Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands.
Tissue Eng Part C Methods. 2010 Jun;16(3):479-85. doi: 10.1089/ten.TEC.2009.0278.
Survival and growth of cellular grafts in tissue engineering (TE) are limited by the rate of oxygen (O(2)) and nutrient diffusion. As such, monitoring the levels of nutrients and O(2) available to the cells is essential to assess the physiology of the cells and to evaluate strategies aiming at improving nutrient availability. In this article, a reporter system containing the luciferase gene driven by a hypoxia responsive promoter was used to monitor cellular hypoxia in a TE context. We report that luciferase activity correlates with the O(2) tension in the cell culture medium. When transgenic cells were seeded onto scaffolds and implanted in immune-deficient mice subcutaneously, luciferase activity was detected. To validate the response to O(2) levels of this reporter system, we cultured transgenic cells on biomaterials in a flow perfusion bioreactor and observed that cells in the bioreactor displayed a drastically lower luciferase activity than conventional static culture, and that higher luciferase activity is observed in the interior of a tissue-engineered construct, illustrating the uneven O(2) distribution in three-dimensional constructs under conventional static culture. We conclude that this reporter system is a versatile tool to investigate cellular O(2) availability in TE both in vitro and in vivo.
细胞移植物在组织工程（TE）中的存活和生长受到氧气（O(2)）和营养物质扩散速率的限制。因此，监测细胞可用的营养物质和 O(2)水平对于评估细胞生理学以及评估旨在提高营养物质可用性的策略至关重要。在本文中，使用含有受缺氧反应性启动子驱动的荧光素酶基因的报告系统来监测 TE 环境中的细胞缺氧。我们报告说，荧光素酶活性与细胞培养介质中的 O(2)张力相关。当转基因细胞接种到支架上并植入免疫缺陷小鼠的皮下时，检测到了荧光素酶活性。为了验证该报告系统对 O(2)水平的反应，我们在流动灌注生物反应器中在生物材料上培养转基因细胞，观察到生物反应器中的细胞的荧光素酶活性明显低于传统的静态培养，并且在组织工程构建体的内部观察到更高的荧光素酶活性，这说明了在传统的静态培养下三维构建体中 O(2)分布不均匀。我们得出结论，该报告系统是一种多功能工具，可用于研究 TE 中细胞的 O(2)可用性，无论是在体外还是体内。
