Scale bars: 200?m. Discussion We optimized a cellular magnetic levitation protocol by negative magnetophoresis suitable for long term cell culture and developed an self-guided cellular assembly model during weightlessness. simulated weightlessness techniques. The low-cost technique presented here may offer a wide range of biomedical applications in several research fields, including mechanobiology, drug discovery and developmental biology. Introduction Cells in living organisms are constantly exposed to varying degrees of mechanical forces, which serve as critical stimuli and influence their fate1C4. Such physical signals are key regulators of organ system maintenance, repair and renewal in mammals5,6. Permanent 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 skeletal muscle8C12. Spaceflight experiments offer great opportunities to improve our understanding on short term and long duration biological effects of weightlessness13C15. Nevertheless, such experiments are rare, expensive to operate and hard to secure, and alternative 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 forces that levitate the subject of interest only act on the surface of the subject and any internal structures are free of those forces28,29. In contrast, levitation through unfavorable magnetophoresis (also referred to as diamagnetophoresis) can exactly mimic weightlessness. During CPA inhibitor unfavorable magnetophoresis, gravitational force on the subject is usually compensated by a counteracting force 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 CPA inhibitor 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 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 solution38 and successfully applied for detection of differences in cell densities at the single-cell level39 and guided assembly of generated spheroids40. However, self-guided assembly of cells during levitation and appropriate Gd3+ based solution for longer term culturing is largely unknown. In this study, we used a magnetic levitation system for cell culture in simulated microgravity. First, we investigated the most appropriate composition and concentration for Gd3+ based solution for weightlessness culturing. Prkwnk1 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 testing 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 In order to select the most appropriate media for cell culture during magnetic levitation, we used a custom made microfluidic levitation device (Fig.?1a, Supplementary Information, Supplementary Fig.?1) to levitate D1 ORL UVA bone marrow mesenchymal stem cells with different Gd-based contrast brokers; gadobutrol (Gd-BT-DO3A), gadopentetate dimeglumine (Gd-DTPA), gadodiamide (Gd-DTPA-BMA), gadoterate meglumine (Gd-DOTA) and gadobenate dimeglumine (Gd-BOPTA) at increasing concentrations (0, 10, 25, 50, 100 and 200?mM) and measured location of cells from bottom surface of capillary after 10?min of levitation to allow cells levitated at lower concentrations of Gd3+ to reach steady state (Fig.?1a,b,d and Supplementary Fig.?2). Irrespective of the chemical composition of the Gd-based agent, increasing concentrations resulted in increased levitation height of cells. Levitation heights of cells at concentration of 100?mM solutions reached more than 80% of the cell heights observed at 200?mM concentrations (86.4, 84.1, 87.1, 88.1 and 88.1% for Gd-BT-DO3A, CPA inhibitor Gd-DTPA, Gd-DTPA-BMA, Gd-DOTA and Gd-BOPTA, respectively). Furthermore, nonionic structure made up of Gd-BT-DO3A and Gd-DTPA-BMA, provided higher levitation heights at Gd3+ concentrations of 100 and 200?mM than ionic structure containing ones (Gd-DTPA, Gd-DOTA and Gd-BOPTA). Levitation heights for.