Continuous Production of Highly Functional Vascularized Hepatobiliary Organoids from Human Pluripotent Stem Cells using a Scalable Microfluidic Platform

“Organoid medicine” has rapidly progressed over the past decade as a new class of therapeutics with high functionality and complexity for addressing unmet medical needs such as effective treatment of patients suffering from chronic liver disease using liver organoids. Here, scalable and xeno-free integrated differentiation platforms are established to generate hepatic progenitors, mesenchymal stromal cells, and endothelial cells using individual human pluripotent stem cell lines as starting cell types for vascularized liver organoids generation. A scalable microfluidic system is developed to continuously generate cells-loaded microcapsules with self-biodegradable 4-arm-PEG-MMP1-sensitive peptide hydrogel as shell material, to support cells proliferation, self-condensation, and liver organoids generation through self-organization. Self-organized vascularized hepatobiliary organoids (VHOs) containing interconnected biliary networks and vascular structures are generated after optimizing the co-culture conditions inside hydrogel microcapsules and transferring the organoids to 3D dynamic suspension culture for further maturation. The VHOs show key functional features similar to the fetal and adult liver tissue including the expression of liver-specific marker genes, the ability to perform main liver metabolic functions, and inducing drug metabolism. The established platforms can be beneficial to the mass production of human liver organoids for liver organoid medicine and the development of safe, effective, and personalized drugs.     

Tissue Beads: Tissue‐Specific Extracellular Matrix Microbeads to Potentiate Reprogrammed Cell‐Based Therapy

Microbeads have been utilized as efficient cell culture carriers and injectable scaffolds for cell transplantation. However, various polymers currently used to generate microbeads have limited applicability due to loss of biological functions and tissue‐specific effects. Here, a tissue bead platform is reported that can provide a tissue‐specific microenvironment to facilitate cell culture and potentiate cell therapy. Using a flow‐focusing microfluidic device, uniform‐sized tissue microbeads are fabricated with extracellular matrix (ECM) from various decellularized tissues. The tissue microbeads are tested for tissue‐specific encapsulation of induced hepatic (iHep), induced cardiac (iCar), and induced myogenic (iMyo) cells, which are directly reprogrammed from mouse primary fibroblasts. Tissue‐specific microbeads significantly enhanced the viability, lineage‐specific maturation, and functionality of each type of reprogrammed cell, as compared to functionality when using conventional microbeads from a single ECM component (collagen). Finally, tissue microbeads are confirmed to mediate the successful in vivo engraftment of reprogrammed cells (iHep and iMyo) after transplantation, potentiating cell therapy and promoting functional tissue regeneration in tissue defective animal models. The study suggests that the use of a decellularized tissue matrix combined with a microfluidic technique can be employed to produce tissue‐specific ECM microbeads with increased versatility and efficacy for reprogrammed cell‐based therapy.     

Microengineered Bioartificial Liver Chip for Drug Toxicity Screening

Microfluidic 3D cell culture is a promising technology for the screening of drug toxicity profiles. In this study, a bioartificial liver consisting of a surface‐engineered microfluidic silicon chip with microtrenches mimicking hepatic sinusoids is shown to extend 3D primary hepatocyte culture and improve in vitro drug screening for hepatotoxicity, with respect to the state‐of‐the‐art literature on this subject. Primary hepatocytes hosted in the 3D heparin‐coated microtrenches (the bioartificial liver) secrete high levels of albumin and urea over 4 weeks. The cytotoxicity of common drugs, namely, acetaminophen, chlorpromazine, and tacrine, was assessed on primary hepatocytes both at day 1 and day 7. The results suggest that mimicking hepatic sinusoids using a microtrench format allows the maintenance of difficult‐to‐culture primary hepatocytes to be extended to 4 weeks and provides an alternative model to animal studies for the screening of the cytotoxicity of new drugs.     

Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research

Two‐dimensional (2D) cell cultures have been the primary screening tools to predict drug impacts in vitro for decades. However, owing to the lack of tissue‐specific architecture of 2D cultures, secondary screening using three‐dimensional (3D) cell culture models is often necessary. A microfluidic approach that facilitates side‐by‐side 2D and 3D cell culturing in a single microchannel and thus combines the benefits of both set‐ups in drug screening; that is, the uniform spatiotemporal distributions of oxygen, nutrients, and metabolic wastes in 2D, and the tissue‐like architecture, cell–cell, and cell–extracellular matrix interactions only achieved in 3D. The microfluidic platform is made from an organically modified ceramic material, which is inherently biocompatible and supports cell adhesion (2D culture) and metal adhesion (for integration of impedance electrodes to monitor cell proliferation). To induce 3D spheroid formation on another area, a single‐step lithography process is used to fabricate concave microwells, which are made cell‐repellant by nanofunctionalization (i.e., plasma porosification and hydrophobic coating). Thanks to the concave shape of the microwells, the spheroids produced on‐chip can also be released, with the help of microfluidic flow, for further off‐chip characterization after culturing. In this study, the methodology is evaluated for drug cytotoxicity assessment on human hepatocytes.     

