DL: Emulating life in 3D with digital and experimental tissue models

Cell cultures are important experimental tools in medical research. When cells are grown or cultured outside the body, they are grown in single layers on hard plastic. In the bodies of humans or animals, however, cells develop in far more complex soft three-dimensional (3D) structures. Consequently, the cells that we grow in cultures outside of the body develop differently from cells in tissues and organs inside the body. This poses a problem: Todays cell cultures are not sufficiently representative experimental models for studying cell and tissue biology, disease progression, and the testing of different medicinal compounds. 3DLife aims to develop novel strategies for making cell cultures that more closely mimic the conditions and environments that are found in the body. Instead of culturing cells in single layer cultures, we aim to generate new growth matrix materials and to employ these materials in advanced cell cultures with three-dimensional structures. High-capacity methods for determining the cells gene expression, meaning how the cells respond to and behave in their new three-dimensional environments, must be developed. Analysis of gene expression results in vast amounts of data. By using these data in computer models, we can identify key factors in making cell cultures that much more accurately resemble the normal cells, tissues and organs found in the body. These tailored cell cultures would serve as tissue and organ models for biomedical research and thus speeding up the development of novel therapeutic approaches. In the first part of the project, we have made new materials with controllable mechanical and biological properties, and adjusted materials and methods for the handling by robots for high-throughput screening. Detailed characterization of the gels includes novel studies by atomic force microscopy that reveals the surface structures and detailed mechanical mapping of the soft hydrogel structures. We have cultured different cell types in the gels and also adjusted materials and methods to be compatible with high cell viability. Fibroblasts, an important cell type for the development of tissue, respond differently to the different materials. The cells adhere to some materials when cultured on top. When the cells are inside the gels, the cell morphology is changing with the materials. We are now evaluating gene expression profiles from the fibroblasts to analyze how the cells are responding to the different hydrogels. Significant differences are found in the expression of the extracellular matrix proteins. By analyzing cells, materials and culture media with proteomics, we also find the same proteins outside the cells, which means that the cells have produced and secreted the proteins. We are also investigating the gene expression profiles of fibroblasts from different parts of the body, from the dermis, the gingiva, and the lung, in our hydrogels. By comparing our data to existing datasets of primary human fibroblasts from different tissue, we will see if the materials influence cell phenotype. We also further develop our hydrogel systems to include structures that is needed for the perfusion of culture medium through the tissue constructs. Both structures and fluid flow will influence the cell responses. The hydrogel structuring form the basis of a perfused device and chip system for tissue-on-chip that will further mimic natural tissue, such as human alveoli.

Betydelig innsats legges ned i både akademia og industrien for å utvikle celle-, vev- og organsystemer som bedre representerer human fysiologi. Dette inkluderer mer komplekse systemer enn dagens kultur av celler og vev i enkeltlag på hard plast med i et statisk næringsmedium. Nye modeller har ofte et mykt biomateriale som med struktur og bioaktivitet tilsvarer ekstracellulær matrise. De har også gjennomstrømning av næringsmedium som representerer gjennomstrømning av væske i vev. Cellene er også primære celler, i motsetning til kreftceller som er mye brukt i medisinsk forskning. Ofte er kulturene satt sammen av flere celler, enten i kokulturer eller at det er utviklet celleklumper eller organliknende strukturer (organoider) basert på stamceller. Bedre modeller muliggjør bedre forståelse av biologien i menneske og i dyr. Det gjør det også mulig å studere sykdom og effekter av medikamenter og behandling mer presist enn tidligere, og gir muligheter for både persontilpassede modeller og medisinering. Modellene gjør også bruk av dyreforsøk mindre relevant, slik at bruk av dyr til dyreforsøk kan reduseres. Næringsmiddel- og farmasiindustrien investerer store summer for å utvikle egne modeller til uttesting av næringsstoffer og medisiner for å underbygge påstander om effekter og for å danne underlag for videre kliniske forsøk. Akademia fokuserer på studier av material-celle og celle-celle interaksjoner, utvikling av vev og organer, samt sykdomsutvikling og publiserer resultater i de høyest rangerte journalene. Resultater fra 3DLife prosjektet, basert på vitenskapelige publikasjoner og presentasjoner, bidrar til kunnskapsbasen for bruk av alginatbaserte og andre myke materialer i slike avanserte cellekultiveringssystemer. Videre bidrar de til kunnskapsbasen rundt primære humane fibroblaster, som er viktige for dannelsen av bindevev i kroppen og bidrar i å bestemme mikromiljø i forskjellig vev. Kultiveringssystemet vi har optimalisert, med humane fibroblaster i tilpassede hydrogeler, danner nå basis for vevsmodeller i seg selv, eller de kan bygges videre på med for eksempel flere celletyper. Metodene og materialene vi har utviklet i prosjektet bruker vi nå videre i utviklingen av vevsmodeller utover humane fibroblaster, som har vært kjernen i 3DLife prosjektet. Gjennom prosjektperioden har vi erfart at det er stor etterspørsel etter mer avanserte vevsmodeller i norske forskningsmiljø. Vi har jobbet sammen med forskjellige forskere ved Universitetet i Bergen i fire prosjekter med støtte fra Senter for Digitalt Liv. I disse prosjektene har vi brukt materialer og metoder fra 3DLife prosjektet for å dyrke fibroblaster fra hval (finnhval), humane neuroner, samt laget et kultiveringssystem for beinmarg og leukemi for videre å se på effekter av medikamenter. I et nytt prosjekt finansiert fra NTNU Helse, vil vi videreføre 3DLife vevsmodeller ved å utvikle komplekse alveolemodeller for å studere aggressiv lungekreft og behandling av disse.

Cell culture-based experiments are important pillars in all medically related research, allowing examination of living cells without the use of research animals or human subjects. However, the commonly used cellular monolayer cultures are a remote reflection of in vivo conditions, due to a lack of the cellular, structural and chemical elements forming the tissue microenvironment. This disparity results in cells losing their tissue-like phenotype over time, limiting the potential of the models for studying tissue biology and disease progression, and for testing pharmaceutic and toxic compounds. 3DLife aims to develop novel strategies for microtissue engineering in 3D, to provide model systems of organ function and bridge the gap to in vivo conditions. To understand how the microenvironment affects cells we will synthesize novel and tuneable extracellular scaffold materials, and develop tools for high-throughput screening (HTS) of 3D cell cultures to assess genetic expression patterns in response to defined scaffold properties. These advances have limited translational potential without a digital approach that can process the vast data output from HTS analyses and provide a systems-level understanding of material-cell interactions. By applying a computational model, we can predict the requirements of organotypic cells to their microenvironment and tailor materials for improved in vivo-like tissue and organ models for research and clinical applications beyond the state of the art. To achieve this ambitious goal, 3DLife brings in expert competence within material engineering, high-throughput analyses, transcriptomics and bioinformatics, cell biology and cultivation, microsystem technology and mathematical and computational modelling from NTNU and SINTEF supported by international academic collaboration. The project will contribute to the Centre for Digital Life Norway (DLN) with new knowledge, materials and methodology with a broad field of application in biotechnology.