Three-Dimensional-Bioprinted Embedded-Based Cerebral Organoids: An Alternative Approach for Mini-Brain In Vitro Modeling Beyond Conventional Generation Methods

Abstract

Cerebral organoids (cORGs) obtained from induced pluripotent stem cells (iPSCs) have become significant instruments for investigating human neurophysiology, with the possibility of simulating diseases and enhancing drug discovery. The current approaches require a strict process of manual inclusion in animal-derived matrix Matrigel^® and are challenged by unpredictability, operators' skill and expertise, elevated costs, and restricted scalability, impeding their extensive applicability and translational potential. In this study, we present a novel method to generate brain organoids that address these limitations. Our approach does not require a manual, operator-dependent embedding. Instead, it employs a chemically defined hydrogel in which the Matrigel^® is diluted in a solution enriched with sodium alginate (SA) and sodium carboxymethylcellulose (CMC) and used as a bioink to print neural embryoid bodies (nEBs). Immunohistochemical, immunofluorescence, and gene expression analyses confirmed that SA-CMC-Matrigel^® hydrogel can sustain the generation of iPSC-derived cortical cORGs as the conventional Matrigel^®-based approach does. By day 40 of differentiation, hydrogel-based 3D-bioprinted cORGs showed heterogeneous and consistent masses, with a cytoarchitecture resembling an early-stage developmental fetal brain composed of neural progenitor cells PAX6⁺/Ki67⁺ organized into tubular structures, and densely packed cell somas with extensive neurites SYP⁺, suggestive of cortical tissue-like neuronal layer formation.

Keywords: 3D bioprinting; cerebral organoids; hydrogel; induced pluripotent stem cells; neural stem cells; tissue engineering.

Figure 1

(A) Schematic workflow of the Matrigel^®-embedded cORGs. (B) Representative images of the main steps of iPSC-derived cORG formation.

Figure 2

(A) Matrigel^®-embedded cORGs microscopic analysis: Hematoxylin and Eosin (H&E), synaptophysin (SYP), paired box 6 (PAX6), and Ki67. Top: magnification 14×, scale bar 200 µm; center: magnification 45×, scale bar 125 µm. (B) Quantitative PCR assay for OCT4, PAX6, SOX2, TUBB3, DCX, MAP2, and GFAP expression in Matrigel^®-embedded cORGs at day 40. Data were normalized on βACTIN and HSP90AB1, and calculated for the parental iPSC line. The bars represent the mean ± standard deviation (n = 3: BJ hiPSC line n = 2 and episomal hiPSC line n = 1). Student’s t-test, * p < 0.05.

Figure 3

(A) Degradation tests performed on SA-CMC-Matrigel^® hydrogel. (B) Viscosity tests performed on SA-CMC-Matrigel^® hydrogel, expressed as shear rate function.

Figure 4

(A) SA-CMC-Matrigel^® hydrogel 3D-printed samples. (B) Swelling ratio. (C) Diffusion tests: 700 ms exposure, 10× magnification, and fluorescence pictures of the bioinks at various times. (D) Diffusion tests: fluorescence intensity graph derived from normalized fluorescence pictures on materials that were not exposed to BSA.

Figure 5

(A) Schematic workflow of the 3D-bioprinted hydrogel-based cORGs. (B) Representative images of the main steps of iPSC-derived cORG formation.

Figure 6

(A) Microscopic analysis: Hematoxylin and Eosin (H&E), synaptophysin (SYP), paired box 6 (PAX6), and Ki67. Top: magnification 18×, scale bar 200 μm; bottom: magnification 45×, scale bar 125 μm. (B) Quantitative PCR assay for OCT4, PAX6, SOX2, TUBB3, DCX, MAP2, and GFAP expression in Matrigel^®-based cORGs at day 40. Data were normalized on βACTIN and HSP90AB1, and calculated for the parental iPSC line (black bars). The bars represent the mean ± standard deviation (n = 3: BJ hiPSC line n = 2 and episomal hiPSC line n = 1). Statistical analyses were performed using Student’s t-test, * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Figure 7

Representative images for Matrigel^®-based and 3D-bioprinted cORGs and immunofluorescence assay: double staining of synaptophysin (SYP) in green, and paired box 6 (PAX6) in red. Nuclei are stained with DAPI in blue. LEFT: magnification 18×, scale bar 200 µm; center: magnification 45×, scale bar 125 µm; right: magnification 150×, scale bar 40 µm.

Figure 8

Quantitative PCR assay for FOXG1, EGR2, FZD9, BCL11B, EMX1, TTR, and TBR1 expression in Matrigel^®-based and 3D-bioprinted cORGs at day 40. Data were normalized on βACTIN and HSP90AB1 and calculated for the parental iPSC line (black bars). The bars represent the mean ± standard deviation (n = 3: BJ hiPSC line n = 2 and episomal hiPSC line n = 1). Statistical analysis was performed using Student’s t-test. * Matrigel^®-based cORGs vs. hydrogel-based cORGs, # * Matrigel^®-based cORGs vs. iPSCs, ° hydrogel-based cORGs vs. iPSCs. *, #, and ° p < 0.05, ## p < 0.01.