Reconstructing the heart using iPSCs: Engineering strategies and applications.
Journal of molecular and cellular cardiology
Induced pluripotent stem cells (iPSCs) have emerged as a key component of cardiac tissue engineering, enabling studies of cardiovascular disease mechanisms, drug responses, and developmental processes in human 3D tissue models assembled from isogenic cells. Since the very first engineered heart tissues were introduced more than two decades ago, a wide array of iPSC-derived cardiac spheroids, organoids, and heart-on-a-chip models have been developed incorporating the latest available technologies and materials. In this review, we will first outline the fundamental biological building blocks required to form a functional unit of cardiac muscle, including iPSC-derived cells differentiated by soluble factors (e.g., small molecules), extracellular matrix scaffolds, and exogenous biophysical maturation cues. We will then summarize the different fabrication approaches and strategies employed to reconstruct the heart in vitro at varying scales and geometries. Finally, we will discuss how these platforms, with continued improvements in scalability and tissue maturity, can contribute to both basic cardiovascular research and clinical applications in the future.
View details for DOI 10.1016/j.yjmcc.2021.04.006
View details for PubMedID 33895197
Flow-enhanced vascularization and maturation of kidney organoids in vitro
2019; 16 (3): 255-+
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.
View details for DOI 10.1038/s41592-019-0325-y
View details for Web of Science ID 000459804000023
View details for PubMedID 30742039
View details for PubMedCentralID PMC6488032
Voxelated soft matter via multimaterial multinozzle 3D printing.
2019; 575 (7782): 330–35
There is growing interest in voxelated matter that is designed and fabricated voxel by voxel1-4. Currently, inkjet-based three-dimensional (3D) printing is the only widely adopted method that is capable of creating 3D voxelated materials with high precision1-4, but the physics of droplet formation requires the use of low-viscosity inks to ensure successful printing5. By contrast, direct ink writing, an extrusion-based 3D printing method, is capable of patterning a much broader range of materials6-13. However, it is difficult to generate multimaterial voxelated matter by extruding monolithic cylindrical filaments in a layer-by-layer manner. Here we report the design and fabrication of voxelated soft matter using multimaterial multinozzle 3D (MM3D) printing, in which the composition, function and structure of the materials are programmed at the voxel scale. Our MM3D printheads exploit the diode-like behaviour that arises when multiple viscoelastic materials converge at a junction to enable seamless, high-frequency switching between up to eight different materials to create voxels with a volume approaching that of the nozzle diameter cubed. As exemplars, we fabricate a Miura origami pattern14 and a millipede-like soft robot that locomotes by co-printing multiple epoxy and silicone elastomer inks of stiffness varying by several orders of magnitude. Our method substantially broadens the palette of voxelated materials that can be designed and manufactured in complex motifs.
View details for DOI 10.1038/s41586-019-1736-8
View details for PubMedID 31723289
Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels.
2019; 5 (9): eaaw2459
Engineering organ-specific tissues for therapeutic applications is a grand challenge, requiring the fabrication and maintenance of densely cellular constructs composed of ~108 cells/ml. Organ building blocks (OBBs) composed of patient-specific-induced pluripotent stem cell-derived organoids offer a pathway to achieving tissues with the requisite cellular density, microarchitecture, and function. However, to date, scant attention has been devoted to their assembly into 3D tissue constructs. Here, we report a biomanufacturing method for assembling hundreds of thousands of these OBBs into living matrices with high cellular density into which perfusable vascular channels are introduced via embedded three-dimensional bioprinting. The OBB matrices exhibit the desired self-healing, viscoplastic behavior required for sacrificial writing into functional tissue (SWIFT). As an exemplar, we created a perfusable cardiac tissue that fuses and beats synchronously over a 7-day period. Our SWIFT biomanufacturing method enables the rapid assembly of perfusable patient- and organ-specific tissues at therapeutic scales.
