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.

Evaluation of Fluid Behaviors in a Pushbutton-Activated Microfluidic Device for User-Independent Flow Control.

Although numerous studies have been conducted to realize ideal point-of-care testing (POCT), the development of a user-friendly and user-independent power-free microfluidic platform is still a challenge. Among various methods, the finger-actuation method shows a promising technique that provides a user-friendly and equipment-free way of delivering fluid in a designated manner. However, the design criteria and elaborate evaluation of the fluid behavior of a pushbutton-activated microfluidic device (PAMD) remain a critical bottleneck to be widely adopted in various applications. In this study, we have evaluated the fluid behavior of the PAMD based on various parameters, such as pressing velocity and depth assisted by a press machine. We have further developed a user-friendly and portable pressing block that reduces user variation in fluid behavior based on the evaluation.

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.

Characterization of PDMS Microchannels Using Horizontally or Vertically Formed 3D-Printed Molds by Digital Light Projection.

Three-dimensional (3D) printing is one of the promising technologies for the fabrication of microstructures due to its versatility, ease of fabrication, and low cost. However, the direct use of 3D-printed microstructure as a microchannel is still limited due to its surface property, biocompatibility, and transmittance. As an alternative, rapid prototyping of poly(dimethylsiloxane) (PDMS) from 3D-printed microstructures ensures both biocompatibility and efficient fabrication. We employed 3D-printed molds fabricated using horizontal and vertical arrangement methods with different slice thicknesses in a digital light projection (DLP)-based 3D printing process to replicate PDMS microchannels. The replicated PDMS structures were investigated to compare their optical transmittances and surface roughness. Interestingly, the optical transmittance of PDMS from the 3D-printed mold was significantly increased via bonding two single PDMS layers. To evaluate the applicability of the replicated PDMS devices from the 3D-printed mold, we performed droplet generation in the PDMS microchannels, comparing the same device from a conventional Si-wafer mold. This study provides a fundamental understanding of prototyping microstructures from the DLP-based 3D-printed mold.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis.

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.

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).

Development of the micro-patterned 3D neuronal-hydrogel model using soft-lithography for study a 3D neural network on a microelectrode array.

In vitro patterned neuronal models have been studied as one of the strategies to investigate the relationship between structural connectivity and functional activity of neural network. Despite the importance of three-dimensional (3D) cell models, most of these studies have been performed on two-dimensional models. In this study, we present a technique to construct the micro-pattern to 3D neuronal-hydrogel model using a micromolding in capillaries (MIMIC) technique on microelectrode array (MEA). Our technique was suitable to prevent the deformation of micro-patterned collagen model against the neuronal contracted tension during the network formation. The relationship between the growth directions of glial cells and micro-pattern direction was investigated. Lastly, we confirmed that our 3D model had synchronized activity among neurons in 3D. This model is expected to be used as a tool to study the relationship between structural connectivity and functional activity in the 3D environment.

Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography.

The highly complex central nervous systems of mammals are often studied using three-dimensional (3D) in vitro primary neuronal cultures. A coupled confocal microscopy and immunofluorescence labeling are widely utilized for visualizing the 3D structures of neurons. However, this requires fixation of the neurons and is not suitable for monitoring an identical sample at multiple time points. Thus, we propose a label-free monitoring method for 3D neuronal growth based on refractive index tomograms obtained by optical diffraction tomography. The 3D morphology of the neurons was clearly visualized, and the developmental processes of neurite outgrowth in 3D spaces were analyzed for individual neurons.

Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip.

Spheroid, a 3D aggregate of tumor cells in a spherical shape, has overcome the limitations of conventional 3D cell models to accurately mimic the in-vivo environment of a human body. The spheroids are cultured with other primary cells and embedded in collagen drops using hang drop plates and low-attachment well plates to construct a spheroid-hydrogel model that better mimics the cell-cell and cell-extracellular matrix (ECM) interactions. However, the conventional methods of culturing and embedding spheroids into ECM have several shortcomings. The procedure of transferring a single spheroid at a time by manual pipetting results in well-to-well variation and even loss or damage of the spheroid. Based on the previously introduced droplet contact-based spheroid transfer technique, we present a poly(dimethylsiloxane) and resin-based drop array chip and a pillar array chip with alignment stoppers, which enhances the alignment between the chips for uniform placement of spheroids. This method allows the facile and stable transfer of the spheroid array and even eliminates the need for a stereomicroscope while handling the cell models. The novel platform demonstrates a homogeneous and time-efficient construction and diverse analysis of an array of fibroblast-associated glioblastoma multiforme spheroids that are embedded in collagen.

