Pekor Christopher, Gerlach Jörg C, Nettleship Ian, Schmelzer Eva
1 Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.
2 Department of Bioengineering, McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.
Tissue Eng Part C Methods. 2015 Jul;21(7):705-15. doi: 10.1089/ten.TEC.2014.0453. Epub 2015 Feb 11.
The development of functional engineered tissue constructs depends on high cell densities and appropriate vascularization. In this study we implemented a four-compartment three-dimensional perfusion bioreactor culture model for studying the effects of medium perfusion on endothelial, hepatic, and hematopoietic cell populations of primary human fetal liver in an in vivo-like environment. Human fetal liver cells were cultured in bioreactors configured to provide either perfusion or diffusion conditions. Metabolic activities of the cultures were monitored daily by measuring glucose consumption and lactate production. Cell viability during culture was analyzed by lactate dehydrogenase activity. Hepatic functionality was determined by the release of albumin and alpha-fetoprotein (AFP) in culture medium samples. After 4 days of culture, cells were analyzed for the expression of a variety of endothelial, hepatic, and hematopoietic genes, as well as the surface marker expression of CD31 and CD34 in flow cytometry. We found that medium perfusion increased the gene expression of endothelial markers such as CD31, von Willebrand factor (vWF), CD140b, CD309, and CD144 while decreasing the gene expression of the erythrocyte-surface marker CD235a. Hepatic differentiation was promoted under perfusion conditions as demonstrated by lower AFP and higher albumin secretion compared with cultures not exposed to medium perfusion. Additionally, cultures exposed to medium perfusion gave higher rates of glucose consumption and lactate production, indicating increased metabolic activity. In conclusion, high-density bioreactors configured to provide constant medium perfusion significantly induced hepatic and endothelial cell differentiation and provided improved conditions for the culture of human fetal liver cells compared with cultures without perfusion.
功能性工程组织构建体的发展依赖于高细胞密度和适当的血管化。在本研究中，我们实施了一种四室三维灌注生物反应器培养模型，用于在类似体内的环境中研究培养基灌注对原代人胎肝内皮细胞、肝细胞和造血细胞群体的影响。人胎肝细胞在配置为提供灌注或扩散条件的生物反应器中培养。通过测量葡萄糖消耗和乳酸产生，每天监测培养物的代谢活性。通过乳酸脱氢酶活性分析培养期间的细胞活力。通过培养基样品中白蛋白和甲胎蛋白（AFP）的释放来确定肝功能。培养4天后，分析细胞中多种内皮、肝和造血基因的表达，以及流式细胞术中CD31和CD34的表面标志物表达。我们发现，培养基灌注增加了内皮标志物如CD31、血管性血友病因子（vWF）、CD140b、CD309和CD144的基因表达，同时降低了红细胞表面标志物CD235a的基因表达。与未暴露于培养基灌注的培养物相比，灌注条件下促进了肝分化，表现为AFP较低和白蛋白分泌较高。此外，暴露于培养基灌注的培养物具有更高的葡萄糖消耗率和乳酸产生率，表明代谢活性增加。总之，与无灌注培养相比，配置为提供恒定培养基灌注的高密度生物反应器显著诱导肝和内皮细胞分化，并为培养人胎肝细胞提供了更好的条件。
