定量量測細胞活動造成的彈性變化

Abstract

細胞重塑造成胞外基質的彈性變化，在生理和病理過程中扮演著舉足輕重的角色。根據許多研究顯示，比起二維的培養環境，將細胞培養在三維環境中將更能貼近細胞在活體內的情況，然而，目前鮮有平台能夠量測細胞活動造成胞外基質硬度的動態改變。超音波式剪切波彈性影像本身有高輸出通量和非接觸式的優點，並且極具潛力能定量量測胞外基質彈性隨時間和空間的變化情形。此篇研究中，我們在三維細胞培養系統架構下，使用剪切波彈性影像定量胞外基質彈性的改變。三維細胞培養系統主要由厚度1-2mm的細胞培養性水膠組成，我們在水膠內混入生物相容性的散射質以提供超音波成像，並將水膠附著在吸收層上來避免邊界的反射。彈性影像的量測上，使用20MHz的超音波探頭聚焦在水膠內，產生聲場輻射力，剪切波的傳遞訊號則由40MHz的探頭接收。將此三維水膠模擬成伏伊特(Voigt) 材料，如此可由剪切波在不同頻率下的相位速度來決定剪切模數。運用此嶄新的量測平台，我們成功展示三維細胞培養系統培養不同種類的癌症細胞和正常細胞，其基質硬度和結構的改變。在三維系統下培養經過一週，癌症細胞 (如：人類肺腺癌細胞) 將基質彈性提升為原來的40-50倍。膠體的硬度和顯著的體積收縮有關，而這兩種參數間存在著冪次現象的關係。分別加入Blebbistatin和Beta-aminopropionitrile到培養系統，以抑制細胞收縮力和基質交聯的生成，結果顯示基質硬化主要來自於細胞的主動收縮。最後，我們的結果證明在三維模型下，使用剪切波彈性影像將有能力進行細胞和胞外基質間機械生物學的相關研究。

The stiffening of extracellular matrix (ECM) resulting from active cell remodeling plays a crucial role in many physiological and pathological processes. Culturing cells in 3D better recapitulates the in vivo conditions than in 2D models. However, there are few platforms allowing measurements of the dynamics of 3D ECM stiffness resulting from cell activities. Ultrasound shear wave elasticity imaging (SWEI) has high-throughput, non-contact nature and greater potential to evaluate the spatiotemporal dynamics of ECM stiffness. In the present work, we evaluate the feasibility of quantifying changes of ECM stiffness in a 3D cell culture system using SWEI. The 3D cell culture system was composed of a cell-culturing hydrogel about 1—2 mm in high. The gel was mixed with biocompatible scatterers to facilitate ultrasound imaging and attached to an absorption layer to avoid wave reflection at boundaries. A 20 MHz ultrasonic transducer was employed to generate radiation forces in the gels and a 40MHz transducer was used to scan the propagating shear waves. The 3D gels were modeled as Voigt materials and the shear moduli were determined from the phase velocities of the shear waves at various frequencies. Using the novel platform, we successfully demonstrated changes of matrix stiffness and structure when culturing different cancer and normal cell lines. After cultured in the system for 1 week, advanced cancer cells such human lung adenocarcinoma cells CL1-5 stiffened the matrix about 40—50 times than the acellular controls. Gels stiffening was always associated with marked volume contraction and there existed a power law between these two variables. After applying blebbsitatin and beta-aminopropionitrile respectively to the culture system to inhibit cell contraction and matrix cross-linking, we showed that the matrix stiffening mainly resulted from cell contraction. Our data support that SWEI is a promising tool to investigate the dynamics of cell-ECM mechanobiology in 3D models.