title: Culture and detection of primary cilia in endothelial cell models.
abstract: BACKGROUND
  The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length.
  
  
  RESULTS
  HUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p < 0.001). HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).
  
  
  CONCLUSIONS
  We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.
authors.affiliation: Auckland Bioengineering Institute, University of Auckland, 70 Symonds Street, Auckland, 1142 New Zealand.
authors.forename: Yi Chung
authors.initials: YC
authors.lastname: Lim
authors.affiliation: Department of Anatomy with Radiology, University of Auckland, 85 Park Road, Auckland, New Zealand.
authors.forename: Sue R
authors.initials: SR
authors.lastname: McGlashan
authors.affiliation: Auckland Bioengineering Institute, University of Auckland, 70 Symonds Street, Auckland, 1142 New Zealand.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Cooling
authors.affiliation: Auckland Bioengineering Institute, University of Auckland, 70 Symonds Street, Auckland, 1142 New Zealand ; Department of Engineering Science, University of Auckland, 70 Symonds Street, Auckland, New Zealand.
authors.forename: David S
authors.initials: DS
authors.lastname: Long
keywords: Cilia incidence; Cobblestone morphology; Foetal bovine serum; HMEC-1; HUVEC; Primary cilium; Serum starvation