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Aims Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell-specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry, and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19. Methods and results In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). Massive analysis of cDNA ends (MACE)-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset ‘Heart Cell Atlas’ and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis. MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value, Graphical Abstract

Myocarditis in response to COVID-19 vaccination has been reported since early 2021. In particular, young male individuals have been identified to exhibit an increased risk of myocardial inflammation following the administration of mRNA-based vaccines. Even though the first epidemiological analyses and numerous case reports investigated potential relationships, endomyocardial biopsy (EMB)-proven cases are limited. Here, we present a comprehensive histopathological analysis of EMBs from 15 patients with reduced ejection fraction (LVEF = 30 (14–39)%) and the clinical suspicion of myocarditis following vaccination with Comirnaty® (Pfizer-BioNTech) (n = 11), Vaxzevria® (AstraZenica) (n = 2) and Janssen® (Johnson & Johnson) (n = 2). Immunohistochemical EMB analyses reveal myocardial inflammation in 14 of 15 patients, with the histopathological diagnosis of active myocarditis according the Dallas criteria (n = 2), severe giant cell myocarditis (n = 2) and inflammatory cardiomyopathy (n = 10). Importantly, infectious causes have been excluded in all patients. The SARS-CoV-2 spike protein has been detected sparsely on cardiomyocytes of nine patients, and differential analysis of inflammatory markers such as CD4+ and CD8+ T cells suggests that the inflammatory response triggered by the vaccine may be of autoimmunological origin. Although a definitive causal relationship between COVID-19 vaccination and the occurrence of myocardial inflammation cannot be demonstrated in this study, data suggest a temporal connection. The expression of SARS-CoV-2 spike protein within the heart and the dominance of CD4+ lymphocytic infiltrates indicate an autoimmunological response to the vaccination.

Aims MicroRNAs (miRNAs) might be used as prospective biomarkers for the identification of unexplained heart failure caused by a viral and/or inflammatory process. The aim of this study was to identify and to evaluate prognostic miRNAs in serum of patients with inflammatory heart diseases diagnosed by endomyocardial biopsies. Methods and results After TaqMan® OpenArray® screening of 754 unique circulating miRNAs in serum of biopsy‐proven patients [184 patients with inflammatory and/or virally induced myocardial diseases (DCMi), 25 patients with dilated cardiomyopathy (DCM), and 25 healthy donors], we identified seven miRNAs of interest (P

Plasminogen activator inhibitor-1 (PAI-1) has a cardioprotective function in mice by repressing cardiac fibrosis through TGF-β and plasminogen-mediated pathways. In addition it is known to be involved in the recruitment and polarization of monocytes/macrophages towards a M2 phenotype in cancer. Here, we investigated the expression of PAI-1 in human dilated cardiomyopathy (DCM) and inflammatory dilated cardiomyopathy (DCMi) and its effect on cardiac fibrosis and macrophage polarization. We retrospectively analyzed endomyocardial biopsies (EMBs) of patients with DCM or DCMi for PAI-1 expression by immunohistochemistry. Furthermore, EMBs were evaluated for the content of fibrotic tissue, number of activated myofibroblasts, TGF-β expression, as well as for M1 and M2 macrophages. Patients with high-grade DCMi (DCMi-high, CD3+ lymphocytes > 30 cells/mm2) had significantly increased PAI-1 levels compared to DCM and low-grade DCMi patients (DCMi-low, CD3+ lymphocytes = 14–30 cells/mm2) (15.5 ± 0.4% vs. 1.0 ± 0.1% and 4.0 ± 0.1%, p ≤ 0.001). Elevated PAI-1 expression in DCMi-high subjects was associated with a diminished degree of cardiac fibrosis, decreased levels of TGF-β and reduced number of myofibroblasts. In addition, DCMi-high patients revealed an increased proportion of non-classical M2 macrophages towards classical M1 macrophages, indicating M2 macrophage-favoring properties of PAI-1 in inflammatory cardiomyopathies. Our findings give evidence that elevated expression of cardiac PAI-1 in subjects with high-grade DCMi suppresses fibrosis by inhibiting TGF-β and myofibroblast activation. Moreover, our data indicate that PAI-1 is involved in the polarization of M2 macrophages in the heart. Thus, PAI-1 could serve as a potential prognostic biomarker and as a possible therapeutic target in inflammatory cardiomyopathies.

