3D bioprinting offers the opportunity to automate the process of tissue engineering, which combines biomaterial scaffolds and cells to generate substitutes for diseased or damaged tissues. These bioprinting methods construct tissue replacements by positioning cells encapsulated in bioinks into specific locations in the resulting constructs. Human induced pluripotent stem cells (hiPSCs) serve as an important tool when engineering neural tissues. These cells can be expanded indefinitely and differentiated into the cell types found in the central nervous systems, including neurons. One common method for differentiating hiPSCs into neural tissue requires the formation of aggregates inside of defined diameter microwells cultured in chemically defined media. However, 3D bioprinting of such hiPSC-derived aggregates has not been previously reported in the literature, as it requires the development of specialized bioinks for supporting cell survival and differentiation into mature neural phenotypes. Here we detail methods including preparing base material components of the bioink, producing the bioink, and the steps involved in printing 3D neural tissues derived from hiPSC-derived neural aggregates using Aspect Biosystems’ novel RX1 printer and their lab-on-a-printer (LOP) technology.

Most neurological diseases and disorders lack true cures, including spinal cord injury (SCI). Accordingly, current treatments only alleviate the symptoms of these neurological diseases and disorders. Engineered neural tissues derived from human induced pluripotent stem cells (hiPSCs) can serve as powerful tools to identify drug targets for treating such diseases and disorders. In this work, we demonstrate how hiPSC-derived neural progenitor cells (NPCs) can be bioprinted into defined structures using Aspect Biosystems’ novel RX1 bioprinter in combination with our unique fibrin-based bioink in rapid fashion as it takes under 5 min to print four tissues. This printing process preserves high levels of cell viability (>81%) and their differentiation capacity in comparison to less sophisticated bioprinting methods. These bioprinted neural tissues expressed the neuronal marker, βT-III (45 ± 20.9%), after 15 days of culture and markers associated with spinal cord (SC) motor neurons (MNs), such as Olig2 (68.8 ± 6.9%), and HB9 (99.6 ± 0.4%) as indicated by flow cytometry. The bioprinted neural tissues expressed the mature MN marker, ChaT, after 30 days of culture as indicated by immunocytochemistry. In conclusion, we have presented a novel method for high throughput production of mature hiPSC-derived neural tissues with defined structures that resemble those found in the SC.

No abstract

Saman Naghieh) and xbc719@mail.usask.ca (Xiongbiao Chen). AbstractLow-concentration hydrogels have favorable properties for many cell functions in tissue engineering but are considerably limited from a scaffold fabrication point of view due to poor three-dimensional (3D) printability. Here, we developed an indirect-bioprinting process for alginate scaffolds and characterized the potential of these scaffolds for nerve tissue engineering applications. The indirect-bioprinting process involves (1) printing a sacrificial framework from gelatin, (2) impregnating the framework with low-concentration alginate, and (3) removing the gelatin framework by an incubation process, thus forming low-concentration alginate scaffolds. The scaffolds were characterized by compression testing, swelling, degradation, and morphological and biological assessment of incorporated or seeded Schwann cells. For comparison, varying concentrations of alginate scaffolds (from 0.5 to 3%) were fabricated and sterilized using either ultraviolet light or ethanol. Results indicated that scaffolds can be fabricated using the indirect-bioprinting process, wherein the scaffold properties are affected by 5 the concentration of alginate and sterilization technique used. These factors provide effective means of regulating the properties of scaffolds fabricated using the indirect-bioprinting process.Cell-incorporated scaffolds demonstrated better cell viability than bulk gels. In addition, scaffolds showed better cell functionality when fabricated with a lower concentration of alginate compared to a higher concentration. The indirect-bioprinting process that we implemented could be extended to other types of low-concentration hydrogels to address the tradeoffs between printability and properties for favorable cell functions.

Low-concentration hydrogels have favorable properties for many cell functions in tissue engineering but are considerably limited from a scaffold fabrication point of view due to poor three-dimensional (3D) printability. Here, we developed an indirect-bioprinting process for alginate scaffolds and characterized the potential of these scaffolds for nerve tissue engineering applications. The indirect-bioprinting process involves (1) printing a sacrificial framework from gelatin, (2) impregnating the framework with low-concentration alginate, and (3) removing the gelatin framework by an incubation process, thus forming low-concentration alginate scaffolds. The scaffolds were characterized by compression testing, swelling, degradation, and morphological and biological assessment of incorporated or seeded Schwann cells. For comparison, varying concentrations of alginate scaffolds (from 0.5 to 3%) were fabricated and sterilized using either ultraviolet light or ethanol. Results indicated that scaffolds can be fabricated using the indirect-bioprinting process, wherein the scaffold properties are affected by the concentration of alginate and sterilization technique used. These factors provide effective means of regulating the properties of scaffolds fabricated using the indirect-bioprinting process. Cell-incorporated scaffolds demonstrated better cell viability than bulk gels. In addition, scaffolds showed better cell functionality when fabricated with a lower concentration of alginate compared to a higher concentration. The indirect-bioprinting process that we implemented could be extended to other types of low-concentration hydrogels to address the tradeoffs between printability and properties for favorable cell functions.