Rods, tori, and honeycombs: the directed self‐assembly of microtissues with prescribed microscale geometries

Abstract

It is thought that, due to energy and surface area:volume minimization, the spheroid is the terminal structure of cellular self-assembly. We investigated whether self-assembly could be directed to generate complex-shaped structures. Using micromolded, nonadhesive agarose hydrogels seeded with rat hepatoma (H35s), human fibroblasts (NHFs), or their mix (1:1), we show that cells can self-assemble rods, tori, and honeycombs. We found that in trough-shaped recesses up to 2.2 mm long, H35s readily formed rod-like structures stable at 49% the recess lengths. They also formed intact tori (88%) and fully intact honeycombs structures with patent lumens (9/9) even when released from the mold. In contrast, NHFs in trough features progressed rapidly to spheroids and formed fewer stable tori (30%) and honeycombs (0/9). The 1:1 mix of cells self-assembled rapidly like NHFs but were able to form more stable structures (tori: 30%, honeycombs: 3/9). Experiments with labeled cells in tori and honeycombs revealed that cells self-segregated in these complex structures, with H35s enveloping NHFs, and that NHFs had different morphologies in taut vs. relaxed structures. These data open new possibilities for in vitro tissue models for embryo- and organogenesis study as well as for tissue engineering applications.

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

“…For example, cell power measurements could be used with selected pharmacologies or genetic tools to tailor the tissue assembly process. Previously, we have shown that H35s will readily form a stable microtissue in the shape of honeycombs or a loop-ended dogbone; however, NHF microtissues in the same configuration will self-assemble, stretch, and ultimately fail (4,5). NHF dogbone structures can be stabilized by treatment with Y-27632 (5).…”

“…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)(4)(5)(6). Thus, self-assembly is a complex process involving multiple protein mechanisms working in concert.…”

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

“…13,15 Of the cell types tested, fibroblasts exert the greatest forces, which is why dogbone tissues often rupture. Other cell types, such as H35s, self-assemble tissues that are stable but are also under tension.…”

“…[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.…”

“…16 In addition to the spheroid geometry, we have shown that cells can self-assemble into microtissues with complex shapes, such as rods, toroids, and honeycombs. 15,[17][18][19] In this process, known as directed self-assembly, cells are seeded into a micro-mold made from a nonadhesive hydrogel that does not interfere with the cell-to-cell interactions that drive self-assembly. The mold does, however, contain nonadhesive obstacles (e.g., posts) that direct the self-organizing tissue toward specific outcomes in terms of size and shape.…”