剪切波影像量測三維細胞環境彈性

Abstract

微環境的硬度由細胞與基質共同塑造，在發育和腫瘤發育等過程中，細胞感受外在硬度而有著不同的行為表現，包含影響幹細胞分化以及癌細胞主動改造基質硬度及並轉移等。硬度在二維環境中已被大量證明其重要性，然而在三維環境中，目前較缺乏適合動態量測的平台與工具，超音波剪切波彈性影像具有一定程度的空間解析度與時間解析度，並具有非接觸式與非入侵式量測的優點。我們在此篇研究中，發展了由聚二甲基矽氧烷(PDMS)作為外在固定的模具去合成三維的膠原蛋白水膠(Collagen)以及基質膠(Matrigel)平台，混入細胞，進行三維細胞培養，並量測細胞在水膠中的活動。我們使用20 MHz的超音波探頭聚焦送入聲場輻射力，並用40 MHz的探頭同步監測剪切波傳遞。我們首先以流變儀測量基質膠、洋菜膠、膠原蛋白水膠，驗證我們系統量測的準確性，接著測試我們的量測參數，證實厚度、溫度、附著底的不同，在一定範圍內都不影響量測。此外我們也以洋菜膠進行空間異質性的測試，結果證實我們的系統具有空間解析度。另外我們隨著時間量測H9C2肌肉母細胞以及CL1-5肺腺癌細胞對於水膠造成的硬度變化和厚度變化，發現H9C2在半天內會讓水膠硬度上升4~5倍，而此硬度變化會被BDM抑制，證明硬度變化和細胞收縮有關。另外CL1-5在兩種膠原蛋白濃度中會有不同反應，在 1 mg/mL(毫克/毫升)的濃度中，經過五天後環境硬度會上升到原本的10倍左右，而在2 mg/mL的環境中則沒有硬度變化，顯示細胞在兩種膠體濃度會有不同的細胞行為。

Microenvironment stiffness was formed by cell and ECM. In the cell development and the tumor progression, cell would sense the local stiffness and then adapt their behavior such as cell differentiation or cancer cell remodeling substrate stiffness even metastasis. Stiffness has been proved its importance in 2D condition. However, we lack suitable approach and platform to dynamically measure the stiffness in 3D condition. Supersonic-based shear wave elasticity imaging has great spatial resolution, temporal resolution, and measure in non-invasive way. In our present work, we developed a device fabricated by PDM to bind 3D collagen gel and matrigel to do 3D cell culture and measure cell activity. We generate the ultrasound radiation force by 20 MHz push transducer and monitor the shear wave propagation by 40 MHz image transducer. At first, we use rheometer as a gold standard technique to validate our system. Secondly, we test our measuring parameter, and found that gel thickness, binding-base, temperature would not significantly affect our measurement. In addition, we could discern spatial patterned heterogeneous agarose gel. Moreover, we found that H9C2 myoblast would stiffen the gel in a half day and it would rebound by BDM blocking which proved that the stiffness change was correlated with cell contraction. On the other hand, CL1-5 would have different behavior in two different concentration of collagen gel. In 1 mg/mL gel, collagen gel would be stiffer but not in 2 mg/mL gel that it may involve ECM remodeling.