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Growing Tissues in Space

Sascha Kopp1*, Marcus Krüger1, Markus Wehland1, Johann Bauer2, Anita Dittrich3, Manfred Infanger1 and Daniela Grimm1, 3
  • 1 Medizinische Fakultät, Universitätsklinikum Magdeburg, Germany
  • 2 Molecular Immunology and Signaltransduction Group, Max Planck Institute of Biochemistry, Germany
  • 3 Aarhus University, Denmark

ABSTRACT Despite the fact that astronauts have various health problems in space, the unique culture condition of microgravity (µg) bears the possibility to grow human cells in a three-dimensional (3D) way without scaffolds or matrices. Using facilities like the International Space Station (ISS) or unmanned satellites, we showed that human thyroid cancer cells, as well as human endothelial cells formed 3D aggregates or spheroids after µg-exposure for up to 14 days. Comparable results were obtained when using ground-based facilities like the Random Positioning Machine (RPM) and fast rotating clinostat (FRC). During spheroid formation, factors involved in angiogenesis, proliferation, cell adhesion, migration, extracellular matrix signalling and others seem to play a crucial role in concert with the initial cell density. These findings will help to engineer human tissues which might be used for transplantation or drug testing. The following proceeding should give a condensed overview of the results obtained so far in real µg (r-µg) and simulated µg (s-µg). INTRODUCTION Tissue engineering is a fast-developing research field. Even though a lot of improvement was achieved during the last years, the use of scaffolds and matrices is still a critical point to be solved (Grimm et al., 2014). In addition, nutrient and waste transport in the engineered tissue is still a problem because the tissues will become necrotic with growing size and culture time (Grimm et al., 2014). The majority of the available techniques to engineer tissues scaffold free are forcing the cells to stick to each other, like the hanging drop or the liquid overlay method. The use of µg allows the cells to form spheroids naturally without any force (Aleshcheva et al., 2016). Ingram et al. had investigated several different human carcinoma cell lines cultivated in a NASA rotating bioreactor, which was developed to simulate aspects of microgravity. The authors found that each cell line formed 3D spheroids, when kept from sedimentation (Ingram et al., 1997). These experiments, however, started with suspended cells floating in the medium. In 2002, Grimm et al. exposed adherent ML-1 thyroid cancer cells to a random positioning machine to check for its possible usability in cancer studies. They found that these cells from multicellular spheroids aside adherently growing cells suggesting that during clinorotation some cells detached from the substrate (Grimm et al., 2002). How and why the cells detach and accumulate into spheroids is still under investigation. Therefore, the objective of this proceeding is to condense recent findings in the field, which were observed when different cell types (thyroid cancer cells and endothelial cells) were exposed to r-µg in space or to s-µg using ground-based facilities. METHODS Follicular thyroid cancer cells (Pietsch et al., 2013; Ma et al., 2014a; Riwaldt et al., 2015) and endothelial cells (Pietsch et al., 2017) were investigated during international spaceflights in orbit or on the International Space Station (ISS) and were cultured in newly developed automated hardware for several days in space. Post-flight the cells were analysed by histological and molecular biological methods (proteomics, gene array, quantitative rtPCR, multianalyte profiling). In addition, cell biological experiments using the 3D RPM or the 2D FRC were performed. RESULTS and DISCUSSION Exposing the cells to r-µg and s-µg revealed the formation of two phenotypes: one part of the cells of both investigated cell types detached from the culture flask bottom and grew in form of 3D multicellular spheroids (MCS; Figure 1), the other one continued growing as a 2D monolayer (Pietsch et al., 2013; Ma et al., 2014a; Warnke et al., 2014; Kopp et al., 2015; Pietsch et al., 2017). Interestingly, this 3D formation occurred scaffold-free. MCS formed by thyroid cancer cells were more round aggregates (Fig. 1 A) (Kopp