Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications
Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of −80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.
1. Qiu B, Bessler N, Figler K, et al., 2020, Bioprinting Neural Systems to Model Central Nervous System Diseases. Adv Funct Mater, 30:1910250. https://doi.org/10.1002/adfm.201910250.
2. Soman SS, Vijayavenkataraman S, 2020, Perspectives on 3D Bioprinting of Peripheral Nerve Conduits. Int J Mol Sci, 21:5792. https://doi.org/10.3390/ijms21165792
3. Yu X, Zhang T, Li Y, 2020, 3D Printing and Bioprinting Nerve Conduits for Neural Tissue Engineering. Polymers (Basel), 12:1637. https://doi.org/10.3390/polym12081637
4. Gao F, Xu Z, Liang Q, et al., 2019, Osteochondral Regeneration with 3D-Printed Biodegradable High-Strength Supramolecular Polymer Reinforced-Gelatin Hydrogel Scaffolds. Adv Sci (Weinh), 6:1900867. https://doi.org/10.1002/advs.201900867
5. Soman SS, Vijayavenkataraman S, 2020, Applications of 3D Bioprinted-Induced Pluripotent Stem Cells in Healthcare. Int J Bioprint, 6:280. https://doi.org/10.18063/ijb.v6i4.280
6. Srubar WV 3rd, 2021, Engineered Living Materials: Taxonomies and Emerging Trends. Trends Biotechnol, 39:574-83. https://doi.org/10.1016/j.tibtech.2020.10.009
7. Lozano R, Stevens L, Thompson BC, et al., 2015, 3D printing of layered brain-like structures using peptide modified gellan gum substrates. Biomaterials, 67:264-73. https://doi.org/10.1016/j.biomaterials.2015.07.022
8. Joung D, Truong V, Neitzke CC, et al., 2018, 3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds. Adv Funct Mater, 28:1801850. https://doi.org/10.1002/adfm.201801850
9. Madhusudanan P, Raju G, Shankarappa S, 2020, Hydrogel systems and their role in neural tissue engineering. J R Soc Interface, 17:20190505. https://doi.org/10.1098/rsif.2019.0505
10. Bsoul A, Pan S, Cretu E, et al., 2016, Design, microfabrication, and characterization of a moulded PDMS/SU-8 inkjet dispenser for a Lab-on-a-Printer platform technology with disposable microfluidic chip. Lab Chip, 16:3351-61. https://doi.org/10.1039/c6lc00636a
11. Park S, Kim D, Park S, et al., 2018, Nanopatterned Scaffolds for Neural Tissue Engineering and Regenerative Medicine. Adv Exp Med Biol, 1078:421-43. https://doi.org/10.1007/978-981-13-0950-2_22
12. Shaqour B, Aizawa J, Guarch-Pérez C, et al., 2021, Coupling Additive Manufacturing with Hot Melt Extrusion Technologies to Validate a Ventilator-Associated Pneumonia Mouse Model. Pharmaceutics, 13:772. https://doi.org/10.3390/pharmaceutics13060772
13. Levato R, Jungst T, Scheuring RG, et al., 2020, From Shape to Function: The Next Step in Bioprinting. Adv Mater, 32:e1906423. https://doi.org/10.1002/adma.201906423
14. Moroni L, Burdick JA, Highley C, et al., 2018, Biofabrication strategies for 3D in vitro models and regenerative medicine. Nat Rev Mater, 3:21-37. https://doi.org/10.1038/s41578-018-0006-y
15. Ouyang L, Highley CB, Rodell CB, et al., 2016, 3D Printing of Shear-Thinning Hyaluronic Acid Hydrogels with Secondary Cross-Linking. ACS Biomater Sci Eng, 2:1743-51. https://doi.org/10.1021/acsbiomaterials.6b00158
16. Assuncao-Silva RC, Gomes ED, Sousa N, et al., 2015, Hydrogels and Cell Based Therapies in Spinal Cord Injury Regeneration. Stem Cells Int, 2015:948040. https://doi.org/10.1155/2015/948040
17. Stolberg S, McCloskey KE, 2009, Can shear stress direct stem cell fate? Biotechnol Prog, 25:10-9. https://doi.org/10.1002/btpr.124
18. Li C, Ouyang L, Armstrong JP, et al., 2021, Advances in the Fabrication of Biomaterials for Gradient Tissue Engineering. Trends Biotechnol, 39:150-64. https://doi.org/10.1016/j.tibtech.2020.06.005
19. De Santis MM, Alsafadi HN, Tas S, et al., 2021, Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue. Adv Mater, 33:e2005476. https://doi.org/10.1002/adma.202005476
