3D Cellular Constructs Research Articles

Cardiac Organoids: A 3D Technology for Disease Modeling and Drug Screening.

Cardiovascular diseases remain the leading cause of death worldwide; therefore, there is increasing attention to developing physiological-related in vitro cardiovascular tissue models suitable for personalized healthcare and preclinical test. Recently, more complex and powerful in vitro models have emerged for cardiac research. Human cardiac organoids (HCOs) are three-dimensional (3D) cellular constructs similar to in vivo organs. They are derived from pluripotent stem cells and can replicate the structure, function, and biogenetic information of primitive tissues. High-fidelity HCOs are closer to natural human myocardial tissue than animal and cell models to some extent, which helps to study better the development process of the heart and the occurrence of related diseases. In this review, we introduce the methods for constructing HCOs and the application of them, especially in cardiovascular disease modeling and cardiac drug screening. In addition, we propose the prospects and limitations of HCOs. In summary, we have introduced the research progress of HCOs and described their innovation and practicality of them in the biomedical field.

Abstract Mo089: Fontan-Associated Liver Disease On A Chip

Introduction: The Fontan procedure is performed in children with single ventricle physiology, and has led to excellent short-term palliation and survival rates but at the cost of long-term complications. Fontan-associated liver disease (FALD) results from hemodynamic changes following the Fontan procedure, leading to elevated central venous pressures, chronic alterations in hepatic perfusion, and resultant hepatic fibrosis. Improved understanding of FALD pathophysiology, which may provide insights for prevention and treatment is challenged by the lack of experimental models. Current in vitro liver models, mostly 2D or suspension hepatic cell cultures, lack biomimicry. This study presents a 3D bioprinted perfusable human liver model for in vitro study of hepatic fibrosis in response to altered flow conditions in FALD patients. Methods: Vascular 3D model of hepatic sinusoid was biofabricated using hybrid bioinks in two distinct peripheral layers and a central lumen. The outer layer was cast with hepatic (HepG2) cells encapsulated in a mixture of gelatin methacrylate (GelMA) and collagen type I. The inner layer consisted of hepatic stellate cells (HSCs) in GelMA. Human endothelial cells (ECs) were seeded onto the central lumen. 3D cellular constructs were cultured in vitro for three weeks in static or dynamic conditions, by circulating the culture media at varying flow conditions to simulate sinusoid pressure characteristic of healthy vs. disease state. Using Volcano system in cath lab, we invasively measured the pressure in mid-channel. Cell viability, proliferation, maturation into functional liver tissue, and tissue stiffness were evaluated via microindentation, flow hemodynamics assays, metabolomic assays, and immunohistochemistry analyses. Results: Bioengineered liver constructs cultured under static and dynamic conditions demonstrated high cell viability and growth and full endothelialization of the sinusoid channel. HepG2 cells exhibited long-term function and maturation, forming clusters. Under FALD flow conditions, there were notable changes in EC-HepG2 cell morphology compared to dynamic flow in the healthy range, accompanied by reduced levels of functional markers. Conclusion: This proof-of-principle study introduces an innovative and highly biomimetic 3D model of hepatic sinusoid, which can serve as a robust platform for in vitro investigations of complex cell-microenvironment interactions in the context of FALD as well as other hepatic disorders.

A 3D-printable gelatin/alginate/ε-poly-l-lysine hydrogel scaffold to enable porcine muscle stem cells expansion and differentiation for cultured meat development

The mass proliferation of seed cells and imitation of meat structures remain challenging for cell-cultured meat production. With excellent biocompatibility, high water content and porosity, hydrogels are frequently-studied materials for anchorage-dependent cell scaffolds in biotechnology applications. Herein, a scaffold based on gelatin/alginate/ε-Poly-l-lysine (GAL) hydrogel is developed for skeletal muscle cells, which has a great prospect in cell-cultured meat production. In this work, the hydrogel GAL-4:1, composed of gelatin (5 %, w/v), alginate (5 %, w/v) and ε-Poly-l-lysine (molar ratio vs. alginate: 4:1) is selected as cell scaffold based on Young's modulus of 11.29 ± 1.94 kPa, satisfactory shear-thinning property and suitable porous organized structure. The commercially available C2C12 mouse skeletal myoblasts and porcine muscle stem cells (PMuSCs), are cultured in the 3D-printed scaffold. The cells show strong ability of attachment, proliferation and differentiation after induction, showing high biocompatibility. Furthermore, the cellular bioprinting is performed with GAL-4:1 hydrogel and freshly extracted PMuSCs. The extracted PMuSCs exhibit high viability and display early myogenesis (desmin) on the 3D scaffold, suggesting the great potential of GAL hydrogel as 3D cellular constructs scaffolds. Overall, we develop a novel GAL hydrogel as a 3D-printed bioactive platform for cultured meat research.

