MICROCARRIER CULTIVATION OF BOVINE AORTIC ENDOTHELIAL-CELLS IN SPINNER VESSELS AND A MEMBRANE STIRRED BIOREACTOR

Primary bovine aortic endothelial cells were cultivated in serum suppl emented medium without any additional growth factors. The anchorage de pendent cells were propagated on Dormacell(R) microcarriers with coval ently 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(R) microcarriers with different ion exchange capacities ranging from 0.3 0 up to 0.65 meg g(-1), corresponding to nitrogen contents from 1.2% t o 2.9%, respectively, optimal attachment and growth of endothelial cel ls were obtained with beads of highest nitrogen content (2.9%). Cells were seeded with ca 5 viable cells per microcarrier being sufficient t o achieve fully confluent microcarriers after 4 to 5 days. Glucose con centrations decreased from 21 mM to uppermost half of the original con centrations. 4 mM glutamine was rapidly consumed and virtually exhaust ed after the cells reached confluency. Lactate concentrations raised t o a maximum of 7 mM in spinner cultures, but was found to be reutilize d in the stationary phase after glutamine limitation occurred. Serine was found to be the second most prominent amino acid being almost exha usted at confluency whereas alanine was produced in noteworthy amounts . Considerable decrease was determined for threonine, lysine and argin ine; low consumption rates were observed for leucine, phenylalanine an d methionine. All other amino acids did not alter significantly throug hout cultivation. These data support that bovine aortic endothelial ce lls are capable to utilize glucose and glutamine as well as lactic aci d (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 endothelia l cells using microcarrier concentrations of 3 g 1(-1).