20. Echeverria Molina MI, Malollari KG, Komvopoulos K, 2021, Design Challenges in Polymeric Scaffolds for Tissue Engineering. Front Bioeng Biotechnol, 9:617141. https://doi.org/10.3389/fbioe.2021.617141
21. He Y, Hou H, Wang S, et al., 2021, From waste of marine culture to natural patch in cardiac tissue engineering. Bioact Mater, 6:2000-10. https://doi.org/10.1016/j.bioactmat.2020.12.011
22. Dunlop MJ, Clemons C, Reiner R, et al., 2020, Towards the scalable isolation of cellulose nanocrystals from tunicates. Sci Rep, 10:19090. https://doi.org/10.1038/s41598-020-76144-9
23. Govindharaj M, Al Hashemi NS, Soman SS, et al., 2022, Bioprinting of bioactive tissue scaffolds from ecologically destructive fouling tunicates. J Clean Prod, 330:129923. https://doi.org/10.1016/j.jclepro.2021.129923
24. Zhu Q, Li M, Yan C, et al., 2017, Directed Differentiation of Human Embryonic Stem Cells to Neural Crest Stem Cells, Functional Peripheral Neurons, and Corneal Keratocytes. Biotechnol J, 12:67. https://doi.org/10.1002/biot.201700067
25. Vijayavenkataraman S, Kannan S, Cao T, et al., 2019, 3D-Printed PCL/PPy Conductive Scaffolds as Three-Dimensional Porous Nerve Guide Conduits (NGCs) for Peripheral Nerve Injury Repair. Front Bioeng Biotechnol, 7:266. https://doi.org/10.3389/fbioe.2019.00266
26. Athukoralalage SS, Balu R, Dutta NK, et al., 2019, 3D Bioprinted Nanocellulose-Based Hydrogels for Tissue Engineering Applications: A Brief Review. Polymers (Basel), 11:898. https://doi.org/10.3390/polym11050898
27. Altman GH, Horan RL, Martin I, et al., 2002, Cell differentiation by mechanical stress. FASEB J, 16:270-2. https://doi.org/10.1096/fj.01-0656fje
28. Duarte Campos DF, Lindsay CD, Roth JG, et al., 2020, Bioprinting Cell- and Spheroid-Laden Protein-Engineered Hydrogels as Tissue-on-Chip Platforms. Front Bioeng Biotechnol, 8:374. https://doi.org/10.3389/fbioe.2020.00374
29. Saldin LT, Cramer MC, Velankar SS, et al., 2017, Extracellular matrix hydrogels from decellularized tissues: Structure and function. Acta Biomater, 49:1-15. https://doi.org/10.1016/j.actbio.2016.11.068
30. Baena JM, Jiménez G, López-Ruiz E, et al., 2019, Volumeby-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration. Exp Biol Med (Maywood), 244:13-21. https://doi.org/10.1177/1535370218821128
31. Sharma R, Smits IP, De La Vega L, et al., 2020, 3D Bioprinting Pluripotent Stem Cell Derived Neural Tissues Using a Novel Fibrin Bioink Containing Drug Releasing Microspheres. Front Bioeng Biotechnol, 8:57. https://doi.org/10.3389/fbioe.2020.00057
32. Skylar-Scott MA, Uzel SG, Nam LL, et al., 2019, Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels. Sci Adv, 5:eaaw2459. https://doi.org/10.1126/sciadv.aaw2459
33. Bernal PN, Delrot P, Loterie D, et al., 2019, Volumetric Bioprinting of Complex Living-Tissue Constructs within Seconds. Adv Mater, 31:e1904209. https://doi.org/10.1002/adma.201904209
34. Keshavarz M, Wales DJ, Seichepine F, et al., 2020, Induced neural stem cell differentiation on a drawn fiber scaffold-toward peripheral nerve regeneration. Biomed Mater, 15:055011. https://doi.org/10.1088/1748-605X/ab8d12
35. Wang J, Kong X, Li Q, et al., 2021, The spatial arrangement of cells in a 3D-printed biomimetic spinal cord promotes directional differentiation and repairs the motor function after spinal cord injury. Biofabrication, 13:ac0c5f. https://doi.org/10.1088/1758-5090/ac0c5f
36. Wen Z, Zheng JQ, 2006, Directional guidance of nerve growth cones. Curr Opin Neurobiol, 16:52-8. https://doi.org/10.1016/j.conb.2005.12.005
37. Murphy SV, De Coppi P, Atala A, 2020, Opportunities and challenges of translational 3D bioprinting. Nat Biomed Eng, 4:370-80. https://doi.org/10.1038/s41551-019-0471-7
38. Vijayavenkataraman S, Yan WC, Lu WF, et al., 2018, 3D bioprinting of tissues and organs for regenerative medicine. Adv Drug Deliv Rev, 132:296-332. https://doi.org/10.1016/j.addr.2018.07.004