A Multichamber Gas System to Examine the Effect of Multiple Oxygen Conditions on Cell Culture

The classic bone tissue engineering model for bone regeneration combines three elements: scaffolds, biomaterials, and mesenchymal stem cells (MSCs). Incorporation of MSCs and growth factors into a scaffold implanted into the area of bone injury is a proven strategy to achieve successful bone regeneration as demonstrated in the literature. However, a major limitation of using bone grafts or scaffolds is oxygen (O₂) deprivation in the inner sections of the construct, due to lack of adequate vascularization. To address this limitation, we proposed two treatment strategies for MSC-seeded constructs or adipose tissue scaffolds before implantation: (1) O₂ enrichment and (2) acclimation to hypoxia. Based on previous studies, the significance of the different O₂ concentrations on MSC biological characteristics remains controversial. Therefore, the optimal O₂ condition for engineered bone tissues should be determined. Thus, we designed an innovative multichamber gas system aimed to simultaneously assess the effects of different O₂ levels on cell culture. This system was assembled using three isolated chambers integrated into a single incubator. To explore the efficacy of our method, we investigated the effect of hyperoxia, normoxia, and hypoxia, (50-60%, 21%, and 5-7.5% O₂, respectively) on the biological characteristics of human adipose-derived MSCs: immunophenotyping, adhesion, proliferation, and osteogenic, and angiogenic differentiation. Our findings demonstrated that hypoxic adipose-derived mesenchymal stem cells (ASCs) conditions exhibited significantly lower levels of CD34 (p = 0.014), with significantly higher osteogenic and angiogenic differentiation capacities (p = 0.023 and p = 0.0042, respectively) than normoxia. Conversely, hyperoxia-cultured ASCs demonstrated significantly higher levels of CD73 and CD90 expression than both normoxic ASCs (p = 0.006 and p = 0.025, respectively) and hypoxic ASCs (p = 0.003 and p = 0.003, respectively). In addition, hyperoxic ASCs showed significantly reduced proliferation capacity by day 11 (p = 0.032) and significantly enhanced migration rates after 48 h (p = 0.044). The newly developed controllable multichamber gas system was cost-effective and easy to use. Different assays can be performed concurrently while preserving all other conditions identical, and the use of other ranges of O₂ concentrations is feasible and also necessary to determine the ideal O₂ concentration. Furthermore, the multichamber gas system has the potential for wide application, including other cell cultures, grafts, or scaffolds for in vitro and in vivo experimentation. This study was approved by the Galilee Medical Center Helsinki Committee (No. 0009-19-NHR). Impact statement The introduced multichamber gas system provides a custom-made setup for simultaneous control of three oxygen (O₂) levels in a single incubator. The use of our innovative multichamber gas system is essential to determine the ideal O₂ levels for engineered tissues by examining multiple O₂ concentrations on cells in vitro. The determined ideal O₂ concentration will then be used through this system to investigate the engrafted cell survival ex vivo, to ensure successful integration of the engineered tissues and tissue regeneration in vivo. Use of this method may promote a therapeutic tool for a major limitation in tissue engineering due to the problematic O₂ insufficiency in tissue scaffolds.