Erythroparvovirus (B19V) genomes have been detected in various organs of infected individuals including endothelial cells of the heart muscle. However, the role of B19V as a causative pathogen of myocardial damage is still unknown. The majority of reports focus on the presence of viral DNA ignoring proof of viral RNAs as important markers for viral activity. During this study, we established (RT-) qPCR to characterize expression of B19V RNAs (NS1 and VP1/2) in endomyocardial biopsies (EMBs) of 576 patients with unexplained heart failure. 403/576 (70%) EMBs were positive for B19V DNA. B19V mRNAs NS1 and/or VP1/2, indicating viral activity, could be detected in 38.5% of B19V DNA positive samples using the newly established B19V RT-PCRs. 22.1% of samples were characterized by only NS1 mRNA detection while 6.0% revealed only VP1/2 mRNA expression. Detection of both intermediates was successful in 10.4% of samples. Applying the molecular testing, our study revealed that a high proportion (38.5%) of B19V DNA positive EMBs was characterized by viral transcriptional activity. Further prospective studies will evaluate relevance of viral transcription intermediates as a diagnostic marker to differentiate between latent B19V infection and clinically relevant transcriptionally active B19V-infection of the heart muscle.

To report the presence of viral ribonucleic acid (RNA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human retina in deceased patients with confirmed novel coronavirus disease 2019 (COVID-19).Fourteen eyes of 14 deceased patients with confirmed COVID-19 disease were enucleated during autopsy. A sample of human retina was secured and fixed in RNAlater™. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect three different viral RNA sequences (RdRp-gene, E-gene and Orf1 gene) of SARS-CoV-2.In three out of 14 eyes SARS-CoV-2 viral RNA was detected in the retina of deceased COVID-19 patients. As analysis for three different sequences (RdRp-gene, E-gene and Orf1 gene) revealed positive results in RT-PCR, the existence of SARS-CoV-2 viral RNA in human retina is proven according to the standards of the World-Health-Organization.Viral RNA of SARS-CoV-2 is detectable in the retina of COVID-19 patients.

Aims Since December 2019, the novel coronavirus SARS‐CoV‐2 has spread rapidly throughout China and keeps the world in suspense. Cardiovascular complications with myocarditis and embolism due to COVID‐19 have been reported. SARS‐CoV‐2 genome detection in the heart muscle has not been demonstrated so far, and the underlying pathophysiological mechanisms remain to be investigated. Methods and results Endomyocardial biopsies (EMBs) of 104 patients (mean age: 57.90 ± 16.37 years; left ventricular ejection fraction: 33.7 ± 14.6%, sex: n = 79 male/25 female) with suspected myocarditis or unexplained heart failure were analysed. EMB analysis included histology, immunohistochemistry, and detection of SARS‐CoV‐2 genomes by real‐time reverse transcription polymerase chain reaction in the IKDT Berlin, Germany. Among 104 EMBs investigated, five were confirmed with SARS‐CoV‐2 infected by reverse real‐time transcriptase polymerase chain reaction. We describe patients of different history of symptoms and time duration. Additionally, we investigated histopathological changes in myocardial tissue showing that the inflammatory process in EMBs seemed to permeate vascular wall leading to small arterial obliteration and damage. Conclusions This is the first report that established the evidence of SARS‐CoV‐2 genomes detection in EMBs. In these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. EMB‐based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated SARS‐CoV‐2‐infection and to design future specific and personalized treatment strategies.

