Self-Assembly and Tissue Fusion of Toroid-Shaped Minimal Building Units

Livoti, C.M., and Morgan, J.R. Self-Assembly and Tissue Fusion of Toroid-Shaped Minimal Building Units. Tissue Eng. 16: 2051-2061, 2010.

Discussion Section
Several groups are using scaffolds as part of a biofabrication or bioprinting approach.11,13–16 This approach is a modification of ink jet printing or rapid prototyping technologies, with tissue constructs made layer-by-layer by printing monodispersed cells or spheroids along with an extracellular-matrix-like material. Computer control of the deposition process facilitates the fabrication of large, complex-shaped structures. In another scaffold-based approach, cells are cast within small (submillimeter) gels and these are used as building units.7–10 HepG2, a human hepatoma cell line, was encapsulated in cylindrical collagen gels and the capsules were subsequently coated with human umbilical vein endothelial cells. These building units were packed into a larger vessel where they created a luminal network via the space between the building units, with the endothelial cells reducing thrombogenicity when the construct was perfused with blood in vitro.7–10 Another group has shown that the shape of cell-containing microgels can help direct the assembly of these building units and their orientations.22
In this article, we used agarose micromolds to direct the self-assembly of monodispersed cells into toroid-shaped multicellular microtissues, and we show that these scaffold-free toroids can be used as building units to form larger tissue structures by the process of tissue fusion. Further, lumen and toroid size can be easily controlled, and their fusion proceeds with predictable kinetics. Unlike the spheroid shape, where diffusion limits its maximum size, toroid building units can be made over a range of diameters without compromising cell viability, provided the thickness of the tissue does not exceed the diffusion limit. The viability of the toroid stems from its open lumen with access to the medium. Although the outermost diameter of the toroid is large, the cross section of the cellular portion of the toroid does not exceed the critical diffusion distance needed to maintain cell viability. If the same number of cells were self-assembled into a single spheroid, its diameter would exceed the critical distance needed to maintain the viability of the cells in the core of the spheroid. The fundamental shape of a toroid, with its open lumen structure, provides new possibilities as a building unit that when fused can produce dense cellular tissues with a network of interconnected lumens.
There are straightforward ways to control the size of the toroid and its lumen. The first step is the design of the micromold, and we were able to produce micromolds where cells self-assembled toroids with lumens that ranged in diameter from 1000μm down to 400μm. Self-assembly by monodispersed cells is rapid and occurs within 48h. Lumen size is controlled by the diameter of the agarose peg in the micromold, and 300–400μm is the smallest diameter that can be reliably made with our rapid prototyping machine. Other technologies, such as photolithography, could make molds with smaller pegs that could create toroids with even smaller lumens.
Thickness of the toroid can be controlled by the number of monodispersed cells seeded into the micromolds. After seeding, the cells self-assemble and contract around the peg, forming the toroid-shaped microtissue, and x–y thickness could be varied from ~250 to ~600μm after 4 days of self-assembly (Fig. 2), depending on the number of cells seeded. There are a minimal number of cells needed to form a toroid, which is probably dependent on cell type and mold design, but we estimate from other studies that this number is in the range of 5–10cells/μm of circumference of the peg.
Once released from the micromolds, the toroids remained intact, but underwent predictable changes to their size and shape. These changes are mediated by cellular processes, and an understanding of the types of changes that occur as well as their kinetics is another level of control over the size and shape of the toroid building unit. After release from the micromold, the lumen diameter of the 600μm toroid narrowed to a minimum of 100μm after 10 days, whereas its outer diameter decreased by only 6%. The largest change in lumen diameter (44%) occurred within the first 24h and is probably due to the release of cellular tension built up as the toroid contracts around the peg. These forces of self-assembly involve not only cell surface adhesion molecules, but also the action of the cytoskeleton.23,24 The H35 toroids do not undergo complete closure in the time frame measured because they are a cell type that has a slow rate of self-assembly.21
