Microcarrier cultivation of bovine aortic endothelial cells in spinner vessels and a membrane stirred bioreactor

摘要

Primary bovine aortic endothelial cells were cultivated in serum supplemented medium without any additional growth factors. The anchorage dependent cells were propagated on Dormacell? microcarriers with covalently bound dimeric DEAE-groups at the surface of the dextrane beads. Cultivations were performed in 200 ml spinner cultures containing 1 g l?1 to 3 g l?1 of microcarriers. Out of five types of Dormacell? microcarriers with different ion exchange capacities ranging from 0.30 up to 0.65 meq g?1, corresponding to nitrogen contents from 1.2% to 2.9%, respectively, optimal attachment and growth of endothelial cells were obtained with beads of highest nitrogen content (2.9%). Cells were seeded withca. 5 viable cells per microcarrier being sufficient to achieve fully confluent microcarriers after 4 to 5 days. Glucose concentrations decreased from 21 mM to uppermost half of the original concentrations. 4 mM glutamine was rapidly consumed and virtually exhausted after the cells reached confluency. Lactate concentrations raised to a maximum of 7 mM in spinner cultures, but was found to be reutilized in the stationary phase after glutamine limitation occurred. Serine was found to be the second most prominent amino acid being almost exhausted at confluency whereas alanine was produced in noteworthy amounts. Considerable decrease was determined for threonine, lysine and arginine; low consumption rates were observed for leucine, phenylalanine and methionine. All other amino acids did not alter significantly throughout cultivation. These data support that bovine aortic endothelial cells are capable to utilize glucose and glutamine as well as lactic acid (after glutamine exhaustion) as energy and/or carbon source. Finally, batch cultures in a 2 liter membrane stirred bioreactor with bubble-free aeration were performed to produce large quantities of endothelial cells using microcarrier concentrations of 3 g l?1.