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

Abstract

When seeded into nonadhesive micro-molds, cells self-assemble three-dimensional (3D) multicellular microtissues via the action of cytoskeletal-mediated contraction and cell-cell adhesion. The size and shape of the tissue is a function of the cell type and the size, shape, and obstacles of the micro-mold. In this article, we used human fibroblasts to investigate some of the elements of mold design and how they can be used to guide the morphological changes that occur as a 3D tissue self-organizes. In a loop-ended dogbone mold with two nonadhesive posts, fibroblasts formed a self-constrained tissue whose tension induced morphological changes that ultimately caused the tissue to thin and rupture. Increasing the width of the dogbone's connecting rod increased the stability, whereas increasing its length decreased the stability. Mapping the rupture points showed that the balance of cell volume between the toroid and connecting rod regions of the dogbone tissue controlled the point of rupture. When cells were treated with transforming growth factor-b1, dogbones ruptured sooner due to increased cell contraction. In mold designs to form tissues with more complex shapes such as three interconnected toroids or a honeycomb, obstacle design controlled tension and tissue morphology. When the vertical posts were changed to cones, they became tension modulators that dictated when and where tension was released in a large self-organizing tissue. By understanding how elements of mold design control morphology, we can produce better models to study organogenesis, examine 3D cell mechanics, and fabricate building parts for tissue engineering.

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs

“…Furthermore, micro-molded technology brings opportunities to a sort of applications including drugs and nanoparticle testing, cell therapy, tissue engineering and biofabrication [34–36], mainly because of the possibility of hundreds of spheroids biofabrication in a unique micro-molded with homogeneous size and shape. Using micro-molded non-adhesive hydrogel also opens the possibility of cell seeding automation, since cell suspension is dispensed in an isolated well of cell culture plate.…”

Bio-Pick, Place, and Perfuse: A New Instrument for Three-Dimensional Tissue Engineering

“…Thus, mold design determines both part shape and the morphological evolution of the part as it self-assembles. We recently reported a finite element simulation based on cell-derived tension that predicts part morphology (41). Moreover, the work or power exerted by cells as they self-assemble a part has been quantified for different cell types as well as cell mixtures (34,42).…”

“…Precision part placement can be improved by more refined control of the gripping head and better definition of the operating parameters of part placement. Although we have described the changes in part morphology during self-assembly and after harvest from the molds, further work is needed to understand the morphological changes that occur when stacks of parts are fused, information useful for part design (32,41). We are currently refining the instrument and investigating long term questions such as viability of the assembled construct and the morphology of its vascular network as a function of perfusion time.…”

3D culture models of tissues under tension

“…By manufacturing T-shaped pillars, the tissue is prevented from slipping off and thus the tissue boundaries are outlined by the spacing of the pillars (Kalman et al, 2016;Legant et al, 2009). However, by introducing conical shaped pillars, one can control where and when the tissue is released from a pillar, and thus change the shape of the microtissue over time (Svoronos et al, 2014).…”

“…Although pillar spacing and profiles are two variables that control tissue geometry, other factors, such as microwell size and shape, still need to be characterized in order to establish the design rules for controlling the assembly and maintenance of stromal microtissues (Svoronos et al, 2014).…”