Modular 3D In Vitro Artery-Mimicking Multichannel System for Recapitulating Vascular Stenosis and Inflammation.

Inflammation and the immune response in atherosclerosis are complex processes involving local hemodynamics, the interaction of dysfunctional cells, and various pathological environments. Here, a modular multichannel system that mimics the human artery to demonstrate stenosis and inflammation and to study physical and chemical effects on biomimetic artery models is presented. Smooth muscle cells and endothelial cells were cocultured in the wrinkled surface in vivo-like circular channels to recapitulate the artery. An artery-mimicking multichannel module comprised four channels for the fabrication of coculture models and assigned various conditions for analysis to each model simultaneously. The manipulation became reproducible and stable through modularization, and each module could be replaced according to analytical purposes. A chamber module for culture was replaced with a microfluidic concentration gradient generator (CGG) module to achieve the cellular state of inflamed lesions by providing tumor necrosis factor (TNF)-α, in addition to the stenosis structure by tuning the channel geometry. Different TNF-α doses were administered in each channel by the CGG module to create functional inflammation models under various conditions. Through the tunable channel geometry and the microfluidic interfacing, this system has the potential to be used for further comprehensive research on vascular diseases such as atherosclerosis and thrombosis.

Droplet contact-based spheroid transfer technique as a multi-step assay tool for spheroid arrays.

Hanging drop plates and low-attachment well plates are suitable for a high throughput screening model of a spheroid, because each drop (or well) contains a single spheroid and the spheroid environment are separated from each other. However, uniform spheroid culture on these devices is difficult as the liquid around the spheroid is replaced by direct pipetting, which can cause spheroid damage or loss, and well-to-well variation. If spheroids need to be cultured for a long time or analyzed through chemical treatment of immunostaining, it becomes a more considerable problem as the number of pipetting action increases. To address these problems, we have developed a poly(dimethylsiloxane) (PDMS)-based drop array chip (DAC) and a pillar array chip (PAC) that can apply a droplet contact-based spheroid transfer (DCST) technique to multiple reagent change or washing steps of spheroid assays. Unlike previous DCST devices, 3D-printed mold-based DCST devices showed stable spheroid manipulation during repetitive drop contact and facile transfer of spheroid arrays to the next reagent-loaded DAC while minimizing cross-contamination of the reagents. Compared to the conventional manual or machine pipetting method, the DCST method showed lower user-to-user variation and a higher spheroid retention rate in the manipulation of the spheroid array. Live/dead staining, hypoxia staining, and immunofluorescence staining of the spheroid array were performed on a breast cancer cell line, BT-474. Furthermore, four clearing methods were applied to the spheroid array as a proof of concept, and we have identified the applicability of the DCST platform as a pretreatment platform for whole spheroid analysis.

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.

Biomarker barcodes: multiplexed microfluidic immunohistochemistry enables high-throughput analysis of tissue microarray.

We present a multiplexed microfluidic immunohistochemistry (IHC) technology that enables high-throughput analysis of tissue microarrays (TMAs) using the patterns of biomarker barcodes, which consist of a series of expressed linear patterns of specific biomarkers. A multichannel poly(dimethylsiloxane) microfluidic device was reversibly assembled by the pressure of simple equipment for multiplexed IHC on each core of TMA or cell microarray (CMA) section slides. By injecting primary antibodies from different biomarkers independently into each channel, multiplexed immunostaining can be performed on each core of TMA. We confirmed the equal immunostaining quality regardless of the channel orders and core positions in the slide. Four different biomarkers (ER, PR, HER2, and Ki67) were used for the demonstration of distinctive expression patterns on CMAs which consist of six different breast cancer cell lines, and it was confirmed that these bar-like signals could be a biomarker barcode for the TMA core. A biomarker barcode of breast cancer patient-derived TMA was quickly scanned by a slide scanner and compared to the conventional method for breast cancer diagnosis. This "barcode-IHC" concept, which has been verified by performing multiplexed microfluidic IHC on CMA and TMA samples, provides high reproducibility and the potential of high-throughput screening with molecular diagnostic capability.

Pushbutton-activated microfluidic cartridge as a user-friendly sample preparation tool for diagnostics.

Microfluidic technologies have several advantages in sample preparation for diagnostics but suffer from the need for an external operation system that hampers user-friendliness. To overcome this limitation in microfluidic technologies, a number of user-friendly methods utilizing capillary force, degassed poly(dimethylsiloxane), pushbutton-driven pressure, a syringe, or a pipette have been reported. Among these methods, the pushbutton-driven, pressure-based method has a great potential to be widely used as a user-friendly sample preparation tool for point-of-care testing or portable diagnostics. In this Perspective, we focus on the pushbutton-activated microfluidic technologies toward a user-friendly sample preparation tool. The working principle and recent advances in pushbutton-activated microfluidic technologies are briefly reviewed, and future perspectives for wide application are discussed in terms of integration with the signal analysis system, user-dependent variation, and universal and facile use.