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration).     

InVADE: Integrated Vasculature for Assessing Dynamic Events

Drug screening with simplified 2D cell culture and relevant animal testing fail to predict clinical outcomes. With the rising cost of drug development, predictive 3D tissue models with human cells are in urgent demand. Establishing vascular perfusion of 3D tissues has always been a challenge, but it is necessary to mimic drug transport and to capture complex interorgan crosstalk. Here, a versatile multiwell plate is presented empowered by built‐in microfabricated vascular scaffolds that define the vascular space and support self‐assembly of various parenchymal tissues. In this configuration, assembly and organ‐specific function of a metabolically active liver, a free‐contracting cardiac muscle, and a metastatic solid tumor are demonstrated, tracking organ function using noninvasive analysis techniques. By linking the 3D tumor and the liver tissue in series, it is demonstrated that the presence of liver tissue is crucial to correctly reveal the efficacy of a chemotherapeutic drug, Tegafur. Furthermore, the complete cancer metastasis cascade is demonstrated across multiple organs, where cancer cells escaping from the solid tumor can invade a distant liver tissue connected through a continuous vascular interface. This combinatory use of microfabricated scaffold onto a standard cell culturing platform can offer important insights into the mechanics of complex interorgan biological events.     

Hierarchical Spiky Microstraws‐Integrated Microfluidic Device for Efficient Capture and In Situ Manipulation of Cancer Cells

Techniques for capturing circulating tumor cells (CTCs) play an important role in cancer diagnosis. Recently, various 3D micro/nanostructures have been applied for effective CTC detection, yet in situ manipulation of the captured cancer cells on micro/nano‐structural substrates is rarely achieved. In this work, a hierarchical spiky microstraw array (HS‐MSA)‐integrated microfluidic device is demonstrated that possessed dual functions of cancer cell capture and in situ chemical manipulations of the captured cells. The 3D micro/nanostructure of HS‐MSA could capture cancer cells with high efficiency (≈84%) and strong specificity. Based on the HS‐MSA‐integrated microfluidic device, extracellular drug delivery to the captured cancer cells is achieved in situ with excellent spatial, dose, and temporal controls. In addition, a drug‐screening assay on the captured cancer cells is implemented to investigate the cell apoptosis behavior under the microstraw‐mediated delivery of staurosporine (STS). This microfluidic system not only presents tremendous potential for CTCs detection technology, but also opens up new opportunities for high‐throughput drug screening on cancer cells and understanding the cellular activity.     

Rapid Biofabrication of Printable Dense Collagen Bioinks of Tunable Properties

Recent convergence of the 3D printing of tissue‐like bioinks and regenerative medicine offers promise in the high‐throughput engineering of in vitro tissue models and organoids for drug screening and discovery research, and of potentially implantable neo‐tissues with tailored structural, biological, and mechanical properties. However, the current printing approaches are not compatible with collagen, the native scaffolding material. Herein, a unique biofabrication approach that uses automated gel aspiration‐ejection (GAE) is reported to potentially overcome these challenges. Automated‐GAE generates highly defined, aligned, dense collagen gel bioinks of various geometries (i.e., cylindrical, quadrangular, and tubular), dimensions, as well as tunable microstructural and mechanical properties that modulate seeded cellular responses. By densifying initial naturally derived reconstituted collagen hydrogels incorporating cells, automated‐GAE generates mini‐tissue building blocks with tailored protein fibril density and alignment, as well as cell loading, density and orientation according to the intended use. Surprisingly, a simple mathematical relationship defining the bioink compaction factor is found to be highly effective in predicting the initial and temporal properties of the bioinks in culture. Therefore, automated‐GAE will potentially also enable a fourth dimension to biofabrication, where cell–cell communications and cell‐extracellular matrix interactions as a function of time in culture can be predicted and modeled.     