Human parvovirus B19 (B19V) is the predominant virus currently detected in endomyocardial biopsies (EMBs). Recent findings indicate that, specifically, transcriptionally active B19V with detectable viral RNA is of prognostic relevance in inflammatory viral cardiomyopathy. We aimed to evaluate B19V replicative status (viral RNA) and beneficial effects in a sub-collective of the prospective randomized placebo-controlled phase II multi-center BICC-Trial (Betaferon In Chronic Viral Cardiomyopathy) after interferon beta-1b (IFN-β) treatment. EMBs of n = 64 patients with B19V mono-infected tissue were retrospectively analyzed. Viral RNA could be detected in n = 18/64 (28.1%) of B19V DNA positive samples (mean age 51.7 years, 12 male), of whom n = 13 had been treated with IFN-ß. Five patients had received placebo. PCR analysis confirmed in follow-up that EMBs significantly reduced viral RNA loads in n = 11/13 (84.6%) of IFN-ß treated patients (p = 0.001), independently from the IFN-ß dose, in contrast to the placebo group, where viral RNA load was not affected or even increased. Consequently, a significant improvement of left ventricular ejection fraction (LVEF) after treatment with IFN-ß was observed (LVEF mean baseline 51.6 ± 14.1% vs. follow-up 61.0 ± 17.5%, p = 0.03). In contrast, in the placebo group, worsening of LVEF was evaluated in n = 4/5 (80.0%) of patients. We could show for the first-time the beneficial effects from treatment with IFN-ß, suppressing B19V viral RNA and improving the hemodynamic course. Our results need further verification in a larger prospective randomized controlled trial.

Scaffold-free tissue formation in microgravity is a new method in regenerative medicine and an important topic in Space Medicine. In this MiniReview, we focus on recent findings in the field of tissue engineering that were observed by exposing cells to real microgravity in space or to devices simulating to at least some extent microgravity conditions on Earth (ground-based facilities). Under both conditions - real and simulated microgravity - a part of the cultured cells of various populations detaches from the bottom of a culture flask. The cells form three-dimensional (3D) aggregates resembling the organs from which the cells have been derived. As spaceflights are rare and extremely expensive, cell culture under simulated microgravity allows more comprehensive and frequent studies on the scaffold-free 3D tissue formation in some aspects, as a number of publications have proven during the last two decades. In this MiniReview, we summarize data from our own studies and work from various researchers about tissue engineering of multi-cellular spheroids formed by cancer cells, tube formation by endothelial cells and cartilage formation by exposing the cells to ground-based facilities such as the 3D Random Positioning Machine (RPM), the 2D Fast-Rotating Clinostat (FRC) or the Rotating Wall Vessel (RWV). Subsequently, we investigated self-organization of 3D aggregates without scaffolds pursuing to enhance the frequency of 3D formation and to enlarge the size of the organ-like aggregates. The density of the monolayer exposed to real or simulated microgravity as well as the composition of the culture media revealed an impact on the results. Genomic and proteomic alterations were induced by simulated microgravity. Under microgravity conditions, adherent cells expressed other genes than cells grown in spheroids. In this MiniReview, the recent improvements in scaffold-free tissue formation are summarized and relationships between phenotypic and molecular appearance are highlighted.

IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be documented in various tissues, but the frequency of cardiac involvement as well as possible consequences are unknown. OBJECTIVE: To evaluate the presence of SARS-CoV-2 in the myocardial tissue from autopsy cases and to document a possible cardiac response to that infection. DESIGN, SETTING, AND PARTICIPANTS: This cohort study used data from consecutive autopsy cases from Germany between April 8 and April 18, 2020. All patients had tested positive for SARS-CoV-2 in pharyngeal swab tests. EXPOSURES: Patients who died of coronavirus disease 2019. MAIN OUTCOMES AND MEASURES: Incidence of SARS-CoV-2 positivity in cardiac tissue as well as CD3(+), CD45(+), and CD68(+) cells in the myocardium and gene expression of tumor necrosis growth factor α, interferon γ, chemokine ligand 5, as well as interleukin-6, -8, and -18. RESULTS: Cardiac tissue from 39 consecutive autopsy cases were included. The median (interquartile range) age of patients was 85 (78-89) years, and 23 (59.0%) were women. SARS-CoV-2 could be documented in 24 of 39 patients (61.5%). Viral load above 1000 copies per μg RNA could be documented in 16 of 39 patients (41.0%). A cytokine response panel consisting of 6 proinflammatory genes was increased in those 16 patients compared with 15 patients without any SARS-CoV-2 in the heart. Comparison of 15 patients without cardiac infection with 16 patients with more than 1000 copies revealed no inflammatory cell infiltrates or differences in leukocyte numbers per high power field. CONCLUSIONS AND RELEVANCE: In this analysis of autopsy cases, viral presence within the myocardium could be documented. While a response to this infection could be reported in cases with higher virus load vs no virus infection, this was not associated with an influx of inflammatory cells. Future investigations should focus on evaluating the long-term consequences of this cardiac involvement.

