Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Tissue Eng Part C Methods. 2013 Feb;19(2):93-100. doi: 10.1089/ten.TEC.2011.0629. Epub 2012 Sep 28.
Many oxygen mass-transfer modeling studies have been performed for various bioartificial liver (BAL) encapsulation types; yet, to our knowledge, there is no experimental study that directly and noninvasively measures viability and metabolism as a function of time and oxygen concentration. We report the effect of oxygen concentration on viability and metabolism in a fluidized-bed NMR-compatible BAL using in vivo ³¹P and ¹³C NMR spectroscopy, respectively, by monitoring nucleotide triphosphate (NTP) and ¹³C-labeled nutrient metabolites, respectively. Fluidized-bed bioreactors eliminate the potential channeling that occurs with packed-bed bioreactors and serve as an ideal experimental model for homogeneous oxygen distribution. Hepatocytes were electrostatically encapsulated in alginate (avg. diameter, 500 μm; 3.5×10⁷ cells/mL) and perfused at 3 mL/min in a 9-cm (inner diameter) cylindrical glass NMR tube. Four oxygen treatments were tested and validated by an in-line oxygen electrode: (1) 95:5 oxygen:carbon dioxide (carbogen), (2) 75:20:5 nitrogen:oxygen:carbon dioxide, (3) 60:35:5 nitrogen:oxygen:carbon dioxide, and (4) 45:50:5 nitrogen:oxygen:carbon dioxide. With 20% oxygen, β-NTP steadily decreased until it was no longer detected at 11 h. The 35%, 50%, and 95% oxygen treatments resulted in steady β-NTP levels throughout the 28-h experimental period. For the 50% and 95% oxygen treatment, a ¹³C NMR time course (∼5 h) revealed 2-¹³C-glycine and 2-¹³C-glucose to be incorporated into [2-¹³C-glycyl]glutathione (GSH) and 2-¹³C-lactate, respectively, with 95% having a lower rate of lactate formation. ³¹P and ¹³C NMR spectroscopy is a noninvasive method for determining viability and metabolic rates. Modifying tissue-engineered devices to be NMR compatible is a relatively easy and inexpensive process depending on the bioreactor shape.
许多氧传质模型研究已经针对各种生物人工肝（BAL）包封类型进行了；然而，据我们所知，目前还没有实验研究能够直接无创地测量随时间和氧浓度变化的活力和代谢。我们报告了在使用体内 ³¹P 和 ¹³C NMR 光谱分别监测核苷酸三磷酸（NTP）和 ¹³C 标记的营养代谢物的情况下，氧浓度对在液体化床 NMR 兼容 BAL 中的活力和代谢的影响。液体化床生物反应器消除了与填充床生物反应器发生的潜在通道化作用，并作为均匀氧分布的理想实验模型。肝细胞通过静电包封在藻酸盐中（平均直径，500μm；3.5×10⁷ 个细胞/mL），并以 3mL/min 的速度在 9cm（内径）圆柱形玻璃 NMR 管中灌注。通过在线氧电极测试和验证了四种氧处理方法：（1）95:5 氧气：二氧化碳（氧合），（2）75:20:5 氮气：氧气：二氧化碳，（3）60:35:5 氮气：氧气：二氧化碳，和（4）45:50:5 氮气：氧气：二氧化碳。在 20%的氧气下，β-NTP 稳步下降，直到 11 小时后不再检测到。在 35%、50%和 95%的氧气处理下，β-NTP 在整个 28 小时的实验期间保持稳定水平。对于 50%和 95%的氧气处理，¹³C NMR 时间过程（约 5 小时）显示 2-¹³C-甘氨酸和 2-¹³C-葡萄糖分别掺入[2-¹³C-甘氨酰]谷胱甘肽（GSH）和 2-¹³C-乳酸，其中 95%的乳酸形成率较低。³¹P 和 ¹³C NMR 光谱是一种非侵入性方法，用于确定活力和代谢率。根据生物反应器的形状，将组织工程设备修改为 NMR 兼容是一个相对简单且廉价的过程。
