Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting
23 Feb 2016-Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A)-Vol. 113, Iss: 8, pp 2206-2211
TL;DR: 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture is presented and finds improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction.
Abstract: The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.
Citations
More filters
TL;DR: The advances in making hydrogels with improved mechanical strength and greater flexibility for use in a wide range of applications are reviewed, foreseeing opportunities in the further development of more sophisticated fabrication methods that allow better-controlled hydrogel architecture across multiple length scales.
Abstract: BACKGROUND Hydrogels are formed through the cross-linking of hydrophilic polymer chains within an aqueous microenvironment. The gelation can be achieved through a variety of mechanisms, spanning physical entanglement of polymer chains, electrostatic interactions, and covalent chemical cross-linking. The water-rich nature of hydrogels makes them broadly applicable to many areas, including tissue engineering, drug delivery, soft electronics, and actuators. Conventional hydrogels usually possess limited mechanical strength and are prone to permanent breakage. The lack of desired dynamic cues and structural complexity within the hydrogels has further limited their functions. Broadened applications of hydrogels, however, require advanced engineering of parameters such as mechanics and spatiotemporal presentation of active or bioactive moieties, as well as manipulation of multiscale shape, structure, and architecture. ADVANCES Hydrogels with substantially improved physicochemical properties have been enabled by rational design at the molecular level and control over multiscale architecture. For example, formulations that combine permanent polymer networks with reversibly bonding chains for energy dissipation show strong toughness and stretchability. Similar strategies may also substantially enhance the bonding affinity of hydrogels at interfaces with solids by covalently anchoring the polymer networks of tough hydrogels onto solid surfaces. Shear-thinning hydrogels that feature reversible bonds impart a fluidic nature upon application of shear forces and return back to their gel states once the forces are released. Self-healing hydrogels based on nanomaterial hybridization, electrostatic interactions, and slide-ring configurations exhibit excellent abilities in spontaneously healing themselves after damages. Additionally, harnessing techniques that can dynamically and precisely configure hydrogels have resulted in flexibility to regulate their architecture, activity, and functionality. Dynamic modulations of polymer chain physics and chemistry can lead to temporal alteration of hydrogel structures in a programmed manner. Three-dimensional printing enables architectural control of hydrogels at high precision, with a potential to further integrate elements that enable change of hydrogel configurations along prescribed paths. OUTLOOK We envision the continuation of innovation in new bioorthogonal chemistries for making hydrogels, enabling their fabrication in the presence of biological species without impairing cellular or biomolecule functions. We also foresee opportunities in the further development of more sophisticated fabrication methods that allow better-controlled hydrogel architecture across multiple length scales. In addition, technologies that precisely regulate the physicochemical properties of hydrogels in spatiotemporally controlled manners are crucial in controlling their dynamics, such as degradation and dynamic presentation of biomolecules. We believe that the fabrication of hydrogels should be coupled with end applications in a feedback loop in order to achieve optimal designs through iterations. In the end, it is the combination of multiscale constituents and complementary strategies that will enable new applications of this important class of materials.
1,588 citations
Cites background from "Deterministically patterned biomime..."
...Using such a manufacturing principle, hydrogel objects with complex 3D architecture andmultiplexedmaterials may be generated with high spatial resolution (100, 117)....
[...]
...For example, almost all organs in the human body contain repeating building units that assemble into distinctive compartments and interfaces [for example, the myocardium, endocardium, and pericardium in the heart (98, 99) and the lobules constituting the liver (99, 100)]....
[...]
TL;DR: The expanding range of printable materials, coupled with the ability to programmably control their composition and architecture across various length scales, is driving innovation in myriad applications.
Abstract: Light- and ink-based three-dimensional (3D) printing methods allow the rapid design and fabrication of materials without the need for expensive tooling, dies or lithographic masks. They have led to an era of manufacturing in which computers can control the fabrication of soft matter that has tunable mechanical, electrical and other functional properties. The expanding range of printable materials, coupled with the ability to programmably control their composition and architecture across various length scales, is driving innovation in myriad applications. This is illustrated by examples of biologically inspired composites, shape-morphing systems, soft sensors and robotics that only additive manufacturing can produce.
1,054 citations
TL;DR: 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro–computed tomography and showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.
Abstract: Collagen is the primary component of the extracellular matrix in the human body. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs. We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves. We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro-computed tomography. Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.
996 citations
TL;DR: The Grigoryan et al. show that natural and synthetic food dyes can be used as photoabsorbers that enable stereolithographic production of hydrogels containing intricate and functional vascular architectures, and establish intravascular and multivascular design freedoms with photopolymerizablehydrogels.
Abstract: A device made from a hydrogel matrix is provided. The hydrogel matrix includes a photoabsorber with a void architecture in the matrix, having a first vessel architecture and a second vessel architecture that are each tubular and branching, wherein the first and second vessel architectures are fluidically independent from each other. A pre-polymerization solution for forming the device, and methods of fabricating such devices are described. A method of fabricating a 3D hydrogel construct is provided. The method includes using a computer-implemented process to create a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices, generate a first vascular component of the model, generate a second vascular component of the model, and combine the first and second vascular components of the model.
