Abstract
Five types of dextran-based microcarriers (Dormacell™, Pfeifer and Langen) with different concentrations of dimeric DEAE anion-exchange groups (nitrogen contents from 1.2 up to 2.9%) were tested as growth substrates for the cultivation of human umbilical vein endothelial cells (HUVECs). All microcarriers were gelatinized before use to improve cell adhesion. The one with the highest DEAE-group density was found to be most suitable for HUVEC propagation reaching final cell densities of 8×105 viable cells ml-1 (95% viability) using microcarrier concentrations of 3 g l−1. Furthermore, metabolic data of glucose/lactate and amino acid metabolism are presented in this study. The concentrations of 18 amino acids were monitored throughout cultivation. A considerable decrease of glutamine and inverse increase of glutamate was observed. Cultivation with initial glucose concentration of 16.5 mmol l−1 resulted in high glutamine consumption rates, whereas high glucose-supplemented starting culture medium (30 mmol l-1) gave considerably lowered rates, indicating altered glutamine metabolism due to different glucose feeding. The glucose consumption and lactate production rates increased 2.6 fold and 3.5 fold, respectively, due to switch over from low to high glucose supplemented cultures. The rate of glucose metabolism was found not to be directly related to cell growth, because almost identical growth rates and doubling times were obtained. Considering the remaining 16 amino acids measured, serine concentrations considerably declined and glycine as well as alanine concentrations raised strongly. Most amino acid values were found insignificantly altered during 14 days of cultivation. Spinner vessel cultures served as inoculum for up scale propagation of HUVECs in membrane stirred 2 liter bioreactors. About 5×109 HUVECs were produced, which were used for the isolation and structural characterization of glycosphingolipids, cell membrane compounds, which are suggested to be involved in e.g. selectin-carbohydrate interaction (cell-cell adhesion), carcinogenesis and atherogenesis.
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Abbreviations
- HUVECs:
-
human umbilical vein endothelial cells
- PBS:
-
phosphate buffered saline
References
Audus KL, Bartel RL, Hidalgo IJ and Borchardt RT (1990) The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm. Res. 7: 435–451.
Bergelson LD (1995) Serum gangliosides as endogenous immunomodulators. Immunol. Today 16: 483–486.
Büntemeyer H, Lütkemeyer D and Lehmann J (1991) Optimization of serum-free fermentation processes for antibody production. Cytotechnology 5: 57–67.
Butler M (1988) A comparative review of microcarriers available for the growth of anchorage-dependent animal cells. In: Spier RE and Griffiths JB (eds.) Animal Cell Biotechnology 3 (pp. 283–303) Academic Press.
Duvar S, Müthing J, Peter-Katalinić J, Mohr H and Lehmann J (1995) Glycosphingolipids of human umbilical vein endothelial cells. Glycoconjugate J. 12: 583.
Fajardo LF (1989) The complexity of endothelial cells. Am. J. Clin. Pathol. 92: 241–50.
Friedl P, Tatje D and Czapla R (1989) An optimized culture medium for human vascular endothelial cells from umbilical cord veins. Cytotechnology 2: 171–179.
Gillard BK, Jones MA and Marcus DM (1987) Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells. Arch. Biochem. Biophys. 256: 435–445.
Gimbrone Jr. MA, Cotran RS and Folkman J (1974) Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Biol. 60: 673–682.
Heidemann R, Riese U, Lütkemeyer D, Büntemeyer H and Lehmann J (1994) The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator. Cytotechnology 14: 1–9.
Hirtenstein MD, Clark JM and Gebb C (1982) A comparison of various laboratory scale culture configurations for microcarrier culture of animal cells. Develop. Biol. Standard. 50: 73–80.
Hoshi H and McKeehan WL (1984) Brain-and liver cell-derived factors are required for growth of human endothelial cells in serum-free culture. Proc. Natl. Acad. Sci. USA 81: 6413–6417.
Jacobs M, Gummich G, Keller I and Schwengers D (1991) Cultivation of CHO-cells on microcarriers Dormacell. In: Spier RE, Griffiths JB and Meignier B (eds.) Production of Biologicals from Animal Cells in Culture (pp. 439–442) Butterworth.
Jaffe EA (1987) Cell biology of endothelial cells. Hum. Pathol. 18: 234–239.
Jaffe EA (1992) Hypoglycemia rapidly develops in cultures of human endothelial cells. In vitro Cell. Dev. Biol. 28A: 73–74.
Jerg KR, Baumann H, Keller R and Friedl P (1990) Fermentation of bovine endothelial cells for preparation of endothelial cell-surface heparan sulphate. Int. J. Biol. Macromol. 12: 153–157.
Kojima N, Shiota M, Sadahira Y, Handa K and Hakomori S-I (1992) Cell adhesion in dynamic flow system as compared to static system. J. Biol. Chem. 267: 17264–17270.
Lasky AL (1992) Selectins: interpreters of cell specific carbohydrate information during inflammation. Science 258: 964–969.
Lehmann J, Piehl GW and Schulz R (1987) Bubble free cell culture aeration with porous moving membranes. Dev. Biol. Standard. 66: 227–240.
Lehmann J, Vorlop J and Büntemeyer H (1988) Bubble free reactors and their development for continuous culture with cell recycle. In: Spier RE and Griffiths JB (eds.) Animal Cell Biotechnology 3 (pp. 221–237) Academic Press.
Müthing J, Duvar S, Nerger S, Büntemeyer H and Lehmann J (1996) Microcarrier cultivation of bovine aortic endothelial cells in spinner vessels and a membrane stirred bioreactor. Cytotechnology 18: 193–206.
Pirt SJ (1985) Principles of microbe and cell cultivation (pp. 209–222) Blackwell Scientific Publication.
Pober JS and Cotran RS (1990) The role of endothelial cells in inflammation. Transplantation 50: 537–544.
Reitzer LJ, Wice BM and Kennell D (1979) Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J. Biol. Chem. 254: 2669–2676.
Rosen SD and Bertozzi CR (1994) The selectins and their ligands. Curr. Opin. Cell Biol. 6: 663–673.
Schrimpf G and Friedl P (1993) Growth of human vascular endothelial cells on various types of microcarriers. Cytotechnology 13: 203–211.
Zielke HR, Sumbilla CM, Zielke CL, Tildon TJ and Ozand PT (1984a) Glutamine metabolism by cultured mammalian cells. In: Häussinger D and Sies H (eds.) Glutamine Metabolism in Mammalian Tissue (pp. 247–254) Springer.
Zielke HR, Zielke CL and Ozand PT (1984b) Glutamine: a major energy source for cultured mammalian cells. Federation Proc. 43: 121–125.
Zimmerman GA, Prescott SM and McIntyre TM (1992) Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol. Today 13: 93–100.
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Duvar, S., Müthing, J., Mohr, H. et al. Scale up cultivation of primary human umbilical vein endothelial cells on microcarriers from spinner vessels to bioreactor fermentation. Cytotechnology 21, 61–72 (1996). https://doi.org/10.1007/BF00364837
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DOI: https://doi.org/10.1007/BF00364837