Marx Kenneth A, Zhou Tiean, Montrone Anne, McIntosh Donna, Braunhut Susan J
Center for Intelligent Biomaterials, University of Massachusetts, Lowell, MA 01854, USA.
Anal Biochem. 2005 Aug 1;343(1):23-34. doi: 10.1016/j.ab.2005.05.013.
A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.
一种石英晶体微天平（QCM）细胞生物传感器，利用附着在金QCM表面的活内皮细胞（ECs）或人乳腺癌细胞（MCF-7），来研究细胞及其下层细胞外基质（ECM）对测量的QCM频率变化量（Δf）和电阻抗变化量（ΔR）的相对贡献。ECM是一种天然生物材料，由细胞合成以使其能够附着于表面。我们采用非蛋白水解方法跟踪ECs或MCF-7细胞从其ECM上的脱离，并能够将生物传感器的总频率降低量（Δf）分配为细胞附着和完整下层ECM的贡献。我们还证明，去除ECs后剩余的Δf变化量对应于通过染色蛋白的光学显微镜观察所确定的ECM。在细胞脱离过程中，我们观察到粘弹性行为出现一种新的瞬时增加，表现为运动阻力参数（ΔR）的瞬时增加。然后，我们通过使用用荧光罗丹明标记的鬼笔环肽（一种肌动蛋白染色剂）染色的ECs进行模拟实验表明，瞬时粘弹性增加与细胞在用乙二醇双（2-氨基乙醚）-N，N，N'，N'-四乙酸去除过程中表现出的细胞应激相关。在细胞从其ECM上抬起之前，附着的ECs首先将其肌动蛋白微丝重排为外周应力纤维，然后重排为内部聚集体，以维持细胞间连接，保持其铺展形态，并试图更紧密地附着于其下层ECM。ΔR在其瞬时上升后下降对应于细胞最终失去其附着焦点并从ECM上抬起。我们还将归一化的频率变化量，-Δ（Δf）（ECM）/附着细胞和-Δ（Δf）（细胞）/附着细胞，表征为附着细胞数量变化的函数。最后，我们证明，与附着在ECM上的细胞相比，所有细胞都已被去除的下层天然ECM生物材料没有表现出任何显著水平的能量耗散。
