Insert-based microfluidics for 3D cell culture with analysis.

We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 µm) and four (channel width = 180 µm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules. Graphical abstract This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules.

Use of electrospinning and dynamic air focusing to create three-dimensional cell culture scaffolds in microfluidic devices.

Organs-on-a-chip has emerged as a powerful tool for pharmacological and physiological studies. A key part in the construction of such a model is the ability to pattern or culture cells in a biomimetic fashion. Most of the reported cells-on-a-chip models integrate cells on a flat surface, which does not accurately represent the extracellular matrix that they experience in vivo. Electrospinning, a technique used to generate sub-micron diameter polymer fibers, has been used as an in vitro cell culture substrate and for tissue engineering applications. Electrospinning of fibers directly into a fully sealed fluidic channel using a conventional setup has not been possible due to issues of confining the fibers into a discrete network. In this work, a dynamic focusing method was developed, with this approach enabling direct deposition of electrospun fibers into a fully sealed fluidic channel, to act as a matrix for cell culture and subsequent studies under continuous flowing conditions. Scanning electron microscopy of electrospun polycaprolactone fibers shows that this method enables the formation of fibrous layers on the inner wall of a 3D-printed fluidic device (mean fiber size = 1.6 ± 0.6 µm and average pore size = 113 ± 19 µm(2)). Cells were able to be cultured in this 3D scaffold without the addition of adhesion proteins. Media was pumped through the channel at high flow rates (up to 400 µL min(-1)) during a dynamic cell culture process and both the fibers and the cells were found to be strongly adherent. A PDMS fluidic device was also prepared (from a 3D printed mold) and coated with polycaprolactone fibers. The PDMS device enables optical detection and confocal imaging of cultured cells on the fibers. Finally, macrophages were cultured in the devices to study how the fibrous scaffold can affect cell behavior. It was found that under lipopolysaccharide stimulation, macrophages cultured on PCL fibers inside of a channel secreted significantly more cytokines than those cultured on a thin layer of PCL in a channel or directly on the inner channel wall. Overall, this study represents a new approach for in vitro cell studies, where electrospinning can be used to easily and quickly create 3D scaffolds that can improve the culture conditions in microfluidic devices.

Evaluation of 3D printing and its potential impact on biotechnology and the chemical sciences.

Nearing 30 years since its introduction, 3D printing technology is set to revolutionize research and teaching laboratories. This feature encompasses the history of 3D printing, reviews various printing methods, and presents current applications. The authors offer an appraisal of the future direction and impact this technology will have on laboratory settings as 3D printers become more accessible.

3D-printed fluidic devices enable quantitative evaluation of blood components in modified storage solutions for use in transfusion medicine.

A fluidic device constructed with a 3D-printer can be used to investigate stored blood components with subsequent high-throughput calibration and readout with a standard plate reader.

Emerging 3D printing technologies and methodologies for microfluidic development.

This review paper examines recent (mostly 2018 or later) advancements in 3D printed microfluidics. Microfluidic devices are widely applied in various fields such as drug delivery, point-of-care diagnosis, and bioanalytical research. In addition to soft lithography, 3D printing has become an appealing technology to develop microfluidics recently. In this work, three main 3D printing technologies, stereolithography, fused filament deposition, and polyjet, which are commonly used to fabricate microfluidic devices, are thoroughly discussed. The advantages, limitations, and recent microfluidic applications are analyzed. New technical advancements within these technology frameworks are also summarized, which are especially suitable for microfluidic development. Next, new emerging 3D-printing technologies are introduced, including the direct printing of polydimethylsiloxane (PDMS), glass, and biopolymers. Although limited microfluidic applications based on these technologies can be found in the literature, they show high potential to revolutionize the next generation of 3D-printed microfluidic apparatus.

A quantitative sensing system based on a 3D-printed ion-selective electrode for rapid and sensitive detection of bacteria in biological fluid.

Bacterial infections, such as urinary tract infections, are crucial health problems. Here, we report a new potentiometric sensor to detect bacteria sensitively, accurately, and quickly. First, a customizable, 3D printed Ag+ selective electrode was fabricated as the probe. Our 3D printed electrode showed sensitive, linear, and selective responses to Ag+. Compared to commercial Ag+ selective electrodes, ours required less sample volume, shorter responding time, and lower costs. Next, a novel potentiometer was developed with Arduino to couple the electrode for data transducing and transferring, which was programmed to transfer results to cell phones wirelessly. Moreover, a filter was designed to quickly remove interfering species in a biofluid sample (e.g., Cl-). By detecting the lost Ag+ taken by bacteria, the bacterial number could be elucidated. With this sensor system, bacteria numbers could be detected as low as 80 CFU/mL (LOD) within 15 min, which is sufficient for many diagnoses (e.g., urinary tract infection >1000 CFU/mL). An amplification method was presented for single-digit bacteria detection. Overall, we are presenting a bacteria detector with three innovative components: the electrode (signal transduction and detection), the potentiometer (transducer and data processing), and the 3D printed filter (sample preparation), which showed robust and improved (than previously reported ones) analytical merits. The low-cost and customizable (the electrode and the open-source coding) nature enhances the transnationality of the system, especially in underdeveloped areas.

