Controlled respiratory gas delivery to embryonic renal epithelial explants in perfusion culture

During generation of artificial tissues high levels of oxygen are usually available whereas after implantation into a recipient's body the implant is not vascularized immediately, which leads to low oxygen partial pressures within the implanted tissue. Under these conditions cells will experience an oxygen shortage, contrasting with the abundance of oxygen during culture. It is uncertain whether tissues can be trained to tolerate such an acute hypoxic situation so that nonphysiological stress reactions and tissue necrosis can be avoided. To investigate the effects of varying oxygen levels on embryonic renal tissue in vitro we have been developing a model system combining continuous medium renewal with the ability to control levels of oxygen and carbon dioxide by gas equilibration through gas-permeable tubing. Renal embryonic tissue from neonatal rabbit was cultured in serum-free Iscove's modified Dulbecco's medium at 45, 90, 115, and 160 mmHg oxygen partial pressure for 14 days under continuous medium exchange in such a setup. After a 14-day culture period tissue sections were analyzed by cell biological methods and compared with fresh tissue histology. Surprisingly, embryonic renal explants survive and maintain good morphology for 14 days under all O(2) conditions tested. Expression of cytokeratin 19 within the established epithelium remains unchanged, indicating a structurally intact tissue. However, Na/K-ATPase is clearly downregulated under low O(2) conditions, whereas COX-2 expression increases drastically. An antiparallel effect of decreased O(2) concentrations on glycoprotein expression can be demonstrated with the lectin Dolichos biflorus agglutinin. Scanning electron microscopy reveals oxygen-dependent changes in cellular surface differentiation of developed collecting duct epithelium.