Bioprinted Multi-Composition Array Mimicking Tumor Microenvironments to Evaluate Drug Efficacy with Multivariable Analysis.

Current organ-on-a-chip technologies confront limitations in effectively recapitulating the intricate in vivo microenvironments and accommodating diverse experimental conditions on a single device. Here, a novel approach for constructing a multi-composition tumor array on a single microfluidic device, mimicking complex transport phenomena within tumor microenvironments (TMEs) and allowing for simultaneous evaluation of drug efficacy across 12 distinct conditions is presented. The TME array formed by bioprinting on a microfluidic substrate consists of 36 individual TME models, each characterized by one of three different compositions and tested under four varying drug concentrations. Notably, the TME model exhibits precise compartmentalization, fostering the development of self-organized vascular endothelial barriers surrounding breast cancer spheroids affecting substance transport. Multivariable screening and analysis of diverse conditions, including model complexity, replicates, and drug concentrations, within a single microfluidic platform, highlight the synergistic potential of integrating bioprinting with microfluidics to evaluate drug responses across diverse TME conditions comprehensively.

Reconfigurable Hanging Drop Microarray Platform for On-Demand Preparation and Analysis of Spheroid Array.

In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.

Fabrication of a self-assembled and vascularized tumor array via bioprinting on a microfluidic chip.

A tumor microenvironment (TME) is a complex system that comprises various components, including blood vessels that play a crucial role in supplying nutrients, oxygen, and growth factors, as well as delivering chemotherapy drugs to the tumor mass through the vascular endothelial barrier. To replicate the TME in vitro, several bioprinting and microfluidic organ-on-a-chip technologies have been developed. However, these technologies have not been fully exploited in terms of potential benefits of bioprinting and microfluidics, such as precise spatial control for biological samples, construction of multiple TMEs per microfluidic device, and the ability to adjust culture environments for better biological similarity. In addition, the complex transport phenomena within the vascular endothelial barrier and the aggregated tumor mass in the TME model should be considered before applying the model to drug treatment and screening. In this study, we describe a novel integrative technology that addresses these issues by introducing a self-organized TME array bioprinted on a microfluidic chip consisting of a vascular endothelial barrier surrounding breast cancer spheroids. To integrate the TME array onto the microfluidic platform, a microfluidic substrate for extrusion bioprinting was developed for a cell culture platform, which enables diffusivity control by microstructures and establishes a perfusion culture environment inside the culture channel. We also analyzed the cellular behaviors within the TME array to investigate the influence of the diffusivity on the self-organization process required to form the vascular endothelial barrier surrounding breast cancer spheroids.

Hybrid Biofabrication of Heterogeneous 3D Constructs Using Low-Viscosity Bioinks.

The application of cytocompatible hydrogels supporting extensive cellular activities to three-dimensional (3D) bioprinting is crucial for recreating complex physiological environments with high biomimicry. However, the poor printability and tunability of such natural hydrogels diminish the versatility and resolution of bioprinters. In this study, we propose a novel approach for the hybrid biofabrication of complex and heterogeneous 3D constructs using low-viscosity bioinks. Poly(lactic acid) (PLA) filament is extruded by fused deposition modeling on a micromesh to create PLA-framed micromesh substrates onto which fibrinogen is printed by microextrusion bioprinting. The micromesh supports the printed hydrogel with a capillary pinning effect to enable high-resolution bioprinting. Accordingly, the micromesh-bioink layers are aligned and stacked to form volumetric constructs. This approach, called the 3D micromesh-bioink overlaid structure and interlocked culture (3D MOSAIC) platform, enables the fabrication of complicated and multimaterial 3D structures, including overhangs and voids. Endothelial cells cultured under vasculogenic conditions in the platform self-organize within the biologically functional hydrogel to form vascular networks, and cancer cell migration can be observed across the layers. The multidisciplinary 3D MOSAIC platform is an important step toward the biofabrication of complex constructs with high biological and structural significance and functionality.

Bioprinting of heterogeneous and multilayered cell-hydrogel constructs using continuous multi-material printing and aerosol-based crosslinking.

Bioprinting is a powerful biofabrication technique that mimics physiological environments and functions. Here, we describe a protocol to set up a continuous multiple-material bioprinting system that can replicate structurally complex and biologically functional microphysiological systems such as a tumor microenvironment. Although this bioprinting system uses a limited crosslinking agent, it is a versatile and advanced continuous multi-material printing technique. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

Multilayered and heterogeneous hydrogel construct printing system with crosslinking aerosol.

Microextrusion bioprinting has been used to recreate the complex architecture and composition of a physiological system through the quick and accurate handling of various biomaterials. However, existing techniques are limited in precisely fabricating complex constructs, including multilayers and heterogeneous patterns with distinct regions, because the extruded bioink spreads rapidly upon contact with the substrate and is partially mixed with subsequently printed bioinks. This issue leads to difficulties in accurately and stably constructing multi-material structures with clear interfaces for prolonged printing before gelation. To fabricate multilayered and heterogeneous constructs, a bioprinting system should be able to continuously extrude various biomaterials and simultaneously crosslink the extruded bioink to stabilize the printed construct. In this study, a multiple-bioink printing system was developed by integrating a multibarrel nozzle for extruding multiple bioinks with a nebulizer for simultaneous crosslinking. The crosslinking aerosol sprayed from the nebulizer was able to gelate the various hydrogel bioinks as they were extruded through the multibarrel nozzle. Such aerosol-based crosslinking improved printing resolution and stability. The developed bioprinting system showed the possibility of recapitulating the physiological complex architecture such as a cancer microenvironment with well-defined interfaces between regions of different mechanical properties and cellular compositions. Using the integrated bioprinting system, a multilayered and heterogeneous construct was printed with four bioinks, including three types of cells (breast cancer cells, stromal cells, and vascular endothelial cells). The printed biological model was characterized by analyzing cancer cell migration and vascular network formation. The developed multiple-bioink printing system is expected to be highly efficient in recapitulating complex tissues and their environments with compartmentalized regions.

Chips-on-a-plate device for monitoring cellular migration in a microchannel-based intestinal follicle-associated epithelium model.

This paper describes a chips-on-a-plate (COP) device for monitoring the migration of Raji cells in the Caco-2/Raji coculture. To generate a model of the human intestinal follicle-associated epithelium (FAE), the coculture method using a conventional Transwell cell culture insert was established. Due to the structural limitations of the Transwell insert, live-cell tracking studies have not been performed previously using the existing FAE model. In this study, we designed a COP device to conduct long-term live-cell tracking of Raji cell migration using a microchannel-based FAE model. The COP device incorporates microfluidic chips integrated on a standard well plate, consistent humidity control to allow live-cell microscopy for 2 days, and microchannels connecting the two cell culture chambers of the COP device, which serve as a monitoring area for cellular migration. Using the COP device, we provide the first analysis of various migratory characteristics of Raji cells, including their chemotactic index in the microchannel-based FAE model. We showed that the migration of Raji cells could be controlled by modulating the geometry of the connecting microchannels. Cellular treatments with cytokines revealed that the cytokines increased the permeability of an FAE model with a detachment of Caco-2 cells. Live-cell monitoring of Raji cells treated with a fluorescent reagent also indicated exocytosis as a key agent of the Caco-2/Raji interaction. The COP device allows live-cell tracking analyses of cocultured cells in the microchannel-based FAE model, providing a promising tool for investigating cellular behavior associated with the recruitment of Raji to Caco-2 cells.