A Biomimetic Polynucleotides–Hyaluronic Acid Hydrogel Promotes the Growth of 3D Spheroid Cultures of Gingival Fibroblasts

(1) Background: Three-dimensional cultures are useful tools to evaluate regenerative approaches in vitro, as they may mimic the spatial arrangement of cells more closely to natural tissues than routine 2D culture methods. (2) Methods: We investigated the effects of a polynucleotide, hyaluronic acid (PN, HA) compound on 3D spheroid cultures of primary gingival fibroblasts, by measuring their morphology over time, cell viability with Calcein-AM, a fluorescent marker, and cell growth potential by re-plating spheroids in attachment-permissive regular culture plates under routine conditions and following them up for 15 days. (3) Results: PN + HA induced an increase in spheroid size and perimeter and a decrease in spheroid circularity, as cells tended to grow and form small peripheral stacks around the spheroid. Levels of cell viability were also higher in this group. After re-plating, only the spheroids previously stimulated with PN + HA dissolved completely during the second week of culture and colonized the plate, thus indicating the retention of a higher level of viability by the cells forming the whole spheroid with this stimulus. (4) Conclusions: Taken together, our data support the idea that the combination of PN and HA has synergic effects on primary fibroblasts and promotes their viability, the growth of 3D cellular constructs, and the retention of a remarkable proliferative potential over the course of the experimental period, making it a promising compound for further investigations.

Dynamic Tumor Perfusion and Real-Time Monitoring in a Multiplexed 3D Printed Microdevice.

Stereolithography based additive manufacturing ("3D printing") has become a useful tool for the development of novel microfluidic in vitro platforms. This method of manufacturing can reduce production time while allowing for rapid design iteration and complex monolithic structures. The platform described in this chapter has been designed for the capture and evaluation of cancer spheroids in perfusion. Spheroids are created in 3D Petri dishes, stained, and loaded into these 3D printed devices and imaged over time under flow conditions. This design allows for active perfusion into complex 3D cellular constructs resulting in longer viability while providing results which better mimic in vivo conditions compared to traditional monolayer static culture.

Biomaterial-based 3D bioprinting strategy for orthopedic tissue engineering.

The advent of three-dimensional (3D) bioprinting has enabled impressive progress in the development of 3D cellular constructs to mimic the structural and functional characteristics of natural tissues. Bioprinting has considerable translational potential in tissue engineering and regenerative medicine. This review highlights the rational design and biofabrication strategies of diverse 3D bioprinted tissue constructs for orthopedic tissue engineering applications. First, we elucidate the fundamentals of 3D bioprinting techniques and biomaterial inks and discuss the basic design principles of bioprinted tissue constructs. Next, we describe the rationale and key considerations in 3D bioprinting of tissues in many different aspects. Thereafter, we outline the recent advances in 3D bioprinting technology for orthopedic tissue engineering applications, along with detailed strategies of the engineering methods and materials used, and discuss the possibilities and limitations of different 3D bioprinted tissue products. Finally, we summarize the current challenges and future directions of 3D bioprinting technology in orthopedic tissue engineering and regenerative medicine. This review not only delineates the representative 3D bioprinting strategies and their tissue engineering applications, but also provides new insights for the clinical translation of 3D bioprinted tissues to aid in prompting the future development of orthopedic implants. STATEMENT OF SIGNIFICANCE: 3D bioprinting has driven major innovations in the field of tissue engineering and regenerative medicine; aiming to develop a functional viable tissue construct that provides an alternative regenerative therapy for musculoskeletal tissue regeneration. 3D bioprinting-based biofabrication strategies could open new clinical possibilities for creating equivalent tissue substitutes with the ability to customize them to meet patient demands. In this review, we summarize the significance and recent advances in 3D bioprinting technology and advanced bioinks. We highlight the rationale for biofabrication strategies using 3D bioprinting for orthopedic tissue engineering applications. Furthermore, we offer ample perspective and new insights into the current challenges and future direction of orthopedic bioprinting translation research.

