Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidi...

Full description

Saved in:
Bibliographic Details
Main Authors: Pang, Kuin T., Ghim, Mean, Arshad, Mehwish, Wang, Xiaomeng, Weinberg, Peter D.
Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
Format: Article
Language:English
Published: 2022
Subjects:
Online Access:https://hdl.handle.net/10356/160745
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-160745
record_format dspace
spelling sg-ntu-dr.10356-1607452023-03-05T16:51:13Z Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies Pang, Kuin T. Ghim, Mean Arshad, Mehwish Wang, Xiaomeng Weinberg, Peter D. Lee Kong Chian School of Medicine (LKCMedicine) Institute of Molecular and Cell Biology, A*STAR Singapore Eye Research Institute Science::Medicine Atherosclerosis Cell Culture Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well. National Medical Research Council (NMRC) Published version The authors gratefully acknowledge a British Heart Foundation project grant (to PDW), a National Medical Research Council Singapore TAAP and DYNAMO Grant (to XW, NMRC/OFLCG/004/2018, NMRC/OFLCG/001/2017), an A*STAR Graduate Scholarship (to KTP), and a British Heart Foundation Center of Research Excellence studentship (to MA). 2022-08-02T03:16:37Z 2022-08-02T03:16:37Z 2021 Journal Article Pang, K. T., Ghim, M., Arshad, M., Wang, X. & Weinberg, P. D. (2021). Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies. Journal of Visualized Experiments (JoVE), 2021(172), 61817-. https://dx.doi.org/10.3791/61817 1940-087X https://hdl.handle.net/10356/160745 10.3791/61817 34152321 2-s2.0-85108189485 172 2021 e61817 en NMRC/OFLCG/001/2017 NMRC/OFLCG/004/2018 Journal of Visualized Experiments (JoVE) © 2021 JoVE Journal of Visualized Experiments. All rights reserved. This paper was published in JoVE Journal of Visualized Experiments and is made available with permission of JoVE Journal of Visualized Experiments. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Medicine
Atherosclerosis
Cell Culture
spellingShingle Science::Medicine
Atherosclerosis
Cell Culture
Pang, Kuin T.
Ghim, Mean
Arshad, Mehwish
Wang, Xiaomeng
Weinberg, Peter D.
Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies
description Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.
author2 Lee Kong Chian School of Medicine (LKCMedicine)
author_facet Lee Kong Chian School of Medicine (LKCMedicine)
Pang, Kuin T.
Ghim, Mean
Arshad, Mehwish
Wang, Xiaomeng
Weinberg, Peter D.
format Article
author Pang, Kuin T.
Ghim, Mean
Arshad, Mehwish
Wang, Xiaomeng
Weinberg, Peter D.
author_sort Pang, Kuin T.
title Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies
title_short Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies
title_full Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies
title_fullStr Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies
title_full_unstemmed Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies
title_sort segmenting growth of endothelial cells in 6-well plates on an orbital shaker for mechanobiological studies
publishDate 2022
url https://hdl.handle.net/10356/160745
_version_ 1759857430648324096