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-SY5

Space Radiobiology

The study of the biologic effects of space radiation is considered a “hot topic,” with increased interest in the past years. In this chapter, the unique characteristics of the space radiation environment will be covered, from their history, characterization, and biological effects to the research that has been and is being conducted in the field. After a short introduction, you will learn the origin and characterization of the different types of space radiation and the use of mathematical models for the prediction of the radiation doses during different mission scenarios and estimate the biological risks due to this exposure. Following this, the acute, chronic, and late effects of radiation exposure in the human body are discussed before going into the detailed biomolecular changes affecting cells and tissues, and in which ways they differ from other types of radiation exposure. The next sections of this chapter are dedicated to the vast research that has been developed through the years concerning space radiation biology, from small animals to plant models and 3D cell cultures, the use of extremophiles in the study of radiation resistance mechanisms to the importance of ground-based irradiation facilities to simulate and study the space environment

BIOLEX – The Biology and Lunar experiment and the LOGOS Cubes

BIOLEX is a concept designed for in situ science on the Moon or in its orbit. As heritage of the polar and space experiment BIOMEX (Biology and Mars Experiment) on the ISS it is a more developed concept. Measurement operations on an exposure platform as well as within a micro-greenhouse device are part of this concept. The goal is to investigate the use of lunar resources as well as to analyse the stability of biomolecules as potential biosignatures serving as reference for future space exploration missions to Mars and the icy ocean moons in the outer solar system. Astrobiological exploration of the solar system is a priority research area such as emphasized by the European Astrobiology Roadmap (AstRoMap). It is focusing on several research topics, such as "Habitability" and on "Biomarkers for the detection of life". Therefore, "space platforms and laboratories", such as the EXPOSE setup installed outside the ISS, are essential to gain more knowledge on space- and planetary environments, which might be an essential basis for improvement of the robotic and human interplanetary exploration (Moon, Mars, Encedalus, Titan and Europa). In reference to these exposure platforms a new generation of hardware is needed to be installed in the lunar orbit or directly on the Moon. The BIOLEX is representing by its LOGOS (Lunar Organisms, Geo-microbiology and Organics Space Experiment) cubes such a concept combining the life detection topics with topics relevant to autonomous life supporting systems. A combination of a sample exposure device and a microhabitat for plants and microorganisms could address a tremendous number of questions from astrobiology and life sciences. The main scientific objectives for the use of BIOLEX-LOGOS cubes are: (i) in situ measurements by spectroscopy methods (such as Raman, IR, UV/VISspectroscopy) for analysis of biosignatures and their stability what is relevant for support of future life detection missions on Mars and the icy moons in the outer solar system); (ii) in situ measurements of environmental conditions (radiation, pressure/vacuum, temperature, pH, humidity) in micro-modules or compartments in reference to planned micro-habitat experiments placed on the Moon or incorporated on an exposure facility in orbit; (iii) in situ measurements of microorganisms’ activity in micro-modules / compartments in reference to planned microhabitat experiments placed on the moon or incorporated in the exposure facility in orbit. In reference to these scientific ideas the Moon is an excellent platform to operate different space experiments which will be of relevance for astrobiology, life sciences and human space missions. BIOLEX tries to fulfil a large number of scientific investigations in reference to these disciplines. The lunar environment is much harsher compared to Mars; and tests on biomolecules in this environment could provide information on their stability and therefore on the value to be used as reference for future space missions to Mars or the icy ocean moons in the outer solar system. Resources of the Moon such as the regolith or the freely available radiation on the surface could be tested by using them in a micro-greenhouse. Within this greenhouse different filters could test the optimal spectra range of the radiation

Intercellular Communication of Tumor Cells and Immune Cells after Exposure to Different Ionizing Radiation Qualities

Ionizing radiation can affect the immune system in many ways. Depending on the situation, the whole body or parts of the body can be acutely or chronically exposed to different radiation qualities. In tumor radiotherapy, a fractionated exposure of the tumor (and surrounding tissues) is applied to kill the tumor cells. Currently, mostly photons, and also electrons, neutrons, protons, and heavier particles such as carbon ions, are used in radiotherapy. Tumor elimination can be supported by an effective immune response. In recent years, much progress has been achieved in the understanding of basic interactions between the irradiated tumor and the immune system. Here, direct and indirect effects of radiation on immune cells have to be considered. Lymphocytes for example are known to be highly radiosensitive. One important factor in indirect interactions is the radiation-induced bystander effect which can be initiated in unexposed cells by expression of cytokines of the irradiated cells and by direct exchange of molecules via gap junctions. In this review, we summarize the current knowledge about the indirect effects observed after exposure to different radiation qualities. The different immune cell populations important for the tumor immune response are natural killer cells, dendritic cells, and CD8+ cytotoxic T-cells. In vitro and in vivo studies have revealed the modulation of their functions due to ionizing radiation exposure of tumor cells. After radiation exposure, cytokines are produced by exposed tumor and immune cells and a modulated expression profile has also been observed in bystander immune cells. Release of damage-associated molecular patterns by irradiated tumor cells is another factor in immune activation. In conclusion, both immune-activating and -suppressing effects can occur. Enhancing or inhibiting these effects, respectively, could contribute to modified tumor cell killing after radiotherapy

