3D-printed airway model as a platform for SARS-CoV-2 infection and antiviral drug testing.

We present a bioprinted three-layered airway model with a physiologically relevant microstructure for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection dynamics. This model exhibited clear cell-cell junctions and mucus secretion with an efficient expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Having infected air-exposed epithelial cells in the upper layer with a minimum multiplicity of infection of 0.01, the airway model showed a marked susceptibility to SARS-CoV-2 within one-day post-infection (dpi). Furthermore, the unique longevity allowed the observation of cytopathic effects and barrier degradation for 21 dpi. The in-depth transcriptomic analysis revealed dramatic changes in gene expression affecting the infection pathway, viral proliferation, and host immune response which are consistent with COVID-19 patient data. Finally, the treatment of antiviral agents, such as remdesivir and molnupiravir, through the culture medium underlying the endothelium resulted in a marked inhibition of viral replication within the epithelium. The bioprinted airway model can be used as a manufacturable physiological platform to study disease pathogeneses and drug efficacy.

3D Inkjet-Bioprinted Lung-on-a-Chip.

There is an urgent need for physiologically relevant and customizable biochip models of human lung tissue to provide a niche for lung disease modeling and drug efficacy. Although various lung-on-a-chips have been developed, the conventional fabrication method has been limited in reconstituting a very thin and multilayered architecture and spatial arrangements of multiple cell types in a microfluidic device. To overcome these limitations, we developed a physiologically relevant human alveolar lung-on-a-chip model, effectively integrated with an inkjet-printed, micron-thick, and three-layered tissue. After bioprinting lung tissues inside four culture inserts layer-by-layer, the inserts are implanted into a biochip that supplies a flow of culture medium. This modular implantation procedure enables the formation of a lung-on-a-chip to facilitate the culture of 3D-structured inkjet-bioprinted lung models under perfusion at the air-liquid interface. The bioprinted models cultured on the chip maintained their structure with three layers of tens of micrometers and achieved a tight junction in the epithelial layer, the critical properties of an alveolar barrier. The upregulation of genes involved in the essential functions of alveoli was also confirmed in our model. Our culture insert-mountable organ-on-a-chip is a versatile platform that can be applied to various organ models by implanting and replacing culture inserts. It is amenable to mass production and the development of customized models through the convergence with bioprinting technology.

Transfer-Tattoo-Like Cell-Sheet Delivery Induced by Interfacial Cell Migration.

A direct transfer of a cell sheet from a culture surface to a target tissue is introduced. Commercially available, flexible parylene is used as the culture surface, and it is proposed that the UV-treated parylene offers adequate and intermediate levels of cell adhesiveness for both the stable cell attachment during culture and for the efficient cell transfer to a target surface. The versatility of this cell-transfer process is demonstrated with various cell types, including MRC-5, HDFn, HULEC-5a, MC3T3-E1, A549, C2C12 cells, and MDCK-II cells. The novel cell-sheet engineering is based on a mechanism of interfacial cell migration between two surfaces with different adhesion preferences. Monitoring of cytoskeletal dynamics and drug treatments during the cell-transfer process reveals that the interfacial cell migration occurs by utilizing the existing transmembrane proteins on the cell surface to bind to the targeted surface. The re-establishment and reversal of cell polarity after the transfer process are also identified. Its unique capabilities of 3D multilayer stacking, freeform design, and curved surface application are demonstrated. Finally, the therapeutic potential of the cell-sheet delivery system is demonstrated by applying it to cutaneous wound healing and skin-tissue regeneration in mice models.

3D pulmonary fibrosis model for anti-fibrotic drug discovery by inkjet-bioprinting.

Pulmonary fibrosis (PF) is known as a chronic and irreversible disease characterized by excessive extracellular matrix accumulation and lung architecture changes. Large efforts have been made to develop prospective treatments and study the etiology of pulmonary fibrotic diseases utilizing animal models and spherical organoids. As part of these efforts, we created an all-inkjet-printed three-dimensional (3D) alveolar barrier model that can be used for anti-fibrotic drug discovery. Then, we developed a PF model by treating the 3D alveolar barrier with pro-fibrotic cytokine and confirmed that it is suitable for the fibrosis model by observing changes in structural deposition, pulmonary function, epithelial-mesenchymal transition, and fibrosis markers. The model was tested with two approved anti-fibrotic drugs, and we could observe that the symptoms in the disease model were alleviated. Consequently, structural abnormalities and changes in mRNA expression were found in the induced fibrosis model, which were shown to be recovered in all drug treatment groups. The all-inkjet-printed alveolar barrier model was reproducible for disease onset and therapeutic effects in the human body. This finding emphasized that thein vitroartificial tissue with faithfully implemented 3D microstructures using bioprinting technology may be employed as a novel testing platform and disease model to evaluate potential drug efficacy.

Effect of Aronia Extract on Collagen Synthesis in Human Skin Cell and Dermal Equivalent.

