Hardware Development for Plant Cultivation Allowing In Situ Fluorescence Analysis of Calcium Fluxes in Plant Roots Under Microgravity and Ground-Control Conditions.

Maintaining an optimal leaf and stem orientation to yield a maximum photosynthetic output is accomplished by terrestrial plants using sophisticated mechanisms to balance their orientation relative to the Earth's gravity vector and the direction of sunlight. Knowledge of the signal transduction chains of both gravity and light perception and how they influence each other is essential for understanding plant development on Earth and plant cultivation in space environments. However, in situ analyses of cellular signal transduction processes in weightlessness, such as live cell imaging of signaling molecules using confocal fluorescence microscopy, require an adapted experimental setup that meets the special requirements of a microgravity environment. In addition, investigations under prolonged microgravity conditions require extensive resources, are rarely accessible, and do not allow for immediate sample preparation for the actual microscopic analysis. Therefore, supply concepts are needed that ensure both the viability of the contained plants over a longer period of time and an unhindered microscopic analysis in microgravity. Here, we present a customized supply unit specifically designed to study gravity-induced Ca2+ mobilization in roots of Arabidopsis thaliana. The unit can be employed for ground-based experiments, in parabolic flights, on sounding rockets, and probably also aboard the International Space Station.

Fluid and Bubble Flow Detach Adherent Cancer Cells to Form Spheroids on a Random Positioning Machine.

In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable low-mass model system, such as cell cultures, these devices simulating microgravity have been shown to produce results similar to those obtained in a space experiment under real microgravity conditions. One of these effects observed under real and simulated microgravity is the formation of spheroids from 2D adherent cancer cell cultures. Since real microgravity cannot be generated in a laboratory on Earth, we aimed to determine which forces lead to the detachment of individual FTC-133 thyroid cancer cells and the formation of tumor spheroids during culture with exposure to random positioning modes. To this end, we subdivided the RPM motion into different static and dynamic orientations of cell culture flasks. We focused on the molecular activation of the mechanosignaling pathways previously associated with spheroid formation in microgravity. Our results suggest that RPM-induced spheroid formation is a two-step process. First, the cells need to be detached, induced by the cell culture flask's rotation and the subsequent fluid flow, as well as the presence of air bubbles. Once the cells are detached and in suspension, random positioning prevents sedimentation, allowing 3D aggregates to form. In a comparative shear stress experiment using defined fluid flow paradigms, transcriptional responses were triggered comparable to exposure of FTC-133 cells to the RPM. In summary, the RPM serves as a simulator of microgravity by randomizing the impact of Earth's gravity vector especially for suspension (i.e., detached) cells. Simultaneously, it simulates physiological shear forces on the adherent cell layer. The RPM thus offers a unique combination of environmental conditions for in vitro cancer research.

Unraveling astrocyte behavior in the space brain: Radiation response of primary astrocytes.

Introduction: Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes' response toward low linear energy transfer (LET) X-rays and high-LET carbon (12C) and iron (56Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Since astronauts are exposed to ionizing radiation and microgravity during space missions, the effect of simulated microgravity on DNA damage induction and repair was investigated. Methods: Primary murine cortical astrocytes were irradiated with different doses of X-rays, 12C and 56Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes' reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X- rays were investigated by mRNA-sequencing. RT-qPCR for several genes of interest was performed with RNA from X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1ß, Il6, and Tgfß1. Levels of the pro inflammatory cytokine IL-6 were determined using ELISA. DNA damage response was investigated after exposure to X-rays followed by incubation on a 2D clinostat to simulate the conditions of microgravity. Results: Astrocytes showed distinct responses toward the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSBs) and the respective repair was dose-, LET- and time-dependent. Simulated microgravity had no significant influence on DNA DSB repair. Proliferation and cell cycle progression was not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. Discussion: In conclusion, primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types.

Differential effects of hypergravity on immune dysfunctions induced by simulated microgravity.