Real and simulated microgravity induce a variety of changes in human cells. Most importantly, changes in the cytoskeleton have been noted, and studies on microtubules have shown that they are gravisensitive. This study focuses on the effects of short-term real microgravity on gene expression, protein content, and cytoskeletal structure of human chondrocytes. We cultivated human chondrocytes, took them along a parabolic flight during the 24th Deutsches Zentrum für Luft- und Raumfahrt Parabolic (DLR) Flight Campaign, and fixed them after the 1st and the 31st parabola. Immunofluorescence microscopy revealed no changes after the 1st parabola, but disruptions of β-tubulin, vimentin, and cytokeratin networks after the 31st parabola. No F-actin stress fibers were detected even after 31 parabolas. Furthermore, mRNA and protein quantifications after the 31st parabola showed a clear up-regulation of cytoskeletal genes and proteins. The mRNAs were significantly up-regulated as follows: TUBB, 2-fold; VIM, 1.3-fold; KRT8, 1.8-fold; ACTB, 1.9-fold; ICAM1, 4.8-fold; OPN, 7-fold; ITGA10, 1.5-fold; ITGB1, 1.2-fold; TGFB1, 1.5-fold; CAV1, 2.6-fold; SOX9, 1.7-fold; BMP-2, 5.3-fold. However, SOX5 (-25%) and SOX6 (-28%) gene expression was decreased. Contrary, no significant changes in gene expression levels were observed during vibration and hypergravity experiments. These data suggest that short-term microgravity affects the gene expression of distinct proteins. In contrast to poorly differentiated follicular thyroid cancer cells or human endothelial cells, chondrocytes only exert moderate cytoskeletal alterations. The up-regulation of BMP-2, TGF-β1, and SOX9 in chondrocytes may play a key role in preventing cytoskeletal alterations.-Aleshcheva, G., Wehland, M., Sahana, J., Bauer, J., Corydon, T. J., Hemmersbach, R., Frett, T., Egli, M., Infanger, M., Grosse, J., Grimm, D. Moderate alterations of the cytoskeleton in human chondrocytes after short-term microgravity produced by parabolic flight maneuvers could be prevented by up-regulation of BMP-2 and SOX-9.

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage / monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage / monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NFκB signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h- and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking.

When incubated under simulated microgravity (s-microg), endothelial cells (EC) form tubular structures that resemble vascular intimas. This delayed formation of 3D EC structures begins between the 5th and 7th day of culturing EC under conditions of s-microg, when double-row cell assemblies become visible. With the aim of learning about this initial phase of tubular structure formation, we found that NFkappaBp65 protein content was similar in all cell populations, but gene and protein expression of phosphokinase A catalytic subunit, phosphokinase Calpha, and extracellular signal-regulated kinases 1 and 2 was altered in cells cultured under s-microg. Apoptosis remained below 30% in all EC cultures. In contrast to controls, the 7-day-old s-microg cultures contained 3D aggregates with proliferating cells, enhanced numbers of necrotic cells, and osteopontin-negative EC as well as supernatants with reduced quantities of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), soluble TNFRSF5, TNFSF5, intercellular adhesion molecule-1, tumor necrosis factor receptor 2, IL-18, complement C3, and von Willebrand factor. VEGF and/or bFGF (10 ng/mL) application influenced the accumulation of proteins in supernatants more profoundly under 1 g than under s-microg. These findings provide evidence that phosphokinase Calpha plays a key role in tube formation. Improving the interaction of VEGF and/or bFGF with EC under s-microg could enhance the engineering of vascular intimas.

