In a recent study published on the bioRxiv* preprint server, researchers fabricated brain cortical tissues in three dimensions (3D) and functionally integrated them into a brain lesion in mice.
Study: Functional integration of 3D printed cerebral cortical tissue in a brain injury. Image credit: Andrus Ciprian/Shutterstock
background
Tissue engineering of cellularly diverse human tissues with desired cellular architectures and functions is challenging. The architecture of cerebral cortical tissues consists of layers of layer-specific neurons arranged in vertically oriented columns that provide high cognition through complex neural circuits. Approaches involving the implantation of brain organoids and neural progenitor cells (NPs) have shown limited success in fully restoring injured cranial tissues, as the implanted cells could not provide cellular anatomy similar to the human brain
About the study
In the present study, the researchers fabricated layered cerebral cortical tissues using droplet and piezoelectricity-based 3D printing and functionally integrated the tissues into a brain lesion in mice.
Human induced pluripotent stem cells (hiPSCs) were differentiated into top-layer NPs (UNPs) and deep-layer NPs (DNPs) and subsequently printed to form two-layer tissues of the cerebral cortex using the method droplet-based printing. The technique produced scaffold-free, structurally defined soft tissues comprising cells and the ECM (extracellular matrix).
The 3D-printed cells had undergone several processes for maturation, including terminal differentiation, process outgrowths, and cell migration. They were subsequently implanted into brain lesion explants of mice to monitor the integration of cellular structures for seven days. Layer-specific neuronal cells, red fluorescent protein (RFP)-labeled UNPs, and unlabeled DNPs were printed to form a 16x8x8 bilayer DRN (droplet network) with a width and height of 500 μm and 1000 μm, respectively, were layer printed. per layer to generate sub-mm-scale cubic structures and cm-scale structures of various shapes.
hiPSCs were reprogrammed from pluripotent somatic cells of a healthy individual and immunostained. A NIM (neural induction medium) containing SMAD (suppressor of maternal anti-decapentaplegic) inhibitors SB431542 and LDN193189 was used for the induction of hiPSC cells in neural ectoderm cells that were subsequently cultured in a NMM (neural maintenance medium) to allow the generation of NPs in vitro.
Cells were matured by seeding NPs and using a γ-secretase inhibitor (DAPT) containing NTM (neural terminal medium). NPs were treated with a cocktail of growth factors, such as EGF, FGF-2, and BDNF, similar to the Boissart protocol. In addition, RT-qPCR (quantitative real-time polymerase chain reaction) gene expression analysis was performed to confirm the identities of UN and DN cells.
Two 8x8x8 DRNs (one containing UNP and the other containing DNP) were printed simultaneously to obtain a 16x8x8 DRN. Host-implant integration was assessed based on the degree of process growth and neuronal migration from the implant site to the host site. The functionality of the implanted tissue was assessed from Ca2+ image analysis using Fluo-4, and calcium ion signals were recorded at the implant/explant interface to assess oscillatory correlations of calcium between the host and the implant.
results
Six-layered cortical structures that resemble the architecture of the human cerebral cortex could be produced using droplet-based 3D printing. NP cells differentiated between printed tissues during post-printing tissue cultures. Of interest, UNPs and DNPs continued to mature in the host, even though they were present in a common growth medium. Producing tissues that include different neuronal types without genetic manipulation reduces safety concerns and could be applied to the construction of tissues with cellular diversity.
Droplet-printed 3D implants could be designed with similar orientation, dimension, structure, and composition to injured/lost tissue, with 100 μm diameter droplets including cells and the ECM. Implantation of the printed and layered cerebral cortical tissues in mouse brain explants showed structural integration based on the growths of the implant-to-host process and neuronal migration and functional integration based on correlated Ca2+ oscillations with the host implant.
NPs mainly differentiated into deep layer neurons (DNs) expressing the deep layer biomarker CTIP2. Immunostained cells showed high pluripotency marker expression NANOG, OCT4 (octamer-binding transcription factor 4), and TRA-1-60 (T cell receptor alpha locus). The superior neuron (UN) morphologically resembled the deep layer neurons (DN); however, elevated expression of top layer markers such as BRN2 and cut-like homeobox (CUX) 1, 2 and the middle layer marker SATB2 (special AT-rich sequence binding protein 2) was observed.
Compared with hiPSCs, growth factor cocktail treatment significantly enhanced CUX1 expression in UNPs and UNs by 20- and 22-fold, respectively. Expression of the neural stem cell marker paired box protein (PAX6) was elevated among DNPs, indicating successful induction of cortical neurons. High expression of NESTIN (neuroepithelial stem cell protein), TUJ1 (beta tubulin class III), and SOX2 (sex-determining region Y-box 2) was observed among the cells, indicating that they were neuronal . High expression of the neuronal marker HNCAM (human neuronal cell adhesion molecule) was observed in the cells of the cortical layer, indicative of neurons of human origin.
conclusion
Overall, the findings of the study showed that the three-dimensional printing technique could be used to produce tissues with simplified double-layered cerebral cortical columns. The structure and identity of the upper and deeper layers were preserved in vitro after imprinting, and process outgrowths and neuronal migration of mature cells from the implant to the host were observed. Implantation of 3D-printed cortical tissue into mouse brain tissue explants showed functional and structural integration of the implant into the host.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.