Ursula Graf-Hausner, Graf3dcellculture, Winterthur, Switzerland

Human three-dimensional (3D) tissue models offer new perspectives as R&D tool for drug development in medicine and pharma. 3D tissue models are used as biologically relevant system for preclinical compound screening and disease research. They help to identify potential toxic liabilities in an early phase of the drug discovery process. Furthermore they provide an insight into pharmacokinetics, physiology and metabolism, effect of protective agents for combinatorial treatment and a lot more. Organoid-based assays present a novel and potentially high-value de-risking strategy, particularly when generated as iPS cell-derived disease models. In addition, 3D tissue models enable the reduction of animal experiments.

Different systems of 3D cell culture technology are established covering a wide range of complexity and application. In scaffold-free microtissue engineering cells produce endogenous extracellular matrix (ECM). Hydrogel-based scaffolds provide natural or synthetic ECM for 3D arrangement and rigid scaffolds like polystyrene or ceramics provide an adhesive biomaterial to enforce 3D. A short comparison of the 3D cell culture formats and few examples will be given as an introduction.

The most important issue of 3D cell culture is a reliable biological relevance. Additional new technologies try to enhance the complexity of the 3D models in order to establish organ-like tissues with multi cell-types and physiological functionality. Organ-like tissue models should simulate the high complexity of our body. In order to meet this goal, innovative technologies like 3D bioprinting and microfluidics have to be integrated into the production and maintenance process of tissue models. A few examples of different approaches will be described, especially the 3D bioprinting technology.

Bioprinting allows the precise deposition of cells, matrix and other bioactive molecules in 3D space and is therefore expected to mirror the in vivo tissue complexity. We developed a bioprinting solution with the following features: i) micro valve-based inkjet printheads for cell jetting and contact printing with needles to print into 96 well plates, ii) a chemically-defined ECM-surrogate BioInk that is print- and cytocompatible, iii) a photopolymerization unit to crosslink the BioInk with UV-LED (365 nm) and iv) a cell mixing unit to avoid cell sedimentation in the print cartridge during printing. For tissue generation one layer of BioInk is printed and polymerized providing a stable support for the subsequent printed cell layer. This process is alternated to produce a multi-layered 3D tissue construct. In a recently finished research project together with 3 industrial partners we developed an in vitro tool for drug assessment to treat muscle-related diseases. The idea is to provide an all-in-one solution to produce and analyse printed in vitro muscle/tendon tissues in a well plate. The customized 24 well plate harbours two posts in each of the wells. The final goal is to print muscle/tendon precursor cells around and in between the posts to induce tissue formation with tendon around the post and muscle fibers between the posts. First, monocultures of primary human myoblasts and primary rat tenocytes were printed separately in a dumbbell-shape around the posts. After cell differentiation the myoblasts were stained positive for myosin heavy chain (MHC) and myotubes developed and for tendon the characteristic collagen I-distribution around the cell nuclei was detected. The printed muscle tissue is contracting on electrical stimulation and shows physiological functionality.

The development of standardized 3D in vitro tissue models combined with read-outs is a prerequisite for the future success of 3D tissues in drug development and substance testing. With the current 3D tissue models established in many labs all over the world we play on the tip of an iceberg. To bring this exciting and promising technology to routine application we have to dig much deeper. Robustness, predictability, complexity versus applicability, cost efficiency, reproducibility and validation will be some of the challenges waiting in the next few years. The competence centre TEDD (Tissue Engineering for Drug Development) promotes this development combining academia, clinicians and industry to bring the 3D cell culture to routine application. www.zhaw.ch/icbt/tedd