Characterizing the Luminal Microenvironment of Human Organoids for Studies of Gastric pH Regulation

The stomach is notorious for having the steepest proton gradient in the body, which pathogenic Helicobacter pylori must traverse to infect the epithelium. This gradient across the 200‐300 μm‐thick gastric mucus layer enables the stomach to maintain a near‐neutral pH at the epithelial surface and a strongly acidic environment in the stomach lumen. In this study, we use human gastric organoids to study the maintenance and regulation of this pH gradient. Human gastric organoids are 3D cellular constructs derived from patient biopsies that mimic the microanatomy and physiology of the stomach, as they contain both mucus and gastric acid. Organoids have been used to investigate H. pylori‐induced gastritis, peptic ulcers, and stomach cancer, which claims 700,000 lives each year. Gastric mucus is critical for protection against H. pylori infection, which is the leading cause of gastric cancer. It is presently unclear how the gastric mucosal pH gradient is maintained, and how closely organoid models can mimic this microenvironment. First, we detected mucus within human gastric organoids using histological staining and bead microrheology. We also used a FITC‐conjugated lectin to visualize the mucosal matrix inside of the organoids. These data were supported by our observation that a clear viscous material was produced on the apical side of two‐dimensional, organoid‐derived cell monolayers. Additionally, we developed a novel method for the measurement of intraluminal pH of the gastric organoids. Using a micromanipulator and stereomicroscope to maneuver a glass microelectrode into the organoid lumen, we measured and manipulated luminal pH using histamine stimulation. We determined that pH remains consistent across organoid lines, and that the intraluminal space is more acidic than the surrounding environment. In ongoing studies, we will develop the spatial resolution of our organoid microprofiling technique to fully characterize the proton gradient within the mucus‐filled lumens gastric organoids. Our studies are the first to utilize pH microsensors in these models, and will lead to improved molecular and biophysical understanding of gastric physiology and potential new treatment approaches for disease conditions (such as peptic ulcers and gastric cancer) that involve dysregulated gastric acid secretion.

Composite Materials by Building Block Chemistry Using Weak Interaction

Abstract Layer-by-Layer (LbL) assembly of interactive polymers onto surfaces leads to the construction of multilayered ultrathin films, which can be done simply by alternately dipping the substrate into various solutions. The range of applications of this LbL assembly can be broadened by introducing molecular recognition mechanisms for polymers and proteins, and by using weak interactions such as van der Waals interactions and biological recognition. As a specific example, it can be applied to the formation of stereocomplexes of poly(methyl methacrylate) (PMMA), poly-lactide (PLA), and fibronectin-collagen as extracellular matrix proteins. In weakly interacting LbL assemblies, the polymer chain tends to be placed in the most structurally stable state. This feature has been successfully used for template polymerization of stereoregular polymers, significant morphological control of biodegradable nanomaterials, and fabrication of three-dimensional (3D) cellular tissue constructs. LbL assembly based on weak interactions is expected to further stimulate interest in the interdisciplinary fields of bioscience and polymer chemistry. Using LbL technology to create functional 3D tissues, such as skin models (LbL-3D Skin) and heart models (LbL-3D Heart), will be a breakthrough in science and technology.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage.

Alginate as a versatile naturally occurring biomaterial has found widespread use in the biomedical field due to its unique features such as biocompatibility and biodegradability. The ability of its semipermeable hydrogels to provide a favourable microenvironment for clinically relevant cells made alginate encapsulation a leading technology for immunoisolation, 3D culture, cryopreservation as well as cell and drug delivery. The aim of this work is the evaluation of structural properties and swelling behaviour of the core-shell capsules for the encapsulation of multipotent stromal cells (MSCs), their 3D culture and cryopreservation using slow freezing. The cells were encapsulated in core-shell capsules using coaxial electrospraying, cultured for 35 days and cryopreserved. Cell viability, metabolic activity and cell–cell interactions were analysed. Cryopreservation of MSCs-laden core-shell capsules was performed according to parameters pre-selected on cell-free capsules. The results suggest that core-shell capsules produced from the low viscosity high-G alginate are superior to high-M ones in terms of stability during in vitro culture, as well as to solid beads in terms of promoting formation of viable self-assembled cellular structures and maintenance of MSCs functionality on a long-term basis. The application of 0.3 M sucrose demonstrated a beneficial effect on the integrity of capsules and viability of formed 3D cell assemblies, as compared to 10% dimethyl sulfoxide (DMSO) alone. The proposed workflow from the preparation of core-shell capsules with self-assembled cellular structures to the cryopreservation appears to be a promising strategy for their off-the-shelf availability.