In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML-1; RO82-W-1), which were exposed to a 2D clinostat (CLINO), a random positioning machine (RPM) and to normal 1g-conditions. After a three (3d)-or seven-day-culture (7d) on the two devices, we found both cell types growing three-dimensionally within multicellular spheroids (MCS) and also cells remaining adherent (AD) to the culture flask, while 1g-control cultures only formed adherent monolayers, unless the bottom of the culture dish was covered by agarose. In this case, the cytokines IL-6 and IL-8 facilitated the formation of MCS in both cell lines using the liquid-overlay technique at 1g. ML-1 cells grown on the RPM or the CLINO released amounts of IL-6 and MCP-1 into the supernatant, which were significantly elevated as compared to 1g-controls. Release of IL-4, IL-7, IL-8, IL-17, eotaxin-1 and VEGF increased time-dependently, but was not significantly influenced by the gravity conditions. After 3d on the RPM or the CLINO, an accumulation of F-actin around the cellular membrane was detectable in AD cells of both cell lines. IL-6 and IL-8 stimulation of ML-1 cells for 3d and 7d influenced the protein contents of beta(1)-integrin, talin-1, Ki-67, and beta-actin dose-dependently in adherent cells. The beta(1)-integrin content was significantly decreased in AD and MCS samples compared with 1g, while talin-1 was higher expressed in MCS than AD populations. The proliferation marker Ki-67 was elevated in AD samples compared with 1g and MCS samples. The beta-actin content of R082-W-1 cells remained unchanged. ML-1 cells exhibited no change in beta-actin in RPM cultures, but a reduction in CLINO samples. Thus, we concluded that simulated microgravity influences the release of cytokines in follicular thyroid cancer cells, and the production of beta(1)-integrin and talin-1 and predicts an identical effect under real microgravity conditions.

Background Chondrocytes are the main cellular component of articular cartilage. In healthy tissue, they are embedded in a strong but elastic extracelluar matrix providing resistance against mechanical forces and friction for the joints. Osteoarthritic cartilage, however, disrupted by heavy strain, has only very limited potential to heal. One future possibility to replace damaged cartilage might be the scaffold-free growth of chondrocytes in microgravity to form 3D aggregates. Results To prepare for this, we have conducted experiments during the 20th DLR parabolic flight campaign, where we fixed the cells after the first (1P) and the 31st parabola (31P). Furthermore, we subjected chondrocytes to isolated vibration and hypergravity conditions. Microarray and quantitative real time PCR analyses revealed that hypergravity regulated genes connected to cartilage integrity (BMP4, MMP3, MMP10, EDN1, WNT5A, BIRC3). Vibration was clearly detrimental to cartilage (upregulated inflammatory IL6 and IL8, downregulated growth factors EGF, VEGF, FGF17). The viability of the cells was not affected by the parabolic flight, but showed a significantly increased expression of anti-apoptotic genes after 31 parabolas. The IL-6 release of chondrocytes cultured under conditions of vibration was not changed, but hypergravity (1.8 g) induced a clear elevation of IL-6 protein in the supernatant compared with corresponding control samples. Conclusion Taken together, this study provided new insights into the growth behavior of chondrocytes under short-term microgravity. Electronic supplementary material The online version of this article (doi:10.1186/s12964-015-0095-9) contains supplementary material, which is available to authorized users.

Many types of cells transit in vitro from a two- to a three-dimensional growth, when they are exposed to microgravity. The underlying mechanisms are not yet understood. Hence, we investigated the impact of microgravity on protein content and growth behavior. For this purpose the human thyroid cancer cells FTC-133 were seeded either in recently developed cell containers that can endure enhanced physical forces and perform media changes and cell harvesting automatically or in T-25 culture flasks. All cells were cultured for 5 days at 1g. Afterwards, a part of the cell containers were flown to the International Space Station, while another part was kept on the ground. T-25 flasks were mounted on and next to a Random Positioning Machine. The cells were cultured for 12 days under the various conditions, before they were fixed with RNAlater. All fixed cultures showed monolayers, but three-dimensional aggregates were not detected. In a subsequent protein analysis, 180 proteins were identified by mass spectrometry. These proteins did not indicate significant differences between cells exposed to microgravity and their 1g controls. However, they suggest that an enhanced production of proteins related to the extracellular matrix could detain the cells from spheroid formation, while profilin-1 is phosphorylated This article is protected by copyright. All rights reserved.