712 citations
20 Feb 2019
TL;DR: The field of 3D printing is continuing its rapid development in both academic and industrial research environments as mentioned in this paper, which offers flexibility over the final properties of the 3D printed materials (such as optical, chemical and mechanical properties) using versatile polymer chemistry.
Abstract: The field of 3D printing is continuing its rapid development in both academic and industrial research environments. The development of 3D printing technologies has opened new implementations in rapid prototyping, tooling, dentistry, microfluidics, biomedical devices, tissue engineering, drug delivery, etc. Among different 3D printing techniques, photopolymerization-based process (such as stereolithography and digital light processing) offers flexibility over the final properties of the 3D printed materials (such as optical, chemical, and mechanical properties) using versatile polymer chemistry. The strategy behind the 3D photopolymerization is based on using monomers/oligomers in liquid state (in the presence of photoinitiators) that can be photopolymerized (via radical or cationic mechanism) upon exposure to light source of different wavelengths (depending on the photoinitiator system). An overview of recent evolutions in the field of photopolymerization-based 3D printing and highlights of novel 3D print...
621 citations
References
More filters
TL;DR: GelMA hydrogels could be useful for creating complex, cell- responsive microtissues, such as endothelialized microvasculature, or for other applications that require cell-responsive microengineered hydrogELs.
1,871 citations
"Deterministically patterned biomime..." refers methods in this paper
...GelMA was synthesized according to previously published procedures (45)....
[...]
TL;DR: This is the first report demonstrating the generation of a functional human organ from pluripotent stem cells by transplantation of liver buds created in vitro (iPSC-LBs), and provides a promising new approach to study regenerative medicine.
Abstract: A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.
1,653 citations
"Deterministically patterned biomime..." refers background or methods in this paper
..., HPCs and HLCs, respectively, have demonstrated their potentials in organizing into vascularized liver buds (11, 13) and in the construction of in vitro models with simple geometry (10, 12, 14)....
[...]
...potentials in forming functional vasculatures as shown and discussed in previous studies (11, 13, 31)....
[...]
TL;DR: An overview of the use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology.
Abstract: Microscale technologies are emerging as powerful tools for tissue engineering and biological studies. In this review, we present an overview of these technologies in various tissue engineering applications, such as for fabricating 3D microfabricated scaffolds, as templates for cell aggregate formation, or for fabricating materials in a spatially regulated manner. In addition, we give examples of the use of microscale technologies for controlling the cellular microenvironment in vitro and for performing high-throughput assays. The use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology. Throughout this review, we will use specific examples where available and will provide trends and future directions in the field.
1,590 citations
TL;DR: Several issues remain to be resolved regarding the catalytic activity of the P-450 3A4 protein, including rate-limiting steps and the need for cytochrome b5, divalent cations, and acidic phospholipid systems for optimal activity.
Abstract: Cytochrome P-450 (P-450) 3A4 is the most abundant P-450 expressed in human liver and small intestine. P-450 3A4 contributes to the metabolism of approximately half the drugs in use today, and variations in its catalytic activity are important in issues of bioavailability and drug-drug interactions. The gene is known to be inducible by barbiturates, glucocorticoids, and rifampicin in humans and in isolated hepatocytes, although the mechanism remains unclear. The 5'-untranslated region includes putative basal transcription element, hepatocyte nuclear factor, p53, AP-3, glucocorticoid regulatory element, pregnane X receptor, and estrogen receptor element sequences. Recently, the GRE element has been shown to act in a classic glucocorticoid response. Several issues remain to be resolved regarding the catalytic activity of the P-450 3A4 protein, including rate-limiting steps and the need for cytochrome b5, divalent cations, and acidic phospholipid systems for optimal activity. Another issue involves the basis of the homotropic and heterotropic cooperativity seen with the enzyme. The in vivo significance of these findings remains to be further established. In addition to more basic studies on P-450 3A4, several areas of practical interest to the pharmaceutical industry require development.
1,202 citations
TL;DR: This work presents a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks and demonstrates utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins.
Abstract: Tissue function depends on hierarchical structures extending from single cells ( approximately 10 microm) to functional subunits (100 microm-1 mm) that coordinate organ functions. Conventional cell culture disperses tissues into single cells while neglecting higher-order processes. The application of semiconductor-driven microtechnology in the biomedical arena now allows fabrication of microscale tissue subunits that may be functionally improved and have the advantages of miniaturization. Here we present a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks. The need for such models is underscored by the high rate of pre-launch and post-market attrition of pharmaceuticals due to liver toxicity. We demonstrate utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins. The combination of microtechnology and tissue engineering may enable development of integrated tissue models in the so-called 'human on a chip'.
1,085 citations
"Deterministically patterned biomime..." refers background or result in this paper
...The greater HPC aggregate size observed in this 3D triculture model further supports the beneficial effects from supporting cells as widely reported in 2D coculture models (14, 42)....
[...]
...The significant induction by rifampicin of CYP3A4, CYP2C9, and CYP2C19 expression levels shows that hiPSC-HPCs after 7 d in 3D triculture are able to respond positively to rifampicin as reported for primary hepatocytes (42), thus potentially improving the metabolism and clearance of the drug....
[...]
...4 D, iv), which was previously reported to be induced by rifampicin in adult hepatocytes (41, 42)....
[...]