Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

• 3D-bioprinting method to produce 3D HepaRG constructs. • 3D constructs are more resistant to aflatoxin B1. • Long-term toxicity assessment is possible. • An alternative method for animal testing relevance.

Development of new anticancer drugs with currently available animal models is hampered by the fact that human cancer cells are embedded in an animal-derived environment. Neuroblastoma is the most common extracranial solid malignancy of childhood. Major obstacles include managing chemotherapy-resistant relapses and resistance to induction therapy, leading to early death in very-high-risk patients. Here, we present a three-dimensional (3D) model for neuroblastoma composed of IMR-32 cells with amplified genes of the myelocytomatosis viral related oncogene MYCN and the anaplastic lymphoma kinase (ALK) in a renal environment of exclusively human origin, made of human embryonic kidney 293 cells and primary human kidney fibroblasts. The model was produced with two pneumatic extrusion printheads using a commercially available bioprinter. Two drugs were exemplarily tested in this model: While the histone deacetylase inhibitor panobinostat selectively killed the cancer cells by apoptosis induction but did not affect renal cells in the therapeutically effective concentration range, the peptidyl nucleoside antibiotic blasticidin induced cell death in both cell types. Importantly, differences in sensitivity between two-dimensional (2D) and 3D cultures were cell-type specific, making the therapeutic window broader in the bioprinted model and demonstrating the value of studying anticancer drugs in human 3D models. Altogether, this cancer model allows testing cytotoxicity and tumor selectivity of new anticancer drugs, and the open scaffold design enables the free exchange of tumor and microenvironment by any cell type.

The 5ʹ-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5ʹ-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stem-loop element. Taken together, our study describes the functional relevance of two alternative 5ʹ-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions

Three-dimensional (3D) tissue culture has attracted a great deal of attention as a result of the need to replace the conventional two-dimensional cell cultures with more meaningful methods, especially for understanding the sophisticated nature of native tumor microenvironments. However, most techniques for 3D tissue culture are laborious, expensive, and limited to spheroid formation. In this study, a low-cost and highly effective nanofibrous scaffold is presented for spontaneous formation of reproducible 3D breast cancer microtissues. Experimentally, aligned and non-aligned chitosan/poly(ethylene oxide) nanofibrous scaffolds were prepared at one of two chitosan concentrations (2 and 4 wt %) and various electrospinning parameters. The resulting fabricated scaffolds (C2P1 and C4P1) were structurally and morphologically characterized, as well as analyzed in silico. The obtained data suggest that the fiber diameter, surface roughness, and scaffold wettability are tunable and can be influenced based on the chitosan concentration, electrospinning conditions, and alignment mode. To test the usefulness of the fabricated scaffolds for 3D cell culture, a breast cancer cell line (MCF-7) was cultured on their surfaces and evaluated morphologically and biochemically. The obtained data showed a higher proliferation rate for cells grown on scaffolds compared to cells grown on two-dimensional adherent plates (tissue culture plate). The MTT assay revealed that the rate of cell proliferation on nanofibrous scaffolds is statistically significantly higher compared to tissue culture plate (P ≤ 0.001) after 14 days of culture. The formation of spheroids within the first few days of culture shows that the scaffolds effectively support 3D tissue culture from the outset of the experiment. Furthermore, 3D breast cancer tissues were spontaneously formed within 10 days of culture on aligned and non-aligned nanofibrous scaffolds, which suggests that the scaffolds imitate the in vivo extracellular matrix in the tumor microenvironment. Detailed mechanisms for the spontaneous formation of the 3D microtissues have been proposed. Our results suggest that scaffold surface topography significantly influences tissue formation and behavior of the cells.

Radiotherapy is an important component in the treatment of lung cancer, one of the most common cancers worldwide, frequently resulting in death within only a few years of diagnosis. In order to evaluate new therapeutic approaches and compare their efficiency with regard to tumour control at a pre-clinical stage, it is important to develop standardized samples which can serve as inter-institutional outcome controls, independent of differences in local technical parameters or specific techniques. Recent developments in 3D bioprinting techniques could provide a sophisticated solution to this challenge. We have conducted a pilot project to evaluate the suitability of standardized samples generated from 3D printed human lung cancer cells in radiotherapy studies. The samples were irradiated at high dose rates using both broad beam and microbeam techniques. We found the 3D printed constructs to be sufficiently mechanically stable for use in microbeam studies with peak doses up to 400 Gy to test for cytotoxicity, DNA damage, and cancer cell death in vitro. The results of this study show how 3D structures generated from human lung cancer cells in an additive printing process can be used to study the effects of radiotherapy in a standardized manner.