Culture and detection of primary cilia in endothelial cell models

Cilia

David S. Long³  Michael T. Cooling¹  Sue R. McGlashan²  Yi Chung Lim¹ 

[1] Auckland Bioengineering Institute, University of Auckland, 70 Symonds Street, Auckland 1142, New Zealand;Department of Anatomy with Radiology, University of Auckland, 85 Park Road, Auckland, New Zealand;Department of Engineering Science, University of Auckland, 70 Symonds Street, Auckland, New Zealand

关键词: Cobblestone morphology;    Cilia incidence;    HMEC-1;    HUVEC;    Serum starvation;    Foetal bovine serum;    Primary cilium;   

Others  :  1232435
DOI  :  10.1186/s13630-015-0020-2

 received in 2015-02-08, accepted in 2015-09-07,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Lim et al.

【 图 表 】

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