Introduction / Background:
Single cells to complex organisms demonstrate changes in physiology and signaling pathways when being exposed to altered gravity conditions. However, open questions concerning the underlying mechanisms of gravi-perception/ signaling still exist. We focus to understand how neuronal cells respond to altered gravitational conditions, since neuronal activity is modulator of behavior, human cognition, learning, memory and motor skills. Changes are highly important for crew performance.

Method:
We developed experiment modules for cell cultivation (temperature, etc.), online electrophysiological measurements and chemical fixation of adherent or suspension cellular or organoid samples. Reusable modules/hardware were designed employing conventional and more sophisticated modelling as well as bio-compatible 3D printing. Development of experiment modules that can be utilized in common gravity research platforms (ZARM Drop Tower in Bremen, Germany; DLR Short-Arm Human Centrifuge in Cologne, Germany; parabolic flights from Bordeaux, France and sounding rockets from ESRANGE, Kiruna, Sweden) plays an increasingly role in data comparability and statistical analysis. In the recently launched DLR rocket campaign MAPHEUS-14, several biological experiments were performed in parallel. Online electrophysiological measurements on a multi-electrode array (MEA) system were recorded (“Mind-G”). In-flight chemical fixation of biological samples (human IPSC-derived motoneurons, primary murine astrocytes) during various acceleration phases was conducted for subsequent proteomic profiling (LIFT). In-flight fixation was performed on mature human IPSC-derived brain organoids for transcriptomic analyses (ROMS). Microgravity studies should always be well prepared and accompanied by studies in ground-based facilities (GBFs). A broad portfolio of facilities for studies in simulated microgravity (which need final verification in real microgravity) as well as hypergravity are used in our studies and are provided to external scientists e.g. by the ESA GBF program.

Results:
All experiment modules functioned flawlessly and environmental data revealed the desired stable conditions throughout the rocket flight. The flight scenario of MAPHEUS-14 equipped with a new motor system provided microgravity time of approximately 6.5 min with optimal conditions for the cell cultures, as revealed by post-flight cellular and organoid morphology as well as by persisting live neuronal activity even after several days after return to Earth.

Conclusion:
The increasingly specialized development of printable materials and the availability of electronic components is opening up a new world of experiment development for space experiments, here demonstrated for sounding rockets. This trend in the growing new space sector also enables the simple and application-based training of young scientists.

Introduction:
Astronauts face a variety of health risks during spaceflight, most of which are due to the absence of gravitational, i.e. mechanical, forces and exposure to space radiation. The musculoskeletal and sensorimotor systems, consisting of sensory and motor neurons, are most affected by microgravity. It is important to study the response of these systems affected by altered gravity to gain a deeper understanding of the effects of spaceflight conditions, to investigate whether reduced neuromuscular transmission in microgravity causes muscle weakness in astronauts, and to identify possible countermeasures.

Methods:
The Laminar Inflight Fixation Technology (LIFT) module is a DLR frequent-flyer payload to enable a fast and reliable chemical fixation of biological samples during different phases of a sounding rocket flight. The samples were cultured on slides and chemically fixed directly after the hypergravity phase (launch) and following the microgravity phase to provide prolonged durations of approx. 6 minutes with respect to potential cellular adaptive processes. Corresponding controls at normal gravity (1g) were fixed during a test-countdown procedure as well as in standard lab conditions. The obtained samples are currently subjected to extensive analyses for proteomic, transcriptomic, and microscopic profiling to investigate changes in cell morphology, gene expression, and protein content.

The MAPHEUS sounding rocket was employed so far for the LIFT module, fixing two models of human- induced pluripotent stem cell (hiPSC)-derived motor neurons, as well as primary murine astrocytes. Analysing the effects of microgravity on these cell types compared to matched 1g ground controls will further our understanding of altered gravity exposure on the brain and central nervous system of astronauts at the cellular level.

Results:
Neural cells subjected to real microgravity on the rocket flights MAPHEUS-13 (05/2023) and -14 (02/2024) have been subjected to preliminary analysis. Post-flight phase contrast microscopy revealed no morphological differences between flight samples and ground controls. We thus were able to verify sample integrity and successful fixation in the LIFT module. The samples were lysed and cryopreserved for further analyses in regards of protein content and gene expression, as well as immunofluorescence analyses to investigate overall cell morphology and ultrastructural changes on a synaptic level.

Conclusion:
The modularity and reliability of the LIFT module were proven with motor neuron and astrocyte cultures during two rocket launches. We are currently investigating potential alterations of motor neurons induced by altered gravity conditions. The technology will be used to further investigate neuromuscular models and more complex co-culture systems.

