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...
| Main Authors: | , , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
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2022
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| Online Access: | https://hdl.handle.net/10356/160745 |
| _version_ | 1826129712012853248 |
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| author | Pang, Kuin T. Ghim, Mean Arshad, Mehwish Wang, Xiaomeng Weinberg, Peter D. |
| 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. |
| author_sort | Pang, Kuin T. |
| collection | NTU |
| 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. |
| first_indexed | 2024-10-01T07:45:00Z |
| format | Journal Article |
| id | ntu-10356/160745 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T07:45:00Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | ntu-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 |
| 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 |
| title | 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_short | 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 |
| topic | Science::Medicine Atherosclerosis Cell Culture |
| url | https://hdl.handle.net/10356/160745 |
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