Biofabrication strategies for 3D in vitro models and regenerative medicine

Abstract

Organs are complex systems composed of different cells, proteins and signalling molecules that are arranged in a highly ordered structure to orchestrate a myriad of functions in our body. Biofabrication strategies can be applied to engineer 3D tissue models in vitro by mimicking the structure and function of native tissue through the precise deposition and assembly of materials and cells. This approach allows the spatiotemporal control over cell-cell and cell-extracellular matrix communication and thus the recreation of tissue-like structures. In this Review, we examine biofabrication strategies for the construction of functional tissue replacements and organ models, focusing on the development of biomaterials, such as supramolecular and photosensitive materials, that can be processed using biofabrication techniques. We highlight bioprinted and bioassembled tissue models and survey biofabrication techniques for their potential to recreate complex tissue properties, such as shape, vasculature and specific functionalities. Finally, we discuss challenges, such as scalability and the foreign body response, and opportunities in the field and provide an outlook to the future of biofabrication in regenerative medicine.

Fig. 1 |. Bioprinting and bioassembly techniques.

a | Selective laser sintering creates scaffolds by scanning a powder bed with a laser beam and by locally sintering the hit grains. b | Stereolithography creates scaffolds by selectively exposing a photopolymer with a light source. c | 3D printing is used to fabricate scaffolds by ejecting a binder onto a powder bed of material. d,e | Fused deposition modelling and 3D plotting fabricate scaffolds by extruding a material (either in filament or pellet form) through a nozzle by pressure. f | Two-photon polymerization is applied to develop scaffolds through focusing a light source on a specific point within a biomaterial. g | Solution and melt electrospinning are used to produce fibrous structures from polymer melts and solutions by applying electric force. Panels a–c and f are adapted from Peltola, S. M. et al. A review of rapid prototyping techniques for tissue engineering purposes, Annals. of Medicine (2008) REF, by permission of Taylor & Francis Ltd. Panels e and g are adapted with permission from REF, Elsevier.

Fig. 2 |. Hydrogel bioprinting.

a | Cell-laden hydrogel scaffolds are created by applying laser light (laser-induced forward transfer) or by extrusion (inkjet printing with or without robotic dispension). b | Light-induced crosslinking strategies for the bioprinting of photocrosslinkable bioinks are shown. Crosslinking can be triggered before (pre-crosslink), after (post-crosslink) or during (in situ crosslink) extrusion. Panel a is adapted with permission from REF, John Wiley and Sons. Panel b is adapted with permission from REF, John Wiley and Sons.

Fig. 3 |. Bioassembly of tissue-like constructs.

Tubular, spherical and casquet-shaped tissue-like structures can be created by automated assembly of cellular spheroids or cell-laden hydrogel building blocks that fuse together because of tissue liquidity principles (cellular spheroids) or secondary interactions (cell-laden microgels). Reproduced with permission from REF, John Wiley and Sons.

Fig. 4 |. Bioprinting in support materials.

Self-healing hydrogels can be used as support media for bioprinting to enable the 3D fabrication of structures. a | A gelatin slurry can be used for the fabrication of hydrogel structures with large void spaces, for example, heart constructs. The hydrogel (green) is extruded and crosslinked within the gelatin slurry support (yellow). The 3D object is then released through melting of gelatin at 37 °C. Using this method, an embryonic chicken heart can be fabricated on the basis of a 3D computer-aided design model. The bioprinted tissue construct made using fluorescent alginate (green) exhibits the same internal trabecular structure as an embryonic chicken heart. b | A granular medium composed of carbomer microgels enables 3D printing of multiscale hierarchical structures, for example, continuous branched tubular networks of hollow vessels. The network of hollow tubes shown in the microscopy images was printed using polyvinyl alcohol, starting from a 25 mm diameter circular base and tapering to 27 capillaries with a diameter of 100 μm and a wall thickness of 100 μm. The insets show confocal cross sections. c | Hydrogels crosslinked by non-permanent, shear-thinning and self-healing bonds support the printing of high-resolution structures. Support material or printed ink can be removed after processing to produce complex structures that are freestanding or that contain voids and channels. The confocal microscopy images show a freestanding 3D tetrahedron made of photocrosslinked methacrylate-modified hyaluronic acid (blue), a rhodamine-labelled spherical structure (red) in an unlabelled support hydrogel and a fluorescein-labelled filament (green) with a rhodamine-labelled spiral (red) in an unlabelled support hydrogel. Panel a is adapted from REF, CC-BY-4.0. Panel b is adapted from REF, CC-BY-4.0. Panel c is adapted with permission from REF, John Wiley and Sons.

