Abstract Space-based platforms currently represent the most accurate means to experimentally assess the influence of the space environment on biological systems. However, performing such experiments remains technically challenging and requires highly specialized instrumentation. This study describes the current development and hardware qualification of ExocubeBio, a miniaturized experimental platform for in-situ biological space exposure. This experiment is scheduled for installation on the exterior of the International Space Station in 2027, as part of Exobio, the European Space Agency’s new generation exobiology exposure facility. ExocubeBio will expose live microbial samples to the low Earth orbit environment, and combine autonomous in-situ optical density and fluorescence measurements, with the capacity to return preserved samples to Earth. Achieving these experimental goals requires a specialized, robust and reliable hardware system. The ExocubeBio hardware testing described here includes assessment of material biocompatibility and durability, functional validation of the miniaturized fluidic system, and optimization of the integrated optical subsystem for optical density and fluorescence measurements. These results demonstrate that the ExocubeBio experimental hardware components can each execute their core functional and operational requirements; subsystems allow for sample exposure, in-situ measurements of microbial cultures, and the chemical preservation of samples for post-flight analysis. As ExocubeBio transitions from hardware development to mission readiness, the results presented here validate the overall design and engineering approaches utilized. By combining the strengths of in-situ monitoring and sample return, ExocubeBio represents a significant advancement in space-based experimentation, and will provide new insights into microbial responses to the space environment.

The spaceflight environment exposes biological systems to microgravity and cosmic radiation, which are factors known to induce oxidative stress and neurodegenerative processes. As the central nervous system is highly susceptible to disruptions in redox homeostasis, the development of effective strategies to safeguard astronaut health and cognitive function during extended space missions has become imperative. In this study, we investigate cerium oxide nanoparticles (nanoceria, NC) as an antioxidant agent for human neuron-like cells aboard the International Space Station. Nanoceria demonstrated excellent biocompatibility, strong antioxidant properties, and the ability to stimulate neurite extension under both Earth gravity and simulated microgravity. Following the return of the samples to Earth, transcriptomic analyses revealed that nanoceria effectively counteracted the detrimental transcriptional alterations triggered by spaceflight stressors, thereby maintaining neuronal homeostasis. Importantly, the expression of genes involved in antioxidant defense, mitochondrial activity, and dopamine metabolism remained stable in nanoceria-treated neurons, in contrast to the dysregulation observed in untreated controls. These findings position cerium oxide nanoparticles as promising antioxidant neuroprotectants for long-duration space missions and related neurodegenerative conditions.

Expanding human space exploration necessitates technologies for sustainable local resource acquisition, to overcome unviable resupply missions. Asteroids, some of which rich in metals like platinum group elements, are promising targets. The BioAsteroid experiment aboard the International Space Station tested the use of microorganisms (bacteria and fungi) to extract 44 elements from L-chondrite asteroidal material under microgravity. Penicillium simplicissimum enhanced the release of palladium, platinum and other elements in microgravity, compared to non-biological leaching. For many elements, non-biological leaching was more effective in microgravity than on Earth, while bioleaching remained stable. Metabolomic analysis revealed distinct changes in microbial metabolism in space, particularly for P. simplicissimum, with increased production of carboxylic acids, and molecules of potential biomining or pharmaceutical interest in microgravity. These results demonstrate the impact of microgravity on bioleaching, highlighting the need for optimal combination of microorganisms, rock substrate, and conditions for successful biomining, in space and Earth.

Exposure to microgravity and cosmic radiation during spaceflight is responsible for oxidative stress onset, contributing to neuronal dysfunction and degeneration. The central nervous system is particularly vulnerable to redox imbalance and requires effective countermeasures to ensure astronaut health and performance on long-duration missions. In this study, the neuroprotective properties of polydopamine nanoparticles (PDNPs), known for their antioxidant activity, are investigated on neuron-like cells exposed to different gravitational and radiation regimes. Culture conditions included administration of PDNPs and permanence aboard the International Space Station (ISS) or on a random positioning machine. Transcriptomic analyses are conducted to assess gene expression alterations associated with oxidative stress, nuclear and mitochondrial integrity, and dopamine metabolism. In-flight, PDNP treatment mitigates the transcriptional changes induced by space stressors, preserving neuronal homeostasis. Notably, expression of key antioxidant defense genes, mitochondrial function markers and dopamine metabolism genes is stabilized in PDNP-treated neurons. This study provides preliminary evidence on the efficacy of PDNPs in protecting neuronal cells from the combined stressors associated with spaceflight: these findings suggest PDNPs as a promising countermeasure for space-induced neurodegeneration and support their potential translational application in the treatment of oxidative stress-related neurodegenerative pathologies on Earth.

