Introduction
High-resolution fluorescence microscopy is a fundamental technology in many research fields such as biology, physiology, immunology and more. While there have been several microscopes on the ISS, none offered the combination of fluorescence microscopy, sustaining life support to cells and the possibility to image while changing the acceleration levels.
The Facility
FLUMIAS is a novel facility for the ISS, developed by the German Space Agency at DLR and built by Airbus DS as a national contribution to ESA´s SciSpace program. ESA will upload and operate the facility under the Op-Nom “Live Cell Imaging”. The core of the facility is an innovative Structured-Illumination Microscope (by TILL I.D., Martinsried, Germany) that is so robust and compact that it can be rotated on a centrifuge. For the first time, it will be possible to image specimen in space with high resolution fluorescence microscopy while gradually altering the acceleration level between 0 g and 1g.
While the main part of the microscope is fixed to the rotating centrifuge plate, the Experiment Blocks that contain the objective for each experiment are stored in the magazine until one EB at a time transferred to the plate for imaging. This modular design makes it possible to adapt each EB to the specific needs of one experiment with respect to microscopy slide, fluorescent dyes and media, temperature control and choice of objective.
Goals
The rationale behind the development of this multi-user facility is to enable a broad range of experiments including but not limited to cell biology, microbiology, biotechnology, immunology and molecular physiology. 10 science proposals for a first round of experiments were selected via an international joint DLR/ESA announcement of opportunity. ESA and DLR both intend to make this facility available also to international partners outside Europe.
Outlook
FLUMIAS is scheduled for launch to the ISS in 2025. Here we will present a design overview of the hardware, science-relevant technical properties, a description of the currently selected experiments and an outlook to possible future applications for Live Cell Imaging in Space.

Space life science research is of paramount importance for the future planned human activities on board Space Stations, Lunar Gateway, or for the colonization of Moon and Mars because either gives insights into the effects of space flight on several biological model systems or provides countermeasures to fight the hostile effects of the space environment on the human body. Kayser develops space hardware systems and provides mission support for investigations on biological systems in space. In particular, several types of space bioreactors have been developed. Among the different type of cell biology experiments performed on many types of eukaryotic cells, especially mammalian cells, and organisms many microbiology experiments are being performed by Kayser in the field of astrobiology/ISRU such as the BioRock and the BioAsteroid experiment onboard ISS, studying the ability of bacterial strain to biomine rare elements from basalt or asteroid’s fragments. Beyond the experimentations performed inside the ISS, Kayser is developing for ESA an Exobiology facility to be installed on an external platform outside the ISS. OREOcube, Exocube (Bio and Chem) and IceCold experiments are currently under development and will evaluate the impact of space environment on different chemical and biological samples.
State of the art experiment hardware proven to be successful in space operations and scientific results is a valuable asset for the support of scientific investigations because it offers solutions that are reliable, cost-affordable, and scientific sound. Such space-proved hardware is also an asset for private entities exploiting research results stemming from the space environment (i.e. commercially-driven activities for utilizing space facilities). Together with the classical approach to perform space experiments (through the support of national or international granting agencies), and driven by current commercial space utilization programs, Kayser has established a partnership with ESA for the commercial exploitation of LEO, a service called BIOREACTOR EXPRESS, aiming to establish an “express” way to perform scientific and/or technological experiments on board the ISS. It offers a large portfolio of bioreactors complemented by a complete set of experiment containers for fully automatic execution of scientific protocols. BIOREACTOR EXPRESS is an end-to-end service that exploits the Kubik incubator facility of ESA which is permanently installed onboard the ISS. BIOREACTOR EXPRESS provides all services and resources necessary to perform the experiment, allowing the user to focus on the exploitation of the research results. Hence, details regarding Kayser activities and future opportunities for life science investigations will be presented.

Introduction

Demographic changes show rapidly increasing numbers of older adults with poor health. Age-related skeletal muscle loss contributes to the development of frailty but the mechanisms by which this occurs are yet to be fully identified. We have demonstrated that muscle from older humans adapts poorly following exercise, compromising the maintenance of muscle mass/function. In a similar yet accelerated manner, the muscles of astronauts exposed to microgravity (µg) rapidly lose muscle mass and are relatively unresponsive to exercise training in spaceflight.

As part of a series of UK Space Agency (UKSA)-funded national missions to the International Space Station (ISS), we performed studies using tissue-engineered, human muscle constructs to determine whether analogous maladaptations to contractile activity occur rapidly under the influence of microgravity as that which occurs in older adults on earth.