Space radiation biology

In this lesson, the interaction of charged particles of space radiation with organisms will be explained on molecular, cellular, tissue, organ and organismal level. First, an overview of possible health effects caused by space radiation will be given considering the radiation exposure during different space missions: cancer, lens opacification (cataract), light flashes, degenerative diseases of various organ systems and acute radiation syndrome. Diving to the molecular level with DNA damage, the specifics of heavy ion-induced DNA damage and sequelae of mis- or unrepaired DNA damage leading to the cancer risk induced by radiation exposure during space missions will be explained in context of the cellular DNA damage response. For other possible late effects of space radiation exposure, cataract and degeneration of the central nervous system, possible mechanisms will be discussed. As acute radiation exposure during a solar particle event and accumulation of high doses could occur in situations of insufficient shielding in deep space, pathogenesis of the acute radiation syndrome will be covered. The countermeasure section of the lecture encompasses basics of radiation protection and radiation risk management including shielding and radiation monitoring and the current stage of prophylactic (focus on cancer prophylaxis) and therapeutic treatment. Requirements of biological experiments at heavy ion accelerators and on spaceflight platforms to answer important open questions in space radiation biology before departing on a long-term interplanetary mission conclude the lecture

Improved cellular survival of bystander cells via NF-κB associated recovery after X-irradiation

Radiation-induced bystander effects (RIBE) are an acknowledged issue of radiation therapy. Radiation of tumor tissue has been shown to affect non-irradiated neighboring cells in a paracrine and endocrine manner. Transduction of bystander signaling though remains to be investigated in detail. A part of the transduction is the receptor-initiated activation of signaling pathways by secreted factors of the irradiated cell during irradiation damage response. This work focusses on the activation of the transcription factor Nuclear Factor kappaB (NF-kappaB) in bystander cells after irradiation. NF-kappaB is a well-known contributor to inflammatory processes like cyto- / chemokine production as well as to stress reactions such as the DNA damage response and cell cycle regulation. Using a mouse embryonic fibroblasts (MEF) in vitro model with a genetic knock-out of an NF-kappaB regulator (NEMO, NF-kappaB essential modulator), clonogenic survival and cell cycle distribution was determined in directly irradiated cells and in cells incubated with conditioned medium from X-irradiated cells (bystander treatment). Directly irradiated NEMO ko cells, plated for clonogenic survival immediately after X-irradiation, display the same dose-effect curve as the wildtype (wt) (a/b NEMO ko = 13.92 ± 2.4 vs. a/b wt = 12.37 ± 2.6). But when allowed to recover for 24 h, the wt cells show a broader shoulder in the curve (a/b = 3.5 ± 2.9), indicating a role of NF-kappaB in the repair of radiation induced DNA damages. Looking into the survival of bystander cells, the survival curves show a statistically different slope, with NEMO ko cells surviving better than wt cells (S16 Gy: NEMO ko = 1.66 vs wt = 0.83). The different behavior may correlate with NF-kappaB dependent DNA repair in bystander cells for NEMO ko and wt cells. Cell cycle analysis revealed a 6 hour delayed arrest in G2/M phase in directly irradiated NEMO ko cells compared to wt cells. This indicates that NF-kappaB regulated DNA repair pathways are important for recovery of radiation induced damages. Bystander NEMO ko show an even further delayed arrest at 48 h, while wt bystander cells show no G2/M arrest at all. This supports the assumption that damages have to overcome a certain threshold to be recognized as repair-worthy. As NF-kappaB has been reported to be involved in homologous recombination; cells with impairment in NF-kappaB pathways, such as NEMO ko, register damages caused by bystander treatment differently from wt cells. This leads to G2/M arrest extending time for repair in NEMO ko bystander cells

