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.

Introduction

Three-dimensional (3-D) in vitro cell culture, in which cells are grown in environments that more closely mimic native tissue architecture and function, have important applications in developmental/cell biology, drug screening, and regenerative medicine (1,2,3,4,5,6,7,8,9). Current methods that employ extracellular matrices, photolithography, cell printing, or laser tweezers are limited by expense and/or difficulty; therefore their use is not widespread (9,10,11). Numerous studies have shown that single-cell suspensions, in the absence of an extracellular matrix, will spontaneously self-assemble spherical microtissues, and mixed cell populations will self-segregate to form multilayered structures (4,12). Unfortunately, the methods to produce these microtissues, mainly spinner culture and hanging drops, have inconvenient design limitations such as spherical geometry, low throughput, or a high shear force environment (4,12,13,14,15,16). We present a straightforward platform that uses micromolded agarose to guide the spontaneous self-assembly of cells into 3-D microtissues. This platform represents significant improvements and additions to our previously published technique, including durable, autoclavable molds, a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1)-based aggregate proliferation assay, as well as methods for viewing self-assembly in the vertical plane, clonal expansion of individual cancer cells into microtumors, and production of multicell-type microtissues with a prescribed shape (17). This micromolded agarose Petri dish is compatible with standard cell culture equipment, biochemical assays, microscopy techniques, and offers new opportunities in microtissue design.

Materials and Methods

Cell Culture

MCF-7 human breast cancer cells (ATCC, Manassas, VA, USA) were expanded in RPMI-1640 medium (GIBCO®; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 1% solution, and 1% L-glutamine. Human umbilical vein endothelial cells (HUVEC; Cambrex, Walkersville, MD, USA) were expanded in endothelial growth medium 2 (EGM2) (Cambrex). Normal human fibroblasts (NHF) derived from neonatal foreskins, rat hepatoma cells (H35), and rat glioblastoma cells (RG2) were expanded in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin. MCF-7, HUVEC, RG2, and H35 were maintained at 37°C in a 5% CO₂-95% air atmosphere in a humidified incubator. NHFs were incubated at 37°C in a 10% CO₂-90% air atmosphere. Growth media were exchanged every other day.

Design and Fabrication of Micromolds

Molds designed using computer-assisted design (CAD; Solid Works, Concord, MA, USA) comprised a cell-seeding chamber, cell aggregation recesses, and medium exchange ports. The cell-seeding chamber is a relatively large rectangular recess that collects the cell suspension and distributes cells into the smaller aggregation recesses as cells settle under gravitational force. Cell aggregation recesses extend downward from the floor of the seeding chamber increasing cell-to-cell contact as cells collect on their concave bottoms. Recess bottoms are located 1.2 mm from the bottom of the agarose gel allowing imaging with inverted microscopes. Medium exchange ports allow room to place a pipet between the hydrogel and the tissue culture plate to change medium without disrupting the cells. Rectangular molds designed for horizontal view microscopy were 3.53 mm in height, 5.9 mm wide, and 17 mm long, and contained a single row of aggregation recesses. CAD files were used to produce wax molds with a ThermoJet® rapid prototyping machine (3D Systems, Valencia, CA, USA). Preliminary testing of the resolution capabilities of the ThermoJet rapid prototyping machine revealed that it could reliably make circular posts as tall 1000 µm with diameters as small as 200 µm and spaced as closely as 200 µm.

Negative replicates were produced by coating wax molds with a fast curing polydimethylsiloxane (PDMS)-based elastomer, Reprorubber (Flexbar, Islandia, NY, USA). After curing, Reprorubber negatives were removed from the wax prototypes, inverted, and sprayed with Epoxy ParFilm release agent (Flexbar). The negatives were filled with SYLGARD® 184 PDMS (Dow Corning, Midland, MI, USA), degassed to remove bubbles, and cured for 1 h at 95°C. PDMS-positive replicates were then released, washed thoroughly with 70% ethanol, rinsed with distilled water, and autoclave-sterilized before each use.