Nieuwoudt Martin, Wiggett Scholtz, Malfeld Susan, van der Merwe Schalk W
Bioengineering Unit, Hepatology Research Laboratory, University of Pretoria, Prinshof Campus, Dr Savage road, Pretoria, South Africa.
J Artif Organs. 2009;12(4):247-57. doi: 10.1007/s10047-009-0480-5. Epub 2009 Dec 25.
In vitro hepatocyte bioreactor functionality depends particularly on maintaining appropriate oxygen levels and exposure to nonparenchymal cells. An attractive solution without immunological consequences to the patient is incorporating a perfluorocarbon oxygen carrier in the circulating medium and co-culturing hepatocytes with stellate cells. Since bioreactors are normally sealed sterile units, demonstrating metabolic functionality is hindered by limited access to the cells after their aggregation in the matrix. A novel possibility is to use positron emission tomography (PET) to image cellular radioactive glucose uptake under O(2)-limited conditions. In this study, primary cell isolation procedures were carried out on eight pigs. Pairs of cell-seeded and cell-free (control) bioreactors with and without perfluorocarbon were cultured under identical conditions and were oxygenated using hypoxic (5% O(2)) and ambient (20% O(2)) gas mixes. Sixteen PET scans were conducted 24 h after cell isolation, the same timescale as that involved in treating a liver failure patient with a primary-cell bioreactor. In all cases, cell-seeded bioreactors without perfluorocarbon were more radioactive, i.e., were more glycolytic, than those with perfluorocarbon. This difference was significant in the hypoxic pair of bioreactors but not in the ambient pair of bioreactors. Additionally, in the same hypoxic bioreactors, circulating extracellular steady-state glucose levels were significantly lower and lactate levels were higher than those in the ambient bioreactors. Similar findings have been made in other in vitro hepatocyte studies investigating the effects of perfluorocarbons. PET is attractive for studying in situ O(2)-dependent bioreactor metabolism because of its visual and numerically quantifiable outputs. Longer-term metabolic studies (e.g., 5-10 days) investigating the effect of perfluorocarbon on bioreactor longevity will complement these findings in the future.
体外肝细胞生物反应器的功能尤其取决于维持适当的氧气水平以及与非实质细胞的接触。一种对患者无免疫后果的有吸引力的解决方案是在循环培养基中加入全氟碳氧载体，并将肝细胞与星状细胞共培养。由于生物反应器通常是密封的无菌单元，在细胞聚集在基质中后，由于难以接触到细胞，因此难以证明其代谢功能。一种新的可能性是使用正电子发射断层扫描（PET）在氧气受限的条件下对细胞摄取放射性葡萄糖进行成像。在本研究中，对八头猪进行了原代细胞分离程序。将有无全氟碳的成对接种细胞和未接种细胞（对照）的生物反应器在相同条件下培养，并使用低氧（5%氧气）和环境（20%氧气）气体混合物进行供氧。在细胞分离后24小时进行了16次PET扫描，这与用原代细胞生物反应器治疗肝功能衰竭患者的时间尺度相同。在所有情况下，未添加全氟碳的接种细胞的生物反应器比添加全氟碳的生物反应器放射性更强，即糖酵解程度更高。这种差异在低氧的一对生物反应器中显著，但在环境条件的一对生物反应器中不显著。此外，在相同的低氧生物反应器中，循环细胞外稳态葡萄糖水平显著低于环境生物反应器，而乳酸水平则更高。在其他研究全氟碳影响的体外肝细胞研究中也有类似发现。PET因其直观且可数值量化的输出结果，对于研究原位氧气依赖的生物反应器代谢具有吸引力。未来开展的研究全氟碳对生物反应器寿命影响的长期代谢研究（例如5 - 10天）将补充这些发现。