A Step Forward for Smart Clothes─Fabric-Based Microfluidic Sensors for Wearable Health Monitoring.

We report the first demonstration of fabric-based microfluidics for wearable sensing. A new technology to develop microfluidics on fabrics, as a part of an undergarment, is described here. Compared to conventional microfluidics from polydimethylsiloxane, fabric-based microfluidics are simple to make, robust, and suitable for efficient sweat delivery. Specifically, acrylonitrile butadiene styrene (ABS) films with precut microfluidic patterns were infused through fabrics to form hydrophobic areas in a specially controlled sandwich structure. Experimental tests and simulations confirmed the sweat delivery efficiency of the microfluidics. Electrodes were screen-printed onto the fabric-based microfluidic. A novel wearable potentiometer based on Arduino was also developed as the transducer and signal readouts, which was low-cost, standardized, open-source, and capable of wireless data transfer. We applied the sensor system as a standalone or as a module of a T-shirt to quantify [Ca2+] in a wearer's sweat, with physiological and accurate results generated. Overall, this work represents a critical step in turning regular undergarments into biochemically smart platforms for health monitoring, which will broadly benefit human healthcare.

A 3D-printed transfusion platform reveals beneficial effects of normoglycemic erythrocyte storage solutions and a novel rejuvenating solution.

A set of 3D-printed analytical devices were developed to investigate erythrocytes (ERYs) processed in conventional and modified storage solutions used in transfusion medicine. During storage, prior to transfusion into a patient recipient, ERYs undergo many chemical and physical changes that are not completely understood. However, these changes are thought to contribute to an increase in post-transfusion complications, and even an increase in mortality rates. Here, a reusable fluidic device (fabricated with additive manufacturing technologies) enabled the evaluation of ERYs prior to, and after, introduction into a stream of flowing fresh ERYs, thus representing components of an in vivo ERY transfusion on an in vitro platform. Specifically, ERYs stored in conventional and glucose-modified solutions were assayed by chemiluminescence for their ability to release flow-induced ATP. The ERY's deformability was also determined throughout the storage duration using a novel membrane transport approach housed in a 3D-printed scaffold. Results show that hyperglycemic conditions permanently alter ERY deformability, which may explain the reduced ATP release, as this phenomenon is related to cell deformability. Importantly, the reduced deformability and ATP release were reversible in an in vitro model of transfusion; specifically, when stored cells were introduced into a flowing stream of healthy cells, the ERY-derived release of ATP and cell deformability both returned to states similar to that of non-stored cells. However, after 1-2 weeks of storage, the deleterious effects of the storage were permanent. These results suggest that currently approved hyperglycemic storage solutions are having adverse effects on stored ERYs used in transfusion medicine and that normoglycemic storage may reduce the storage lesion, especially for cells stored for longer than 14 days.

3D-Printed, Modular, and Parallelized Microfluidic System with Customizable Scaffold Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells.

Because dysfunctions of endothelial cells are involved in many pathologies, in vitro endothelial cell models for pathophysiological and pharmaceutical studies have been a valuable research tool. Although numerous microfluidic-based endothelial models have been reported, they had the cells cultured on a flat surface without considering the possible three-dimensional (3D) structure of the native extracellular matrix (ECM). Endothelial cells rest on the basement membrane in vivo, which contains an aligned microfibrous topography. To better understand and model the cells, it is necessary to know if and how the fibrous topography can affect endothelial functions. With conventional fully integrated microfluidic apparatus, it is difficult to include additional topographies in a microchannel. Therefore, we developed a modular microfluidic system by 3D-printing and electrospinning, which enabled easy integration and switching of desired ECM topographies. Also, with standardized designs, the system allowed for high flow rates up to 4000 µL/min, which encompassed the full shear stress range for endothelial studies. We found that the aligned fibrous topography on the ECM altered arginine metabolism in endothelial cells and thus increased nitric oxide production. There has not been an endothelial model like this, and the new knowledge generated thereby lays a groundwork for future endothelial research and modeling.