Pulsed laser assisted high-throughput intracellular delivery in hanging drop based three dimensional cancer spheroids.

Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core-shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm-2), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 1010 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.

Regulation of Stem Cell Function in an Engineered Vocal Fold-Mimetic Environment

Human mesenchymal stem cells (hMSCs) have been proposed as therapeutic cells for the treatment of vocal fold (VF) scarring. Although functional recovery was observed in animal models after stem cell injection, it is not clear how injected stem cells interact locally with the extracellular matrix (ECM) of the lamina propria (LP) and how such interactions affect stem cell behaviors to improve function. Herein, we developed an in vitro cell culture platform where hMSCs were encapsulated in a LP-mimetic matrix, derived from hyaluronic acid (HA), poly(ethylene glycol) (PEG) and collagen, and cultured dynamically in a custom-designed VF bioreactor. The cell culture system was characterized by oscillatory shear rheology, laser doppler vibrometry (LDV), and digital image correlation (DIC). A constitutive finite element analysis (FEA) model was further developed to predict vibratory responses of the hydrogel. LDV analysis demonstrated an average displacement of 47 μm in the center of the hydrogel construct at 200 Hz applied frequency without any harmonics. The predicted strains throughout the hydrogel ranged from 0 to 0.03, in good agreement with reported values for the VF. The 3D cellular construct was subjected to vibrational stimulations at 200 Hz for an optimized duration of 1 h, as confirmed by a maximal c-Fos upregulation at the transcript level. Vibrational culture over a 3-day period with a 1h-on/1h-off pattern did not compromise the overall cell viability, but resulted in a significant downregulation of fibrogenic markers and diminished staining for alpha smooth muscle actin (αSMA). Collectively, high frequency mechanical loading resulted in the loss of myofibrogenic potential and a shift away from a fibrotic phenotype.

Multi-layer pre-vascularized magnetic cell sheets for bone regeneration

The lack of effective strategies to produce vascularized 3D bone transplants in vitro, hampers the development of thick-constructed bone, limiting the translational of lab-based engineered system to clinical practices. Cell sheet (CS) engineering techniques provide an excellent microenvironment for vascularization since the technique can maintain the intact cell matrix, crucial for angiogenesis. In an attempt to develop hierarchical vascularized 3D cellular constructs, we herein propose the construction of stratified magnetic responsive heterotypic CSs by making use of iron oxide nanoparticles previously internalized within cells. Magnetic force-based CS engineering allows for the construction of thick cellular multilayers. Results show that osteogenesis is achieved due to a synergic effect of human umbilical vein endothelial cells (HUVECs) and adipose-derived stromal cells (ASCs), even in the absence of osteogenic differentiating factors. Increased ALP activity, matrix mineralization, osteopontin and osteocalcin detection were achieved over a period of 21 days for the heterotypic CS conformation (ASCs/HUVECs/ASCs), over the homotypic one (ASCs/ASCs), corroborating our findings. Moreover, the validated crosstalk between BMP-2 and VEGF releases triggers not only the recruitment of blood vessels, as demonstrated in an in vivo CAM assay, as well as the osteogenesis of the 3D cell construct. The in vivo angiogenic profile also demonstrated preserved human vascular structures and human cells showed the ability to migrate and integrate within the chick vasculature.