Three-dimensional multicellular spheroids (MCS) of human cells are important in cancer research. We investigated possible mechanisms of MCS formation of thyroid cells. Both, normal Nthy-ori 3–1 thyroid cells and the poorly differentiated follicular thyroid cancer cells FTC-133 formed MCS within 7 and 14 days of culturing on a Random Positioning Machine (RPM), while a part of the cells continued to grow adherently in each culture. The FTC-133 cancer cells formed larger and numerous MCS than the normal cells. In order to explain the different behaviour, we analyzed the gene expression of IL6, IL7, IL8, IL17, OPN, NGAL, VEGFA and enzymes associated cytoskeletal or membrane proteins (ACTB, TUBB, PFN1, CPNE1, TGM2, CD44, FLT1, FLK1, PKB, PKC, ERK1/2, Casp9, Col1A1) as well as the amount of secreted proteins (IL-6, IL-7, IL-8, IL-17, OPN, NGAL, VEGFA). Several of these components changed during RPM-exposure in each cell line. Striking differences between normal and malignant cells were observed in regards to the expression of genes of NGAL, VEGFA, OPN, IL6 and IL17 and to the secretion of VEGFA, IL-17 and IL-6. These results suggest several gravi-sensitive growth or angiogenesis factors being involved in 3D formation of thyroid cells cultured under simulated microgravity.

Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-beta(1), osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-beta(1) was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

Human follicular thyroid cancer cells were cultured in Space to investigate the impact of microgravity on 3D growth. For this purpose, we designed and constructed a cell container that can endure enhanced physical forces, is connected to fluid storage chambers, performs media changes and cell harvesting automatically and supports cell viability. The container consists of a cell suspension chamber, two reserve tanks for medium and fixative and a pump for fluid exchange. The selected materials proved durable, non-cytotoxic, and did not inactivate RNAlater. This container was operated automatically during the unmanned Shenzhou-8 Space mission. FTC-133 human follicular thyroid cancer cells were cultured in Space for 10 days. Culture medium was exchanged after 5 days in Space and the cells were fixed after 10 days. The experiment revealed a scaffold-free formation of extraordinary large three-dimensional aggregates by thyroid cancer cells with altered expression of EGF and CTGF genes under real microgravity.

It is known that exposing cell lines in vitro to parabolic flights changes their gene expression and protein production patterns. Parabolic flights and spaceflight in general are accompanied by transient hypergravity and vibration, which may impact the cells and therefore, have to be considered too. To estimate the possible impact of transient hypergravity and vibration, we investigated the effects of these forces separately using dedicated ground-based facilities. We placed follicular thyroid ML-1 and CGTH W-1 cancer cells in a specific centrifuge (MuSIC Multi Sample Incubator Centrifuge; SAHC Short Arm Human Centrifuge) simulating the hypergravity phases that occur during one (P1) and 31 parabolas (P31) of parabolic flights, respectively. On the Vibraplex device, the same cell lines were treated with vibration waves corresponding to those that occur during a whole parabolic flight lasting for two hours. After the various treatments, cells were harvested and analyzed by quantitative real-time PCR, focusing on the genes involved in forming (ACTB, MYO9, TUBB, VIM, TLN1, and ITGB1) and modulating (EZR, RDX, and MSN) the cytoskeleton, as well as those encoding growth factors (EGF, CTGF, IL6, and IL8) or protein kinases (PRKAA1 and PRKCA). The analysis revealed alterations in several genes in both cell lines; however, fewer genes were affected in ML-1 than CGTH W-1 cells. Interestingly, IL6 was the only gene whose expression was changed in both cell lines by each treatment, while PKCA transcription remained unaffected in all experiments. We conclude that a PKCa-independent mechanism of IL6 gene activation is very sensitive to physical forces in thyroid cells cultured in vitro as monolayers.