Page 558 - IJB-10-6

International Journal of Bioprinting Internally-crosslinked ADA/Alg/Gel bioinks

2.7. In vitro biological characterization layered grid structures (10 × 10 mm ) with 2 mm strand
2
spacing were printed using a RegenHU-3DDiscovery
TM
2.7.1. Cell culture bioprinter (RegenHU, Switzerland) with optimized
AHCFs were purchased from Lonza (CC-2903; Lonza, parameters, including the printability window. Cell
Switzerland) and maintained in culture using Fibroblasts viability was investigated using the live/dead assay at 1
Growth Medium-3 (PromoCell, Germany) containing and 24 h post-printing by culturing constructs in complete
10% FBS, 1% (v/v) insulin, 1% (v/v) human basal fibroblast media at 37°C with 5% CO .
growth factor (hFGF-B), and 1% (v/v) gentamicin. Cells 2
were expanded until the fourth passage and then used for As a proof-of-concept for cardiac TE, bioprinting of
experiments. H9C2 heart myoblasts were purchased from ADA/Alg/Gel_50/25/25 embedding H9C2 was investigated.
the American Type Culture Collection (ATCC, USA) and Cultured H9C2 were detached using 0.05% Trypsin/EDTA
maintained in culture using DMEM, supplemented with and centrifuged. The cell pellet was resuspended in PBS
10% FBS, 2% L-glutamine, 1% sodium pyruvate, and and incorporated into bioinks at a concentration of 5 ×
5
1% penicillin/streptomycin. 10 cells/mL. Subsequently, two-layered grid structures
(10 × 10 mm ) with 5 mm strand spacing were printed
2
In vitro experiments with AHCFs were conducted in using the RegenHU-3DDiscovery bioprinter (RegenHU,
TM
complete medium composed of DMEM, supplemented with Switzerland) with optimized parameters, including the
10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin, printability window. Cell viability was investigated using
at 37°C in 5% CO atmosphere. In vitro experiments with the live/dead assay at 1 and 24 h post-printing by culturing
2
H9C2 were conducted in a complete medium composed of constructs in complete media at 37°C with 5% CO .
DMEM, supplemented with 10% FBS, 2% L-glutamine, 1% 2
sodium pyruvate, and 1% penicillin/streptomycin. 2.8. Statistical analysis
All measurements were made in triplicate, and data
2.7.2. Indirect cell viability and cytotoxicity assays are presented as the mean ± standard deviation. One-
In vitro cell viability by indirect contact with ADA/Alg/ way analysis of variance (ANOVA) was performed with
Gel_50/50/0, ADA/Alg/Gel_50/40/10, and ADA/Alg/ GraphPad Prism 6 software; differences were considered
Gel_50/25/25 hydrogels was studied using AHCFs following statistically significant at p < 0.05. Tukey post-hoc test
ISO 10993. AHCFs were seeded in a tissue culture 96-well was conducted between two or more groups to determine
at a cell density of 6 × 10 cells/well in a complete medium if there were statistically significant differences between
3
and maintained in a humified incubator at 37°C and 5% populations. In particular, *p < 0.05, **p < 0.01, ***p < 0.001,
CO . For each sample, hydrogels with 6.4 mm diameter and ****p < 0.0001.
2
and 2 mm thickness were prepared and incubated (37°C
and 5% CO ) with 500 μL medium per 100 mg hydrogel for 3. Results and discussion
2
24 h. Eluates were then collected and added to the AHCF
cultures. Then, AHCFs were cultured for 24 h at 37°C with This work aimed to develop novel bioinks based on a
5% CO . After 24 h, cell viability and cytotoxicity were combination of ADA with Alg and Gel, exploiting an
2
measured using Cell TiterBlue and CytoTox-ONE assay, internal crosslinking mechanism to achieve 3D-printed
TM
respectively. For cell viability, the control group consists of cell-supporting and self-standing constructs without
cells cultured in fresh complete medium. For cytotoxicity, the need for a support bath or external crosslinking.
the control group consists of cells cultured in fresh complete Alg-based hydrogels were selected, as Alg has already
medium and treated with CytoTox-ONE lysis solution been widely investigated for biomedical applications,
TM
(to achieve 100% cell death) according to manufacturer specifically in cardiac regeneration. 12,20 In this work,
instructions. Samples were analyzed using a Varioskan ADA was employed to improve the degradability of
spectrophotometer (Thermofisher, Italy) at excitation and Alg. Firstly, ADA was combined with Alg, and internal
emission wavelengths of 560 and 590 nm, respectively. crosslinking was exploited to achieve cardiac tissue-like
viscoelastic properties, tailoring both the ADA/Alg ratio
2.7.3. Cell printing and calcium ion (CaCO ) content. GDL content was then
3
Bioprinting of ADA/Alg/Gel_50/50/0, ADA/Alg/ optimized to reduce potential detrimental effects on cell
Gel_50/40/10, and ADA/Alg/Gel_50/25/25 hydrogels viability caused by environmental acidification. Then, the
embedding AHCFs was investigated. Briefly, cultured incorporation of Gel within the ADA/Alg hydrogels was
AHCFs were detached using 0.05% Trypsin/EDTA and optimized to improve cytocompatibility and impart cell
centrifuged. The cell pellet was resuspended in PBS and adhesive properties. Printability of ADA/Alg/Gel bioinks
incorporated into bioinks at a concentration of 5 × 10 was investigated over time to evaluate the effect of time-
5
cells/mL. Subsequently, for each bioink composition, two- dependent pH-triggered release of calcium ions. Finally,

Volume 10 Issue 6 (2024) 550 doi: 10.36922/ijb.4014