A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton's jelly mesenchymal stromal cells for cell therapy and tissue engineering

Cristina Blanco-Elices; Roke Iñaki Oruezabal; David Sánchez-Porras; Jesús Chato-Astrain; Fernando Campos; Miguel Alaminos; Ingrid Garzón; Antonio Campos

doi:10.3389/fbioe.2023.1235161

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton's jelly mesenchymal stromal cells for cell therapy and tissue engineering

Front Bioeng Biotechnol. 2023 Aug 10:11:1235161. doi: 10.3389/fbioe.2023.1235161. eCollection 2023.

Authors

Cristina Blanco-Elices^{1

2}, Roke Iñaki Oruezabal³, David Sánchez-Porras^{1

2}, Jesús Chato-Astrain^{1

2}, Fernando Campos^{1

2}, Miguel Alaminos^{1

2}, Ingrid Garzón^{1

2}, Antonio Campos^{1

2}

Affiliations

¹ Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain.
² Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain.
³ Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

Abstract

Purpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells. Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton's jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D). Results: Our results showed a significant increase in the number of cells generated after 7 days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules. Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures.

Keywords: biofabrication; cell culture; cell therapy; human Wharton’s jelly mesenchymal stromal cells; three-dimensional; tissue engineering.

Grants and funding

Supported by grant PSETC-19-001 (Fibrigar3D) by Plan Propio de Investigación y Transferencia 2019, Universidad de Granada, Spain. Supported by Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation, Plan Estatal de Investigación Científica y Técnica y de Innovación (I+D+i), grants FIS PI18/0331, FIS PI21/0980, FIS PI22/0059, and FIS PI20/0317 and co-funded by FEDER funds, European Union, Una manera de hacer Europa. Supported by grant PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain and grant B-CTS-450-UGR20 (Programa Operativo FEDER Andalucía 2014–2020, University of Granada and Consejería de Transformación Económica, Industria, Conocimiento y Universidades). Cofinanced by the European Regional Development Fund (ERDF) through the “Una manera de hacer Europa” program.