et al., 2015), whereas endothelial cells exhibited a tube formation (intima constructs) in addition to 3D MCS (Fig. 1 B) (Ma et al., 2013; Ma et al., 2014b). The density of the monolayers exposed to microgravity revealed an impact on the results as during CELLBOX1 the cells were overgrown before launch and no spheroids were formed (Riwaldt et al., 2015). Genomic and proteomic alterations were induced by altered gravity conditions (Pietsch et al., 2010; Pietsch et al., 2011; 2012). Biological processes such as proliferation, migration, the composition of the extracellular matrix proteins (ECM), cell adhesion, focal adhesions, and apoptosis are influencing 3D growth under µg conditions (Pietsch et al., 2013; Ma et al., 2014a; Riwaldt et al., 2015). Growth factors, cell adhesion molecules and cytokines such as vascular endothelial growth factor A (VEGFA), epidermal growth factor (EGF), connective tissue growth factor (CTGF), fibronectin, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin-6 (IL6), IL8, caveolin-1 (CAV1), monocyte chemoattractant protein-1 (MCP1) and intercellular adhesion molecule 1 (ICAM1) and others have shown to be involved in spheroid formation in µg in follicular thyroid cancer cells and normal thyrocytes (Riwaldt et al., 2015; Warnke et al., 2017). Human follicular epithelial thyroid cells (Nthy-ori 3-1) exposed to the RPM formed MCS within 24h. Cytokines and focal adhesion proteins play a key role in the early phase of MCS formation (Warnke et al., 2017). Moreover, RPM-exposed FTC-133 monolayer cells or MCS incorporate vinculin, paxillin, focal adhesion kinase 1, and adenine diphosphate (ADP)-ribosylation factor 6 in different ways into the focal adhesion complex (Bauer et al., 2017). Further studies have to be performed to investigate how these factors exert their effect on cell detachment and aggregation in adherent human cells. CONCLUSION Cultivation of human cells in space or on a µg-simulator induced 3D growth (Fig. 2). Different phenotypes occurred and MCS showed a different gene expression profile involving important biological processes compared to monolayer cells. Tissue engineering under r-µg and s-µg conditions represents a new technology in gravitational biology and translational regenerative medicine which can be beneficial in cancer research and drug screening as well as tissue engineering and can reduce the use of laboratory animals (Fig. 2). Figure Legends Figure 1 | Cells build up 3D spheres when exposed to the Random Positioning Machine. While FTC-133 thyroid cancer cells produce round spheres (A) (here after a 7-day-exposure), endothelial cells grow in form of tubular structures resembling the inner layer (intima) of blood vessels (B) (here after a 23-day-exposure). Scale bars: 100 µm Figure 2 | In microgravity, cells of various tissue origin are detaching from their substrate and start floating in the supernatant. While being suspended, they start forming 3D spheres which resemble tissue-specific features without the need of adding scaffolds or matrices. They can be used for neoangiogenesis, metastasis and radiation models. In addition, this new technique can benefit tissue engineering as well as drug screening and can reduce the need of lab animals erasing the black box of artificial species-reactivity.

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Acknowledgements

This work was supported by the German Space Agency (DLR), BMWi project 50WB1524 (DG). We like to thank Dr. Markus Braun and Dr. Michael Becker (German Space Agency, DLR).

References

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Keywords: Tissue Engineering, Microgravity (μg), Spheroids culture, vascular constructs, 3D growth

Conference: 39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.

Presentation Type: Extended abstract

Topic: Biology and Cells Models

Citation: Kopp S, Krüger M, Wehland M, Bauer J, Dittrich A, Infanger M and Grimm D (2019). Growing Tissues in Space. Front. Physiol. Conference Abstract: 39th ISGP Meeting & ESA Life Sciences Meeting. doi: 10.3389/conf.fphys.2018.26.00014

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Received: 02 Dec 2018; Published Online: 16 Jan 2019.

* Correspondence: Mr. Sascha Kopp, Medizinische Fakultät, Universitätsklinikum Magdeburg, Magdeburg, Germany, sascha.kopp@med.ovgu.de