Human space exploration faces different challenges. Topics like Bioregenerative Life Support Systems, In Situ Resource Utilization, and radiation protection, still require for more suitable solutions to be applied in long-term space exploration. Synthetic biology could be a powerful tool for enabling human exploration of space and planets. This paper explores key topics including resource utilization, life support systems, radiation protection, and human health, providing recommendations for short-, mid-, and long-term advancements in space exploration.

AbstractMicrogravity (µG) experienced during space flights promotes adaptation in several astronauts’ organs and tissues, with skeletal muscles being the most affected. In response to reduced gravitational loading, muscles (especially, lower limb and antigravity muscles) undergo progressive mass loss and alteration in metabolism, myofiber size, and composition. Skeletal muscle precursor cells (MPCs), also known as satellite cells, are responsible for the growth and maintenance of muscle mass in adult life as well as for muscle regeneration following damage and may have a major role in µG-induced muscle wasting. Despite the great relevance for astronaut health, very few data are available about the effects of real µG on human muscles. Based on the MyoGravity project, this study aimed to analyze: (i) the cellular and transcriptional alterations induced by real µG in human MPCs (huMPCs) and (ii) the response of human skeletal muscle to normal gravitational loading after prolonged exposure to µG. We evaluated the transcriptomic changes induced by µG on board the International Space Station (ISS) in differentiating huMPCs isolated from Vastus lateralis muscle biopsies of a pre-flight astronaut and an age- and sex-matched volunteer, in comparison with the same cells cultured on the ground in standard gravity (1×g) conditions. We found that huMPCs differentiated under real µG conditions showed: (i) upregulation of genes related to cell adhesion, plasma membrane components, and ion transport; (ii) strong downregulation of genes related to the muscle contraction machinery and sarcomere organization; and (iii) downregulation of muscle-specific microRNAs (myomiRs). Moreover, we had the unique opportunity to analyze huMPCs and skeletal muscle tissue of the same astronaut before and 30 h after a long-duration space flight on board the ISS. Prolonged exposure to real µG strongly affected the biology and functionality of the astronaut’s satellite cells, which showed a dramatic reduction of responsiveness to activating stimuli and proliferation rate, morphological changes, and almost inability to fuse into myotubes. RNA-Seq analysis of post- vs. pre-flight muscle tissue showed that genes involved in muscle structure and remodeling are promptly activated after landing following a long-duration space mission. Conversely, genes involved in the myelination process or synapse and neuromuscular junction organization appeared downregulated. Although we have investigated only one astronaut, these results point to a prompt readaptation of the skeletal muscle mechanical components to the normal gravitational loading, but the inability to rapidly recover the physiological muscle myelination/innervation pattern after landing from a long-duration space flight. Together with the persistent functional deficit observed in the astronaut’s satellite cells after prolonged exposure to real µG, these results lead us to hypothesize that a condition of inefficient regeneration is likely to occur in the muscles of post-flight astronauts following damage.