The regulation of collagen synthesis, which occurs in fibroblasts in the dermal layer, is a key process in dermis regeneration and skin reconstruction. Herein, we investigated whether Aronia melanocarpa extract affects the human skin condition. We focused on type I collagen synthesis using two different types of model systems: a monolayer of cells and a bioprinted 3D dermal equivalent. The Aronia extract showed no cytotoxicity and increased cell proliferation in neonatal human dermal fibroblasts. Treatment with Aronia extract increased the transcription of COL1A1 mRNA in direct proportion to the extract concentration without causing a decrease in COL1A1 mRNA degradation. Additionally, the Aronia extract inhibited the expression of MMP1 and MMP3, and an increase in type I collagen was observed along with a decrease in MMP1 protein. We also fabricated dermal equivalents from type I collagen (the major component of the dermis) and dermal fibroblasts by bioprinting. In the 3D dermis model, the compressive modulus directly affected by collagen synthesis increased in direct proportion to the Aronia extract concentration, and expression levels of MMP1 and MMP3 decreased in exactly inverse proportion to its concentration. The findings that the Aronia extract increases synthesis of type I collagen and decreases MMP1 and MMP3 expression suggest that this extract may be useful for the treatment of damaged or aged skin.

All-Inkjet-Printed 3D Alveolar Barrier Model with Physiologically Relevant Microarchitecture.

With the outbreak of new respiratory viruses and high mortality rates of pulmonary diseases, physiologically relevant models of human respiratory system are urgently needed to study disease pathogenesis, drug efficacy, and pharmaceutics. In this paper, a 3D alveolar barrier model fabricated by printing four human alveolar cell lines, namely, type I and II alveolar cells (NCI-H1703 and NCI-H441), lung fibroblasts (MRC5), and lung microvascular endothelial cells (HULEC-5a) is presented. Automated high-resolution deposition of alveolar cells by drop-on-demand inkjet printing enables to fabricate a three-layered alveolar barrier model with an unprecedented thickness of ≈10 µm. The results show that the 3D structured model better recapitulate the structure, morphologies, and functions of the lung tissue, compared not only to a conventional 2D cell culture model, as expected, but also a 3D non-structured model of a homogeneous mixture of the alveolar cells and collagen. Finally, it is demonstrated that this thin multilayered model reproduce practical tissue-level responses to influenza infection. Drop-on-demand inkjet-printing is an enabling technology for customization, scalable manufacturing, and standardization of their size and growth, and it is believed that this 3D alveolar barrier model can be used as an alternative to traditional test models for pathological and pharmaceutical applications.

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.

Self-Organization of Fibroblast-Laden 3D Collagen Microstructures from Inkjet-Printed Cell Patterns.

One of the major challenges encountered in engineering complex tissues in vitro is to increase levels of complexity at the micron scale in 3D structures. Here, a strategy to create self-organized 3D collagen microstructures by 2D micropatterning of fibroblasts is developed. Drop-on-demand inkjet printing is used to pattern fibroblast cells on a collagen substrate in pre-designed patterns and with controlled density. It is found that cell-to-ECM interaction promotes cellular self-organization of 3D microstructures on collagen hydrogel, whereas the formation of 3D microstructure is inhibited by disruption of actin polymerization. Using this phenomena, the controlled sizes and morphologies of the 3D collagen microstructures is demonstrated by manipulating the designs of cell patterns and the density of cells. Finally, this technique is applied to build a human skin model with papillary microstructures at the dermo-epidermal junction. This approach to create 3D cell-laden collagen microstructures by cell patterning provides a simple and powerful way to mimic the structures and functions of complex tissues and organs, and can make a contribution to reduce the gap between the human body and in vitro tissue models.

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.

Heterogeneous nuclear ribonucleoprotein A1 regulates rhythmic synthesis of mouse Nfil3 protein via IRES-mediated translation.

Nuclear factor, interleukin 3, regulated (Nfil3, also known as E4 Promoter-Binding Protein 4 (E4BP4)) protein is a transcription factor that binds to DNA and generally represses target gene expression. In the circadian clock system, Nfil3 binds to a D-box element residing in the promoter of clock genes and contributes to their robust oscillation. Here, we show that the 5'-untranslated region (5'-UTR) of Nfil3 mRNA contains an internal ribosome entry site (IRES) and that IRES-mediated translation occurs in a phase-dependent manner. We demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds to a specific region of Nfil3 mRNA and regulates IRES-mediated translation. Knockdown of hnRNP A1 almost completely abolishes protein oscillation without affecting mRNA oscillation. Moreover, we observe that intracellular calcium levels, which are closely related to bone formation, depend on Nfil3 levels in osteoblast cell lines. We suggest that the 5'-UTR mediated cap-independent translation of Nfil3 mRNA contributes to the rhythmic expression of Nfil3 by interacting with the RNA binding protein hnRNP A1. These data provide new evidence that the posttranscriptional regulation of clock gene expression is important during bone metabolism.