Pushbutton-activated microfluidic dropenser for droplet digital PCR.

Here, we report a portable microfluidic device to generate and dispense droplets simply operated by pushbutton for droplet digital polymerase chain reaction (ddPCR), which is named pushbutton-activated microfluidic dropenser (droplet dispenser) (PAMD). After loading the PCR mixtures and the droplet generation oil to PAMD, digitized PCR mixtures are prepared in PCR tubes after the actuation of a pushbutton. Multiple droplet generation units are simultaneously operated by a single pushbutton, and the size of droplets is controllable by adjusting the geometry of the droplet generation channel. To examine the performance of PAMD, digitized PCR mixtures containing genomic DNA of Escherichia coli (E. coli) O157:H7 prepared by PAMD were assessed by a fluorescence signal analyzer after PCR with a thermal cycler. As a result, PAMD can produce analytical droplets for ddPCR as much as a conventional droplet generator even though any external equipment is not required.

Inertial Microfluidics-Based Separation of Microalgae Using a Contraction-Expansion Array Microchannel.

Microalgae separation technology is essential for both executing laboratory-based fundamental studies and ensuring the quality of the final algal products. However, the conventional microalgae separation technology of micropipetting requires highly skilled operators and several months of repeated separation to obtain a microalgal single strain. This study therefore aimed at utilizing microfluidic cell sorting technology for the simple and effective separation of microalgae. Microalgae are characterized by their various morphologies with a wide range of sizes. In this study, a contraction-expansion array microchannel, which utilizes these unique properties of microalgae, was specifically employed for the size-based separation of microalgae. At Reynolds number of 9, two model algal cells, Chlorella vulgaris (C. vulgaris) and Haematococcus pluvialis (H. pluvialis), were successfully separated without showing any sign of cell damage, yielding a purity of 97.9% for C. vulgaris and 94.9% for H. pluvialis. The result supported that the inertia-based separation technology could be a powerful alternative to the labor-intensive and time-consuming conventional microalgae separation technologies.

Controlled 3D co-culture of beta cells and endothelial cells in a micropatterned collagen sheet for reproducible construction of an improved pancreatic pseudo-tissue.

The co-culture of beta cells and endothelial cells in constructing a pancreatic pseudo-tissue can provide a functional advancement for in vitro diabetic-related drug testing and biological studies or in vivo transplantation. In order to mimic the pancreatic tissue more similar to in vivo, it is necessary to control the microenvironment, including cell-cell and cell-extracellular matrix interactions. In this study, we report a geometrically controlled three-dimensional (3D) pancreatic model where MIN6 and MS1 cells are co-cultured within a micropatterned collagen sheet. In 4-10 days, depending on the cell seeding concentration, the MIN6 cells formed islet-like clusters surrounded by an endothelial MS1 cell monolayer. The MS1 cells also formed monolayers at the edge of the micropatterns connecting between the clusters, resulting in a blood vessel-like structure in which no cells were found. It was confirmed that the 3D co-culture structure was not formed in a non-patterned sheet and the structure also helped insulin secretion of MIN6 cells. By simply embedding the cell mixture and the hexagonal micropattern into the collagen sheet, we were also able to achieve the highly reproducible fabrication of a 3D pancreatic pseudo-tissue construct for in vivo and in vitro applications.

Microfluidic channel-integrated hanging drop array chip operated by pushbuttons for spheroid culture and analysis.

Although the hanging drop methods have a number of advantages for spheroid culture, they suffer from reagent exchange procedures that depend on tedious and accurate liquid handling by manual pipetting or robotic arms. To simplify these procedures, we developed a method for liquid handling in a hanging drop array (HDA) chip for spheroid culture and analysis by integrating microfluidic channels operated by pushbuttons. Six finger-actuated microfluidic pumping units connected to a 3 × 3 HDA can draw or replenish reagents in an HDA chip without any external equipment. The initial cell seeding, medium exchange, and staining for further analysis can be simply done by pushing the buttons in the programmed order. After the assessment of the reagent exchange ratio of the device, BT474 spheroids of various sizes were cultured in the device for 7 days by exchanging the medium once a day and stained on the same device by exchanging the medium with staining reagents for the LIVE/DEAD assay. Furthermore, the cultured spheroids were embedded into collagen by exchanging the medium with a collagen solution to mimic a cancer metastasis environment.