IntroductionAstroBio CubeSat (ABCS) is an Italian Space Agency (ASI) 3U CubeSat (100x100x340 mm) selected by European Space Agency (ESA) to be launched with the Vega C qualification maiden flight, as piggy back of the ASI LARES2 main satellite, by the end of 2020. ABCS will be deployed in an approximately circular orbit, with about 5900 km altitude and 70° of inclination. It implies that ABCS will spend a significant part of the orbital period within the internal Van Allen belt, close to its maximum. The radiation environment is characterized by a very high flux of charged particles, which have a significant effect on electronic components in terms of permanent damages due to accumulated dose effects and single events. Considering the extremely harsh space conditions, the estimated mission lifetime useful to perform the payload experiments should be defined in 3 months.ABCS Project is funded and managed by ASI in cooperation with INAF-Astrophysical Observatory of Arcetri, that will coordinate the scientific and engineering team. Partners of the projects are the School of Aerospace Engineering of Sapienza University of Rome, the University of Bologna, the University of Torino, and Kayser Italia.ABCS PayloadABCS will host a mini laboratory payload based on an innovative lab-on chip technology suitable for research in astrobiology. The objective is to test in space environments an automatic laboratory able to provide a highly integrated in-situ multiparameter platform that uses immunoassay tests exploiting chemiluminescence detection by means of on-chip a-Si:H photodiodes. The experiment will consist in a set of lateral flow immunoassays (LFIA) on nitrocellulose support where target biomolecules are immobilized in specific test areas. Reagents are deposited in a non-permanent fashion and in a dry form in the initial part (starting area) of the microfluidic path. When the reagents-delivery-system provides a volume of liquid reagent to the starting pad, capillary forces will guide the reagents through the LFIA microfluidic pathway. During the flow, liquid reagents will solubilize and transport along the path the deposited reagents, triggering specific reactions and allowing the chemiluminescence detection by the photodiodes.ABCS also mounts an ancillary radiation dose payload, to investigate the degradation of of electronic components exposed to the space environment. The device has twin components protected by established radiation screens, kindly provided by Thales Alenia Space Italia and by CESI, so that the degradation can be assessed on the basis of the difference between the observed currents.ABCS architecture and payload are based on the strong heritage gained by the research team with the ground validation of the PLEIADES (Planetary Life Explorer with Integrated Analytical Detection and Embedded Sensors) instrument, an R&D ASI project recently concluded.Enviromental challengesThe main challenges of the project are to mitigate the effects of the expected very high flux of charged particles, keeping the correct temperature (4°C/25°C) and pressure (about 1 atm) range for the payload to prevent reagents degradation. This invoked a series of technological solution to protect the payload. The pressurized environment is ensured by an inner aluminium box, hosting both the experiment and the main subsystems (batteries, on-board data handling, telemetry, tracking and control) hermetically sealed and providing shielding from radiation and charged particles. A thermal control system, including a passive control multi-layer insulation and an active heather mounted inside the pressurized box, maintain the temperature in the desired range.ConclusionABCS mission aims at evaluating the overall system functionality (delivery of reagents, mixing of chemicals, LoC characterization, detection of emitted photons, readout noise, etc.) such as the chemicals and biomolecules stability (reagents and antibodies employed in the experiment) in the extremely harsh environment.The in-orbit validation of the proposed technology would represent a significant breakthrough for autonomous execution of bio-analytical experiments in space with potential application in search for signs of life in planetary exploration missions, space biolabs without human support, health monitoring in manned missions.

Expanding human space exploration beyond Earth’s orbit necessitates efficient technologies for self-sustainable acquisition of local resources to overcome unviable resupply missions from Earth. Potential source of materials are asteroids, some of which contain valuable metals, such as platinum group elements. The BioAsteroid experiment, performed onboard the International Space Station, tested the use of microorganisms (bacteria and fungi) to carry out mining of useful elements from asteroidal material (L-chondrite) under microgravity, in support of a long-term human presence in space. The fungus Penicillium simplicissimum , enhanced the mean release of palladium, platinum and other elements from the meteorite material in microgravity, compared to non-biological leaching. However, there was large variability in the results. For many elements, non-biological leaching under microgravity was enhanced compared to terrestrial gravity, while bioleaching was unaffected. Metabolomics results revealed clear patterns that highlight the influence of space conditions on the microbial metabolism, particularly for P. simplicissimum . We identified the presence of carboxylic acids, and molecules of potential biomining and pharmaceutical interest, enhanced in microgravity. These results show a non-trivial effect of microgravity on bioleaching, highlighting the requirement of an optimal combination of microorganism(s), rock substrate, and conditions for successful biomining, both in space and Earth. ### Competing Interest Statement AM, MB and GN are employees of Kayser Italia L.t.d. All other authors declare no competing interests. * ISS : International Space Station, under microgravity condition Earth : Ground controls, under terrestrial gravity condition µg : Microgravity PGEs : Platinum group elements REEs : Rare earth elements ISRU : In situ resource utilisation BMR : BioMining Reactor EC : Experiment Container EU : Experiment Unit %NB : Element amount as percentage of sample / non-biological sample %M : Element amount as percentage of sample / total in the meteorite rock

