Advanced Polymers for Three-Dimensional (3D) Organ Bioprinting

Abstract

Three-dimensional (3D) organ bioprinting is an attractive scientific area with huge commercial profit, which could solve all the serious bottleneck problems for allograft transplantation, high-throughput drug screening, and pathological analysis. Integrating multiple heterogeneous adult cell types and/or stem cells along with other biomaterials (e.g., polymers, bioactive agents, or biomolecules) to make 3D constructs functional is one of the core issues for 3D bioprinting of bioartificial organs. Both natural and synthetic polymers play essential and ubiquitous roles for hierarchical vascular and neural network formation in 3D printed constructs based on their specific physical, chemical, biological, and physiological properties. In this article, several advanced polymers with excellent biocompatibility, biodegradability, 3D printability, and structural stability are reviewed. The challenges and perspectives of polymers for rapid manufacturing of complex organs, such as the liver, heart, kidney, lung, breast, and brain, are outlined.

Keywords: biomaterials; organ manufacturing; polymers; stem cells; three-dimensional (3D) printing.

Figure 1

Different levels of materials (or molecules) existing in the human body, from small organic molecules or compounds to larger polymers (i.e., macromolecules or biomacromolecules), cells, tissues, organs, and systems in organisms.

Figure 2

Relationships of organ manufacturing, including three-dimensional (3D) bioprinting, with other pertinent fields of science and technology.

Figure 3

Graphical description of 3D bioprinting types [10,11,12,13,14,15,16,17,18,19,20]. Images reproduced with permission from [10,11,12,13,14,15,16,17,18,19,20].

Figure 4

Polymers that have been used for tissue and 3D organ printing.

Figure 5

Basic requirements for selecting a polymer for 3D bioprinting of bioartificial organs.

Figure 6

3D bioprinting of chondrocytes, cardiomyocytes, hepatocytes, and adipose-derived stem cells (ASCs) into living tissues/organs using a pioneering 3D bioprinter made in Prof. Wang’s laboratory at Tsinghua University: (a) the pioneering 3D bioprinter; (b) schematic description of a cell-laden gelatin-based hydrogel being printed into an one-layer grid lattice using the 3D bioprinter; (c) schematic description of the cell-laden gelatin-based hydrogel being printed into large-scale 3D construct using the 3D bioprinter; (d) 3D printing process of a chondrocyte-laden gelatin-based construct; (e) a grid 3D construct made from a cardiomyocyte-laden gelatin-based hydrogel; (f) hepatocytes encapsulated in a gelatin-based hydrogel after 3D printing; (g) hepatocytes in a gelatin-based hydrogel after 3D printing; (h) a gelatin-based hydrogel after 3D printing; (i) hepatocytes in a 3D printed construct after a certain period of in vitro culture; (j) a magnified photo of (i); (k) the shape of the hepatocyte-laden grid construct maintained well after certain period of in vitro culture; (l) a magnified photo of (k), showing hepatocytes formed vortex in the hydrogel; (m) hepatocytes in a 3D printed construct after a longer period of in vitro culture; (n) a magnified photo of (m), showing the vortex structure was still there; (o) immunostaining of the hepatocyte-laden 3D construct after certain period of in vitro culture, showing new hepatic tissue formed in the gelatin-based hydrogel with close cell-cell connection or tight junction; (p) a dark-field microscopy of (o). Images reproduced with permission from [7,22].

Figure 7

A large-scale 3D printed bioartificial organ with vascularized liver tissue constructed through the double-nozzle 3D bioprinter created in Prof. Wang’s laboratory at Tsinghua University: (a) the double-nozzle 3D bioprinter; (b) a computer-aided design (CAD) model containing a branched vascular network; (c) a CAD model containing the branched vascular network; (d) a few layers of the 3D bioprinted construct containing both ASCs encapsulated in a gelatin/alginate/fibrin hydrogel and hepatocytes encapsulated in a gelatin/alginate/chitosan hydrogel; (e–h) 3D printing process of a semielliptical construct containing both ASCs and hepatocytes encapsulated in different hydrogels; (i–l) hepatocytes encapsulated in the gelatin-based hydrogels after 3D bioprinting and different periods of in vitro cultures; (m–p) ASCs encapsulated in the gelatin-based hydrogels after 3D bioprinting and different periods of in vitro cultures as well as growth factor inductions. Images reproduced with permission from [15,32].