Introduction:
Cyanobacteria, also referred to as blue-green algae, are phototrophic bacteria able to perform photosynthesis and use carbon dioxide to create organic matter. These bacteria are great O2 producers and CO2 consumers, which makes them very good candidates to recycle CO2 and produce O2 in closed and highly constrained environments such as the ISS (International Space Station) or long-term space journeys. Our objective is to evaluate a cultivation technique for cyanobacteria using acoustic levitation to optimize light penetration and possibly stimulate the cyanobacteria to enhance their O2 production, which is closely associated with light exposure. Acoustic levitation is a technique that allows the manipulation of small objects and cells (from 1 µm to a few hundreds µm depending on the acoustic parameters) using ultrasounds (Jeger-madiot et al., 2022). It allows easy manipulation of micro-algae without any direct physical contact, making it particularly suitable for micro-gravity environments.

Methods:
Experiments were conducted during the CNES parabolic flights campaign in spring 2024 to determine optimal acoustic levitation parameters for cyanobacteria during 0g-phases. Our approach involves growing cyanobacteria in thin layers and subjecting them to an acoustic field. We present findings on the dynamics of acoustic trapping in micro-gravity, aiming for a comprehensive understanding of cyanobacteria manipulation in a weightless environment and for a minimization of energy consumption, which is also critical in space.
Cyanobacteria were cultivated in multi-node PDMS (polydimethylsiloxane) chips at room temperature and exposed to an ultrasonic standing wave generated by a piezoelectric transducer. Side view visualizations were used to measure oscillation velocities at various amplitude of the acoustic force.

Results:
The study identified a minimum voltage required to allow the acoustic manipulation of cyanobacteria in weightless conditions. A minimum voltage of 0.4 V was needed to move the cyanobacteria using the acoustic force. This has been compared to the minimum amplitude of 2 to 3 V needed to move the cyanobacteria on earth. It confirms that acoustic manipulation will need much less energy in space than on earth.
Furthermore, a relationship between cyanobacteria layer thickness and the amplitude of the acoustic force was established. These findings contribute to optimizing cyanobacteria cultivation techniques for space applications, particularly within the MELiSSA (Micro-Ecological Life Support System Alternative) program (Farges et al., 2008). By leveraging acoustic levitation in microgravity, we aim to enhance cyanobacteria's oxygen production capacity, facilitating sustainable life support systems for future space exploration missions.

Introduction
The impact of altered gravitational conditions, such as hyper-gravity or micro-gravity, on the neurological health of astronauts presents a significant challenge for extended space missions, whether destined for the Moon, Mars, or prolonged stays aboard the International Space Station (ISS). Extensive research has underscored the critical importance of monitoring brain activity, both pre- and post-mission, revealing profound and enduring effects on the cognitive functions of astronauts [1]. These findings demonstrate the intricate relationship between gravitational variations and brain physiology. It becomes necessary to understand the real impact of microgravity down to the cellular level to ensure the well-being and performance of space travelers during extended voyages beyond Earth's orbit.

Methods
In this talk, we focus on the influence of gravity variations on the activity of cellular spheroids of primary hippocampal neurons, which were trapped and maintained in acoustic levitation (Fig. 2). Indeed in a Bulk Acoustic Wave resonator, spherical objects, such as particles in suspension in a fluid, can be moved toward an acoustic pressure node where they can be maintained in an equilibrium position, between the walls of a resonant cavity. In the so-called acoustic levitation plane, the gravity is counterbalanced by the Acoustic Radiation Force (ARF) created when the resonance condition is respected (ℎ = 1/2 λ_a, with h the height of the cavity and λ_a the acoustic wavelength) [2].

A dedicated setup has been designed and built to perform live calcium imaging during parabolic flights (Fig. 1) funded by the CNES (Centre National d’Etudes Spatiales) and organized by Novespace. The setup has already been used to monitor 2D neuronal networks showing an influence of gravity variations on the neurons activity [3].

Results
Using this imaging platform, we were able to record the electrical activity of 3D neurons spheroids trapped in acoustic levitation during gravity changes over different parabolas. The analysis of these experiments will allow us to better understand the impact of gravity on neuronal activity in 3D.

