ZHAW Waedenswil : a new approach in the fight against cancer

A happy coincidence brought Dr Markus Rimann from ZHAW Wädenswil together with Dr Andreas Meyer from the start-up FGen and PD Dr Emanuela Felley-Bosco, Molecular Oncologist at Zurich University Hospital, to develop a technology platform for the manufacture and high throughput analysis of single mesothelioma spheroids. Armin Picenoni, former student in Chemistry for the Life Sciences at ZHAW, confirmed everything in writing his Master Thesis on this Innosuisse project

Evaluation of 3D hepatic tissue models for bioprinting

Introduction: Drug-induced liver injury is the leading cause of acute liver failure and post-market drug withdrawals. In vivo animal studies cannot be totally translated to humans; therefore, there’s huge demand of novel in vitro human models. 3D culture conditions would increase models longevity while bioprinting technology is expected to improve their functionality by the in vivo-like cell spatial patterning. Aim: The present project aims at the establishment and characterization of printed liver tissue-like models as co-culture of human cells such as hepatocytes, stellate and endothelial cells in order to reproduce a functional liver sinusoid. 3D equivalents were printed to assess hepatic cells printability (printing test), while high-density hepatocytes models were manually produced and characterized in order to simulate in-vivo cellular density conditions and define experimental parameters for bioprinting process. Materials and methods: - Printing test: HepG2 cells were mixed 6*106 cells/ml with bioink, PEG-based ink produced in house. Matrigel solution was added to further improve cell viability during the printing process. Cell mixture was printed by direct dispensing in a spiral pattern and polymerized at 365nm wavelength. Models were analysed up to 7 days for cell proliferation, viability and morphology. - High-density 3D models: High-density HepG2 discs and drop models were manually produced mixing 2:1 HepG2 “paste” with ink (supplemented with matrigel). Models were polymerized by exposure to 365nm wavelength for few seconds and cultivated up to 28 days. The equivalents were analysed for cell viability and albumin secretion as well as processed for histological analyses (cell proliferation, tissue-like intercellular tight junctions and lipid storage investigation). Results and discussion: - Bioprinting as well as ink is suitable for HepG2 viability and proliferation up to 7 days (15.84 ± 1.46 fold change increase day 7 vs day1). Printed HepG2 are homogenously distributed and round-shaped, forming agglomerates that increase in size over time. - HepG2-ink-matrigel equivalents are long-term stable characterized by high and constant cell viability up to 28 days. The models resemble native liver with respect to the high cell density and due to the ink supplementation can easily be printed into tissue-like models with exact cell patterning and defined structure. Models are currently under investigation for tissue functionality and morphology and will be subsequently printed. Conclusions: Bioprinting shows high potential for the manufacture of high-density liver tissue-like models. The printing of different cell types (hepatocytes, stellate and endothelial cells) will allow producing organotypical 3D liver equivalents

TEDD Competence Centre Report 2019

Copyright ©Swiss Chemical Society: CHIMIA, Volume 74, Number 5, 2020, pp. 426-428, 10.2533/chimia.2020.426TEDD (Tissue Engineering for Drug Development and Substance Testing) is an education, R&D and networking platform promoting the application of 3D organotypic technologies, with the core goal of replacing animal experimentation for therapy development. Based in Switzerland, TEDD is one of its kind in Europe focused on 3D for 3Rs. The community is composed of international members from academia, clinics, industry and non-profits. Training of members is achieved through regular events at the national and international level, including workshops, symposia, company visits, scientific reviews and by providing the platform to generate research consortia, projects and grant applications

Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression

Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmySΔS, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-α) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (PPIR8) placed 5′ of desired transgenes; (iii) within modified enhancer-free 3′-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct PPIR-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell line

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

Advances in the biofabrication of 3D skin in vitro : healthy and pathological models

The relevance for in vitro three-dimensional (3D) tissue culture of skin has been present for almost a century. From using skin biopsies in organ culture, to vascularized organotypic full-thickness reconstructed human skin equivalents, in vitro tissue regeneration of 3D skin has reached a golden era. However, the reconstruction of 3D skin still has room to grow and develop. The need for reproducible methodology, physiological structures and tissue architecture, and perfusable vasculature are only recently becoming a reality, though the addition of more complex structures such as glands and tactile corpuscles require advanced technologies. In this review, we will discuss the current methodology for biofabrication of 3D skin models and highlight the advantages and disadvantages of the existing systems as well as emphasize how new techniques can aid in the production of a truly physiologically relevant skin construct for preclinical innovation

Advanced modular self‐inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult‐to‐transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit‐like self‐inactivating lentiviral expression vector family which is compatible to state‐of‐the‐art packaging and pseudotyping technologies and contains, besides essential cis‐acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate‐early promoter (PhCMV) or the human elongation factor 1α (PhEF1α), (iii) PhCMV- or PPGK- (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein‐based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size‐optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5′U3, R‐U5‐ψ+‐SD, cPPT‐RRE‐SA and 3′LTRΔU3 modules or placed within the 5′U3 (-78 bp) and 3′LTRΔU3 (8666 bp). pLentiModule enables straightforward cassette‐type module swapping between lentiviral expression vector family members and facilitates the design of Tat‐independent (replacement of 5′LTR by heterologous promoter elements), regulated and self‐excisable proviruses (insertion of responsive operators or LoxP in the 3′LTRΔU3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi‐purpose construction kit‐like vector series described here is compatible with itself as well as many other (non‐viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human disease

Standardized 3D bioprinting of soft tissue models with human primary cells

Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application

In vitro characterization of a new composite material for biomedical applications and 3D (bio)printing

Study goal: The present project aims at evaluating the cytocompatibility and printability of a new composite material, based on a mixture of a new methacrylate-based monomer developed within a CTI project (18514.1 PFLS-LS) and glass-ceramic powder supplemented with co- and photo-initiators (patent in preparation). This study is the basis to demonstrate the suitability of the biomaterial, for biomedical applications, such as stent, orthopedic implants and hearing aid components, as well as for 3D (bio)printing Key findings: - Cultivation, proliferation and differentiation of three different human cell types were successfully established on composite material discs (1cm diameter, 1mm height). Biological activity was shown - The material is suitable for 3D (bio)printing, printing protocols were established - The new composite material is suitable for cell and tissue interaction in biomedical application