View details for DOI 10.1126/sciadv.aaw2459
View details for PubMedID 31523707
View details for PubMedCentralID PMC6731072
In Vitro Human Tissues via Multi-material 3-D Bioprinting
ATLA-ALTERNATIVES TO LABORATORY ANIMALS
2018; 46 (4): 209–15
This paper highlights the foundational research on multi-material 3-D bioprinting of human tissues, for which the Lewis Bioprinting team at Harvard University was awarded the 2017 Lush Science Prize. The team's bioprinting platform enables the rapid fabrication of 3-D human tissues that contain all of the essential components found in their in vivo counterparts: cells, vasculature (or other tubular features) and extracellular matrix. The printed 3-D tissues are housed within a customised perfusion system and are subjected to controlled microphysiological environments over long durations (days to months). As exemplars, the team created a thick, stem cell-laden vascularised tissue that was controllably differentiated toward an osteogenic lineage in situ, and a 3-D kidney tissue that recapitulated the proximal tubule, a subunit of the nephron responsible for solute reabsorption. This highly versatile platform for manufacturing 3-D human tissue in vitro opens new avenues for replacing animal models used to develop next-generation therapies, test toxicity and study disease pathology.
View details for DOI 10.1177/026119291804600404
View details for Web of Science ID 000451747100003
View details for PubMedID 30365335
3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds.
Science (New York, N.Y.)
2018; 362 (6420): 1281–85
Lithographic nanofabrication is often limited to successive fabrication of two-dimensional (2D) layers. We present a strategy for the direct assembly of 3D nanomaterials consisting of metals, semiconductors, and biomolecules arranged in virtually any 3D geometry. We used hydrogels as scaffolds for volumetric deposition of materials at defined points in space. We then optically patterned these scaffolds in three dimensions, attached one or more functional materials, and then shrank and dehydrated them in a controlled way to achieve nanoscale feature sizes in a solid substrate. We demonstrate that our process, Implosion Fabrication (ImpFab), can directly write highly conductive, 3D silver nanostructures within an acrylic scaffold via volumetric silver deposition. Using ImpFab, we achieve resolutions in the tens of nanometers and complex, non-self-supporting 3D geometries of interest for optical metamaterials.
View details for DOI 10.1126/science.aau5119
View details for PubMedID 30545883
View details for PubMedCentralID PMC6423357
- Multi-photon microfabrication of three-dimensional capillary-scale vascular networks SPIE-INT SOC OPTICAL ENGINEERING. 2017
Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips
2016; 6: 34845
Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.
View details for DOI 10.1038/srep34845
View details for Web of Science ID 000385242900001
View details for PubMedID 27725720
View details for PubMedCentralID PMC5057112
Laser-assisted direct ink writing of planar and 3D metal architectures
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (22): 6137–42
The ability to pattern planar and freestanding 3D metallic architectures at the microscale would enable myriad applications, including flexible electronics, displays, sensors, and electrically small antennas. A 3D printing method is introduced that combines direct ink writing with a focused laser that locally anneals printed metallic features "on-the-fly." To optimize the nozzle-to-laser separation distance, the heat transfer along the printed silver wire is modeled as a function of printing speed, laser intensity, and pulse duration. Laser-assisted direct ink writing is used to pattern highly conductive, ductile metallic interconnects, springs, and freestanding spiral architectures on flexible and rigid substrates.
View details for DOI 10.1073/pnas.1525131113
View details for Web of Science ID 000376784600032
View details for PubMedID 27185932
View details for PubMedCentralID PMC4896727
Guided Homing of Cells in Multi-Photon Microfabricated Bioscaffolds
ADVANCED HEALTHCARE MATERIALS
2016; 5 (10): 1233–43
Tissues contain exquisite vascular microstructures, and patterns of chemical cues for directing cell migration, homing, and differentiation for organ development and function. 3D microfabrication by multi-photon photolithography is a flexible, high-resolution tool for generating 3D bioscaffolds. However, the combined fabrication of scaffold microstructure simultaneously with patterning of cues to create both geometrically and chemically defined microenvironments remains to be demonstrated. This study presents a high-speed method for micron-resolution fabrication of scaffold microstructure and patterning of protein cues simultaneously using native scaffold materials. By the simultaneous microfabrication of arbitrary microvasculature geometries, and patterning selected regions of the microvasculature with the homing ligand P-selectin, this study demonstrates adhesion, rolling, and selective homing of cells in defined 3D regions. This novel ability to generate high-resolution geometries replete with patterned cues at high speed enables the construction of biomimetic microenvironments for complex 3D assays of cellular behavior.
View details for DOI 10.1002/adhm.201600082
View details for Web of Science ID 000377528300015
View details for PubMedID 27059425
View details for PubMedCentralID PMC6070392
Three-dimensional bioprinting of thick vascularized tissues
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (12): 3179–84
The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.
View details for DOI 10.1073/pnas.1521342113
View details for Web of Science ID 000372488200037
View details for PubMedID 26951646
View details for PubMedCentralID PMC4812707