Making quantitative biomicrofluidics from microbore tubing and 3D-printed adapters.

Microfluidic technology has tremendously facilitated the development of in vitro cell cultures and studies. Conventionally, microfluidic devices are fabricated with extensive facilities by well-trained researchers, which hinder the widespread adoption of the technology for broader applications. Enlightened by the fact that low-cost microbore tubing is a natural microfluidic channel, we developed a series of adaptors in a toolkit that can twine, connect, organize, and configure the tubing to produce functional microfluidic units. Three subsets of the toolkit were thoroughly developed: the tubing and scoring tools, the flow adaptors, and the 3D cell culture suite. To demonstrate the usefulness and versatility of the toolkit, we assembled a microfluidic device and successfully applied it for 3D macrophage cultures, flow-based stimulation, and automated near real-time quantitation with new knowledge generated. Overall, we present a new technology that allows simple, fast, and robust assembly of customizable and scalable microfluidic devices with minimal facilities, which is broadly applicable to research that needs or could be enhanced by microfluidics.

Electrospun Microfibers Modulate Intracellular Amino Acids in Liver Cells via Integrin ß1.

Although numerous recent studies have shown the importance of polymeric microfibrous extracellular matrices (ECMs) in maintaining cell behaviors and functions, the mechanistic nexus between ECMs and intracellular activities is largely unknown. Nevertheless, this knowledge will be critical in understanding and treating diseases with ECM remodeling. Therefore, we present our findings that ECM microstructures could regulate intracellular amino acid levels in liver cells mechanistically through integrin ß1. Amino acids were studied because they are the fundamental blocks for protein synthesis and metabolism, two vital functions of liver cells. Two ECM conditions, flat and microfibrous, were prepared and studied. In addition to characterizing cell growth, albumin production, urea synthesis, and cytochrome p450 activity, we found that the microfibrous ECM generally upregulated the intracellular amino acid levels. Further explorations showed that cells on the flat substrate expressed more integrin ß1 than cells on the microfibers. Moreover, after partially blocking integrin ß1 in cells on the flat substrate, the intracellular amino acid levels were restored, strongly supporting integrin ß1 as the linking mechanism. This is the first study to report that a non-biological polymer matrix could regulate intracellular amino acid patterns through integrin. The results will help with future therapy development for liver diseases with ECM changes (e.g., fibrosis).

Microfibrous Extracellular Matrix Changes the Liver Hepatocyte Energy Metabolism via Integrins.

Cell line-based liver models are critical tools for liver-related studies. However, the conventional monolayer culture of hepatocytes, the most widely used in vitro model, does not have the extracellular matrix (ECM), which contributes to the three-dimensional (3D) arrangement of the hepatocytes in the liver. As a result, the metabolic properties of the hepatocytes in the monolayer tissue culture may not accurately reflect those of the hepatocytes in the liver. Here, we developed a modular platform for 3D hepatocyte cultures on fibrous ECMs produced by electrospinning, a technique that can turn a polymer solution to the micro/nanofibers and has been widely used to produce scaffolds for 3D cell cultures. Metabolomics quantitation by liquid chromatography-mass spectrometry (LC-MS) indicated that Huh7 hepatocytes grown in microfibers electrospun from silk fibroin exhibited reduced glycolysis and tricarboxylic acid (TCA) cycle, as compared to the cells cultured as a monolayer. Further mechanistic studies suggested that integrins were correlated to the ECM's effects. This is the first time to report how an ECM scaffold could affect the fundamental metabolism of the hepatocytes via integrins.

3D-Printed Microfluidic Devices for Enhanced Online Sampling and Direct Optical Measurements.

3D printing has emerged as a robust technique to fabricate reliable and reproducible microfluidic devices. However, a limitation of 3D-printed devices has been the low transparency even when printed in a "clear" material. There are currently no reports regarding direct optical measurements through a 3D-printed device. Here, we present for the first time that the printing orientation can affect the transparency of a 3D-printed object. With the optimal orientation, we printed a microfluidic detector that was sufficiently transparent (transmittance ≈ 80%) for optical quantitation. This finding is significant because it shows the feasibility to directly 3D-print optical components for analytical applications. In addition, we created a novel microfluidic dialysis device via 3D printing, which enabled higher flow rates (for sampling with high temporal resolution) and increased extraction efficiency than commercially available ones. By coupling the microfluidic detector and dialysis probe, we successfully measured the release kinetics of indole from biofilms in a continuous, automated, and near real-time fashion. Indole is an intercellular signaling molecule in biofilms, which may regulate antibiotic resistance. The release kinetics of this molecule had not been quantitated likely because of the lack of a suitable analytical tool. Our results fill this knowledge gap.

