SCALE-UP CULTIVATION OF PRIMARY HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS ON MICROCARRIERS FROM SPINNER VESSELS TO BIOREACTOR FERMENTATION

Five types of dextran-based microcarriers (Dormacell(TM), Pfeifer and Langen) with different concentrations of dimeric DEAE anion-exchange g roups (nitrogen contents from 1.2 up to 2.9%) were tested as growth su bstrates for the cultivation of human umbilical vein endothelial cells (HUVECs). All microcarriers were gelatinized before use to improve ce ll adhesion. The one with the highest DEAE-group density was found to be most suitable for HUVEC propagation reaching final cell densities o f 8 x 10(5) viable cells ml(-1) (95% viability) using microcarrier con centrations of 3 g l(-1). Furthermore, metabolic data of glucose/lacta te and amino acid metabolism are presented in this study. The concentr ations of 18 amino acids were monitored throughout cultivation. A cons iderable decrease of glutamine and inverse increase of glutamate was o bserved. Cultivation with initial glucose concentration of 16.5 mmol l (-1) resulted in high glutamine consumption rates, whereas high glucos e-supplemented starting culture medium (30 mmol l(-1)) gave considerab ly lowered rates, indicating altered glutamine metabolism due to diffe rent glucose feeding. The glucose consumption and lactate production r ates increased 2.6 fold and 3.5 fold, respectively, due to switch over from low to high glucose supplemented cultures. The rate of glucose m etabolism was found not to be directly related to cell growth, because almost identical growth rates and doubling times were obtained. Consi dering the remaining 16 amino acids measured, serine concentrations co nsiderably declined and glycine as well as alanine concentrations rais ed strongly. Most amino acid values were found insignificantly altered during 14 days of cultivation. Spinner vessel cultures served as inoc ulum for up scale propagation of HUVECs in membrane stirred 2 liter bi oreactors. About 5 x 10(9) HUVECs were produced, which were used for t he isolation and structural characterization of glycosphingolipids, ce ll membrane compounds, which are suggested to be involved in e.g. sele ctin-carbohydrate interaction (cell-cell adhesion), carcinogenesis and atherogenesis.