Greaney Aisling J, McCarthy Clíona M, Vethil Jishnu Padacherri, Abubaker Mannthalah, Reardon Erin C, Crowley Frederick D, Cunnane Eoghan M, Mulvihill John J E
School of Engineering, University of Limerick, Limerick, Ireland.
Bernal Institute, University of Limerick, Limerick, Ireland.
PLoS One. 2025 May 12;20(5):e0323283. doi: 10.1371/journal.pone.0323283. eCollection 2025.
Cells exhibit remarkable sensitivity to the mechanical properties of their surrounding matrix, particularly stiffness changes, a phenomenon known as cellular mechanotransduction. In vivo, tissues exhibit a wide range of stiffness, from kilopascals (kPa) to megapascals (MPa), which can alter with aging and disease. Traditional cell culture methods employ plastic substrates with stiffness in the gigapascal range, which does not accurately mimic the physiological conditions of most biological tissues. Therefore, employing substrates that can be engineered to span a wide range of stiffnesses, closely resembling the native tissue environment, is crucial for obtaining results that more accurately reflect cellular responses in vivo. Polydimethylsiloxane (PDMS) substrates are widely used in cell culture for their ability to simulate tissue stiffness, but their optimization presents several challenges. Fabrication requires precise control over mixing, weighing, and curing to ensure reproducible mechanical properties. Inconsistent preparation can lead to improperly cured PDMS substrates, compromising experimental outcomes. Additionally, PDMS's inherent hydrophobicity poses challenges for cell attachment, necessitating surface modifications to enhance adhesion. Moreover, the risk of contamination during the sterilization process necessitates stringent protocols to maintain cell culture integrity. These challenges are further compounded by substrate autofluorescence which can cause difficulties when imaging cells. The aim of this study is to develop a standardized method for fabricating PDMS substrates with tuneable stiffness, ranging from kPa to MPa, suitable for diverse cell types using standard laboratory equipment. This method aims to minimize the complexity and equipment required for PDMS fabrication, ensuring reproducibility and ease of use. Achieving consistent and contaminant-free PDMS substrates will facilitate a broader adoption of these substrates in mechanobiology research and improve the relevance of in vitro models to in vivo conditions. Ultimately, contributing to a more comprehensive understanding of cellular responses to mechanical cues in health and disease.
细胞对其周围基质的力学性质表现出显著的敏感性，尤其是硬度变化，这种现象被称为细胞机械转导。在体内，组织表现出广泛的硬度范围，从千帕（kPa）到兆帕（MPa），并且会随着衰老和疾病而改变。传统的细胞培养方法使用硬度在吉帕范围的塑料基质，这不能准确模拟大多数生物组织的生理条件。因此，使用能够被设计成跨越广泛硬度范围、与天然组织环境非常相似的基质，对于获得更准确反映体内细胞反应的结果至关重要。聚二甲基硅氧烷（PDMS）基质因其能够模拟组织硬度而被广泛用于细胞培养，但其优化存在几个挑战。制造过程需要对混合、称重和固化进行精确控制，以确保可重复的力学性能。制备不一致可能导致PDMS基质固化不当，从而影响实验结果。此外，PDMS固有的疏水性对细胞附着构成挑战，需要进行表面改性以增强附着力。而且，灭菌过程中的污染风险需要严格的方案来维持细胞培养的完整性。这些挑战因基质自身荧光而进一步加剧，这在对细胞成像时可能会造成困难。本研究的目的是开发一种标准化方法，使用标准实验室设备制造具有可调硬度（范围从kPa到MPa）的PDMS基质，适用于多种细胞类型。该方法旨在最小化PDMS制造所需的复杂性和设备，确保可重复性和易用性。实现一致且无污染物的PDMS基质将促进这些基质在力学生物学研究中的更广泛应用，并提高体外模型与体内条件的相关性。最终，有助于更全面地理解细胞在健康和疾病状态下对机械信号的反应。