From cells-on-a-chip to organs-on-a-chip: scaffolding materials for 3D cell culture in microfluidics.

It is an emerging research area to integrate scaffolding materials in microfluidic devices for 3D cell culture (organs-on-a-chip). The technology of organs-on-a-chip holds the potential to obviate the gaps between pre-clinical and clinical studies. As accumulating evidence shows the importance of extracellular matrix in in vitro cell culture, significant efforts have been made to integrate 3D ECM/scaffolding materials in microfluidics. There are two families of materials that are commonly used for this purpose: hydrogels and electrospun fibers. In this review, we briefly discuss the properties of the materials, and focus on the various technologies to obtain the materials (e.g. extraction of collagen from animal tissues) and to include the materials in microfluidic devices. Challenges and potential solutions of the current materials and technologies were also thoroughly discussed. At the end, we provide a perspective on future efforts to make these technologies more translational to broadly benefit pharmaceutical and pathophysiological research.

Image Representations with Spatial Object-to-Object Relations for RGB-D Scene Recognition.

Scene recognition is challenging due to the intra-class diversity and inter-class similarity. Previous works recognize scenes either with global representations or with the intermediate representations of objects. In contrast, we investigate more discriminative image representations of object-to-object relations for scene recognition, which are based on the triplets of obtained with detection techniques. Particularly, two types of representations, including co-occurring frequency of object-to-object relation (denoted as COOR) and sequential representation of object-to-object relation (denoted as SOOR), are proposed to describe objects and their relative relations in different forms. COOR is represented as the intermediate representation of co-occurring frequency of objects and their relations, with a three order tensor that can be fed to scene classifier without further embedding. SOOR is represented in a more explicit and freer form that sequentially describe image contents with local captions. And a sequence encoding model (e.g., recurrent neural network (RNN)) is implemented to encode SOOR to the features for feeding the classifiers. In order to better capture the spatial information, the proposed COOR and SOOR are adapted to RGB-D data, where a RGB-D proposal fusion method is proposed for RGB-D object detection. With the proposed approaches COOR and SOOR, we obtain the state-of-the-art results of RGB-D scene recognition on SUN RGB-D and NYUD2 datasets.

Class Agnostic Image Common Object Detection.

Learning similarity of two images is an important problem in computer vision and has many potential applications. Most of previous works focus on generating image similarities in three aspects: global feature distance computing, local feature matching and image concepts comparison. However, the task of directly detecting class agnostic common objects from two images has not been studied before, which goes one step further to capture image similarities at region level. In this paper, we propose an end-to-end Image Common Object Detection Network (CODN) to detect class agnostic common objects from two images. The proposed method consists of two main modules: locating module and matching module. The locating module generates candidate proposals of each two images. The matching module learns the similarities of the candidate proposal pairs from two images, and refines the bounding boxes of the candidate proposals. The learning procedure of CODN is implemented in an integrated way and a multi-task loss is designed to guarantee both region localization and common object matching. Experiments are conducted on PASCAL VOC 2007 and COCO 2014 datasets. Experimental results validate the effectiveness of the proposed method.

Learning Effective RGB-D Representations for Scene Recognition.

Deep convolutional networks (CNN) can achieve impressive results on RGB scene recognition thanks to large datasets such as Places. In contrast, RGB-D scene recognition is still underdeveloped in comparison, due to two limitations of RGB-D data we address in this paper. The first limitation is the lack of depth data for training deep learning models. Rather than fine tuning or transferring RGB-specific features, we address this limitation by proposing an architecture and a twostep training approach that directly learns effective depth-specific features using weak supervision via patches. The resulting RGBD model also benefits from more complementary multimodal features. Another limitation is the short range of depth sensors (typically 0.5m to 5.5m), resulting in depth images not capturing distant objects in the scenes that RGB images can. We show that this limitation can be addressed by using RGB-D videos, where more comprehensive depth information is accumulated as the camera travels across the scenes. Focusing on this scenario, we introduce the ISIA RGB-D video dataset to evaluate RGB-D scene recognition with videos. Our video recognition architecture combines convolutional and recurrent neural networks (RNNs) that are trained in three steps with increasingly complex data to learn effective features (i.e. patches, frames and sequences). Our approach obtains state-of-the-art performances on RGB-D image (NYUD2 and SUN RGB-D) and video (ISIA RGB-D) scene recognition.

Use of 3D Printing and Modular Microfluidics to Integrate Cell Culture, Injections and Electrochemical Analysis.

