The development of 3D Cell-printing technology contributes to the application of tissue constructs in vitro in neuroscience. Collecting neural cells from patients is an efficient way of monitoring health of an individual target, which, in turn, benefits the enhancement of medicines. The fabricated sample of neural cells is exposed to potential drugs for the analysis of neuron responses. 3D cell-printing as an emerging biofabrication technology has been widely used to mimic natural 3D models in in vitro tissue research, especially in vitro brain-like tissue constructs in neuroscience. Fabricated brain-like tissue constructs provide a 3D microenvironment for primary neural cells to grow within. After more than several weeks of in vitro culturing, the formation of neural circuits in structures equips them with the capability of sensitively responding to a stimulus. In this study, an in vitro layered brain-like tissue construct is first proposed and later developed by 3D cell-printing technology. The layered structure is systematically analyzed, starting from printing parameter optimization to biological functionality performance. The optimized diameter of printing nozzle and printing speed are 0.51 mm and 5 µl s −1 , respectively, and the elastic modulus is approximately 6 kPa. Live/dead and immunostaining imaging is used to verify the growth of neural cells in the printed structure. The survival rate of neural cells in 2D and 3D samples is compared, and the results demonstrate that the 3D-printed structures exhibit a better artificial culturing environment and a higher survival rate. Both 2D and 3D samples are directly cultured in a 4 × 4 multiple electrode array. Local field potentials are collected and validated by the Med64 recording system. Tetrodotoxin is used to test the drug sensitivity of the printed structure, and the excitatory postsynaptic potential signals of the physiological performance indicate that the 3D-printed structure has great potential as a drug testing model in the pharmaypeceutical study.

In tissue engineering studies, scaffolds play a very important role in offering both physical and chemical cues for cell growth and tissue regeneration. However, in some cases, tissue regeneration requires scaffolds with high mechanical properties (e.g., bone and cartilage), while cells need a soft mechanical microenvironment. In this study, to mimic the heterogenous mechanical properties of a spinal cord tissue, a biomimetic rat tissue construct is fabricated. A collagen‐coated poly(lactic‐co‐glycolic acid) scaffold is manufactured using thermally induced phase separation casting. Primary rat neural cells (P01 Wistar rat cortex) with soft hydrogels are later printed within the scaffold using an image‐guided intrascaffold cell assembly technique. The scaffolds have unidirectional microporous structure with parallel axial macrochannels (260 ± 4 µm in diameter). Scaffolds showed mechanical properties similar to rat spine (ultimate tensile strength: 0.085 MPa, Young's modulus [stretch]: 0.31 MPa). The bioink composed of gelatin/alginate/fibrinogen is precisely printed into the macrochannels and showed mechanical properties suitable for neural cells (Young's modulus [compressive]: 3.814 kPa). Scaffold interface, cell viability, and immunostaining analyses show uniform distribution of stable, healthy, and elongated neural cells and neurites over 14 culture days in vitro. The results demonstrated that this method can serve as a valuable tool to aid manufacturing of tissue constructs requiring heterogenous mechanical properties for complex cell and/or biomolecule assembly.

For tissue engineering and regenerative medicine, creating thick and heterogeneous scaffold-based tissue constructs requires deep and precise multicellular deposition. Traditional cell seeding strategies lack the ability to create multicellular tissue constructs with high cell penetration and distribution, while emerging strategies aim to simultaneously combine cell-laden tissue segments with scaffold fabrication. Here we describe a technique that allows for three-dimensional (3D) intrascaffold cell assembly in which scaffolds are prefabricated and pretreated, followed by accurate cell distribution within the scaffold using an image-guided technique. This two-step process yields less limitation in scaffold material choice as well as additional treatments, provides accurate cell distribution, and has less potential to harm cells. The image processing technique captures a 2D geometric image of the scaffold, followed by a series of processes, mainly including grayscale transformation, threshold segmentation, and boundary extraction, to ultimately locate scaffold macropore centroids. Coupled with camera calibration data, accurate 3D cell assembly pathway plans can be made. Intrascaffold assembly parameter optimization and complex intrascaffold gradient, multidirectional, and vascular structure assembly were studied. Demonstration was also made with path planning and cell assembly experiments using NIH3T3-cell-laden hydrogels and collagen-coated poly­(lactic-co-glycolic acid) (PLGA) scaffolds. Experiments with CellTracker fluorescent monitoring, live/dead staining, and phalloidin–F-actin/DAPI immunostaining and comparison with two control groups (bioink manual injection and cell suspension static surface pipetting) showed accurate cell distribution and positioning and high cell viability (>93%). The PrestoBlue assay showed obvious cell proliferation over seven culture days in vitro. This technique provides an accurate method to aid simple and complex cell colonization with variant depth within 3D-scaffold-based constructs using multiple cells. The modular method can be used with any existing printing platform and shows potential in facilitating direct spatial organization and hierarchal 3D assembly of multiple cells and/or drugs within scaffolds for further tissue engineering studies and clinical applications.

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