Methods

Muscle constructs were fabricated using immortalised human myoblasts encapsulated in fibrin hydrogels and anchored onto bespoke, 3D-printed scaffolds. Scaffolds were integrated into bespoke ‘life-support’ systems, designed in collaboration with Kayser Space Ltd, to autonomously perform fluid exchanges, electrical stimulations to initiate contractions and monitor contractions whilst in spaceflight. Upon return to earth, a ground-matched study was performed. Muscle constructs underwent LC-MS using a timsTOF mass spectrometer in dia-PASAF mode. Bioinformatic analyses determined differential expression patterns and gene ontology (GO) term functional enrichment. Culture medium samples were recovered and analysed using a Bio 27-Plex immunoassay panel for human cytokines.

Results

In the proteomic analysis, 2934 human proteins were identified across all samples. When determining the effects of microgravity on the proteome at rest, there were 541 proteins differentially expressed (DE) compared with the ground-matched samples (287 upregulated and 254 downregulated, p <0.05). When performing function enrichment analyses, pathways such as mitochondrial metabolism/gene expression were highlighted. Electrical stimulation of the muscle constructs on ground resulted in the enrichment of pathways associated with muscle adaptive responses. However, these responses were perturbed in microgravity.
Fifteen cytokines/chemokines were released from the muscle constructs and data supported the hypothesis that exposure to µg resulted in a pro-inflammatory response and suppressed beneficial contraction-induced responses observed in the ground reference experiments.

Conclusion

In summary, the study demonstrated major modification to the muscle construct proteome and secretome at rest and in response to electrical stimulation in microgravity compared to those seen on ground. These results have led to a subsequence UKSA-funded mission to the ISS, MicroAge II. Due to be flown in 2025.

The teaching of space biology is essential to prepare future generations of scientists to tackle the challenges of space exploration and to understand the effects of space on living organisms. Yet, the opportunity to study space biology in an organized curriculum is very limited in Europe and overseas. The United Nations Office for Outer Space Affairs (UNOOSA) program “Access to Space 4 all” has a webinar series on the subject in its Education Component, but Space Biology is only 1 topic of seven in one of the 5 major fields on UNOOSA’s Education Curricula. In 2022 the European Space Policy Institute (ESPI) published a report on Space Education that although cited “Astrobiology” 3 times in its 40 pages but failed to cite “Space Biology”. This suggests the authors were not aware of the differences between the terms or that there was no dedicated Space Biology training at the time in the region. The European Space Agency (ESA) declared the intention to increase the number of students in STEM by 20%, to offer talents with attractive opportunities and have put “Life Sciences” as one of its major disciplines. But Space Biology training made accessible via ESA Academy has been limited and a majority of students in space fields report difficulty finding a job in the area.
But since 2021 the International Space University (ISU) have pioneered the teaching of Space Omics in Europe. Space Omics is the application of large-scale biological data acquisition and analysis – e.g. Genomics, Transcriptomics and others – to Space Education. ISU maintains, so far, the only dedicated education initiative in this field in the whole continent. This initiative comprises a module of the Master of Studies Program, and workshops have been taught in France, Portugal, Spain, Norway and online. The training is interdisciplinary, making sure the topics covered are of relevance for students from diverse backgrounds (engineering, physics, business, humanities and life sciences). This interdisciplinarity puts this course in a strong position to contribute to the Focus Areas of the ESA Life Science Industry Accelerator program, being strategic for ESA to fulfil it intentions in STEM Education and the creation of opportunities. We look forward to engaging a larger portion of the community to make this possible.

Intracellular viscosity, a key mechanical property of cells, can significantly influence biochemical diffusion rates. Changes in cellular viscosity have been found in various human diseases, including diabetes and neurodegenerative diseases like Parkinson's disease, and are also linked to cancer cell migration. Cell behaviour changes in altered gravity and is suspected to contribute to the macroscale symptoms seen in astronauts. However, research on intracellular viscosity in altered gravity is limited. Therefore, in preparation for the ESA's MechanoCell project, this study investigates whether hypergravity affects the viscosity of HeLa cells and if this can be measured with Single Particle Tracking (SPT). The SPT software developed by Schmidt Lab was employed to track endogenous particles in the cells subjected to hypergravity ranging from 1 to 20g in the Large Diameter Centrifuge (LDC) at ESA ESTEC. The software tracks the endogenous particles in the HeLa cells by comparing the video frames captured by the EVOS microscope in the LDC while in hypergravity. The trajectories of the particles allow the calculation of the Mean Square Displacement (MSD) and subsequently the intracellular viscosity. Results show measurable changes in viscosity, although further research is needed to confirm these findings and account for other influencing factors. Future recommendations include investigating more cell types, considering time-dependent mechanical properties, optimising experimental conditions, and examining the cytoskeleton's role in viscosity changes.