Fabrication of microchip-based devices using 3-D printing technology offers a unique platform to create separate modules that can be put together when desired for analysis. A 3-D printed module approach offers various advantages such as file sharing and the ability to easily replace, customize, and modify the individual modules. Here, we describe the use of a modular approach to electrochemically detect the ATP-mediated release of nitric oxide (NO) from endothelial cells. Nitric oxide plays a significant role in the vasodilation process; however, detection of NO is challenging due to its short half-life. To enable this analysis, we use three distinct 3-D printed modules: cell culture, sample injection and detection modules. The detection module follows a pillar-based Wall-Jet Electrode design, where the analyte impinges normal to the electrode surface, offering enhanced sensitivity for the analyte. To further enhance the sensitivity and selectivity for NO detection the working electrode (100 µm gold) is modified by the addition of a 27 µm gold pillar and platinum-black coated with Nafion. The use of the pillar electrode leads to three-dimensional structure protruding into the channel enhancing the sensitivity by 12.4 times in comparison to the flat electrode (resulting LOD for NO = 210 nM). The next module, the 3-D printed sample injection module, follows a simple 4-Port injection rotor design made of two separate components that when assembled can introduce a specific volume of analyte. This module not only serves as a cheaper alternative to the commercially available 4-Port injection valves, but also demonstrates the ability of volume customization and reduced dead-volume issues with the use of capillary-free connections. Comparison between the 3-D printed and a commercial 4-Port injection valve showed similar sensitivities and reproducibility for NO analysis. Lastly, the cell culture module contains electrospun polystyrene fibers with immobilized endothelial cells, resulting in 3-D scaffold for cell culture. With the incorporation of all 3 modules, we can make reproducible ATP injections (via the 3-D printed sample injection module) that can stimulate NO release from endothelial cells cultured on a fibrous insert in the cell culture module which can then be quantitated by the pillar WJE module (0.19 ± 0.03 nM/cell, n = 27, 3 inserts analyzed each day, on 9 different days). The modular approach demonstrates the facile creation of custom and modifiable fluidic components that can be assembled as needed.

Microchip-based 3D-Cell Culture Using Polymer Nanofibers Generated by Solution Blow Spinning.

Polymer nano/micro fibers have found many applications including 3D cell culture and the creation of wound dressings. The fibers can be produced by a variety of techniques that include electrospinning, the primary disadvantage of which include the requirement for a high voltage supply (which may cause issues such as polymer denaturation) and lack of portability. More recently, solution blow spinning, where a high velocity sheath gas is used instead of high voltage, has been used to generate polymer fibers. In this work, we used blow spinning to create nano/microfibers for microchip-based 3D cell culture. First, we thoroughly investigated fiber generation from a 3D printed gas sheath device using two polymers that are amenable to cell culture (polycaprolactone, PCL and polystyrene, PS) as well as the parameters that can affect PCL and PS fiber quality. Using the 3D printed sheath device, it was found that the pressure of the sheath N2 and the concentration of polymer solutions determine if fibers can be produced as well as the resulting fiber morphology. In addition, we showed how these fibers can be used for 3D cell culture by directly depositing PCL fibers in petri dishes and well plates. It is shown the fibers have good compatibility with RAW 264.7 macrophages and the PCL fiber scaffold can be as thick as 178 ± 14 µm. PCL fibers created from solution blow spinning (with the 3D printed sheath device) were then integrated with a microfluidic device for the first time to fabricate a 3D cell culture scaffold with a flow component. After culturing and stimulating macrophages on the fluidic device, it was found that the integrated 3D fibrous scaffold is a better mimic of the extracellular matrix (as opposed to a flat, 2D substrate), with enhanced nitrite accumulation (product of nitric oxide release) from macrophages stimulated with lipopolysaccharide. PS fibers were also made and integrated in a microfluidic device for 3D culture of endothelial cells, which stayed viable for at least 72 hours (48 hours under the flowing conditions). This approach will be useful for future studies involving more realistic microchip-based culture models for studying cell-to-cell communication.

3D-printed Microfluidic Devices: Fabrication, Advantages and Limitations-a Mini Review.

A mini-review with 79 references. In this review, the most recent trends in 3D-printed microfluidic devices are discussed. In addition, a focus is given to the fabrication aspects of these devices, with the supplemental information containing detailed instructions for designing a variety of structures including: a microfluidic channel, threads to accommodate commercial fluidic fittings, a flow splitter; a well plate, a mold for PDMS channel casting; and how to combine multiple designs into a single device. The advantages and limitations of 3D-printed microfluidic devices are thoroughly discussed, as are some future directions for the field.