Biofabrication of HepG2 Cells-Laden 3D Structures Using Nanocellulose-Reinforced Gelatin-Based Hydrogel Bioinks: Materials Characterization, Cell Viability Assessment, and Metabolomic Analysis

abstract

The successful replication of the intricate architecture of human tissues remains a major challenge in the biomedical area. Three-dimensional (3D) bioprinting has emerged as a promising approach for the biofabrication of living tissue analogues, taking advantage of the use of adequate bioinks and printing methodologies. Here, a hydrogel bioink based on gelatin (Gel) and nanofibrillated cellulose (NFC), cross-linked with genipin, was developed for the 3D extrusion-based bioprinting of hepatocarcinoma cells (HepG2). This formulation combines the biological characteristics of Gel with the exceptional mechanical and rheological attributes of NFC. Gel/NFC ink formulations with different Gel/NFC mass compositions, viz., 90:10, 80:20, 70:30, and 60:40, were prepared and characterized. The corresponding cross-linked hydrogels were obtained using 1.5% (w/w) genipin as the cross-linking agent. The rheological and mechanical performances of the inks were enhanced by the addition of NFC, as evidenced by the rise in the yield stress from 70.9 +/- 28.6 to 627.9 +/- 74.8 Pa, compressive stress at 80% strain from 0.5 +/- 0.1 to 1.5 +/- 0.2 MPa, and Young's modulus from 4.7 +/- 0.9 to 12.1 +/- 1.1 MPa, for 90:10 and 60:40 inks, respectively. Moreover, higher NFC contents translated into 3D structures with better shape fidelity and the possibility of printing more intricate structures. These hydrogels were noncytotoxic toward HepG2 cells for up to 48 h, with cell viabilities consistently above 80%. The ink 70:30 was loaded with HepG2 cells (2 x 106 cells mL-1) and bioprinted. Cell viability remained elevated (90 +/- 4%) until day 14 postbioprinting, with cells maintaining their metabolic activity shown by 1H NMR metabolomics, proving the enormous potential of Gel/NFC-based bioinks for bioprinting HepG2 cells without jeopardizing their viability and metabolism.

keywords

GENIPIN; LIVER; SCAFFOLDS

subject category

Materials Science

authors

Lameirinhas, NS; Teixeira, MC; Carvalho, JPF; Valente, BFA; Luís, JL; Duarte, IF; Pinto, RJB; Oliveira, H; Oliveira, JM; Silvestre, AJD; Vilela, C; Freire, CSR

Groups

G4 - Renewable Materials and Circular Economy
G5 - Biomimetic, Biological and Living Materials

Projects

CICECO - Aveiro Institute of Materials (UIDB/50011/2020)

CICECO - Aveiro Institute of Materials (UIDP/50011/2020)

Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)

Rede Nacional de Ressonância Magnética Nuclear (PTNMR)

acknowledgements

This work was developed within the scope of the projects CICECO-Aveiro Institute of Materials, UIDB/50011/2020 (DOI 10.54499/UIDB/50011/2020), UIDP/50011/2020 (DOI 10.54499/UIDP/50011/2020) & LA/P/0006/2020 (DOI 10.54499/LA/P/0006/2020), LAQV-REQUIMTE (LA/P/0008/202, DOI 10.54499/LA/P/0008/2020; UIDP/50006/2020, DOI 10.54499/UIDP/50006/2020; UIDB/50006/2020, DOI10.54499/UIDB/50006/2020) and CESAM (UIDP/50017/2020 & UIDB/50017/2020 & LA/P/0094/2020), financed by national funds through the FCT/MEC (PIDDAC). It was also financially supported by the project I&D "NANOBIOINKS-Engineering bio-based nanofibers for the development of high-performance nanostructured bioinks for 3-D bioprinting, CENTRO-01-0145-FEDER-031289" funded by the Operational Program of the Center Region, in its FEDER/FNR component, and by national funds (OE), through FCT/MCTES. The NMR spectrometer is part of the National NMR Network (PTNMR) and is partially supported by Infrastructure Project no. 022161. FCT is acknowledged for the doctoral grants to N.S.L. (SFRH/BD/140229/2018) and J.P.F.C (2020.09018.BD) and the research contracts under Scientific Employment Stimulus to C.S.R.F. (CEECIND/00464/2017), C.V. (2021.01571.CEECIND), H.O. (DOI 10.54499/CEECIND/04050/2017/CP1459/CT0023), and I.F.D. (CEECIND/02387/2018). The research contract of R.J.B. Pinto was funded by national funds (OE), through FCT in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. Confocal fluorescence image acquisition was performed in the LiM facility of iBiMED, a node of PPBI (Portuguese Platform of BioImaging): POCI-01-0145-FEDER-022122.