Sedimentation study of bioink containing living cells

3D bioprinting utilizes a cell-laden bioink to fabricate 3D cellular constructs for a variety of biomedical applications. The printing process typically takes hours to fabricate heterogeneous artificial tissues with multiple types of cells, different types of extracellular matrices, and interconnected vascular networks. During the printing process, the suspended cells sediment within the bioink with time, resulting in inhomogeneous cell concentration, which significantly affects the printing reliability and accuracy. This paper is the first study to quantify the cell sedimentation process in the bioink containing living cells. In this study, the effects of polymer concentration and standing time on the cell sedimentation velocity and cell concentration have been systematically investigated. The main conclusions are (1) the cell sedimentation velocity is almost constant at different standing times, because the cell gravitational force is balanced by the cell buoyant force and the drag force; (2) with the increase of the polymer concentration, the cell sedimentation velocity decreases, while the cell mass density increases due to less water absorbed; (3) with the increase of the standing time, the cell concentration near the bottom of the bioink reservoir increases linearly. With the increase of the polymer concentration, this linear increase of the cell concentration with the standing time significantly slows down due to a significant decrease of the cell sedimentation velocity; and (4) for the bioink with a low sodium alginate concentration, cell concentration near the bottom of the bioink reservoir is not uniform, and cell aggregates are observed.

A novel Roll Porous Scaffold 3D bioprinting technology

Biomanufacturing is a novel promising technology and an important field of research, offering hope for bridging the gap between organs shortage and transplantation needs. The paper describes the ways to overcome the main technological barriers and to accelerate biofabrication of 3D cellular tissue constructs greatly, to build multi-cell implantations of high density and accuracy for vascular system. It should be noted that though the Roll Porous Scaffold (RPS) 3D is a novel technology and never being used but derived from the time-proved bioprinting methods and components. The potential of the RPS are micron and submicron precision, performance of up to 5 l per hour with 20 μm layer thickness, which makes it more applicable than traditional methods. Features of the RPS are based on employing the Archimedean spiral and manufacturing 3D objects layer-by-layer with their transformation into a support ribbon. RPS may be one of the possible ways to solve the serious problem of “3D bioprinting” for tissue engineering and regenerative medicine in the nearest future.

Fabrication of perfusable 3D hepatic lobule-like constructs through assembly of multiple cell type laden hydrogel microstructures

The in vitro reproduction of three-dimensional (3D) cellular constructs to physiologically mimic human liver is highly desired for drug screening and clinical research. However, the fabrication of a liver-mimetic 3D model using traditional bottom-up technologies is challenging owing to the complex architecture and specific functions of real liver tissue. This work proposes a versatile strategy for spatially assembling gear-like microstructures encapsulating multiple cell types, and reorganizing them into 3D lobule-like micro-architecture with physiological relevance to native liver tissue. Gear-like microstructures were fabricated by photo-crosslinking poly(ethylene glycol) diacrylate (PEGDA) hydrogel mixed with hepatocytes and fibroblasts, in a digital micromirror device (DMD)-based microfluidic channel. The microstructures were assembled through coordinated micromanipulation based on local fluid force, and spatially self-aligned through hydrophilic–hydrophobic interactions into a 3D integrated construct with lobule-like morphology and a perfusable central lumen. The resulting 3D lobule-like constructs allowed long-term co-culture of hepatocytes and fibroblasts with high cell viability. The co-cultured constructs enhanced hepatocyte proliferation and spreading, as well as liver functions including a 50% increase in albumin secretion and urea synthesis. For hepatotoxicity assessment, the 3D lobule-like construct enabled drug perfusion through its built-in lumen for simulation of drug diffusion in the liver, which could improve the response sensitivity and efficiency to hepatotoxic drug. These results demonstrated that this method provides a valuable 3D co-culture model with perfusable lobule-like architecture and physiological functions, which has potential applications in drug discovery and tissue engineering applications.

Spheroids Formation on Non-Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches.

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre-clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell-cell and cell-extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold-based and -free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high-throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold-free techniques used for the spheroids formation are also addressed. Highlights • 2D cell culture drawbacks are summarized; • spheroids mimic the features of human tissues; • scaffold-based and scaffold-free technologies for spheroids production are discussed; • advantages of LOT over other scaffold-free techniques are highlighted; • LOT advances, practical approaches and troubleshooting are underlined.

Roles of Diffusion Dynamics in Stem Cell Signaling and Three-Dimensional Tissue Development.

