Creation of bladder assembloids mimicking tissue regeneration and cancer.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

Use of inkjet-printed single cells to quantify intratumoral heterogeneity.

Quantification of intratumoral heterogeneity is essential for designing effective therapeutic strategies in the age of personalized medicine. In this study, we used a piezoelectric inkjet printer to enable analysis of intratumoral heterogeneity in a bladder cancer for the first time. Patient-derived tumor organoids were dissociated into single cell suspension and used as a bioink. The individual cells were precisely allocated into a microwell plate by drop-on-demand inkjet printing without any additive or treatment, followed by culturing into organoids for further analysis. The sizes and morphologies of the organoids were observed, so as the expression of proliferation and apoptotic markers. The tumor organoids also showed heterogeneous responses against chemotherapeutic agent. Further, we quantified mRNA expression levels of representative luminal and basal genes in both type of tumor organoids. These results verify the heterogeneous expression of various genes among individual organoids. This study demonstrates that the fully automated inkjet printing technique can be used as an effective tool to sort cells for evaluating intratumoral heterogeneity.

Immobilization of planktonic algal spores by inkjet printing.

The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.

Inkjet-Spray Hybrid Printing for 3D Freeform Fabrication of Multilayered Hydrogel Structures.

Here, a new bioprinting process by combining drop-on-demand inkjet printing with a spray-coating technique, which enables the high-resolution, high-speed, and freeform fabrication of large-scale cell-laden hydrogel structures is reported. Hydrogel structures with various shapes and composed of different materials, including alginate, cellulose nanofiber, and fibrinogen, are fabricated using the inkjet-spray printing. To manufacture cell-friendly hydrogel structures with controllable stiffness, gelatine methacryloyl is saponified to stabilize jet formation and is subsequently mixed with sodium alginate to prepare blend inks. The hydrogels crosslinked from the blend inks are characterized by assessing physical properties including the microstructure and mechanical stiffness and cellular responses including the cell viability, metabolic activity, and functionality of human dermal fibroblasts within the hydrogel. Cell-laden hydrogel structures are generated on a large scale and collagen type I secretion and spreading of cells within the hydrogels are assessed. The results demonstrate that the inkjet-spray printing system will ensure the formation of a cell-laden hydrogel structure with high shape fidelity in a rapid and reliable manner. Ultimately, the proposed printing technique and the blend bioink to be used to fabricate 3D laminated large-scale tissue equivalents that potentially mimic the function of native tissues is expected.

Drop-on-demand inkjet-based cell printing with 30-µm nozzle diameter for cell-level accuracy.

We present drop-on-demand inkjet-based mammalian cell printing with a 30-µm nozzle diameter for cell-level accuracy. High-speed imaging techniques have been used to analyze the go-and-stop movement of cells inside the nozzle under a pulsed pressure generated by a piezo-actuator and the jet formation after ejection. Patterning of an array of 20 × 20 dots on a glass substrate reveals that each printed drop contains 1.30 cells on average at the cell concentration of 5.0 × 106 cells ml-1 for the very small nozzle, whereas larger nozzles with the diameter of 50 and 80 µm deliver 2.57 and 2.88 cells per drop, respectively. The effects of the size and concentration of printed cells on the number of cells have also been investigated. Furthermore, the effect of the nozzle diameter on printed cells has been evaluated through an examination of viability, proliferation, and morphology of cells by using a live/dead assay kit, CCK-8 assay, and cellular morphology imaging, respectively. We believe that the 30-µm inkjet nozzle can be used for precise cell deposition without any damages to the printed mammalian cells.