Nanowires: Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells (Adv. Mater. 21/2016).

Quantitative probing of the Cu(2+) ions naturally present in single living cells is accomplished by a probe made from a quantum-dot-embedded-nanowire waveguide. After inserting the active nanowire-based waveguide probe into single living cells, J. H. Je and co-workers directly observe photoluminescence (PL) quenching of the embedded quantum dots by the Cu(2+) ions diffused into the probe as described on page 4071. This results in quantitative measurement of intracellular Cu(2+) ions.

Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells.

Quantitative probing of Cu(2+) ions naturally present in single living cells is realized by developing a quantum-dot-embedded nanowire-waveguide probe. The intracellular Cu(2+) ion concentration is quantified by direct monitoring of photoluminescence quenching during the insertion of the nanowire in a living neuron. The measured intracellular Cu(2+) ion concentration is 3.34 ± 1.04 × 10(-6) m (mean ± s.e.m.) in single hippocampal neurons.

Heterogeneous ribonucleoprotein R regulates arylalkylamine N-acetyltransferase synthesis via internal ribosomal entry site-mediated translation in a circadian manner.

Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA.

AUF1 contributes to Cryptochrome1 mRNA degradation and rhythmic translation.

In the present study, we investigated the 3' untranslated region (UTR) of the mouse core clock gene cryptochrome 1 (Cry1) at the post-transcriptional level, particularly its translational regulation. Interestingly, the 3'UTR of Cry1 mRNA decreased its mRNA levels but increased protein amounts. The 3'UTR is widely known to function as a cis-acting element of mRNA degradation. The 3'UTR also provides a binding site for microRNA and mainly suppresses translation of target mRNAs. We found that AU-rich element RNA binding protein 1 (AUF1) directly binds to the Cry1 3'UTR and regulates translation of Cry1 mRNA. AUF1 interacted with eukaryotic translation initiation factor 3 subunit B and also directly associated with ribosomal protein S3 or ribosomal protein S14, resulting in translation of Cry1 mRNA in a 3'UTR-dependent manner. Expression of cytoplasmic AUF1 and binding of AUF1 to the Cry1 3'UTR were parallel to the circadian CRY1 protein profile. Our results suggest that the 3'UTR of Cry1 is important for its rhythmic translation, and AUF1 bound to the 3'UTR facilitates interaction with the 5' end of mRNA by interacting with translation initiation factors and recruiting the 40S ribosomal subunit to initiate translation of Cry1 mRNA.

MicroRNA-185 oscillation controls circadian amplitude of mouse Cryptochrome 1 via translational regulation.

Mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, a master pacemaker, but also throughout the peripheral tissues. Investigation of the regulation of clock gene expression has mainly focused on transcriptional and posttranslational modifications, and little is known about the posttranscriptional regulation of these genes. In the present study, we investigate the role of microRNAs (miRNAs) in the posttranscriptional regulation of the 3'-untranslated region (UTR) of the mouse Cryptochrome 1 (mCry1) gene. Knockdown of Drosha, Dicer, or Argonaute2 increased mCry1-3'UTR reporter activity. The presence of the miRNA recognition element of mCry1 that is important for miR-185 binding decreased mCRY1 protein, but not mRNA, level. Cytoplasmic miR-185 levels were nearly antiphase to mCRY1 protein levels. Furthermore, miR-185 knockdown elevated the amplitude of mCRY1 protein oscillation. Our results suggest that miR-185 plays a role in the fine-tuned regulation of mCRY1 protein expression by controlling the rhythmicity of mCry1 mRNA translation.

Modulation of exosome-mediated mRNA turnover by interaction of GTP-binding protein 1 (GTPBP1) with its target mRNAs.

Eukaryotic mRNA turnover is among most critical mechanisms that affect mRNA abundance and are regulated by mRNA-binding proteins and the cytoplasmic exosome. A functional protein, guanosine-triphosphate-binding protein 1 (GTPBP1), which associates with both the exosome and target mRNAs, was identified. The overexpression of GTPBP1 accelerated the target mRNA decay, whereas the reduction of the GTPBP1 expression with RNA interference stabilized the target mRNA. GTPBP1 has a putative guanosine-triphosphate (GTP)-binding domain, which is found in members of the G-protein family and Ski7p, a well-known core factor of the exosome-mediated mRNA turnover pathway in yeast. Analyses of protein interactions and mRNA decay demonstrated that GTPBP1 modulates mRNA degradation via GTP-binding-dependent target loading. Moreover, GTPBP1-knockout models displayed multiple mRNA decay defects, including elevated nocturnal levels of Aanat mRNA in pineal glands, and retarded degradation of TNF-α mRNA in lipopolysaccharide-treated splenocytes. The results of this study suggest that GTPBP1 is a regulator and adaptor of the exosome-mediated mRNA turnover pathway.