Platform for High-Throughput Testing of the Effect of Soluble Compounds on 3D Cell Cultures

Abstract

In vitro 3D culture could provide an important model of tissues in vivo, but assessing the effects of chemical compounds on cells in specific regions of 3D culture requires physical isolation of cells, and thus currently relies mostly on delicate and low-throughput methods. This paper describes a technique (“cells-in-gels-in-paper” CiGiP) that permits rapid assembly of arrays of 3D cell cultures, and convenient isolation of cells from specific regions of these cultures. The 3D cultures were generated by stacking sheets of 200-μm-thick paper, each sheet supporting 96 individual “spots” (thin circular slabs) of hydrogels containing cells, separated by hydrophobic material (wax, PDMS) impermeable to aqueous solutions, and hydrophilic and most hydrophobic solutes. A custom-made 96-well holder isolated the cell-containing zones from each other. Each well contained media to which a different compound could be added. After culture, and disassembly of the holder, peeling the layers apart ‘sectioned’ the individual 3D cultures into 200-μm-thick sections which were easy to analyze using 2D imaging (e.g., with a commercial gel scanner). This 96-well holder brings new utilities to high-throughput, cell-based screening, by combining the simplicity of CiGiP with the convenience of a microtiter plate. This work demonstrated the potential of this type of assays by examining the cytotoxic effects of phenylarsine oxide (PAO) and cyclophosphamide (CPA) on human breast cancer cells positioned at different separations from culture media in 3D cultures.

Fabrication of Hydrogel Particles of Defined Shapes Using Superhydrophobic‐Hydrophilic Micropatterns

“…The shape of particles will be important for constructing macro assemblies with diverse architectures, inter-particulate distances or particle-to-particle interactions. This method can be used as a toolbox of cell bearing hydrogel bricks of different shapes to enable diversity of multi-particle assemblies for modular tissue engineering 29 . In addition, shape of such building blocks can be used to encode information about the composition of the individual blocks without the need for separate fluorescent labeling (vide infra).…”

“…25,26 Micro-patterned arrays have been generated by immobilizing hydrogels on different micro-domains within a plane surface, and they have been used for disease diagnosis, prognosis, biochemical analysis, and therapeutic regimes, and have become an alternative approach for high-throughput multiplexed assays. [27][28][29][30] Strategies avoiding such 2D fixation of various hydrogels have employed shape-coded hydrogel particles as a suspension microarray format for multiplexed bioassays. 31,32 Here we describe a method for the rapid fabrication of alginate hydrogel particles of defined sizes and shapes using the effect of discontinuous dewetting on an SH-SL microarray combined with the sandwiching method to achieve the simultaneous gelation of the pre-hydrogel droplets.…”

Multilayer three-dimensional filter paper constructs for the culture and analysis of aortic valvular interstitial cells

“…The method avoids the difficulty of analyzing each 3D well separately by imaging each of the constructs with a gel scanner. Subsequent studies from the same research group have expanded the filter paper method to include a polyethylene (terephthalate) substrate and to have the ability to analyze the effects of soluble compounds on cell behavior [22,23]. An additional study used the system to study ischemia in cardiomyocytes [24].…”

Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures

“…These systems provide a powerful method for manipulating and analyzing 3D cell cultures. [17,18,19] Here we report the use of disulfide-functionalized PGMA-PHPMA worm gels (Figure 1) as an alternative to Matrigel for embedding cells in sheets of polymer-based mesh.…”

“…[17,18,19] Upon spotting with a micropipette, a cell suspension in Matrigel at 4 °C wicks through the sheets of paper or mesh. Upon incubation in media at 37 °C, the suspension forms a gel in the voids of the sheets.…”