3D Bioprinting: Bioink Selection Guide

What Is 3D Bioprinting?

3D bioprinting enables the generation of precisely controlled 3D cell models and tissue constructs, by engineering anatomically-shaped substrates with tissue-like complexity. Due to the high degree of control on structure and composition, 3D bioprinting has the potential to solve many critical unmet needs in medical research, including applications in cosmetics testing, drug discovery, regenerative medicine, and functional organ replacement.¹ Personalized models of disease can be created using patient-derived stem cells, such as induced pluripotent stem cells (iPS cells) or mesenchymal stem cells. Depending on the application, a range of materials, methods, and cells can be used to yield the desired tissue construct (Figure 1). For more in-depth information, including expert review articles on 3D Bioprinting, protocols, and related products, please explore our 3D Bioprinting Handbook.

Figure 1. 3D Bioprinting of tissue and organs. Bioinks are created by combining cultured cells and various biocompatable materials. Bioinks can then be 3D bioprinted into functional tissue constructs for drug screening, disease modeling, and in vitro transplantation.

What Are Bioinks?

Bioinks contain living cells and biomaterials that mimic the extracellular matrix environment, supporting cell adhesion, proliferation, and differentiation after printing. In contrast to tradtional 3D printing materials, bioinks must have:

• Print temperatures that do not exceed physiological temperatures
• Mild cross-linking or gelation conditions
• Bioative components that are non-toxic and able to be modified by the cells after printing

Bioinks for extrusion-based printing: Cell-encapsulating hydrogels

Cell-encapsulating hydrogels are used in 3D bioprinting to create living tissue structures by forming multicellular bioprinting building blocks. Cell encapsulation allows for precise control over cell attachment and the spatial distribution of the cells and biomolecules within the scaffold, in comparison to other methods and materials.¹ Combining multiple cell types and growth factors in a prescribed pattern allows for the generation of highly-complex tissue constructs.³ In addition to biocompatibility, bioprinting materials used  for cellular encapsulation must feaure high water content and porosity, allowing encapsulated cells to receive nutrients and remove waste.¹ As water-swollen, porous networks, hydrogels are ideal materials for cell-encapsulation, tissue engineering, and 3D bioprinting applications. Hydrogels for 3D bioprinting must also feature tunable substrate stiffness and allow for network remodeling post-printing, so cells can spread, migrate, proliferate, and interact.⁹ While a wide variety of materials are used for bioinks, the most popular materials include gelatin methacrylol (GelMA), collagen, poly(ethylene glycol) (PEG), Pluronic^®, alginate, and decellularized extracellular matrix (ECM)-based materials (Table 1).

Featured bioink material: Gelatin Methacryloyl (GelMA)

Gelatin MethacryloylGelatin methacryloyl (GelMA) can be used to form crosslinked hydrogels for tissue engineering and 3D printing. GelMA-based bioinks feature excellent cytocompatibility, tunable substrate stiffness, improved printability, and rapid crosslinking with exposure to UV or visible light (depending on the identity of the photoinitiator)¹¹. GelMA has been used in endothelial cell morphogenesis, cardiomyocytes, epidermal tissue, injectable tissue constructs, bone differentiation, and cartilage regeneration. Gelatin methacryloyl has also been used in microspheres and hydrogels for drug delivery applications.

Figure 2. Gelatin Methacryloyl

Acellular materials: Structural scaffolds and polymers

In addition to bioinks, acellular materials are also used in 3D bioprinted structures.² Acellular materials typically provide structural support for tissue constructs and when utilized with bioinks, can generate functional, bioprinted tissues. Acellular materials are porous structures that recapitulate both mechanical and biochemical properties of the native extracellular matrix (ECM)⁴. Porosity enables cell migration, tissue growth, vascular formation, and cell viability within these structural constructs.⁶  In addition, acellular materials must also have the necessary surface chemistry for cell attachment, proliferation, and differentiation.⁵ Popular acellular materials include: collagen, fibrin, chitosan, nanocellulose, poly(lactic acid) (PLA), polycaprolactone (PCL), hydroxyapatite (HA), and β-tricalcium phosphate (β-TCP) (Table 1).

Bioink Material Building Blocks

Table 1 Biomaterials commonly used in 3D bioprinting.