A Non‐Mulberry Silk Fibroin Protein Based 3D In Vitro Tumor Model for Evaluation of Anticancer Drug Activity

The limitations of clinical chemotherapy are credited primarily to drug resistance. Effective development and screening of new drugs require appropriate in vitro tumor models that resemble the in vivo situation to evaluate drug efficiency and to decrease the use of experimental animals. 3D in vitro model systems that are able to mimic in vivo microenvironments are now highly sought after in cancer research. Here, the characteristics of breast cancer cell line MDA‐MB‐231 cells on 3D, and 2D Antheraea mylitta silk matrices and tissue culture plates are compared. After long term culture of breast cancer cells in the silk scaffold, the engineered tumor construct shows different zones of cell proliferation, such as an avascular tumor. Silk fibroin matrix 3D tumor models are studied for the evaluation of various anticancer drugs. The cytotoxic effects of three different drugs (Paclitaxel, Celecoxib, and ZD6474) at different concentrations are evaluated for MDA‐MB‐231 grown on 2D films as well as on a 3D fibroin scaffold. Higher drug concentrations are required to achieve a comparable reduction in cell viability and invasive potential in 3D culture. Combinatorial treatment of drugs at IC₅₀ concentrations result in up to 84% death of cancer cells. The results indicate that 3D in vitro tumor models may be better systems to evaluate cancer treatment strategies.     

Screening of Nanocomposite Scaffolds Arrays Using Superhydrophobic‐Wettable Micropatterns

Platforms containing multiple arrays for high‐throughput screening are demanded in the development of biomaterial libraries. Here, an array platform for the combinatorial analysis of cellular interactions and 3D porous biomaterials is described. Using a novel method based on computer‐aided manufacturing, wettable regions are printed on superhydrophobic surfaces, generating isolated spots. This freestanding benchtop array is used as a tool to deposit naturally derived polymers, chitosan and hyaluronic acid, with bioactive glass nanoparticles (BGNPs) to obtain a scaffold matrix. The effect of fibronectin adsorption on the scaffolds is also tested. The biomimetic nanocomposite scaffolds are shown to be osteoconductive, non‐cytotoxic, promote cell adhesion, and regulate osteogenic commitment. The method proves to be suitable for screening of biomaterials in 3D cell cultures as it can recreate a multitude of combinations on a single platform and identify the optimal composition that drives to desired cell responses. The platforms are fully compatible with commercially routine cell culture labware and established characterization methods, allowing for a standard control and easy adaptability to the cell culture environment. This study shows the value of 3D structured array platforms to decode the combinatorial interactions at play in cell microenvironments.     

Microphysiological Systems as Enabling Tools for Modeling Complexity in the Tumor Microenvironment and Accelerating Cancer Drug Development

Tumor cell heterogeneity with distinct phenotypes, genotypes, and epigenetic states as well as the complex tumor microenvironment is major challenges for cancer diagnosis and treatment. There have been substantial advances in our knowledge of tumor biology and in the capabilities of available biological analysis tools; however, the absence of physiologically relevant in vitro testing platforms limits our ability to gain an in‐depth understanding of the role of the tumor microenvironment in cancer pathology. In this review, recent advances in engineered tumor microenvironments to advance cancer research and drug discovery are presented, including tumor spheroids, microfluidic chips, paper scaffolds, hydrogel‐based engineered tissues, 3D bioprinted scaffolds, and multiscale topography. Furthermore, how these technologies address the specific characteristics of the native tumor microenvironment is described. Through the comparison of these biomimetic 3D tumor models to conventional 2D culture models, the validity and physiological relevance of these platforms for fundamental in vitro studies of the tumor biology, as well as their potential use in drug screening applications, is also discussed.     

Implantable Vascularized Liver Chip for Cross‐Validation of Disease Treatment with Animal Model

Artificial liver models have been extensively developed for pathological modeling and toxicological studies. However, the prediction of existing in vitro liver models rarely corresponds to what is consequently observed in vivo owing to the structural and functional complexity of the liver. Here, a new liver model designed to enable the implantation and maintenance of liver buds in perfusable 3D hydrogels where a microvascular network develops within a 200 µm diffusion limit is developed. This system replicates inflammation, lipid accumulation, and fibrosis during the progressive processes of nonalcoholic fatty liver disease, in which this model predicted the results from a mouse model. This model reveals that a hepatic steatosis‐reducing drug restored mitochondrial activities with significant reduction of inflammation, oxidative stress, and lipid accumulation. This liver model is not only highly predictive but also scalable and easy to apply to high‐throughput drug screening and implantation studies, suggesting a promising alternative to animal models.     

