Culturing hMSCs and OBs under hypoxia in X3 Hypoxia hood and culture combo-Xvivo system

Bony defects may be found as a result of congenital anomalies, trauma, neoplasms, or infectious conditions. There is a growing interest in the use of different cell culture approaches to realize in vitro 3D bone microenvironments mimicking the physiological context in order i. to increase our understanding about bone responses in health and disease, and ii. to produce implantable “tissue engineered construct” that will promote in vivo bone healing and regeneration. The ideal candidates for cell-based bone tissue regeneration should be cells with a certain degree of plasticity able to respond to signals that guide them to form extracellular mineral matrix, but also to preserve some stemness properties. In this regard, despite the large amount of literature data, the choice of cells to be used, mesenchymal stem cells (MSCs) rather than mature cells (osteoblasts, OBs), still remains an open question. Another interesting issue regards preconditioning the cells with specific treatments to improve their retention after transplantation in damaged tissues, and maintain their ability to secrete appropriate factors. A number of studies have shown increased regenerative and repair potentials of preconditioned cells. It is well known that oxygen concentration is a critical environmental factor that affects MSCs, and recent studies demonstrated that hypoxic culture conditions have beneficial effects on MSCs. However, most of the studies are conducted using conventional O2/CO2 hypoxic incubators that do not guarantee the maintenance of a stable condition. Moreover, considering that many hypoxia regulated genes are immediately turned off by brief exposures to ambient air, traditional systems may compromise the reproducibility of the results and the validity of information. Here, we studied preconditioning of hMSCs from bone marrow and hOBs from vertebral bone (lamina) with a low percentage of O2, culturing cells in a reliable innovative experimental device represented by “X3 HYPOXIA HOOD AND CULTURE COMBO – Xvivo System model X3” workstation. This sophisticated hypoxia cell culture system allows incubation and handling of the cells in a stable hypoxic environment which provides full-time, uninterruptible optimization of hypoxic conditions. The cells were cultured under hypoxia (hypoMSCs, hypoOBs 1.5 - 5% O2) at different time of exposure, and the following parameters were measured: - morphology and proliferation rate: cells grew under hypoxia likewise under normoxia during the first two days of culture, showing the same morphology as evidenced by the analysis of cytoskeleton organization (Alexa-488-Phalloidin) and nuclei size (DAPI). Cell viability was also assessed by Annexin V/Propidium iodide analysis demonstrating the absence of apoptotic/necrotic events - ROS production (DFCDA kit): preliminary experiments demonstrated that hypoMSCs and hypoOBs didn’t retain any significant increase in cytosolic ROS content - gene expression: in the absence of osteogenic inducers the expression of osteogenic markers (assessed by western blot analysis) including Runx2 and Collagen type XV increased in hypoMSCs. This effect correlated with a remarkable increase of mineralized matrix deposition ability (ARS staining area) evaluated after short-term (48h) or prolonged hypoxia exposure (20 days) in osteogenic medium. The optimization of cell culture conditions is of great importance to obtain high quality cells for more successful application in bone or cartilage tissue regeneration. Here, we introduced a novel equipment that can provide stable hypoxic culture condition for cells mimicking a physiologic niche microenvironment. Considering that the ability of cells to respond within a short time to in vitro treatments should be predictive of their ability to perceive the physiological microenvironment and the effectiveness of their potential use in vivo, our data provide a promising solution to obtain very performant cells for transplantation in a bone defect