Cross-industrial applications of organotypic models.

Recent advances in microphysiological systems (MPS) promise a global paradigm shift in drug development, diagnostics, disease prevention, and therapy. The expectation is that these systems will model healthy and various diseased stages and disease progression to predict toxicity, immunogenicity, ADME profiles, and treatment efficacies. MPS will provide unprecedented human-like physiological properties of in vitro models, enabling their routine application in the pharma industry and thus reducing drug development costs by lowering the attrition rate of compounds. We showcased MPS application diversity across different industries during the TEDD Annual Meeting on 14th October 2021 in Wädenswil, Switzerland. The goal was to promote cross-sectoral collaboration of academia and industry to further pave the way for developing next-generation MPS based on 3D cell culture, organoid, and organ-on-chip technology and their widespread exploitation. To enable visionary projects and radical innovations, we covered multidisciplinary fields and connected different industry sectors, like pharma, medtech, biotech, cosmetics, diagnostics, fragrances, and food, with each other.

TEDD Annual Meeting with 3D Bioprinting Workshop.

Bioprinting is the technology of choice for realizing functional tissues such as vascular system, muscle, cartilage and bone. In the future, bioprinting will influence the way we engineer tissues and bring it to a new level of physiological relevance. That was the topic of the 2017 TEDD Annual Meeting at ZHAW Waedenswil on 8th and 9th November. In an exciting workshop, the two companies regenHU Ltd. and CELLINK gave us an insight into highly topical applications and collaborations in this domain.

Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion.

The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid's surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor.

Quantification of collagen contraction in three-dimensional cell culture.

Many different cell types including fibroblasts, smooth muscle cells, endothelial cells, and cancer cells exert traction forces on the fibrous components of the extracellular matrix. This can be observed as matrix contraction both macro- and microscopically in three-dimensional (3D) tissues models such as collagen type I gels. The quantification of local contraction at the micron scale, including its directionality and speed, in correlation with other parameters such as cell invasion, local protein or gene expression, can provide useful information to study wound healing, organism development, and cancer metastasis. In this article, we present a set of tools to quantify the flow dynamics of collagen contraction, induced by cells migrating out of a multicellular cancer spheroid into a three-dimensional (3D) collagen matrix. We adapted a pseudo-speckle technique that can be applied to bright-field and fluorescent microscopy time series. The image analysis presented here is based on an in-house written software developed in the Matlab (Mathworks) programming environment. The analysis program is freely available from GitHub following the link: http://dx.doi.org/10.5281/zenodo.10116. This tool provides an automatized technique to measure collagen contraction that can be utilized in different 3D cellular systems.

Filtration of dermal fibroblast-conditioned culture media is required for the reliable quantitation of cleaved carboxy-terminal peptide of collagen type I (CICP) by ELISA.

Cleavage of the collagen type I carboxy-terminal peptide (CICP) from the procollagen molecule is an essential step in collagen biosynthesis. The commercial CICP ELISA (Quidel Corporation, USA), developed for quantifying CICP in serum in clinical monitoring, is often also applied to cellular studies as a measure of collagen synthesis. However, unlike in serum samples, which contain only cleaved CICP, cell-conditioned culture media also contains "uncleaved CICP", namely procollagen, and there is no specific guidance on how to interpret the ELISA data obtained with such samples. Here we attempted to reliably quantify cleaved CICP in human dermal fibroblast-conditioned cell culture media using the CICP ELISA. CICP concentration was determined in the parent and filtered samples of culture media of dermal fibroblasts (CCD-25SK). Gel-separated samples were also subjected to protein staining or analyzed by Western blot using the anti-CICP antibodies supplied in the ELISA kit. The derived concentrations of CICP in the filtered aliquots and the parent unfiltered samples increased over time. The increase in CICP in the unfiltered samples was not proportional to the increase seen in the filtered aliquot. CICP ELISA antibodies recognized both the cleaved CICP trimer and procollagen molecule. The data presented show that (a) the commercial CICP ELISA recognizes both procollagen and cleaved CICP in cell-conditioned culture media and thus attention should be paid in interpreting data from cell culture studies using this ELISA and (b) the filtration method described herein can be used to exclusively and reliably monitor cleaved CICP.

Synthesis of new analogs of benzotriazole, benzimidazole and phthalimide--potential inhibitors of human protein kinase CK2.

New derivatives of 4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), and N-substituted tetrabromophthalimides were synthesized and their effect on the activity of human protein kinase CK2 was examined. The most active were derivatives with N-hydroxypropyl substituents (IC(50) in 0.32-0.54 microM range) whereas derivatives of phthalimide were almost ineffective.

Searching for a new anti-HCV therapy: synthesis and properties of tropolone derivatives.

Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication.

Synthesis and biological activity of 1H-benzotriazole and 1H-benzimidazole analogues--inhibitors of the NTpase/helicase of HCV and of some related Flaviviridae.

To improve anti-helical activity of analogues of 1H-benzotriazole and 1H-benzimidazole their N-alkyl derivatives were synthesized and tested for antihelicase activity against enzymes of selected Flaviviridae including hepatitis C virus (HCV), West Nile virus (WNV), Dengue virus (DENV) and Japanese encephalitis virus (JEV). 1- and 2-alkyl derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole were obtained by direct alkylation of 4,5,6,7-tetrabromo-1H-benzotriazole with the use of respective alkyl halides in the presence of KOH in methanol, to give a mixture of 1- and 2- isomers, which was separated by flash column chromatography in good yield. The proportion of isomers strongly depended on the reaction time and temperature. 1- and 2-hydroxyethyl and 1- and 2-chloroethyl derivatives of the tetrabromobenzo-triazole were synthesized with the use of 2-bromoethanol and 1-bromo-2-chloroethane respectively as alkylating agents. N-alkylation of this benzotriazole compound enhanced inhibitory activity and selectivity towards the helicase activity of HCV NTPase/helicase. The most active were the 2-methyl, 2-ethyl and 2-propyl derivatives (IC50 approximately 6.5 microM in the presence of DNA as a substrate). Derivatives of the benzotriazole in which hydroxyethyl or chloroethyl replaced the alkyl substituents lost their inhibitory activity. Brominated or methylated benzotriazole N(1) ribosides also did not exert helicase inhibitory activity. Although a number of N(1) and N(2) alkyl derivatives exerted good HCV and WNV helicase inhibitory activity when DNA was used as substrate, the activity was strongly decreased or even disappeared when RNA was used as substrate. The cytotoxicity tests in Vero and HeLa Tat cells showed a substantial decrease of cytotoxicity of N-alkyl derivatives as compared to the parent benzotriazole.

Synthesis and activity of 1H-benzimidazole and 1H-benzotriazole derivatives as inhibitors of Acanthamoeba castellanii.

Chloro-, bromo- and methyl- analogues of 1H-benzimidazole and 1H-benzotriazole and their N-alkyl derivatives have been synthesized and tested in vitro against the protozoa Acanthamoeba castellanii. The results indicate that 5,6-dimethyl-1H-benzotriazole (11) and 5,6-dibromo-1H-benzotriazole (14) have higher efficacy than the antiprotozoal agent chlorohexidine.