2D and 3D Hybrid Systems for Enhancement of Chondrogenic Differentiation of Tonsil‐Derived Mesenchymal Stem Cells

2D/3D hybrid cell culture systems are constructed by increasing the temperature of the thermogelling poly(ethylene glycol)‐poly(l ‐alanine) diblock copolymer (PEG‐l ‐PA) aqueous solution in which tonsil tissue‐derived mesenchymal stem cells and graphene oxide (GO) or reduced graphene oxide (rGO) are suspended, to 37 °C. The cells exhibit spherical cell morphologies in 2D/3D hybrid culture systems of GO/PEG‐l ‐PA and rGO/PEG‐l ‐PA by using the growth medium. The cell proliferations are 30%–50% higher in the rGO/PEG‐l ‐PA hybrid system than in the GO/PEG‐l ‐PA hybrid system. When chondrogenic culture media enriched with TGF‐β3 is used in the 2D/3D hybrid systems, cells extensively aggregate, and the expression of chondrogenic biomarkers of SOX 9, COL II A1, COL II, and COL X significantly increases in the GO/PEG‐l ‐PA 2D/3D hybrid system as compared with the PEG‐l ‐PA 3D systems and rGO/PEG‐l ‐PA 2D/3D hybrid system, suggesting that the GO/PEG‐l ‐PA 2D/3D hybrid system can be an excellent candidate as a chondrogenic differentiation platform of the stem cell. This paper also suggests that a 2D/3D hybrid system prepared by incorporating 2D materials with various surface biofunctionalities in the in situ forming 3D hydrogel matrix can be a new cell culture system.     

High Throughput,Polymeric Aqueous Two‐Phase Printing of Tumor Spheroids

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti‐cancer drugs. Two immiscible polymeric aqueous solutions are used and a submicroliter drop of the “patterning” phase containing cells is microprinted into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well‐defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, a phase diagram is established to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long‐term incubation and dose‐dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically relevant 3D tumor models.     

Versatile Microfluidic Platforms Enabled by Novel Magnetorheological Elastomer Microactuators

Microfluidic systems enable rapid diagnosis of diseases, biological analysis, drug screening, and high‐precision materials synthesis. In spite of these remarkable abilities, conventional microfluidic systems are microfabricated monolithically on a single platform and their operations rely on bulky expensive external equipment. This restricts their applications outside of research laboratories and prevents development and assembly of truly versatile and complex systems. Here, novel magnetorheological elastomer (MRE) microactuators are presented including pumps and mixers using an innovative actuation mechanism without the need of delicate elements such as thin membranes. Modularized elements are realized using such actuators, which can be easily integrated and actuated using a single self‐contained driving unit to create a modular, miniaturized, and robust platform. The performance of the microactuators is investigated via a series of experiments and a proof‐of‐concept modular system is developed to demonstrate the viability of the platform for self‐contained applications. The presented MRE microactuators are small size, simple, and efficient, offering a great potential to significantly advance the current research on complex microfluidic systems.     

Advances in Spheroids and Organoids on a Chip

Multicellular spheroids and organoids are promising in vitro 3D models in personalized medicine and drug screening. They replicate the structural and functional characteristics of human organs in vivo. Microfluidic technology and micro-nano fabrication can fulfill the high requirement of the engineering approach in the growing research interest in spheroids and organoids. In this review, spheroids and organoids are comparatively introduced. Then it is illustrated how spheroids- and organoids-on-a-chip technology facilitates their establishment, expansion, and application through spatial-temporal control, mechanical cues modeling, high-throughput analysis, co-culture, multi-tissue interactions, biosensing, and bioimaging integration. The potential opportunities and challenges in developing spheroids- and organoids-on-a-chip technology are finally outlooked.     

Engineered 3D Silk‐Based Metastasis Models: Interactions Between Human Breast Adenocarcinoma,Mesenchymal Stem Cells and Osteoblast‐Like Cells

Bone metastasis occurs in 70% of breast cancer patients and is a frequent cause of morbidity in cancer patients. A delicate balance exists in the bone microenvironment, but the functional dynamics underlying the tumor cell‐microenvironment interactions remain poorly understood. 3D in vitro model systems of metastasis can throw new light on this phenomenon. Silk protein fibroin scaffolds, are cytocompatible for 3D cancer cell culture. They are structurally more resistant to protease degradation than other native biomaterials making these matrices suitable for cancer modeling. In this report, human breast adenocarcinoma cells, human osteoblast like cells and mesenchymal stem cells are co‐cultered. Cancer cells and osteoblast‐like cells are found to interact through secreted products. Decreased population of osteoblast‐like cells and mineralization of extracellular matrix are observed as a result of co‐culture. Significantly increased migration of breast cancer cells is observed in the bone‐like constructs than in non‐seeded scaffolds. The co‐culture constructs show significant increase in drug resistance, invasiveness and angiogenicity. Co‐culture of breast cancer cells with osteoblast like cells and mesenchymal stem cells also indicate that the interaction of cancer cells with bone microenvironment varies with spatial organization, presence of osteogenic factors as well as stromal cell type. Here, results show that 3D in vitro co‐culture models is possibly a better system to study and target cancer progression.     