Introduction Astrobiology is an interdisciplinary field covered by only a few CubeSat missions so far. Moreover, no CubeSat mission has ever mounted miniaturized technology for the purpose of searching for molecular evidences of life in space. AstroBio CubeSat (ABCS) is a 3U CubeSat selected by the European Space Agency (ESA) to be launched in spring 2022 with the Vega C maiden flight, as piggy back passenger of the ASI LARES2 mission. ABCS will host a payload assembly based on Lab-on-Chip (LoC) technology for biomarkers detection and will be deployed along a circular orbit with altitude of about 5900 km and inclination of 7°, therefore crossing the inner Van Allen belt where the radiation flux is close to its maximum. Due to the harsh environment, ABCS payload and subsystems will be likely exposed to damages and degradations of electronics and performances, thus the payload assembly and the operational architecture were designed to be as much dependable as possible. This approach should constitute the first step to implement a mature technology with the aim to check the stability of chemicals and biomolecules involved in space experiments. This work reports an overview of ABCS architecture and the approach chosen for its operational design. ABCS Architecture ABCS objective is to test in space an automatic in-situ multiparameter LoC [1], which exploits luminol injection and enzymatic bio-mimicking assays on a functionalized 3D wax-printed origami. Luminol will be transported by capillarity to reaction sites with immobilized biomolecules targets where the reactions will trigger chemiluminescence, detected by means of hydrogenated amorphous silicon (a-Si:H) photodiodes deposited on a borosilicate glass substrate and connected to a photocurrent readout board [2]. The described payload consists in an experiment board hosting the LoC and a support board containing peristaltic pumps for luminol injection, drivers for pumps, radiation field effect transistors (RADFETs) and pressure/temperature sensors. The LoC architecture allows to repeat the experiment up to six times. In addition to RADFETs, ABCS mounts an ancillary radiation dose sensor (ARDS), developed by Thales Alenia Space, with the aim to assess the radiation effects. The ARDS is able to measure different amounts of current, until its failure, depending on the dose acquired. To mitigate the effects of the expected very high flux of charged particles, an extra tungsten layer shielding was mounted on each side panel and all the main subsystems (experiment and support board, batteries and EPS board, on-board computer (OBC), telemetry, tracking and control board), were placed inside a 5 mm thick aluminium box. At the same time, to keep the temperature range (from 4°C to 20°C) and operative pressure (about 1 atm) required to allow the LoC capillarity effect and to prevent reagents degradation, the box was sealed and a thermal control system, composed by a multi-layer insulation and an active heather mounted inside the box, was implemented. ABCS Mission Design ABCS will be deployed in an approximately circular orbit at about 5900 km altitude and 70° of inclination, spending a significant amount of the orbital period within the inner Van Allen belt, very close to its radiation peak point. ABCS ground operations will be mainly performed from the School of Aerospace Engineering (SIA) Ground Station. Simulations show that SIA will have access to ABCS 4 times a day, with an average duration of about 65 minutes. For this reason, a network of radioamateurs and third part ground stations will be involved for supporting the collection of the telemetry and science data packages and possibly uplink commands. ABCS Operations The assumption we made is that ABCS should be able to perform the payload operations in a completely autonomous manner. As we know, radiation flux will most likely induce several errors on electronics and performances, causing potential mission failure due to the fact that payload operations may not start because the OBC fails to send the command to start the experiment. A possible way to reduce failure is to perform ABCS experiments where the proton flux is lower. Simulations shows that this happens when ABCS is at polar latitudes, namely outside the range [-60°; 60°]. For this reason, the payload operations, based on redundant checks and triggers, were implemented accordingly. The purpose is to automatically determine if ABCS is at a latitude useful to perform the experiments and verifying this condition by means of multiple triggers, time or position based. Each trigger is used for scheduling purposes only if the ones with higher priority are unreliable. If all the triggers are not reliable, payload operations are forced to begin, as it is better to perform eventually degraded payload operations rather than performing no payload operations at all. Conclusions ABCS is required to operate in an extremely harsh environment where radiation fluxes are likely to degrade the electronic devices. Operations should be scheduled in order to reduce the time needed to perform all the experiments. The chosen approach will lead ABCS to complete the payload operations in three orbital periods, reducing the total ionizing dose absorbed and guaranteeing the higher system reliability. Acknowledgments ABCS AstroBio-CubeSat is supported by ASI - Italian Space Agency ASI/INAF Agreement n. 2019-30-HH.0. References [1] Iannascoli, L. et al. 2020, "Astrobio cubesat: Enabling technologies for astrobiology research in space", Proceedings of the International Astronautical Congress, IAC. [2] Mirasoli M, et al. 2014. Multiwell cartridge with integrated array of amorphous silicon photosensors for chemiluminescence detection: development, characterization and comparison with cooled-CCD luminograph. Anal Bioanal Chem. Sep;406(23):5645-56.