Bioink Material	Overview	Advantage	Disadvantage
Agarose	Polysaccharide extracted from seaweed	Non-toxic crosslinking High stability	Not degradable; Poor cell adhesion
Alginate	Naturally-derived biopolymer from brown algae	Mild crosslinking conditions (Ca2+) Rapid gelation High biocompatibility	Slow degradation kinetics; Poor cell adhesion
Chitosan	Polysaccharide obtained from the outer skeleton of shellfish (e.g. shrimp). Non-animal derived chitosan can be obtained from fungal fermentation.	High biocompatibility Antibacterial properties	Slow gelation rate
Collagen	Primary structural protein found in skin and other connective tissues	High biological relevance	Acid-soluble
Decellularized ECM	Isolated extracellular matrix of a tissue from inhabiting native cells	High biological relevance Tissue-specific High cell survival	Undefined and inconsistent; Loss of native ECM organization; Low stability
Fibrin/Fibrinogen	Insoluble protein formed during blood clotting	High biological relevance Rapid gelation	Limited printability
Gelatin	Protein substance derived from partial hydrolysis of collagen	High biocompatibility High water solubility Thermally reversible gelation	Poor shape fidelity; Limited rigidity
Graphene	Carbon-based material that can be viewed as a one atom thick sheet of graphite	Flexible Electrically-conductive	Low biological relevance
Hyaluronic Acid (HA)	Non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues.	Fast gelation Promotes cell proliferation	Poor stability
Hydroxyapatite	Naturally-occurring mineral form of calcium apatite found in teeth and bones	High strength and rigidity	Low printability; Limited tissue specificity
PCL/PLA/PLGA	Biodegradable, thermoplastic polymers and/or copolymers	High strength and rigidity	Low cell adhesion and proliferation
Pluronic® F127	Poly(ethylene oxide) and poly(propylene oxide) block copolymer	Printable at room temperatures Shear thinning material	Not suitable for long-term cell culture

What 3D Bioprinting Method Should Be Used?

Depending on the type of ink (bioink or acellular materials) selected and complexity of the final tissue construct, different 3D printing methods can be used (Figure 1). Advantages and disadvantages of common methods can be found in the table below (Table 2).

Table 2. Summary of 3D bioprinting methods.

Printing method	Advantages	Disadvantages	Cell compatible?
Extrusion-based	Printing speed and structures can be highly controlled	Shear stress can impact cell viability	Yes
Inkjet-based	Fast printing speed Compatible with biological components Low cost	Requires low viscosity materials	Yes
Stereolithography (SLA)	High resolution	Requires large amounts of material Long processing time Long printing times can decrease cell viability	Yes
Laser-based	Can be used with viscous materials Highly accurate	Heat generated by laser can impact cells	Yes
Fused-deposition modeling (FDM)	Yields highly porous structures	Materials must exhibit a molten phase Heat used to melt materials not compatible with cells Difficult to make complex geometries	No
Selective laser sintering (SLS)	Capable of making complex structures Yields better bonding between each printed layer	Heat generated by laser not compatible with cells	No

In addition to ink type, the bioprinting method can also be dictated by the end application of the printed construct (Table 3).

Tissue Engineering Applications

Table 3. 3D Bioprinting of tissue constructs.

Tissue Model	Cells Used	Bioprinter Used	Bioink Material Used	Reference
Cartilage	Mesenchymal Stem Cells	HP® Deskjet 500 printer	PEG diacrylate	Inkjet‐bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging
	iPS Cells/ Chondrocytes	3DDiscovery™	Alginate/ Nanocellulose	Cartilage tissue engineering by the 3D bioprinting of iPS cells in a nanocellulose/alginate bioink
	Chondrocytes	ITOP	PCL/Pluronic®	A 3D bioprinting system to produce human-scale tissue constructs with structural integrity
Bone	MC3T3-E1	In-House	Alginate/ Polyvinyl alcohol/ Hydroxyapatite	Development of a novel alginate-polyvinyl alcohol-hydroxyapatite hydrogel for 3D bioprinting bone tissue engineered scaffolds
	Mesenchymal Stem Cells	HP® Deskjet 500 printer	GelMA	Improved properties of bone and cartilage tissue from 3D inkjet-printed human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA
Skin	Mesenchymal Stem Cells	In-House	Fibrin/Collagen I	Bioprinted amniotic fluid‐derived stem cells accelerate healing of large skin wounds
	Keratinocytes/ Fibroblasts	In-House	Collagen I	Design and fabrication of human skin by three-dimensional bioprinting
Blood Vessel	HUVEC/HUVSMC/ Fibroblasts	In-House	Agarose	Scaffold-free vascular tissue engineering using bioprinting
Muscle	Muscle Derived Stem Cells	In-House	Fibrin	Microenvironments engineered by inkjet bioprinting spatially direct adult stem cells toward muscle‐ and bone‐like subpopulations
Brain	Neural Stem Cell	In-House	Polyurethane	3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair
Liver	iPS Cells	Nanolitre Dispensing System	Alginate-RGD	Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D
Lung	A549/HUVEC	BioFactory™	ECM Gel	Engineering an in vitro air-blood barrier by 3D bioprinting
Kidney	Immortalized PTECs/Primary RPTECs	In-House	Gelatin/Fibrin	Bioprinting of 3D convoluted renal proximal tubules on perfusable chips
Heart	HUVEC/Neonatal Cardiomyocytes	NovoGen MMX	Alginate/GelMA	Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip
	Mesenchymal Stem Cells/iPSC Derived Neurons	3D-BioPlotter®	Graphene/ PLGA	Three-Dimensional printing of high-content graphene scaffolds for electronic and biomedical applications

Conclusion

3D bioprinting allows for the spatially-controlled placement of cells in a defined 3D microenvironment. Bioinks are formed by combining cells and various biocompatible materials, which are subsequently printed in specific shapes to generate tissue-like, 3D structures. Combing our expertise in materials science and cell biology, we offer a variety of solutions to simplify the 3D bioprinting workflow.

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