Electroaddressing Functionalized Polysaccharides as Model Biofilms for Interrogating Cell Signaling

Bacteria often reside at surfaces as complex biofilms in which an exopolysaccharide matrix entraps the population while allowing access to its chemical environment. There is a growing awareness that the biofilm structure and activity are integral to a wide array of properties important to health (the microbiome), disease (drug resistance) and technology (fouling). Despite the importance of bacterial biofilms, few experimental platforms and systems are available to assemble complex populations and monitor their activities. Here, a functionalized alginate composite material for creating in vitro model biofilms suitable for cell‐cell signaling studies by entrapping bacterial cells in situ is reported. Biofilm assembly is achieved using device‐imposed electrical signals to electrodeposit the stimuli‐responsive polysaccharide alginate. This electrodeposition mechanism is versatile in that it allows control of the bacterial population density and distribution. For instance, it is demonstrated that a mixed population can be homogeneously distributed throughout the biofilm or can be assembled as spatially segregated populations within a stratified biofilm. The “electroaddressable” biofilms are visualized using both a planar 2D chip with patterned electrodes and a microfluidic bioMEMS device with sidewall electrodes. Specifically, it is observed that bacteria entrapped within the model biofilm recognize and respond to chemical stimuli imposed from the fluidic environment. Finally, reporter cells are used to demonstrate that bacteria entrapped within this model biofilm engage in intercellular quorum sensing. This work demonstrates the functionality of the stimuli‐responsive polysaccharide by biofabricating pseudo‐3D cell‐gel biocomposites, mimicking the formation of biofilms, for interrogating phenotypes of E. coli bacterial populations. In addition to controlling assembly, the microfluidic device allows the biofilm to be monitored through the fluorescence methods commonly used in biological research. This platform technology should be able to be exploited for monitoring biofilm development, as well as for extending the understanding of the interactions between various bacterial species arranged in controlled patterns.     

Surface Textured Polymer Fibers for Microfluidics

This article introduces surface textured polymer fibers as a new platform for the fabrication of affordable microfluidic devices. Fibers are produced tens of meters‐long at a time and comprise 20 continuous and ordered channels (equilateral triangle grooves with side lengths as small as 30 micrometers) on their surfaces. Extreme anisotropic spreading behavior due to capillary action along the grooves of fibers is observed after surface modification with polydopamine (PDA). These flexible fibers can be fixed on any surface—independent of its material and shape—to form three‐dimensional arrays, which spontaneously spread liquid on predefined paths without the need for external pumps or actuators. Surface textured fibers offer high‐throughput fabrication of complex open microfluidic channel geometries, which is challenging to achieve using current photolithography‐based techniques. Several microfluidic systems are designed and prepared on either planar or 3D surfaces to demonstrate outstanding capability of the fiber arrays in control of fluid flow in both vertical and lateral directions. Surface textured fibers are well suited to the fabrication of flexible, robust, lightweight, and affordable microfluidic devices, which expand the role of microfluidics in a scope of fields including drug discovery, medical diagnostics, and monitoring food and water quality.     

Engineering Tridimensional Hydrogel Tissue and Organ Phantoms with Tunable Springiness

Biomimicking organ phantoms with vivid biological structures and soft and slippery features are essential for in vitro biomedical applications yet remain hither to unmet challenges in their fabrication such as balancing between spatial structural complexity and matchable mechanical properties. Herein, 3D printable tissue-mimicking elastomeric double network hydrogels with tailorable stiffness are evolved to idiosyncratically match diverse biological soft tissues by regulating the compositions of hydrogel matrix and the density of metal coordination bonds. Relying on digital light processing 3D printing, various mechanically tunable biomimetic volumetric hydrogel organ constructs with structural complexity and fidelity, including kidney, brain, heart, liver, stomach, lung, trachea, intestine, and even the intricate vascularized tissues, are fabricated faultlessly. Proof-of-concept 3D printed hydrogel heart and liver phantoms provide sophisticated internal channels and cavity structures and external realistic anatomical architectures that more closely mimic native organs. For the in vitro application demonstration, a 3D printed hydrogel brain phantom with tortuous cerebral arteries and slippery characters serves as an effective neurosurgical training platform for realistic simulation of endovascular interventions. This platform offers a means to construct mechanically precisely tunable hydrogel-based biomimetic organ phantoms that are expected to be used in surgical training, medical device testing, and organs-on-chips.