
- Discovery Biology: Supports hypothesis testing of TGF-β signaling effects on cellular mechanics and contractility.
- Screening: Enables high-throughput, reproducible quantification of traction forces across multi-well formats for dose-response analysis.
- Analytics: Generates contractility metrics (force magnitude, distribution) that allow comparison of TGF-β concentrations and exposure times.
- Translational Research: Connects in vitro EMT induction to measurable biomechanical changes, supporting preclinical model relevance.
- Enterprise Reuse: Establishes a reusable traction force platform applicable across cell types and mechanobiology targets in oncology and fibrosis.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing ambiguity in mechanobiological phenotypes.
- Operational Value: Enhances reproducibility and scalability through standardized PDMS substrates and multi-well compatibility.
- Strategic Value: Improves capital efficiency by enabling parallel condition testing and reducing reagent consumption per data point.
- Portfolio Impact: Facilitates risk-adjusted prioritization of targets based on quantitative contractility shifts during pathogenic transitions.
Implementation Considerations
- Requires expertise in cell culture, TFM image analysis, and PDMS substrate preparation.
- Depends on rheometry for substrate characterization and fluorescence microscopy for traction force quantification.
- Necessitates standardization of seeding density, substrate functionalization, and imaging conditions across wells.
- Adaptation to different cell types may require optimization of substrate stiffness and adhesion ligands.
- Practical limitations include substrate thickness uniformity and potential autofluorescence of PDMS at certain wavelengths.
