Bioink Composition and Printing Parameters for 3D Modeling Neural Tissue

Abstract

Neurodegenerative diseases (NDs) are a broad class of pathologies characterized by the progressive loss of neurons in the central nervous system. The main problem in the study of NDs is the lack of an adequate realistic experimental model to study the pathogenic mechanisms. Induced pluripotent stem cells (iPSCs) partially overcome the problem, with their capability to differentiate into almost every cell types; even so, these cells alone are not sufficient to unveil the mechanisms underlying NDs. 3D bioprinting allows to control the distribution of cells such as neurons, leading to the creation of a realistic in vitro model. In this work, we analyzed two biomaterials: sodium alginate and gelatin, and three different cell types: a neuroblastoma cell line (SH-SY5Y), iPSCs, and neural stem cells. All cells were encapsulated inside the bioink, printed and cultivated for at least seven days; they all presented good viability. We also evaluated the maintenance of the printed shape, opening the possibility to obtain a reliable in vitro neural tissue combining 3D bioprinting and iPSCs technology, optimizing the study of the degenerative processes that are still widely unknown.

Keywords: 3D bioprinting; 3D cell culture; bioink; cell culture; disease modeling; gelatin; iPSC; neural stem cell; neuroblastoma cell line; sodium alginate.

Figure 1

Timetable of the peripheral blood mononuclear cells (PBMCs) reprogramming protocol (in red) and of induced pluripotent stem cell (iPSC) differentiation in neural stem cells (NSCs) (in blue), with the characteristic checkpoints used in the protocol.

Figure 2

Repeatability tests using samples listed in Table 1: (a) CAD image of a grid with gap dimension and side dimension; (b) Dimension of side of samples shown in Table 1. Both sample 1 and sample 3 show a statistically significant difference compared to sample 4 (* p < 0.05). Dotted line indicates the reference value; (c) Dimension of gap of samples shown in Table 1. Sample 1 shows a statistically significant difference compared to sample 4 (* p < 0.05). Dotted line indicates the reference value. Error bars indicate SD. Data were analyzed by ANOVA (n = 9), followed by Newman-Keuls Multiple Comparison Test; * p < 0.05.

Figure 3

Viability test for SH-SY5Y cell line after 5 days of culture in the bioink. On the y-axis, the viability of cells is expressed as a percentage. Error bars indicate SD.

Figure 4

Crosslinked structure printed with Cellink INKREDIBLE+ (a) Crosslinked grid after 5 min in a bath with 2% CaCl₂ (diluted in distilled water); (b) Phase-contrast microscope image captured at 4× (EVOS XL Core Cell Imaging System, Thermo Fisher, USA) of a printed grid with Cellink INKREDIBLE+ bioplotter. The presence of bubbles in the bioink does not interfere with the stability of the 3D printed structures; (c) 10× magnification of phase-contrast image.

Figure 5

Immunofluorescence images of SH-SY5Y in 6% SA and 4% GEL. (a) Image of SH-SY5Y after 1 day of culture in 6% SA and 4% GEL, indicating the distribution of single cells. (b) The image shows the presence of several nuclei, indicating a good proliferation and a characteristically three-dimensional organization; GAPDH (green) labels cells and DAPI (blue) labels the nuclei; (b) Example of 3D reconstruction using ImageJ software (version 1.50i, NIH) of the confocal image: GAPDH (green) labels cells and DAPI (blue) labels the nuclei at day 5 of culture; (c–d) Reconstruction in three dimensions of a Z-stack obtained from Axio Imager 2 (Axiocam Mrm) in which cells were labeled with DAPI. The image shows the formation of two aggregation clusters to indicate cellular proliferation within 5 days of culture within bioink.

Figure 6

Phase-contrast image of PBMCs-derived iPSCs colonies after day 20, 27, and 30 after transduction. At day 20, cells started to grow adherent to vitronectin-coated plates, and organized themselves in colony, visible already at day 27. At day 30, cell reprogramming is evident: cells are organized in a well-defined perimeter lane, and they are concentrated into colonies. Cells that were not de-differentiated died because of the presence of a selective medium for iPSCs.

Figure 7

Characterization of 2D PBMCs reprogramming into iPSCs. (a) PCR of the viral RNA (SeV) and the three transgenes used during transduction: KOS, Klf4, and c-Myc. Sample at day 3 after transduction express all the transgenes, confirming the success of the transfection. (b,c) Immunofluorescence images of PBMCs-derived iPSCs using four stemness marker: SSEA4 and OCT4 (b), SOX2 and TRA1-60 (c), and DAPI (blue) labels the nuclei.

Figure 8

Relative cell viability of iPSCs encapsulated into 6% SA and 4% GEL bioink, at day 0, 3, and 7. The relative cell viability was expressed in percentage of day 0 (y-axis). Error bars indicate SD.

Figure 9

Characterization of iPSCs-derived NSC. (a) Phase-contrast image of NSC at day 3 of differentiation: cells show little cytoplasmic extension; (b) Phase-contrast image of NSC at day 10 of differentiation: cells were organized like half-moon shape (indicated with white arrow; (c) RT-qPCR of four genes: Nestin, SOX2, SOX1, and PAX6 conducted at day 7 since the beginning of the differentiation to neural stem cells; (d,e) Immunofluorescence images of iPSCs-derived NSC using four differentiation marker: Nestin and SOX2 (d), SOX1 and PAX6 (e), and DAPI (blue) labels the nuclei. Error bars indicate SD.

Figure 10

Relative cellular viability of NSCs encapsulated into 6% SA and 4% GEL bioink at day 0, 3, and 7. The relative cell viability is expressed as percentage (y-axis) of day 0. Both at day 3 and day 7, relative cell viability shows a statistically significant increase compared to day 0 (** p < 0.01; * p < 0.05, respectively). Error bars indicate SD. Data were analyzed by ANOVA (n = 3), followed by Newman–Keuls multiple comparison test; * p < 0.05 and ** p < 0.01.