Directing fibroblast self-assembly to fabricate highly-aligned, collagen-rich matrices.

Extracellular matrix composition and organization play a crucial role in numerous biological processes ranging from cell migration, differentiation, survival and metastasis. Consequently, there have been significant efforts towards the development of biomaterials and in vitro models that recapitulate the complexity of native tissue architecture. Here, we demonstrate an approach to fabricating highly aligned cell-derived tissue constructs via the self-assembly of human dermal fibroblasts. By optimizing mold geometry, cell seeding density, and media composition we can direct human dermal fibroblasts to adhere to one another around a non-adhesive agarose peg to facilitate the development of cell-mediated circumferential tension. By removing serum and adding ascorbic acid and l-proline, we tempered fibroblast contractility to enable the formation of stable tissue constructs. Similarly, we show that the alignment of cells and the ECM they synthesize can be modulated by changes to seeding density and that constructs seeded with the lowest number of cells have the highest degree of fibrillar collagen alignment. Finally, we show that this highly aligned, tissue engineered construct can be decellularized and that when re-seeded with fibroblasts, it provides instructive cues which enable cells to adhere to and align in the direction of the remaining collagen fiber network. STATEMENT OF SIGNIFICANCE: Cell and extracellular matrix organization is directly related to biological function including cell signaling and tissue mechanics. Changes to this organization are often associated with injury or disease. The majority of in vitro tissue engineering models investigating cell and matrix organization rely on the addition of stress-shielding exogenous proteins and polymers and, or the application of external forces to promote alignment. Here we present a completely cell-based approach that relies on the development of cell-mediated tension to direct anisotropic cellular alignment and matrix synthesis using human dermal fibroblasts. A major challenge with this approach is excessive cellular contractility that results in necking and failure of the tissue construct. While other groups have tried to overcome this challenge by simply adding more cells, here we show that matrix alignment is inversely related to cell seeding density. To engineer tissue constructs with the highest degree of alignment, we optimized media components to reduce cellular contractility and promote collagen synthesis such that fibroblast toroids remained stable for at least 28 days in culture. We subsequently showed that these collagen-rich tissue constructs could be decellularized while maintaining their collagen microstructure and that cells adhered to and responded to the decellularized cell-derived matrix by aligning and elongating along the collagen fibers. The complexity of cell-derived matrices has been shown to better recapitulate in vivo tissue architecture and composition. This study provides a straight-forward approach to fabricating instructive cell-derived matrices with a high degree of uniaxial alignment generated purely by cell-mediated tension.