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.

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.

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.

Astronauts are exposed to considerable doses of space radiation during long-term space missions. As complete shielding of the highly energetic particles is impracticable, the cellular response to space-relevant radiation qualities has to be understood in order to develop countermeasures and to reduce radiation risk uncertainties. The transcription factor Nuclear Factor κB (NF-κB) plays a fundamental role in the immune response and in the pathogenesis of many diseases. We have previously shown that heavy ions with a linear energy transfer (LET) of 100–300 keV/µm have a nine times higher potential to activate NF-κB compared to low-LET X-rays. Here, chemical inhibitor studies using human embryonic kidney cells (HEK) showed that the DNA damage sensor Ataxia telangiectasia mutated (ATM) and the proteasome were essential for NF-κB activation in response to X-rays and heavy ions. NF-κB’s role in cellular radiation response was determined by stable knock-down of the NF-κB subunit RelA. Transfection of a RelA short-hairpin RNA plasmid resulted in higher sensitivity towards X-rays, but not towards heavy ions. Reverse Transcriptase real-time quantitative PCR (RT-qPCR) showed that after exposure to X-rays and heavy ions, NF-κB predominantly upregulates genes involved in intercellular communication processes. This process is strictly NF-κB dependent as the response is completely absent in RelA knock-down cells. NF-κB’s role in the cellular radiation response depends on the radiation quality.

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

MiniFix, a syringe-based biological fixation system (SBBFS) is a versatile, fully 3D-printed syringe-driven, stepper motor-operated platform designed for the chemical fixation of biological samples in space-based research. Unlike conventional systems, it leverages additive manufacturing to provide modularity and customizability, enabling manipulation and a chemical fixation of biological samples under altered gravity conditions. MiniFix has performed five successful missions on the MAPHEUS sounding rocket and has demonstrated its reliability and adaptability. The integrated thermal management system uses waste heat from the stepper motors to maintain accurate sample temperature and in turn reduces power consumption and weight. MiniFix is particularly notable for its flexibility, allowing adaptation to diverse biological model systems, from simple organisms to more complex tissue cultures. Its modular design and 3D-printing process enable quick, cost-effective adjustments for different experimental setups. It was successfully printed with three different materials– PLA (Polylactic acid), PETG (Polyethylene terephthalate glycol-modified), and the biodegradable GreenTEC Pro. Its ability to integrate modifications such as illumination further enhances its adaptability for future space missions, for instance with photosynthetic organisms. By offering reliability, modular flexibility, and adaptation to a broad range of biological research goals, the SBBFS represents a new approach to construct flexible hardware for space and gravitational biology.