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).

 

3D Bioprinting of tissue and organs

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.

Accellular materials: Structural scaffolds and polymers

In addition to bioinks, accellular materials are also used in 3D bioprinted structures.2 Accellular 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

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

Table 1. Biomaterials commonly used in 3D bioprinting.

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).

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

    Table 2. Summary of 3D bioprinting methods.

     

    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

    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/
    Polyinvl 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

    Table 3. 3D Bioprinting of tissue constructs.

    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, Sigma-Aldrich offers a variety of solutions to simplify the 3D bioprinting workflow.

    Legal information
    Pluronic® is a registered trademark of BASF
    3D-BioPlotter® is a registered trademark of EnvisionTec
    HP® is a registered trademark of Hewlett-Packard Inc.

     

    Materials

         

    References

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