Quantification of the forces driving self-assembly of three-dimensional microtissues

Abstract

In a nonadhesive environment, cells will self-assemble into microtissues, a process relevant to tissue engineering. Although this has been recognized for some time, there is no basis for quantitative characterization of this complex process. Here we describe a recently developed assay designed to quantify aspects of the process and discuss its application in comparing behaviors between cell types. Cells were seeded in nonadhesive micromolded wells, each well with a circular trough at its base formed by the cylindrical sidewalls and by a central peg in the form of a right circular cone. Cells settled into the trough and coalesced into a toroid, which was then driven up the conical peg by the forces of self-assembly. The mass of the toroid and its rate of upward movement were used to calculate the cell power expended in the process against gravity. The power of the toroid was found to be 0.31 ± 0.01 pJ/h and 4.3 ± 1.7 pJ/h for hepatocyte cells and fibroblasts, respectively. Blocking Rho kinase by means of Y-27632 resulted in a 50% and greater reduction in power expended by each type of toroid, indicating that cytoskeletal-mediated contraction plays a significant role in the self-assembly of both cell types. Whereas the driving force for self-assembly has often been viewed as the binding of surface proteins, these data show that cellular contraction is important for cell-cell adhesion. The power measurement quantifies the contribution of cell contraction, and will be useful for understanding the concerted action of the mechanisms that drive self-assembly. T he self-assembly of cells into microtissues, often viewed as a passive process driven by chemical forces resulting from binding of cadherins expressed on cell surfaces (1, 2), is now thought to be a more complex process. Recent work has shown that the cytoskeleton also plays an important role in force generation, stability, and self-sorting of microtissues (3-6). Thus, self-assembly is a complex process involving multiple protein mechanisms working in concert.Self-assembly and the cell-cell adhesion that drives it are relevant to tissue formation, morphogenesis, and disease. However, little quantitative data are available on the forces driving self-assembly. At the molecular level, precise measurements of the binding strength and energy of adhesion of surface proteins (7,8) as well as the force of contraction of the actin cytoskeleton (9, 10) have been made. On the cellular level, traction-force microscopy has been used to measure the adhesion forces of single cells on flat substrates (11-13), and fibroblast-populated collagen gels have been used in the study of cell-matrix interactions (14). However, cell-cell interactions in a 3D environment have not been fully investigated. Doing so requires a quantitative systems biology approach that can be used not only to quantify the aggregation and self-assembly of groups of cells but also to quantify the contributions of specific proteins and protein systems to the process.In the past, we have reported observations on ...

Passive and active contributions to generated force and retraction in heart valve tissue engineering

“…In literature, the reported active and passive cellular contributions to generated force are much higher. The active cellular contribution to force of fibroblasts in collagen gels was 35-65% (Marquez et al 2009;Youssef et al 2011;John et al 2010;Legant et al 2009) and lysation caused an additional 30% reduction of force below the level observed with CytoD treatment (Wakatsuki et al 2000). To measure a decrease in force within our model system, bending of the leaf springs must become less and this requires elongation of the TE constructs.…”

“…Active and passive phenomena have been investigated thoroughly in short-term studies with fibroblast-populated collagen and fibrin gel systems Marquez et al 2009;John et al 2010;Youssef et al 2011;Tomasek et al 1992;Grouf et al 2007;Balestrini and Billiar 2009). In tissue engineered heart valves, the ECM is developed during 4 weeks of tissue culture and is more mature than the generally investigated collagen and fibrin gels.…”

Micro-Mold Design Controls the 3D Morphological Evolution of Self-Assembling Multicellular Microtissues

“…Most of our dogbones ruptured even when the trough width was uniform throughout the design, suggesting that the uniaxial and circumferential forces of our most stable design were not perfectly balanced for fibroblasts, a cell type we have shown exerts the greatest forces during self-assembly. 13 After the initial aggregation phase, the most stable dogbone tissue had the following dimensions: uniform tissue width of *650 mm, rod length of *3.6 mm, and inner circumference around post of *2.5 mm. When we increased the length of the rod, we found that longer rods ruptured sooner than shorter rods, presumably because longer rods have a greater cell volume, which exerts greater forces versus the cell volume surrounding the posts.…”

“…[2][3][4][5][6][7][8] Self-assembly is driven by the concerted actions of various surface adhesion molecules (e.g., cadherins, connexons) along with cytoskeletal-mediated contraction, and it is thought to mimic the aspects of embryogenesis, morphogenesis, and organogenesis. [9][10][11][12][13][14] Different cell types vary with regard to their rate and extent of self-assembly. 13,15 Movement of individual cells occurs as a loose layer of cells aggregate and form a 3D multicellular tissue with substantial x, y, and z dimensions.…”

“…[9][10][11][12][13][14] Different cell types vary with regard to their rate and extent of self-assembly. 13,15 Movement of individual cells occurs as a loose layer of cells aggregate and form a 3D multicellular tissue with substantial x, y, and z dimensions. In addition to climbing over one another, cell movements can be quite specific, as evidenced by the self-sorting that can occur when a mixture of two cell types self-assemble.…”