Microgravity (µg) is among the major stressors in space causing immune cell dysregulations. These are frequently expressed as increased pro-inflammatory states of monocytes and reduced activation capacities in T cells. Hypergravity (as artificial gravity) has shown to have beneficial effects on the musculoskeletal and cardiovascular system both as a countermeasure option for µg-related deconditioning and as "gravitational therapy" on Earth. Since the impact of hypergravity on immune cells is sparsely explored, we investigated if an application of "mild" mechanical loading of 2.8 g is able to avoid or treat µg-mediated immune dysregulations. For this, T cell and monocyte activation states and cytokine pattern were first analyzed after whole blood antigen incubation in simulated µg (s-µg) by using the principle of fast clinorotation or in hypergravity. Subsequent hypergravity countermeasure approaches were run at three different sequences: one preconditioning setting, where 2.8 g was applied before s-µg exposure and two therapeutic approaches in which 2.8 g was set either intermediately or at the end of s-µg. In single g-grade exposure experiments, monocyte pro-inflammatory state was enhanced in s-µg and reduced in hypergravity, whereas T cells displayed reduced activation when antigen incubation was performed in s-µg. Hypergravity application in all three sequences did not alleviate the increased pro-inflammatory potential of monocytes. However, in T cells the preconditioning approach restored antigen-induced CD69 expression and IFNγ secretion to 1 g control values and beyond. This in vitro study demonstrates a proof of concept that mild hypergravity is a gravitational preconditioning option to avoid adaptive immune cell dysfunctions induced by (s-)µg and that it may act as a booster of immune cell functions.

Human neural network activity reacts to gravity changes in vitro.

During spaceflight, humans experience a variety of physiological changes due to deviations from familiar earth conditions. Specifically, the lack of gravity is responsible for many effects observed in returning astronauts. These impairments can include structural as well as functional changes of the brain and a decline in cognitive performance. However, the underlying physiological mechanisms remain elusive. Alterations in neuronal activity play a central role in mental disorders and altered neuronal transmission may also lead to diminished human performance in space. Thus, understanding the influence of altered gravity at the cellular and network level is of high importance. Previous electrophysiological experiments using patch clamp techniques and calcium indicators have shown that neuronal activity is influenced by altered gravity. By using multi-electrode array (MEA) technology, we advanced the electrophysiological investigation covering single-cell to network level responses during exposure to decreased (micro-) or increased (hyper-) gravity conditions. We continuously recorded in real-time the spontaneous activity of human induced pluripotent stem cell (hiPSC)-derived neural networks in vitro. The MEA device was integrated into a custom-built environmental chamber to expose the system with neuronal cultures to up to 6 g of hypergravity on the Short-Arm Human Centrifuge at the DLR Cologne, Germany. The flexibility of the experimental hardware set-up facilitated additional MEA electrophysiology experiments under 4.7 s of high-quality microgravity (10-6 to 10-5 g) in the Bremen drop tower, Germany. Hypergravity led to significant changes in activity. During the microgravity phase, the mean action potential frequency across the neural networks was significantly enhanced, whereas different subgroups of neurons showed distinct behaviors, such as increased or decreased firing activity. Our data clearly demonstrate that gravity as an environmental stimulus triggers changes in neuronal activity. Neuronal networks especially reacted to acute changes in mechanical loading (hypergravity) or de-loading (microgravity). The current study clearly shows the gravity-dependent response of neuronal networks endorsing the importance of further investigations of neuronal activity and its adaptive responses to micro- and hypergravity. Our approach provided the basis for the identification of responsible mechanisms and the development of countermeasures with potential implications on manned space missions.

Hypergravity Attenuates Reactivity in Primary Murine Astrocytes.

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approaches.

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies.

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson's disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y.

In Prostate Cancer Cells Cytokines Are Early Responders to Gravitational Changes Occurring in Parabolic Flights.

