including skin, bone, cartilage, heart tissues, blood vessels, etc., have successfully been recreated in vitro, [3-5] their clinical application is limited. Most tissues are highly vulnerable to ischemia, [6] hindering the survival of clinically relevant tissue volumes after implantation in vivo of engineered constructs. The inability to deliver oxygen and nutrients to cells within scaffolds is thought to result in cell death and lack of efficiency of transplanted cells. [7-9] Indeed, oxygen and nutrient supply in bioreactors is only possible through the movement of the culture medium through their structure and is limited within the tissue by diffusion. [10] Furthermore, such constructs do not possess a functioning blood vessel network that could anastomose to the host vasculature upon implantation. Without an integrated vascularization, their in vivo survival depends on the host's ability to vascularize the graft and previous research has shown that the complete vascularization of a 3 mm tissue engineered construct could take between one and two weeks. [11,12] During this time, cells embedded deep within these constructs become hypoxic and in some cases necrotic. [7,9] This also hampers important cellular functions such as cell proliferation and neo-vascularization. [13] Mesenchymal stem cells (MSCs) hold great potential in tissue engineering application due mainly to the beneficial factors they secrete and to their stem cell properties. Some of these include proangiogenic and immunosuppressive functions, the ability to differentiate into multiple cell lineages, and the ability to interact and regulate cells within both the innate and adaptive immune systems. [14-19] Through cell to cell communication, they also help mediate release of specific growth factors and cytokines that promote cell survival. [20-25] In vitro MSC are routinely cultured in "normoxia," i.e., 20% oxygen, namely atmospheric air and 5% CO 2 , but the physoxic levels are estimated to be 3-6% for human stem cell niches and many studies have indicated differences in behavior when cultured in normoxia, physoxia, and hypoxia. We sought to determine whether MSC could be cultured in the absence of oxygen, and whether glucose availability affected long term anoxic survival (Figure 1). Erythropoietin (EPO) producing MSC allowed us to measure protein production and also to determine if EPO, a protein reported to improve anoxic survival preclinically, [26] could have any effect on anoxic MSC survival. This study determines whether the viability of mesenchymal stem cell (MSC) in vitro is most sensitive to oxygen supply, energetic substrate supply, or accumulation of lactate. Mouse unmodified (wild type (WT)) and erythropoietin (EPO) gene-modified MSC is cultured for 7 days in normoxic (21%) and anoxic conditions. WT-MSC is cultured in anoxia for 45 days in high and regular glucose media and both have similar viability when compared to their normoxic controls at 7 days. Protein production of EPO-MSC is unaffected by the absence of oxygen. MSC doubling ti...
