A Rapid Fabrication Technique for Self-Assembled Collagen Based Multicellular, Heterogeneous, 3D Tissue Constructs

Although monolayer cell culture models are considered to be the gold standard for in vitro modeling of pathophysiological events, they cannot reconstruct in vivo like gradient of gases and nutrients and lack proper cell-cell and cell-matrix interactions. Spherical cellular aggregates, otherwise known as multicellular spheroids, are widely used as three-dimensional in vitro models to mimic natural in vivo cellular microenvironment for applications such as drug screening. Although very useful, the previously established techniques are limited to low cell numbers, usually slow, and sometimes have limitations in terms of the cell type that can be used. Here, a versatile technique based on rapid self-assembly of cells and extracellular matrix materials and shapes using microfabricated molds is introduced to form multicellular tissue constructs. The self-assembly process takes less than 6 hrs and produces a mechanically robust tissue construct that could be handled easily. We demonstrate that a variety of shapes including spherical, cuboidal, dumbbell- and cross-like could be fabricated using this approach. Interestingly, the structures formed with non-spherical shapes were able to retain that shape even after removal from the molds and during long term cell culture. This versatile approach is applicable to a variety of cell types (breast cancer cell lines MCF-7, MDA-MB-321, Hs-578T; osteosarcoma cell line SaOS-2; endothelial cell line HUVEC) as well as a range of cell numbers (104-106). Furthermore, we also show that the constructs could be spatially patterned to position various cell types in a precisely controlled way. Such heterogeneous constructs that are formed provide physiologically relevant cell densities, 3D structure as well as close positioning of multiple types of cells that are not possible using other fabrication approaches. This fabrication approach will find significant applications in developing 3D cell culture models for drug discovery as well as tissue grafts for implantation.