Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells

Inês Pereira; Maria J Lopez-Martinez; Aranzazu Villasante; Clelia Introna; Daniel Tornero; Josep M Canals; Josep Samitier

doi:10.3389/fbioe.2023.1110547

Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells

Front Bioeng Biotechnol. 2023 Mar 3:11:1110547. doi: 10.3389/fbioe.2023.1110547. eCollection 2023.

Authors

Inês Pereira¹, Maria J Lopez-Martinez^{1

2

3}, Aranzazu Villasante^{1

2}, Clelia Introna^{4

5

6}, Daniel Tornero^{6

7}, Josep M Canals^{4

5

6}, Josep Samitier^{1

2

3}

Affiliations

¹ Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.
² Department of Electronic and Biomedical Engineering, University of Barcelona, Barcelona, Spain.
³ Biomedical Research Networking, Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain.
⁴ Laboratory of Stem Cells and Regenerative Medicine, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, Barcelona, Spain.
⁵ Creatio - Production and Validation Center of Advanced Therapies, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain.
⁶ Research Foundation Clinic Barcelona-August Pi i Sunyer Biomedical Research Institute (FRCB-IDIBAPS), Barcelona, Spain.
⁷ Laboratory of Neuronal Stem Cells and Cerebral Damage, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, Barcelona, Spain.

Abstract

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge. Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture. Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of β-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca²⁺ imaging.

Keywords: biomaterials; bioprinting; differentiation; in vitro models; neural progenitor cells; neuronal models.

Grants and funding

This work was supported by the Networking Biomedical Research Center (CIBER) of Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III (RD16/0006/0012), with the support of the European Regional Development Fund, CERCA Programme, and the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2021SGR015452), and the Spanish Ministry of Economy and Competitiveness (MINECO) through the projects AlterNED (PLEC 2022-009401) and NEUR-ON-A-CHIP (RTI 2018-097038-B-C21 and RTI 2018-097038-B-C22). This study was developed in the context of AdvanceCat and Base3D with the support of ACCIÓ (Catalonia Trade and Investment; Generalitat de Catalunya) under the Catalonian ERDF operational program (European Regional Development Fund) 2014–2020. The authors wish to acknowledge the MicroFabSpace and Microscopy Characterisation Facility, Unit 7 of ICTS “NANBIOSIS” from the CIBER-BBN at IBEC. IP and CI are fellows of the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Innovative Training Networks (ASCTN Training Network, grant agreement No. 813851). AV was supported by the Spanish Association Against Cancer (AECC), the Association of Families and Friends of Children with Neuroblastoma (NEN), and the Spanish National R + D + I plan PID 2020-117977RA-I00. DT was supported by the European Union’s Horizon 2020 research and innovation program under the grant agreement NEUChiP No. 964877. JC is supported by grants from the Ministerio de Ciencia e Innovación (PID 2021-126961OB-I00); Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades, and European Regional Development Fund (ERDF) (Red de Terapias Avanzadas, RD21/0017/0020); and Generalitat de Catalunya (2021SGR-01094), Spain; and European Union (ASCTN Training Network, grant agreement No. 813851).