关键词: 生物反应器, 组织工程, 血管平滑肌细胞, 聚乙醇酸, 溶解氧, 细胞增殖, 有氧糖酵解

Abstract: BACKGROUND: Seed cells are seeded on three-dimensional scaffold materials, and three-dimensional culture in bioreactors is a common in vitro tissue engineering culture method, but the changes in cell proliferation and metabolic patterns in bioengineered blood vessel construction are still unclear.
OBJECTIVE: To explore the metabolic changes of cells such as oxygen consumption and their causes during the whole process of biological vascular tissue construction by in vitro bioreactor.
METHODS: The self-built vascular bioreactor system was used as the platform; bovine vascular smooth muscle cells were used as the seed cells, and a conventional CO2 incubator provided the external gas environment for the cultivation process. Seed cells were seeded on a tubular porous polyglycolic acid scaffold material for three-dimensional culture, and the whole process included a one-week resting period and a seven-week pulsating tensile stress stimulation loading period. A non-invasive monitoring system was built, and the optical dissolved oxygen patch method was used to monitor the changes of dissolved oxygen in the culture solution in the reactor, and the glucose consumption and lactic acid production were measured by regular sampling. CCK-8 assay was used to determine the proliferation of smooth muscle cells on polyglycolic acid three-dimensional scaffold materials. Nicotinamide adenine dinucleotide oxidation state and reduction state ratio (NAD+/NADH) was utilized to understand cell proliferation and metabolism in the early stage of culture. RT-qPCR and western blot assay were applied to detect the expression of proliferation-related genes (Ki67) and glycolysis-related genes (GLUT-1, LDHA).
RESULTS AND CONCLUSION: (1) The dissolved oxygen level in the culture solution was (4.314±0.380) mg/L from the cell injection to the end of the resting period (the first week), and gradually stabilized at (1.960±0.866) mg/L after the tensile stress stimulation (the last seven weeks); the two had significant changes (P < 0.05). (2) The ratio of glucose consumption to lactic acid production in the cell culture medium YL/G increased rapidly after the cells were injected, and the highest value was above 1 on the fifth day, and then slow down to 0.5 (The mean value of YL/G in the resting period was 0.89 and the mean value in the pressurized period was 0.57, P < 0.05). (3) CCK-8 assay results showed that A450 value gradually increased after the cells were injected, and reached the highest value on the fifth day, reaching 3.17, and then slowly decreased. At the same time, it was found that Ki67 mRNA was up-regulated on the third day of culture, and then declined. The expression level of Ki67 protein was higher from the third day to the fifth day. (4) The detection of NAD+/NADH showed that the increase was obvious from the fifth to the seventh day after the injection of cells, and the expression of glycolysis-related genes (GLUT-1 and LDHA) was up-regulated and changed synchronously, and the relative expression was higher in the first five days. (5) The results showed that the tissue-engineered blood vessels were constructed using the vascular bioreactor and the smooth muscle cells in the early stage mainly proliferated and exhibited a metabolic feature of low oxygen consumption. The metabolic characteristics of high oxygen consumption were observed during the pulsatile tensile stress stimulation stage. 

Key words: bioreactor, tissue engineering, vascular smooth muscle cell, polyglycolic acid, dissolved oxygen, cell proliferation, aerobic glycolysis