RADIATION RESPONSE OF PORCINE LENS EPITHELIAL CELLS AND EYE LENSES IN ORGAN-CULTURE

Astronauts on long-term space missions have a higher risk for the expression of radiation late effects such as cancer or sub-capsular cortical eye lens opacities. This is due to higher dose and different patterns of cellular energy deposition from high-linear-energy-transfer (LET) components of galactic cosmic radiation in space than that of terrestrial low-LET radiation on Earth. The eye lens is a radiation sensitive organ with radiation induced cataract to occur with a threshold absorbed dose of 0.5 Gy (0 - 1 Gy) of sparsely ionizing radiation. Doses perceived by astronauts on the International Space Station (ISS) are in average 150 mSv per year (Cucinotta et al. (2001) Radiat Res. 156:460-466). Radiationinduced lens opacification is assumed to initiate from post irradiation proliferative activity of genetically damaged lens epithelial cells with alterations in cell cycle control, apoptosis, differentiation, and cellular disorganization, or other pathways controlling lens fiber cells’ differentiation. As the porcine eye lens is similar to the human lens in size and anatomy, DNA damage response was investigated in ex-vivo porcine lenses in organ culture, in in-vitro cultivated lens epithelial slabs (ES) and in porcine lens epithelial cells (pLEC). Cell survival of proliferative cells was calculated from colony forming ability (CFA) assay. The phosphorylated form of H2AX (γH2AX) was used as a molecular marker to visualize DNA double strand breaks (DSB) and their repair. Propidium iodide based DNA staining for cellular DNA content marked radiation-induced cell cycle disturbances. In pLEC the cell survival curve of immediate plated cells and after a recovery period of 24 h follow the equation S=1.40xD+ln 1.47 and S=1.59xD+ln 1.79, respectively. DNA DSB are induced in a dose-dependent manner ( 18 DSB/cell/Gy) and repaired during successive recovery ( 5 DSB/cell/Gy residual damage after 24 h). For doses >2 Gy a cell cycle arrest in G2 phase occurred 24 h after X-irradiation and persisted up to 72 h post-irradiation. DNA DSB induction and repair could as well be documented for ES and whole lenses after X-irradiation. In whole lenses, the amount of residual damage (after 24 h and 48 h) was highest in the equatorial zone while in the central epithelial zone DSB repair seemed to proceed with time in a manner comparable to in-vitro cultivated pLEC. Lens organ culture allows cellular metabolism and DNA synthesis in whole lenses. Repair of DNA DSB takes place in the central epithelial layer and is reduced in the equatorial region of cultivated lenses

STARLIFE-An International Campaign to Study the Role of Galactic Cosmic Radiation in Astrobiological Model Systems.

In-depth knowledge regarding the biological effects of the radiation field in space is required for assessing the radiation risks in space. To obtain this knowledge, a set of different astrobiological model systems has been studied within the STARLIFE radiation campaign during six irradiation campaigns (2013-2015). The STARLIFE group is an international consortium with the aim to investigate the responses of different astrobiological model systems to the different types of ionizing radiation (X-rays, γ rays, heavy ions) representing major parts of the galactic cosmic radiation spectrum. Low- and high-energy charged particle radiation experiments have been conducted at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. X-rays or γ rays were used as reference radiation at the German Aerospace Center (DLR, Cologne, Germany) or Beta-Gamma-Service GmbH (BGS, Wiehl, Germany) to derive the biological efficiency of different radiation qualities. All samples were exposed under identical conditions to the same dose and qualities of ionizing radiation (i) allowing a direct comparison between the tested specimens and (ii) providing information on the impact of the space radiation environment on currently used astrobiological model organisms. Key Words: Space radiation environment-Sparsely ionizing radiation-Densely ionizing radiation-Heavy ions-Gamma radiation-Astrobiological model systems. Astrobiology 17, 101-109

STARLIFE—An International Campaign to Study the Role of Galactic Cosmic Radiation in Astrobiological Model Systems

In-depth knowledge regarding the biological effects of the radiation field in space is required for assessing the radiation risks in space. To obtain this knowledge, a set of different astrobiological model systems has been studied within the STARLIFE radiation campaign during six irradiation campaigns (2013–2015). The STARLIFE group is an international consortium with the aim to investigate the responses of different astrobiological model systems to the different types of ionizing radiation (X-rays, γ rays, heavy ions) representing major parts of the galactic cosmic radiation spectrum. Low- and high-energy charged particle radiation experiments have been conducted at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. X-rays or γ rays were used as reference radiation at the German Aerospace Center (DLR, Cologne, Germany) or Beta-Gamma-Service GmbH (BGS, Wiehl, Germany) to derive the biological efficiency of different radiation qualities. All samples were exposed under identical conditions to the same dose and qualities of ionizing radiation (i) allowing a direct comparison between the tested specimens and (ii) providing information on the impact of the space radiation environment on currently used astrobiological model organisms