Chemomechanics of attached and suspended cells

[摘要] Chemomechanical coupling in single eukaryotic animal cells is investigated in the con- text of the attached (substratum-adhered) and the suspended (free-floating) states. These dichotomous configurations determine behavioral differences and commonalities relevant to therapeutic reimplantation of stem cells and to our general under- standing of the cell as an animate material. Analytical, simulation, and experimental techniques are applied to key questions including: (1) How deep can mechanosensitive attached cells ;;feel;; into the adjacent environment? (2) In what manner do suspended cells deform, absent the prominent actomyosin stress fibers that arise upon attachment to a rigid substratum? (3) What explains the remarkable mechanical heterogeney among single cells within a population? (4) Can we leverage putative mechanical markers of useful stem cells to sort them before reimplantation in tissue generation therapies? Attached cells are found to barely detect an underlying rigid base more than 10 micrometers below the surface of a compliant coating. This conclusion, based on ex- tensions to the Boussinesq problem of elasticity theory, is validated by observations of cell morphology on compliant polyacrylamide coatings in a range of thicknesses. Analytical equations are developed for estimating the effective stiffness sensed by a cell atop a compliant layer. We also identify and consider conceptualizations of a ;;critical thickness,;; representing the minimum suitable thickness for a specific application. This parameter depends on the cell behavior of interest; the particular case of stem cell culture for paracrine extraction is presented as a case study. Suspended cells are found to exhibit no single characteristic time scale during de- formation; rather, they behave as power-law (or ;;soft glassy;;) materials. Here, optical stretching is used as a non-contact technique to show that stress fibers and probe-cell contact are not critical in enabling power-law rheological behavior of cells. Further- more, suspended cell fluidity, as characterized by both the hysteresivity of complex modulus and the power-law exponent of creep compliance, is found to be unaffected by adenosine triphosphate (ATP) depletion, showing that ATP hydrolysis is not the origin of fluidity in cells during deformation. However, ATP depletion does reduce the natural variation in hysteresivity values among cells. This finding, and the finding that changes in the power-law exponent and stiffness of single cells are correlated upon repeated loading, motivates study of how and why these parameters are coupled. To further explore this coupling, chemomechanical cues are applied to cell populations to elucidate the origin of the wide, right-skewed distribution of stiffness values that is consistently observed. The distribution and width are found to be not detectably dependent on cell-probe contact, cell lineage, cell cycle, mechanical perturbation, or fixation by chemical crosslinking. However, ATP depletion again reduces heterogeneity, now in the case of cell stiffness values. It is further found analytically that a postulated Gaussian distribution of power-law exponent values leads naturally to the log-normal distribution of cell stiffness values that is widely observed. Based on these connections, a framework is presented to improve our understanding of the appearance of mechanical heterogeneity in successively more complex assemblies of cell components. Two case studies are described to explore the implications of unavoidable intrinsic variation of cell stiffness in diagnostic and therapeutic applications. Finally, all the single-cell mechanical parameters studied so far (stiffness during creep and recovery, stiffness heterogeneity among cells, and power-law exponents in creep and recovery) are characterized in mesenchymal stem cells during twenty population doublings with the aim of developing a high-throughput sorting tool. How- ever, mechanical and structural changes that are observed in the attached state during this culture time are not observed after cell detachment from the substratum. The absence in the suspended state of these alterations indicates that they manifest themselves through stress fiber arrangement rather than cortical network arrangement. While optical stretching under the present approach does not detect mechanical markers of extended passaging that are correlated with decreased differentiation propensity, the technique is nevertheless found capable of investigating another structural transition: mechanical stiffening over tens of minutes after adherent cells are suspended. This previously unquantified transition is correlated with membrane resorption and reattachment to the cortex as the cell ;;remodels;; after substratum detachment. Together, these quantitative studies and models of attached and suspended cells de- fine the extremes of the extracellular environment while probing mechanisms that con- tribute to cellular chemomechanical response. An integration of the results described above shows that no one existing model can describe cell chemomechanics. However, the cell can be usefully described as a material -- one in which animate mechanisms such as active contraction will generally, but not invariably, need to be considered as augmenting existing viscoelastic theories of inanimate matter.

[发布机构] Massachusetts Institute of Technology