Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels.

Techniques that allow cells to self-assemble into three-dimensional (3-D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular--especially in fields such as stem cell research, tissue engineering, and cancer biology. Unfortunately, caveats involving scale, expense, geometry, and practicality have hindered the widespread adoption of these techniques. We present an easy-to-use, inexpensive, and scalable technology for production of complex-shaped, 3-D microtissues. Various primary cells and immortal cell lines were utilized to demonstrate that this technique is applicable to many cell types and highlight differences in their self-assembly phenomena. When seeded onto micromolded, nonadhesive agarose gels, cells settle into recesses, the architectures of which optimize the requisite cell-to-cell interactions for spontaneous self-assembly. With one pipeting step, we were able to create hundreds of uniform spheroids whose size was determined by seeding density. Multicellular tumor spheroids (MCTS) were assembled or grown from single cells, and their proliferation was quantified using a modified 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. Complex-shaped (e.g., honeycomb) microtissues of homogeneous or mixed cell populations can be easily produced, opening new possibilities for 3-D tissue culture.

Dynamics of the self-assembly of complex cellular aggregates on micromolded nonadhesive hydrogels.

The process by which cells self-assemble to form three-dimensional (3D) structures is central to morphogenesis and development of living tissues and hence is of growing interest to the field of tissue engineering. Using rapid prototyping technology we made micromolded nonadhesive hydrogels to study the dynamics of self-assembly in a low-shear environment with simple spherical geometries as well as more complex geometries such as a toroid. Aggregate size, shape, and composition were easily controlled; aggregates were easily retrieved; and the dynamics of the assembly process were readily observed by time-lapse microscopy. When two cell types, normal human fibroblasts (NHFs) and human umbilical vein endothelial cells (HUVECs), were seeded together, they self-segregated into multilayered spherical microtissues with a core of NHFs enveloped by a layer of HUVECs. Surprisingly, when a single cell suspension of NHFs was added to 7-day-old HUVEC spheroids, the HUVEC spheroid reorganized such that NHFs occupied the center and HUVECs coated the outside, demonstrating that self-assembly is not terminal and that spheroids are fluid structures that retain the ability to reassemble. We also showed that cells can self-assemble to form a complex toroid shape, and we observed several phenomena indicating that cellular contraction and tension play a significant role in the assembly process of complex shapes.

Porous titanium bases for osteochondral tissue engineering.

Tissue engineering of osteochondral grafts may offer a cell-based alternative to native allografts, which are in short supply. Previous studies promote the fabrication of grafts consisting of a viable cell-seeded hydrogel integrated atop a porous, bone-like metal. Advantages of the manufacturing process have led to the evaluation of porous titanium as the bone-like base material. Here, porous titanium was shown to support the growth of cartilage to produce native levels of Young's modulus, using a clinically relevant cell source. Mechanical and biochemical properties were similar or higher for the osteochondral constructs compared to chondral-only controls. Further investigation into the mechanical influence of the base on the composite material suggests that underlying pores may decrease interstitial fluid pressurization and applied strains, which may be overcome by alterations to the base structure. Future studies aim to optimize titanium-based tissue engineered osteochondral constructs to best match the structural architecture and strength of native grafts. STATEMENT OF SIGNIFICANCE: The studies described in this manuscript follow up on previous studies from our lab pertaining to the fabrication of osteochondral grafts that consist of a bone-like porous metal and a chondrocyte-seeded hydrogel. Here, tissue engineered osteochondral grafts were cultured to native stiffness using adult chondrocytes, a clinically relevant cell source, and a porous titanium base, a material currently used in clinical implants. This porous titanium is manufactured via selective laser melting, offering the advantages of precise control over shape, pore size, and orientation. Additionally, this manuscript describes the mechanical influence of the porous base, which may have applicability to porous bases derived from other materials.