We will present in this talk experimental evidences showing that indeed ARF can be used to move and levitate large objects in weightlessness conditions. We will also show that it is indeed possible to monitor calcic activity of neuronal spheroids in acoustic levitation (Fig 3). Preliminary results after this campaign show that indeed the calcic activity of neuronal spheroids seems to be influenced by the variation of gravity, from hyper-gravity (1.8g) to micro-gravity (0g).

TTo explore new worlds we must ensure humans can survive and thrive in the space environment. Incidence of kidney stones in astronauts is a major risk factor associated with long-term missions, caused by increased blood calcium levels due to bone demineralisation triggered by microgravity and space radiation. Transcriptomic changes have been observed in other tissues during spaceflight, including the kidney. We analysed kidney transcriptome patterns in two different strains of mice flown on the International Space Station, C57BL/6J and BALB/c. Transcriptomic data related to kidney tissue obtained in the missions Rodent Research-1 (RR-1), Rodent Research-3 (RR-3), and Rodent Research-7 (RR-7) were obtained from NASA’s GeneLab Platform from dataset identifiers OSDR-102, OSDR-163 and OSDR-253. R Studio and the package DESeq2 were used to perform differential gene expression analysis comparing Flight to Ground Control. Overrepresentation analysis was done using WebGestalt and Gene set enrichment analysis was performed using the program GSEA. The set of non-synonymous genetic differences between C57BL/6J and BALB/c was taken from Timmermans and collaborators, 2017. We found that the expression of genes involved in lipid metabolism, extracellular matrix and TGF- β signalling are affected in the kidney by spaceflight and the overrepresentation analysis showed that spaceflight is associated with positive enrichment of cholesterol metabolic pathways and negative enrichment of ECM pathways in the kidney. Interestingly we found diverging patterns of lipid synthesis, protein synthesis and circadian rhythm in response to spaceflight in C57BL/6J and BALB/c mice determined by overrepresentation analysis and that C57BL/6J (RR-1) and BALB/c (RR-3) mice present genetic background differences in lipid and extracellular matrix metabolism was reflected in the transcriptomics difference encountered. Overall, we found a stronger response was seen in C57BL/6J mice than BALB/c. Genetic differences in hyaluronan metabolism between strains may confer protection against extracellular matrix remodelling through the downregulation of epithelial-mesenchymal transition. We intend for our findings to contribute to the development of new countermeasures against kidney disease in astronauts and people here on Earth. We would expect that mice exposed to the same length of spaceflight would have similar responses to the stress, nonetheless, we encountered a very different response between strains. Their different genetic background is potentially a factor in explaining this difference.

The International Space Station (ISS) has long been a hub for bio-experiments in microgravity, but challenges such as human error, labor costs, and experiment restrictions have prompted a shift towards artificial satellite-based missions. Researchers are exploring bio-experimental missions to be conducted via satellites, aiming to automate procedures and reduce human intervention. This shift becomes more crucial with the retirement of the ISS, expecting an increase in satellite-based bio-experiments.

To facilitate this transition, IDDK is collaborating with satellite manufacturers to streamline the development of bio-experimental equipment and artificial satellites. Their focus lies in microscopic observation, a vital aspect of bio-experiments, and they introduce the micro imaging device (MID) technology to address challenges related to weight, vibration, and space availability.

The MID technology revolutionizes traditional microscopy by utilizing semiconductor-based systems. Unlike conventional microscopes, MID requires no optical magnification, eliminating the need for individual adjustments of the optical lens system. This ensures a resolution dependent on the size of the semiconductor mesh, providing a lightweight and ultra-compact solution for microscopic observation.

In a microscopic observation method using MID, the specimen is placed on the surface (or placement unit set as necessary) of an MID imaging area (imaging device array) and is illuminated. The light information from the specimen is then directly collected in the MID. In MID, multiple pixels, including an optical element that collects light and a light receiving unit (photodiode) that receives the light collected by the optical element, are arranged at predetermined intervals. This method requires no optical magnification, and hence images can be acquired with a resolution dependent on the size of the semiconductor mesh without loss of light information due to the optical path. Therefore, unlike conventional microscopes, no individual adjustment is required for the optical lens system to form an image of light from the object of interest at the desired position and magnification.

The MID technology's versatility is demonstrated through sample images, showcasing the ease of capturing microscopic images without the need for focusing. The images include zooplankton and lysozyme crystals, highlighting the technology's capabilities in observing dynamic samples.

Moving forward, IDDK is developing a Micro Bio Space Lab, a compact system for real-time microscopic observation of cultured cells or microorganisms in space. This lab aims to provide an integrated solution, incorporating environmental sensors and a solution tank for various applications, including cell culture.