Loss of skeletal muscle mass and function is the major factor in increasing weakness in older people, and in astronauts exposed to microgravity during spaceflight. Best practice for maintaining muscle in astronauts on the International Space Station (ISS) and older people on earth requires exercise, although current protocols cannot fully maintain muscle mass in either situation. There is a need to understand the mechanisms leading to loss of muscle mass and function in ageing and in microgravity in order to optimise preserving muscle mass in both situations.
Altered mitochondrial morphology and increased mitochondrial formation of Reactive Oxygen Species (ROS), particularly hydrogen peroxide (H2O2) are associated with ageing in skeletal muscle. ROS are critical signalling molecules that modulate changes in muscle homeostasis and increased mitochondrial ROS generation is proposed to play a key role in pathological changes in muscle loss in microgravity. Unfortunately, studies are inconclusive, in part because of the lack of definitive methods to study specific ROS in biological tissues under microgravity and previous limitations to studies in space.
The new FLUMIAS microscope installed on the ISS will, for the first, time allow definitive studies to take place under microgravity conditions. This study will examine the role that mitochondrial dysfunction and H2O2 production plays in loss of muscle mass in microgravity and how this relates to muscle loss in older people on earth.
The concentration of individual ROS is low in cells and it has not been possible to measure individual species in biological systems. HyPer is a specific probe for H2O2 that can be localised to mitochondria. Mitochondria form a dynamic interconnected reticulum which can be imaged in confocal microscopy. Mitochondrial dynamics are regulated by fission and fusion. Studies indicate that in ageing and in microgravity, mitochondria may change to reflect modified fusion with increased H2O2 production and disorganisation of the actin cytoskeleton, creating a functional link between cytoskeletal-mitochondrial dynamics and muscle loss.
This study will use the FLUMIAS microscope (https://www.dlr.de/en/research-and-transfer/projects-and-missions/horizons/flumias) to test the hypothesis that increased H2O2 plays a key role in mediating loss of muscle mass that occurs rapidly following exposure to microgravity and over a longer period of time during ageing on earth with disrupted mitochondria as the key intracellular site for increased H2O2 generation. Data will demonstrate the ability to monitor H2O2 generation in mitochondria on the ISS and the process to determine associated changes in the structure and function of the mitochondrial reticulum.

Introduction / Background:

The project LAARA (Live Assessment of Astrocytic Reactivity under Space Conditions) aims to investigate how astrocytes, the most prominent type of glial cell in the brain, adapt to altered gravity using the automated FLUMIAS-ISS live-cell fluorescence microscope. Astrocyte reactivity has been shown to be influenced by altered gravitational loads (Lichterfeld et al., 2022) and is thus relevant for astronaut health as it is thought to play an important role in brain physiological changes in prolonged space missions.

Method / Experiment:

FLUMIAS (FLUorescence MIcroscopic Analysis in Space) is a newly developed research platform, which combines an automated microscope with a life support system and a centrifuge to apply variable gravitational loads up to 1g onboard the ISS. The FLUMIAS microscope enables live visualization of astrocytic adaptation processes and sensitivity thresholds at altered gravity conditions. To increase the knowledge on astrogliosis and the impact of space conditions on the reactive state of astrocytes, cytoskeletal dynamics and mitochondrial activity will be investigated. To prepare for this unique and complex microscopy platform, extensive testing protocols and procedures in ground-based facilities have been devised and are currently ongoing.

Result:

As ground-based preparation studies for the LAARA mission on FLUMIAS-ISS, we have established various pretesting protocols for our primary murine astrocyte samples which can be generalized for future missions on FLUMIAS. The imaging properties of the FLUMIAS science-reference model (SRM) and engineering model (EM) have been tested with mission-relevant samples and compared to other standard microscope systems. Biological samples and culturing protocols have to be adapted and tested with the FLUMIAS-specific microslides and fluid system to verify their viability under the challenging conditions of an ISS mission.

Conclusion:

The FLUMIAS microscope enables fast live-cell microscopy with confocal-like resolution. Living samples can be grown on FLUMIAS microslides and survive under simulated upload conditions as well as simulated on-orbit life-support system operations. Staining protocols and imaging procedures have been adapted to work with the FLUMIAS life-support system and microscope facility. The testing protocols will be published and recommended for other science teams utilizing FLUMIAS.