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

Tissue Engineering - the gateway to regenerative medicine

Tissue Engineering as an emerging biotechnology sector aims at the in vitro regeneration of diseased tissues and promises to profoundly change medical practice, offering the possibility of regenerating tissues and organs instead of just repairing them (regenerative medicine). Improved healing processes and a higher quality of life are the expected results. This article gives an overview of different technologies for regenerative medicine and presents results of our own current applied research and development. A recent project was successfully closed with the development of a natural biomaterial for soft tissue oral defects. The establishment of an in vitro bioreactor system enabled us to simulate the mechanical and biological environment in a healing wound and to investigate the suitability of different implant materials for the oral tissue regeneration. Moreover, focusing the attention on an alternative method for the intervertebral disc (IVD) regeneration, we established a new tissue engineered approach, based on the three-dimensional (3D) culture of autologous human IVD cells into a polyurethane (PU)-fibrin composite. IVD cells were able to proliferate and, thanks to the 3D conditions, to differentiate expressing the typical native tissue markers. The development of an automated platform was the goal of an additional project, to standardize the cell culture technology, increase the bio-safety and reduce the production costs, moving tissue engineering nearer to clinical application

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

Automation of 3D cell culture using cellulose-based scaffolds

Introduction: It is recognized that screening of drugs on 2D models is unable to precisely select clinically active medicaments; therefore, 3D culture systems are emerging and show potential for better simulating the in vivo tumour microenvironment and for eliminating the species differences to allow drug testing directly in human systems before drugs move into clinical trials. The purpose of this study was to automate the production and cultivation of human primary osteogenic sarcoma cells line, SaOS-2, in scaffold-based, multiple spheroids in GrowDex® (GDS) and scaffold-free systems, single spheroids (SS) for high-throughput screening. Methods: For scaffold-based models, SAOS-2 were embedded in nanofibrillar cellulose hydrogel, GrowDex®, in flat ultra-low attachment (ULA) 96-well plates while for the scaffold-free system cells were let to form spheroids in U-bottom ULA 96-well plates. Experiments were conducted on the Fluent® 780 automation workstation. The scripts for automated model production and media exchange were established in the FluentControlTM. Technical parameters such as aspiration and dispensing speed as well as XYZ dispensing positions were empirically defined and optimized i) to allow production of stable models, ii) to avoid disturbing during the medium exchange neither the GDS nor the GrowDex structure/shape, iii) to avoid SS displacement and aspiration. GDS and SS models were cultivated up to 11 days and were characterized for their viability (ATP assay), morphology and size (MTT assay). Dose-response tests with taxol and doxorubicin were carried out for both system types. Drugs were automated dispensed on the 3D models at day 4 for a treatment time of 72h. Cells were analysed for cell viability, measuring ATP levels, morphology, and size. Results: The established scripts allowed the formation of multiple spheroids in GrowDex as well as the aggregation of SAOS-2 in single spheroids in the U-bottom ULA plates. 70% of medium could be successfully automated exchanged maintaining unaltered the shape and the position of the models in the wells. GDS and SS remained stable for up to 11 days and increased in size over time, showing a similar growth rate. Viable GDS populated the entire model at different Z values with a compact morphology, parameter that characterized the SS too. GDS showed, as expected, a wide size distribution while SS were bigger and more homogenous in size in comparison to GDS (SS area 10-fold bigger than GDS area). SAOS-2 responded in both systems to taxol and doxorubicin, showing higher IC50 values for GDS compared to SS. Taxol (fig.1) and doxorubicin were 3.5- and 4.5-fold more potent in SS than in GDS. Collapsed morphology with a viable and compact core and a loose cell layer around border at high drugs concentrations characterized exclusively SS. Discussion & Conclusions: The automation protocols were successfully established allowing the reproducible production and maintenance of GrowDex multiple spheroids and scaffold-free models. Although its viscosity, GrowDex is automation compatible, and the results obtained in this project shows its high potential for high-throughput drug screening. Acknowledgements: The ZHAW authors would like to thank the Tecan for the technical support and the consumables, and the UPM Biomedicals for its contribution

Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures

The screening of large cell libraries is an important process in pharmaceutical discovery and R&D, e.g. to define drug targets or develop effective medicines. The goal of this project is the implementation of a screening platform based on 3D cultivation of primary human mesothelioma cells encapsulated in alginate hydrogels. To this end, new hydrogel compositions will be designed, tested and finally utilized in the Nanoliter Reactor (NLR) cultivation system that enables high throughput analysis of 3D cell cultures

Cell-seeded thermoreversible hydrogel-polyurethane composites for nucleus pulposus augmentation

Tissue engineering represents an alternative approach to the current invasive surgical procedures for the intervertebral disc (IVD) repair. The combination of injectable hydrogels and elastic biomaterials allow three-dimensional cell cultures and provide mechanical stability. In the present study a thermoreversible hyaluronan (HA) hydrogel as well as fibrin glue were mixed with polyurethane (PU) and their effect was investigated on the proliferation and differentiation of human IVD (hIVD cells) and mesenchymal stem cells (hMSCs) by in vitro and ex-vivo experiments