Paper-supported 3D cell culture for tissue-based bioassays

Abstract

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the ''tissue'', whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overexpressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the O2 and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue-and organ-level functions.diffusion ͉ hypoxia ͉ molecular gradients ͉ multilayer constructs

Configurable 2D and 3D spheroid tissue cultures on bioengineered surfaces with acquisition of epithelial–mesenchymal transition characteristics

“…Limitations to the present method include: (1) microfluidic patterning is incompatible with certain complex 3D structures, such as layer-by-layer assembly to mimic in vivo-like tissues; 16,17 (2) dynamic and continuous nutrient supply and waste removal to mimic the physiological microenvironments could not be easily achieved with the current proposed design (although this drawback could be overcome by integrating with an evaporation-based micropump 36 ). However, the new capabilities allowed by the combination of techniques reported here (for example, the perforated membrane deterministically enabling cell trapping and in-situ generation of uniformly sized spheroids) may enable studies in 3D modeling of complex metastatic cascade (see results for the migration of tumor cells in 3D matrices in Supplementary Figure S7) and drug screening for metastatic cancers in 3D (via delivery of drugs diffused across the perforated cell culture areas 9 ).…”

“…5,25,29 Furthermore, the passive method that enables the pumping of multiple fluids using pipettes has been widely adopted, which has the advantages of ease of use, accessibility and a reduction of the complexity of working with pumps and tubing. 11,12,16,37 Here, we successfully adapt these methods in the microchip, which indicates that the microchip could be adopted for use in existing, widely used high-throughput screening instruments, such as liquid-handling robots. 3 This easy, flexible and effective approach may be used for a range of applications in drug discovery, tissue engineering or stem cell research, and should be feasible to integrate with other biological assay components into a monolithic system.…”

“…10 Various techniques for patterning cells in 2D or 3D tissue culture have demonstrated the ability to direct cells onto selected areas of a surface, including microcontact printing 8 or soft lithography, 1 microfluidic-or microstructure-based methods, 11,12 active control of cell attachment or suspension by physical forces [13][14][15] and layer-bylayer assembly. 16,17 However, challenges still remain. For example, some techniques typically require a substantial amount of cells to seed onto open surfaces and wash away numerous unattached cells after the cell-patterning step has been executed.…”

Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates

“…As a key component for cellular energy generation through oxidative phosphorylation, local oxygen depletion impairs cell survival when sustained over critical periods of time [5], even independent of local nutrient concentrations [6].…”

Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system

“…To address the issues in 3D cultures for radiation biology, we adapted Cells-in Gels-in-Paper, or CiGiP, [45][46][47] to evaluate the response of cells to increasing doses of ionizing radiation. CiGiP is a 3D cell culture system consisting of a stack of layers of paper, which are patterned to contain hydrophilic zones surrounded with hydrophobic borders.…”