The high mortality in men with metastatic prostate cancer (PC) establishes the need for diagnostic optimization by new biomarkers. Mindful of the effect of real microgravity on metabolic pathways of carcinogenesis, we attended a parabolic flight (PF) mission to perform an experiment with the PC cell line PC-3, and submitted the resulting RNA to next generation sequencing (NGS) and quantitative real-time PCR (qPCR). After the first parabola, alterations of the F-actin cytoskeleton-like stress fibers and pseudopodia are visible. Moreover, numerous significant transcriptional changes are evident. We were able to identify a network of relevant PC cytokines and chemokines showing differential expression due to gravitational changes, particularly during the early flight phases. Together with differentially expressed regulatory lncRNAs and micro RNAs, we present a portfolio of 298 potential biomarkers. Via qPCR we identified IL6 and PIK3CB to be sensitive to vibration effects and hypergravity, respectively. Per NGS we detected five upregulated cytokines (CCL2, CXCL1, IL6, CXCL2, CCL20), one zink finger protein (TNFAIP3) and one glycoprotein (ICAM1) related to c-REL signaling and thus relevant for carcinogenesis as well as inflammatory aspects. We found regulated miR-221 and the co-localized lncRNA MIR222HG induced by PF maneuvers. miR-221 is related to the PC-3 growth rate and MIR222HG is a known risk factor for glioma susceptibility. These findings in real microgravity may further improve our understanding of PC and contribute to the development of new diagnostic tools.

The MAPHEUS module CellFix for studying the influence of altered gravity on the physiology of single cells.

Gravity is the only constant stimulus during the evolution of life. To investigate the impact of the absence of gravity on living systems, their molecular and morphological status has to be studied under microgravity conditions. The experiment unit CellFix was developed in order to provide the possibility of exposure and chemical fixation of small biological systems, such as neurons, stem cells, small animals, yeast cultures, plants, etc., at dedicated time points during a sounding rocket flight. The current version of CellFix consists of two culture bags containing cell cultures in a temperature-controlled pressure vessel. The biosystems in the culture bags can be fixed by pumping the fixative [e.g., paraformaldehyde (PFA), methanol, RNAlater, or others] from a connected bag into the cell suspension. The mechatronic basis of the experiment unit is constructed from compartments of the shelf parts. Open source microcontroller systems (Arduino) or gear pumps, accumulators, etc., from the model making sector are affordable and reliable components to build up an experiment on an unmanned space mission such as a sounding rocket flight. Also, new technologies such as fused deposition modeling were used to construct structures and brackets, which were tested successfully in environmental tests and real space flights (MAPHEUS 7 and 8 sounding rocket missions). In combination with the possibility to handle the experiment as a late access insert in a standardized rocket compartment, CellFix provides a multiusable experiment unit for performing life science experiments in space.

Short-Term Microgravity Influences Cell Adhesion in Human Breast Cancer Cells.

With the commercialization of spaceflight and the exploration of space, it is important to understand the changes occurring in human cells exposed to real microgravity (r-µg) conditions. We examined the influence of r-µg, simulated microgravity (s-µg, incubator random positioning machine (iRPM)), hypergravity (hyper-g), and vibration (VIB) on triple-negative breast cancer (TNBC) cells (MDA-MB-231 cell line) with the aim to study early changes in the gene expression of factors associated with cell adhesion, apoptosis, nuclear factor "kappa-light-chain-enhancer" of activated B-cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. We had the opportunity to attend a parabolic flight (PF) mission and to study changes in RNA transcription in the MDA-MB cells exposed to PF maneuvers (29th Deutsches Zentrum für Luft- und Raumfahrt (DLR) PF campaign). PF maneuvers induced an early up-regulation of ICAM1, CD44 and ERK1 mRNAs after the first parabola (P1) and a delayed upregulation of NFKB1, NFKBIA, NFKBIB, and FAK1 after the last parabola (P31). ICAM-1, VCAM-1 and CD44 protein levels were elevated, whereas the NF-κB subunit p-65 and annexin-A2 protein levels were reduced after the 31st parabola (P31). The PRKCA, RAF1, BAX mRNA were not changed and cleaved caspase-3 was not detectable in MDA-MB-231 cells exposed to PF maneuvers. Hyper-g-exposure of the cells elevated the expression of CD44 and NFKBIA mRNAs, iRPM-exposure downregulated ANXA2 and BAX, whereas VIB did not affect the TNBC cells. The early changes in ICAM-1 and VCAM-1 and the rapid decrease in the NF-κB subunit p-65 might be considered as fast-reacting, gravity-regulated and cell-protective mechanisms of TNBC cells exposed to altered gravity conditions. This data suggest a key role for the detected gravity-signaling elements in three-dimensional growth and metastasis.