Fig. 5 |. Bioassembly of macroscopic tissue structures.

a | Cell-laden beads are assembled by moulding in poÌy(dimethyÌsiÌoxane). The microscopy image shows a human doll-shaped tissue made of fluorescently labelled fibroblasts (green) and collagen beads. b | Point-shaped cell-laden structures containing human epithelial cells transfected with green fluorescent protein and human embryonic kidney cells transfected with red fluorescent protein can be delivered and subsequently assembled by microfluidic flow, c | Optically induced dielectrophoretic force-based manipulation for the assembly of point-shaped cell-laden structures can also be used. The device consists of a top glass substrate with transparent and conductive indium oxide (ITO) coating, a working chamber and a bottom ITO glass substrate coated with a thin photoconductive hydrogenated amorphous silicon (a-Si:H) film. The microscopy image shows assembled point-shaped structures containing fibroblasts (green), human embryonic kidney cells (blue) and human metastatic mammary carcinoma cells (red). d | Assembly of line-shaped cell-laden structures is also used. Helical tubes are formed by reeling of a hepatocyte-laden and a fibroblast-laden fibre with a rod. A T-shirt-shaped structure is formed by weaving cell-laden fibres with fibroblasts (green), hepatocytes (red) and small lung carcinoma cells (blue). Blood vessellike structures can be fabricated by dissolving smooth muscle cell-laden and endothelial cell-laden alginate gel fibres in a collagen block. e | Assembly of plane-shaped cell-laden structures is also used. Cell-laden sheets are stacked by sandwiching a hepatocyte-laden sheet (green) between endothelial cell-laden sheets (red). Tubular structures are created by rolling of a cell-laden sheet containing endothelial cells (green), smooth muscle cells (blue) and fibroblasts (magenta). The tubular structure has multiple cell layers. Panel a is reproduced from REF, Macmillan Publishers Limited. Panel b is adapted with permission from REF, John Wiley and Sons. Panel c is adapted from REF, Macmillan Publishers Limited. Panel d is adapted with permission from REF, American Chemical Society Panel e is adapted with permission from REFS^,, John Wiley and Sons.

Fig. 6 |. 3D bioprinting of tissues and organs.

Biomedical applications based on design concept and printing resolution. Constructs of various shapes and sizes can be made: human-scale bone, ear-shaped and nose-shaped structures can be fabricated. At the level of tissue organization, cellular alignment can be achieved for skeletal and cardiac muscle constructs. Composite tissues, such as osteochondral (bone–cartilage) and musculotendinous (muscle–tendon), can be fabricated by sequentially patterning multiple components. Functional inner structures, such as microvasculature and nephrons, are required for whole organ bioprinting. The ear and skeletal muscle images are reproduced from REF, Macmillan Publishers Limited. The osteochondral tissue image is reproduced by permission from authors Francois Berthiaume and Jeffrey Morgan, 3D tissue engineering, Norwood, MA: Artech House, Inc., (2010). Copyright 2010 by Artech House, Inc. The musculotendinous tissue image is reproduced with permission from REF, IOP Publishing. The microvasculature image is reproduced with permission from REF, John Wiley and Sons. The nephron image is reproduced from REF, CC-BY-4.0.

Fig. 7 |. Stereolithography and continuous liquid interface production.

a | A 3D computer-aided design (CAD) file is first created for a given structure and then sliced. b | Continuous liquid interface production (CLIP) requires fewer steps than stereolithography (SLA) to assemble the designed structure. c The fabrication process includes placement of the build elevator on the resin, subsequent UV exposure to selectively cure the resin, separation of the cured resin from the oxygen-impermeable window, mechanical recoating of the resin and, finally, repositioning of the build elevator to repeat the process until the part is fully printed. CLIP uses a constant liquid interface enabled by an oxygen-permeable window, which eliminates the need for the last three steps. Adapted with permission from REF, Proceedings of the National Academy of Sciences.

Fig. 8 |. Bioacoustic levitation.

a | A densitometry platform for the bioacoustic levitation of cells is shown. b | Owing to magnetic induction (B) and gravity (g), cells are levitated in the channel and focused in a plane in which magnetic forces (F_mag) and buoyancy forces (F_b) are in equilibrium. The magnetic susceptibility of the medium (X_m) has to be substantially larger than the magnetic susceptibility of the cells (X_c), such that different cell types with different densities (tumour cells (TCs), white blood cells (WBCs) and red blood cells (RBCs)) can be separated. c A bioacoustic levitation bioprinting process to construct 3D neural constructs is shown. Neuroprogenitor cells in a fibrin hydrogel are placed in the levitation chamber. An acoustic ceramic generates incident waves, which coherently interfere with the waves reflected from the glass reflector, which is placed on top of the chamber. The resultant standing waves induce cells to levitate, resulting in 3D multilayer constructs of differentiated neural cells. Panels a and b are reproduced with permission from REF., Proceedings of the National Academy of Sciences. Panel c is adapted with permission from REF, John Wiley and Sons.