Recent advancements in the ability to construct three-dimensional (3D) tissues and organoids from stem cells and biomaterials have not only opened abundant new research avenues in disease modeling and regenerative medicine but also have ignited investigation into important aspects of molecular diffusion in 3D cellular architectures. This article describes fundamental mechanics of diffusion with equations for modeling these dynamic processes under a variety of scenarios in 3D cellular tissue constructs. The effects of these diffusion processes and resultant concentration gradients are described in the context of the major molecular signaling pathways in stem cells that both mediate and are influenced by gas and nutrient concentrations, including how diffusion phenomena can affect stem cell state, cell differentiation, and metabolic states of the cell. The application of these diffusion models and pathways is of vital importance for future studies of developmental processes, disease modeling, and tissue regeneration.

Collagen Microparticle-Mediated 3D Cell Organization: A Facile Route to Bottom-up Engineering of Thick and Porous Tissues.

In closely packed artificial 3D cellular constructs, cells located near the center of the constructs are not functional because of the limited supply of oxygen and nutrition. Here we describe a simple, unique, and highly versatile approach to organizing cells into thick but porous 3D tissues, using cell-sized collagen microparticles as particulate scaffolds. When cells and particles are mixed and seeded in a noncell-adhesive planar chamber, they gather to form sheet-shaped structures with a thickness of 100-150 μm. In the construct, uniformly distributed particles work as a binder between cells and modulate the strong intercellular contraction. We confirmed that several factors, including the particle/cell ratio and particle size, critically affect the stability and shrink behaviors of porous tissues prepared using mouse embryonic fibroblasts (NIH-3T3 cells). Cross-sectional observation, together with cell proliferation and viability assays, revealed that the cells composing the tissues are functional primarily because interior pores between cells/particles worked as a path for efficient molecular transport. Furthermore, we prepared thick cell tissues of a liver model using human hepatocarcinoma cells (HepG2 cells), and confirmed that liver-specific functions were upregulated when composite tissues were formed using collagen microparticles prepared with several different stabilization protocols by glutaraldehyde, genipin, and methyl acetate). The process presented would be highly useful in enabling one-step production of thick cellular constructs in which porosity and morphology are tunable.

Generating Multicompartmental 3D Biological Constructs Interfaced through Sequential Injections in Microfluidic Devices.

A novel technique is presented for molding and culturing composite 3D cellular constructs within microfluidic channels. The method is based on the use of removable molding polydimethylsiloxane (PDMS) inserts, which allow to selectively and incrementally generate composite 3D constructs featuring different cell types and/or biomaterials, with a high spatial control. The authors generate constructs made of either stacked hydrogels, with uniform horizontal interfaces, or flanked hydrogels with vertical interfaces. The authors also show how this technique can be employed to create custom-shaped endothelial barriers and monolayers directly interfaced with 3D cellular constructs. This method dramatically improves the significance of in vitro 3D biological models, enhancing mimicry and enabling for controlled studies of complex biological districts.

Three-dimensional hepatic lobule-like tissue constructs using cell-microcapsule technology.

The proper functioning of the liver and tissues containing hepatocytes greatly depends upon the intricate organization of the cells. Consequently, controlling the shape of three-dimensional (3D) cellular constructs is an important issue for in vitro applications of fabricated artificial livers. However, the precise control of tissue shape at the microscale cannot be achieved with various commonly used 3D tissue-engineered building units, such as spheroids. Here, we present the fabrication of hepatic lobule-shaped microtissue (HLSM) containing rat liver (RLC-18) cells. By using cell-microcapsule technology, RLC-18 cells were encapsulated in the core region of poly-l-lysine-alginate microcapsules. After 14days of long-term cultivation, RLC-18 cells self-assembled into HLSM, and the cells fully occupied the microcapsule. By monitoring the cell number and albumin secretion during culture and characterizing the dimensions of the fabricated tissue, we demonstrated that the HLSM showed higher hepatic function as compared with normal cell spheroids. We also showcased the assembly of these microtissues into a 3D four-layered hepatic lobule model by a facile micromanipulation method. Our technology for fabricating 3D multilayer hepatic lobule-like, biofunctional tissue enables the precise control of tissue shape in three dimensions. Furthermore, these constructs can serve as tissue-engineered building blocks for larger organs and cellular implants in clinical treatment.