The toroid’s lumen undergoes significant narrowing, but changes to its outer diameter are minimal due to the fact that the toroid’s x–y thickness increases. When first harvested, the z dimension of the 600μm toroid was 113.6±21.2μm and the thickness in the x–y dimensions was 246.3±26.8μm. Over 10 days, the x–y thickness increased steadily to a maximum of 479.2±23.6μm. This thickening may be due to cellular migration and spreading of the toroid and/or cell proliferation. The rate of thickening was nearly the same for the 600μm and the 1000μm toroids, suggesting that the process is independent of toroid diameter. Cell density in the toroid was initially uniform, but with time, cell density increased in a nonuniform way with high cell density localized to a central ring closer to the lumen rather than the outermost circumference. It would be interesting to determine whether cell migration and/or proliferation vary at these locations.
Critical to the usefulness of a building unit is its ability to be used to build larger structures. In biofabrication, spheroids, along with an extracellular-matrix-like material, are melded together to build a tissue, and our toroids may be useful in this approach. Scaffold-free building units, such as spheroids, can also undergo cell-mediated fusion to build larger structures.12,19,25 Our data show that toroids can fuse at points of contact along their outer rim as well as their top and bottom surfaces. The kinetics of fusion shows that this process is largely complete after 48–72h with some compaction when fusion occurs along the top and bottom surfaces. Similar to spheroid fusion, our fluorescent-labeled toroids show that fusion occurs with minimal cell mixing between the building units.25 Cell–cell adhesion is critical to fusion,26 and surface adhesion molecules, such as cadherins and integrins, are involved as well as the cytoskeleton to which these proteins are linked.27
As a proof of principle, we show that toroids can be fused to form a large multitorus structure. Toroids were added to a single well and allowed to settle to form a random pile of toroids; however, the settling was not random. Overlap of toroids was random, but the majority of toroids had their lumens oriented along the z axis. This bias is probably due to the shape of the toroid and would provide a dominant orientation to the network of lumens created after fusion. Moreover, toroids that are randomly overlapped would create a range of lumen sizes after fusion, all smaller than the lumen of the building unit. Although this approach may not be useful for creating lumens that approximate neither the density nor the diameter of capillaries (~10μm), it may be useful for mimicking the range of vessels that connect capillaries to small-diameter arteries and veins (~0.1–5.0mm).28 However, strategies to endothelialize the lumens of these structures are needed.
Future work with toroid building units can take advantage of the fact that it is possible to make toroids with two or more different cell types. Previously, we have shown that when two cell types are mixed and added to the micromolds, the cells will self-assemble a mixed cell toroid.21 The cells also self-sorted so that one cell type was located in the inner core of the toroid and the other cell type formed an outer coating. The self-sorting phenomenon has been observed for many years in spheroids and patterns vary with cell types.29,30 Another variation on the toroid building unit is a larger structure with multiple lumens. We have shown that cells can self-assemble a stable honeycomb structure with 13 lumens.21,31 Like toroids, these honeycomb parts were self-assembled within 48h and could likely be fused with other parts in a similar time frame. Parts of different sizes, geometries, and cell types could be mixed or directed by secondary molds to control the size, shape, cell position, and lumen sizes of a large tissue construct.
In summary, we have shown that monodispersed cells can be self-assembled into scaffold-free multicellular building units in the shape of a toroid. The diameter and lumen size of these units are controlled by micromold geometry, and both undergo predictable changes when harvested. By the process of tissue fusion, these building units can be assembled into larger structures with interconnected lumens.

3D Petri Dishes™ used in this paper (please click catalog numbers for detailed product descriptions):
Catalog #12-36TO is the MicroTissues Inc product used to form multi-cellular microtissues in the shape of a toroid. Catalog # 12-256, and 24-96 are used to grow small spheroids. Catalog # 12-81 and 24-35 are used to grow larger spheroids.

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