Figure 8

3D bioprinting of ASC-laden gelatin/alginate/fibrin hydrogel for organ manufacturing in Prof. Wang’s laboratory at Tsinghua University: (a) a pioneering double-nozzle 3D bioprinter made in this laboratory; (b) schematic description of the cell-laden gelatin/alginate/fibrin hydrogel and pancreatic islets being printed into a grid construct using the 3D bioprinter; (c) a large-scale 3D printed grid construct containing ASC-laden gelatin/alginate/fibrin hydrogel cultured in a plate; (d) a grid ASC-laden gelatin/alginate/fibrin construct after being cultured for one month; (e) a multicellular construct after three weeks of culture, containing both ASCs encapsulated in the gelatin/alginate/fibrin hydrogel before epidermal growth factor (EGF) engagement and relatively integrated pancreatic islets seeding in the predefined channels (immunostaining with anti-insulin in green); (f) some envelopes of the islets were broken after one month of culture; (g) immunostaining of the 3D construct with mAbs for CD31⁺ cells (i.e., mature endothelial cells from the ASC differentiation after three days of culture with EGF added in the culture medium) in green, having a fully confluent layer of endothelial cells (i.e., endothelium) on the surface of the predefined channels; (h) a vertical image of the 3D construct showing the fully confluent endothelium (formed from endothelial cells) and the predefined go-through channels; (i) immunostaining of the 3D construct with mAbs for CD34⁺ cells (i.e., endothelial cells) in green and pyrindine (PI) for cell nuclei (nucleus) in red; (j) immunostaining of the 3D construct with mAbs for CD34⁺ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium; (k) immunostaining of the 3D construct with mAbs for CD31⁺ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture with EGF added in the culture medium; (l) a control of (k), immunostaining of the 3D construct with mAbs for CD31⁺ endothelial cells differentiated from the ASCs in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium; (m) immunostaining of the 3D construct with mAbs for CD31⁺ cells in green and Oil Red O staining for adipocytes in red, showing both the heterogeneous tissues coming from the ASC differentiation after a cocktail growth factor engagement (i.e., on the surface of the channels, the endothelium coming from the ASCs differentiation after being treated with EGF for 3 days; deep inside the gelatin/alginate/fibrin hydrogel, the adipose tissue coming from the ASC differentiation after being subsequently treated with insulin, dexamethasone, and isobutylmethylxanthine (IBMX) for another three days); (n) a control of (m), showing all the ASCs in the 3D construct differentiated into target adipose tissue after three days of treatment with insulin, dexamethasone, and IBMX but no EGF; (o) immunostaining of two-dimensional (2D) cultured ASCs with mAbs for CD31⁺ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture with EGF added in the culture medium; (p) immunostaining of 2D cultured ASCs with mAbs for CD31⁺ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium. Images reproduced with permission from [36].

Figure 9

A combined four-nozzle 3D organ bioprinting technology created in Prof. Wang’s laboratory at Tsinghua University in 2013 [20,50]: (a) equipment of the combined four-nozzle 3D organ bioprinter; (b) working state of the combined four-nozzle 3D organ printer; (c) a CAD model representing a large-scale vascularized and innervated hepatic tissue; (d) a semielliptical 3D construct containing a poly(lactic-co-glycolic acid) (PLGA) overcoat, a hepatic tissue made from hepatocytes in a gelatin/chitosan hydrogel, a branched vascular network with fully confluent endothelialized ASCs on the inner surface of the gelatin/alginate/fibrin hydrogel, and a hierarchical neural (or innervated) network made from Shwann cells in the gelatin/hyaluronate hydrogel; the maximal diameter of the semiellipse can be adjusted from 1 mm to 2 cm according to the CAD model; (e) a cross section of (d), showing the endothelialized ASCs and Schwann cells around a branched channel; (f) a large bundle of nerve fibers formed in (d); (g) hepatocytes underneath the PLGA overcoat; (h) an interface between the endothelialized ASCs and Schwann cells in (d); (i) some thin nerve fibers.

Figure 10

A large-scale 3D printed complex organ with vascularized liver tissue constructed through the double-nozzle low-temperature 3D bioprinter created in Prof. Wang’s laboratory at Tsinghua University: (a) the double-nozzle low-temperature 3D bioprinter; (b) working principle of an elliptical hybrid hierarchical polyurethane-cell/hydrogel construct built via the double-nozzle low-temperature 3D bioprinter; (c) a CAD model containing the branched vascular network; (d) a cross section of the CAD model containing five sub-branched channels; (e) working platform of the 3D bioprinter containing two nozzles and the 3D printed semielliptical hybrid hierarchical polyurethane-cell/hydrogel construct; (f) an elliptical sample containing both a cell-laden natural hydrogel and a synthetic polyurethane (PU) overcoat; (g) several layers of the elliptical sample in the middle section containing a hepatocyte-laden gelatin-based hydrogel and a PU overcoat; (h) several layers of the elliptical sample in the middle section containing an ASC-laden gelatin-based hydrogel and a PU overcoat; (i) hepatocytes encapsulated in the gelatin-based hydrogel; (j) a magnified photo of (i), showing the alginate/fibrin fibers around the hepatocytes; (k) ASCs encapsulated in the gelatin-based hydrogel growing into the micropores of the PU layer; (l) ASCs on the inner surface of the branched channels; (m) pulsatile culture of two elliptical samples; (n) two samples cultured in the bioreactor; (o) static culture of the ASCs encapsulated in the gelatin-based hydrogel; (p) pulsatile culture of the ASCs encapsulated in the gelatin-based hydrogel. Images reproduced with permission from [28,49].