In contrast, levitation through unfavorable magnetophoresis (also referred to as diamagnetophoresis) can exactly mimic weightlessness. developmental biology. Introduction Cells in living organisms are constantly exposed to varying degrees of mechanical causes, which serve as crucial stimuli and influence their fate1C4. Such physical signals are key regulators of organ system maintenance, repair and renewal in mammals5,6. Permanent IKK-beta or temporary reduction of mechanical stimulations, as experienced during spaceflight, immobilization, paralysis and bed rest, cause deteriorations in the human body7, especially in the musculoskeletal system such as demineralization of bones and mass loss of Ombrabulin skeletal muscle mass8C12. Spaceflight experiments offer great opportunities to improve our understanding on short term and long period biological effects of weightlessness13C15. Nevertheless, such experiments are rare, expensive to operate and hard to secure, and option ground-based techniques have hence been developed to simulate the weightlessness environment16. The most commonly used devices to study simulated weightlessness are the rotating-wall vessel (RWV) platform17C19, 2D clinostats20C22 and Random Positioning Machines (RPM)20,23,24. However, these devices create fluid shear stress on the cells due to rotation and this can interrupt the response of cells to a randomized gravity vector25,26. Furthermore, both the clinostat and the RPM requires time for randomization of gravity vector and therefore they are not convenient for relatively rapidly occurring cellular processes. One of the most recent ground based technology to mimic the biological effects of weightlessness is usually magnetic levitation technique27. Magnetic levitation can be applied via positive or unfavorable magnetophoresis, however positive magnetophoresis (i.e. magnetic bead labeling technique) cannot simulate weightlessness because acting causes that levitate the subject of interest Ombrabulin only take action on the surface of the subject and any internal structures are free of those causes28,29. In contrast, levitation through unfavorable magnetophoresis (also referred to as diamagnetophoresis) can exactly mimic weightlessness. During unfavorable magnetophoresis, gravitational pressure on the subject is usually compensated by a counteracting pressure that induces weightlessness. In contrast to other ground-based methods, magnetic levitation allows the investigation of relatively fast cellular processes. In this technique, diamagnetic objects (i.e. almost all cells) are guided towards regions of low magnetic field in a magnetic field gradient and the process is usually resulted in stable magnetic levitation and the simulation of weightlessness environment as long as the gradient is usually intact30C32. Such a strategy requires high magnitude magnetic fields that can be detrimental to biological subjects33. In order to reduce the magnitude of magnetic fields, it is possible Ombrabulin to increase the magnetic susceptibility of medium by using paramagnetic solutions34C36 or ferrofluids37. Recently an inexpensive strategy has been exhibited for label-free cell levitation in gadolinium (Gd3+) based answer38 and successfully applied for detection of differences in cell densities at the single-cell level39 and guided assembly of generated spheroids40. However, self-guided Ombrabulin assembly of cells during levitation and appropriate Gd3+ based answer for longer term culturing is largely unknown. In this study, we used a magnetic levitation system for cell culture in simulated microgravity. First, we Ombrabulin investigated the most appropriate composition and concentration for Gd3+ based answer for weightlessness culturing. Further, we documented the self-assembly pattern of cells and controlling of cluster size with initial cell number. Finally, we applied our previous findings to determine the possibility of coculture and biofabrication of novel cellular patterns. Our study established the possibility of levitation through diamagnetophoresis as a powerful biomedical tool that will allow screening of molecular and cellular level hypotheses on biological effects of weightlessness in a single cell level that is not possible with current methods simulating weightlessness. Results Short-term levitation of cells with different Gd-based solutions.