title: Establishment of a microcarrier culture system with serial sub-cultivation for functionally active human endothelial cells.
abstract: A microcarrier culture system was established for a large-scale production of functional human endothelial cells. It has been difficult to cultivate human endothelial cells in large quantities for the reasons that specific growth factor and extracellular matrix are required for the survival and proliferation of the cells and the life span of the primary cells are limited. A lot of studies have reported that the shear stress gives significant influences on the structure, growth rate and biological functions of endothelial cells. We aimed to develop a convenient microcarrier culture system for human endothelial cells which can reproduce the flow effects experienced in vivo or in vitro. In 200 mL volume culture, human umbilical vein endothelial cells (HUVEC) could be serially sub-cultivated by optimizing the culture conditions such as shear strength, growth factor, beads and seeding cell concentration, serum concentration, and passage timing. The growth rate was enhanced depending on the shear strength and the life span of the cells was elongated until over 43PDL which is much longer than those of monolayer cultures. The cells maintained the diploidy of over 80% without obvious abnormal changes in the chromosomes. The serially sub-cultured microcarrier cells maintained various endothelial cell functions such as the syntheses of von Willebrand factor (vWf), prostacyclin and other biological substances, the expression of CD31, and the VEGF(165) dependent growth characteristic. The synthesis of biological products was affected by shear strength. In the case of prostacyclin, a different synthesis response was observed between steady flow and transiently reduced shear strength. The synthesis of endothelin-1 (ET-1) was down-regulated by increase of shear strength different from those of other products. The culture system was scaled up until 2 L volume under the optimum DO control. The cells synthesized IL-6 in response to shear strength. These results indicate that the established microcarrier system might be able to contribute to the supply of functional human endothelial cells for various medical applications such as the reconstruction of injured blood vessels caused by atherosclerosis or restenosis of coronary arteries after angioplasty, and the construction of an anti-coagulable artificial blood vessel or an artificial skin with good transplant-ability.
authors.affiliation: Department of Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Tokyo 108-8639, Japan.
authors.forename: Shinnya
authors.initials: S
authors.lastname: Tashiro
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Emiko
authors.initials: E
authors.lastname: Sano
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*