title: Effect of cell-seeding density on the proliferation and gene expression profile of human umbilical vein endothelial cells within ex vivo culture.
abstract: OBJECTIVE
  Characterization of endothelial cell-biomaterial interaction is crucial for the development of blood-contacting biomedical devices and implants. However, a crucial parameter that has largely been overlooked is the cell-seeding density.
  
  
  METHODS
  This study investigated how varying cell-seeding density influences human umbilical vein endothelial cell (HUVEC) proliferation on three different substrata: gelatin, tissue culture polystyrene (TCPS) and poly-l-lactic acid (PLLA).
  
  
  RESULTS
  The fastest proliferation was seen on gelatin, followed by TCPS and PLLA, regardless of seeding density. On both TCPS and gelatin, maximal proliferation was attained at an initial seeding density of 1000 cells/cm(2). At seeding densities above and below 1000 cells/cm(2), the proliferation rate decreased sharply. On PLLA, there was a decrease in cell numbers over 7 days of culture, below a certain threshold seeding density (c. 2500-3000 cells/cm(2)), which meant that some of the cells were dying off rather than proliferating. Above this threshold seeding density, HUVEC displayed slow proliferation. Subsequently, quantitative real-time polymerase chain reaction (RT-qPCR) analysis of eight gene markers associated with adhesion and endothelial functionality (VEGF-A, integrin-α5, VWF, ICAM1, ICAM2, VE-cadherin, endoglin and PECAM1) was carried out on HUVEC seeded at varying densities on the three substrata. A significant downregulation of gene expression was observed at an ultralow cell-seeding density of 100 cells/cm(2). This was accompanied by an extremely slow proliferation rate, probably because of an acute lack of intercellular contacts and paracrine signaling.
  
  
  CONCLUSIONS
  Hence, this study demonstrates that seeding density has a profound effect on the proliferation and gene expression profile of endothelial cells seeded on different biomaterial surfaces.
authors.affiliation: School of Materials Science and Engineering, Nanyang Technological University, Singapore. Alexishengbc@ntu.edu.sg
authors.forename: Boon Chin
authors.initials: BC
authors.lastname: Heng
authors.forename: Paula Porto
authors.initials: PP
authors.lastname: Bezerra
authors.forename: Peter Rainer
authors.initials: PR
authors.lastname: Preiser
authors.forename: S K Alex
authors.initials: SK
authors.lastname: Law
authors.forename: Yun
authors.initials: Y
authors.lastname: Xia
authors.forename: Freddy
authors.initials: F
authors.lastname: Boey
authors.forename: Subbu S
authors.initials: SS
authors.lastname: Venkatraman
mesh_terms: D002452:Cell Count; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D011091:Polyesters; D011108:Polymers / Q000378:metabolism; D011137:Polystyrenes / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology*
chemical_list: D011091:Polyesters; D011108:Polymers; D011137:Polystyrenes; D019344:Lactic Acid; C033616:poly(lactide); D005780:Gelatin