Poster M14 - Impact of Microgravity Environment on Bioprinted Cardiac Tissue Constructs

Introduction: Microgravity is one of the most significant stress factors experienced by living organisms during spaceflight. Controlled in vitro studies of cells and tissues can improve our understanding of gravity sensing, transduction, and responses in living cells and tissues. Cells exposed to microgravity show reorganization of the cytoskeletal system, altered proliferation, and increased apoptosis. Most culture systems used to study the effects of microgravity are limited by the 2D confines of the culture dish. This limits the proliferation and differentiation of cells in 2D, which could potentially restrict cell-cell interactions that are important for developing the level of organization in tissue obtained in vivo. 3D culture in microgravity can profoundly modulate cell proliferation and survival by allowing cells to self-organize by aggregation and facilitate spatially unrestricted interactions between cells and their surroundings. Microgravity is the most significant stress factor experienced by living organisms during spaceflight. Microgravity can significantly impact the cell cycle dynamics of endothelial cells, potentially inducing a hibernation-like state where cell growth and division slow down [1]. These conditions also lead to microvascular endothelial cell apoptosis, which contributes to developing endothelium-dependent vasodilator dysfunction [2]. Studies utilizing the head-down tilt model to simulate microgravity's effects on cardiovascular function are important as these simulations are critical for understanding the mechanotransduction pathways in cardiac cells - how cells convert mechanical stimuli into chemical activity [3]. In vitro studies using 3D bioprinted tissue models in microgravity are expected to help in identifying mechanisms related to gravity sensing and transduction in biological organisms.

Materials and

Methods: Cellink BioX6 3D Bioprinter has been used to print a cardiac tissue construct having a rectilinear grid pattern with channels for perfusion. The channels are coated with five ug/ml of fibronectin and seeded with endothelial cells to mimic in vivo vascular tissue construct. These bioprinted tissue constructs maintained in 6 well plates have been exposed to a Clinostat Gravite microgravity simulator system (Figure 1a) and Random Positioning Machine (Figure 1b) available at NASA KSC over a period of 24-72 hours. Following exposure, biochemical analysis is performed by measuring the availability of nitric oxide (NO) and reactive oxygen species (ROS) generation. NO and ROS measurements are conducted by embedding the tissue constructs in Tissue-tek and sectioning thin samples (5 μm) followed by incubation using a fluorescence probe 4-Amino-5-Methylamino-2', 7’-Difluorofluorescein Diacetate (DAF-FM) which produces a green fluorescence (488nm) and Dihydroethidium (DHE) which is analyzed using the red fluorescence filter (518/606nm) respectively. The images are processed using a laser scanning confocal microscope.

Results, Conclusions, and Discussions: Results from simulated microgravity for a period of 48 hrs demonstrate increased ROS generation compared to control samples, as shown in confocal microscopy images (Figure 2a). Thus, an increase in oxidative stress is observed in bioprinted tissue constructs, as shown in Figure 2b, resulting in endothelial dysfunction. Biochemical characterization of the 3D constructs will help in understanding the underlying mechanisms that lead to vascular dysfunction due to increased oxidative stress post-exposure to microgravity or simulated microgravity environment. Further research can involve analyzing the effects of microgravity by performing monochrome multiplex q-PCR to measure the telomere length (TL) on selected genes.
These findings provide key insights into how microgravity modulates endothelial cell behavior and function. In vitro, 3D bioprinted tissue models allow for controlled studies elucidating the impact of spaceflight conditions on vascular cell physiology, which has implications for long-duration space missions and understanding mechanisms of cardiovascular disease on Earth.

References:
1. Sokolovskaya, A. A., Ignashkova, T. I., Bochenkova, A. V., Moskovtsev, A. A., Baranov, V. M., & Kubatiev, A. A. (2014). Effects of simulated microgravity on cell cycle in human endothelial cells. Acta astronautica, 99, 16-23.
2. Vion, A. C., Ramkhelawon, B., Loyer, X., Chironi, G., Devue, C., Loirand, G., ... & Boulanger, C. M. (2013). Shear stress regulates endothelial microparticle release. Circulation research, 112(10), 1323-1333.

Acknowledgements (Optional): The authors would like to acknowledge funding support from National Science Foundation – Grant Number CBET 2317757.