Introduction: Long-term space missions trigger a prolonged neuroendocrine stress response leading to immune system dysregulation evidenced by susceptibility to infections, viral reactivation, and skin irritations. However, due to existing technical constraints, real-time functional immune assessments are not currently available to crew inflight. The in vitro cytokine release assay (CRA) has been effectively employed to study the stimulated cytokine response of immune cells in whole blood albeit limited to pre- and post-flight sessions. A novel two-valve reaction tube (RT) has been developed to enable the execution of the CRA on the International Space Station (ISS).Methods: In a comprehensive test campaign, we assessed the suitability of three materials (silicone, C-Flex, and PVC) for the RT design in terms of biochemical compatibility, chemical stability, and final data quality analysis. Furthermore, we thoroughly examined additional quality criteria such as safety, handling, and the frozen storage of antigens within the RTs. The validation of the proposed crew procedure was conducted during a parabolic flight campaign.Results: The selected material and procedure proved to be both feasible and secure yielding consistent and dependable data outcomes. This new hardware allows for the stimulation of blood samples on board the ISS, with subsequent analysis still conducted on the ground.Discussion: The resultant data promises to offer a more accurate understanding of the stress-induced neuroendocrine modulation of immunity during space travel providing valuable insights for the scientific community. Furthermore, the versatile nature of the RT suggests its potential utility as a testing platform for various other assays or sample types.

One of the main challenges to be faced in deep space missions is to protect the health and ensure the maximum efficiency of the crew by preparing methods of prevention and in situ diagnosis. Indeed, the hostile environment causes important health problems, ranging from muscle atrophy, osteopenia, and immunological and metabolic alterations due to microgravity, to an increased risk of cancer caused by exposure to radiation. It is, therefore, necessary to provide new methods for the real-time measurement of biomarkers suitable for deepening our knowledge of the effects of space flight on the balance of the immune system and for allowing the monitoring of the astronaut’s health during long-term missions. APHRODITE will enable human space exploration because it fills this void that affects both missions in LEO and future missions to the Moon and Mars. Its scientific objectives are the design, production, testing, and in-orbit demonstration of a compact, reusable, and reconfigurable system for performing the real-time analysis of oral fluid samples in manned space missions. In the frame of this project, a crew member onboard the ISS will employ APHRODITE to measure the selected target analytes, cortisol, and dehydroepiandrosterone sulfate (DHEA-S), in oral fluid, in four (plus one additional desired session) separate experiment sessions. The paper addresses the design of the main subsystems of the analytical device and the preliminary results obtained during the first implementations of the device subsystems and testing measurements on Earth. In particular, the system design and the experiment data output of the lab-on-chip photosensors and of the front-end readout electronics are reported in detail along with preliminary chemical tests for the duplex competitive CL-immunoassay for the simultaneous detection of cortisol and DHEA-S. Different applications also on Earth are envisaged for the APHRODITE device, as it will be suitable for point-of-care testing applications (e.g., emergency medicine, bioterrorism, diagnostics in developing countries, etc.).

This study is preliminary to an experiment to be performed onboard the International Space Station (ISS) and on Earth to investigate how low gravity influences the healing of sutured human skin and vein wounds. Its objective was to ascertain whether these tissue explants could be maintained to be viable ex vivo for long periods of time, mimicking the experimental conditions onboard the ISS. We developed an automated tissue culture chamber, reproducing and monitoring the physiological tensile forces over time, and a culture medium enriched with serelaxin (60 ng/mL) and (Zn(PipNONO)Cl) (28 ng/mL), known to extend viability of explanted organs for transplantation. The results show that the human skin and vein specimens remained viable for more than 4 weeks, with no substantial signs of damage in their tissues and cells. As a further clue about cell viability, some typical events associated with wound repair were observed in the tissue areas close to the wound, namely remodeling of collagen fibers in the papillary dermis and of elastic fibers in the vein wall, proliferation of keratinocyte stem cells, and expression of the endothelial functional markers eNOS and FGF-2. These findings validate the suitability of this new ex vivo organ culture system for wound healing studies, not only for the scheduled space experiment but also for applications on Earth, such as drug discovery purposes.

Long-time exposure to the microgravity conditions experienced during space flights induces alterations in the homeostasis of organs and tissues, including skeletal muscles, which undergo atrophy with the loss of mass and strength due to decreased size and altered composition of myofibers. Microgravity conditions can also affect the functionality of satellite cells, i.e., the adult stem cells providing the muscle precursors that are responsible for the growth and maintenance of muscle mass in adult life, as well as for muscle regeneration following a damage. The MyoGravity project, funded by Agenzia Spaziale Italiana (ASI), aimed to send human muscle precursor cells (huMPCs) on board the International Space Station (ISS) in order to study the effects of real microgravity on the differentiation capacity of this cell type. To this end, it was necessary to use a methodology to cultivate huMPCs inside dedicated space bioreactor devices (Experiment Units, EUs) specifically designed to cultivate cell cultures and perform scientific protocols in the space environment of the ISS. Here, we report the setting of several cell culture parameters to convert the EUs into suitable devices for biomedical experiments using huMPCs for space flight purposes.