hiPSCs-derived Vascular Organ-on-Chip Model Under Unidirectional Controlled Flow

Importance of Unidirectional Flow in Vascular Organ-on-Chip Models  

Vessel-on-Chip (VoC) and other vascularized in vitro models aim to replicate stable vascular physiology, where endothelial cells are continuously exposed to unidirectional shear stress (1). In vivo, this mechanical stimulus is essential for maintaining mature endothelial phenotype. 

In contrast: 

• Static culture fails to provide mechanical stimulation, resulting in non-aligned, immature endothelial cells. Whereas, rocking platforms, mimics disturbed back and forth flow. These varying flow patterns typically associated with abnormal conditions rather than healthy vasculature.(2) 

For hiPSC-derived endothelial cells in particular, flow directionality plays a significant role (3) in: 

• Cell elongation and alignment parallel to flow 
• Golgi–nucleus polarization 
• Barrier integrity and functional maturation 

However, implementing unidirectional flow with recirculation using traditional microfluidic systems could often be complex and difficult to maintain over extended periods.  

Experimental Workflow and Quantitative Readouts

This application note provides a step-by-step experimental workflow, from cell seeding to quantitative image analysis. 

You will find: 

• A 5-day unidirectional flow protocol for hiPSC-derived endothelial cells 
• Omi™ setup and recirculation workflow 
• Comparison of static, bidirectional, and unidirectional flow conditions
• Quantitative analysis of cell alignment and polarization using PolarityJam 
• Evidence of stable flow maintenance over prolonged culture 

👉 Download the application note to explore the full methodology and results. 

Results: Stable Unidirectional Flow Drives hiPSC-derived Endothelial Cells Alignment and Polarization 

Omi™ maintained the preconfigured unidirectional flow profiles over a continuous 5-day culture period. The recirculation functionality enabled continuous perfusion without the need for additional medium replenishment, thereby minimizing experimental variability and reducing medium consumption. 

Under unidirectional shear stress, hiPSC-derived endothelial cells (hiPSC-ECs) underwent pronounced morphological and organizational changes characteristic of a mature endothelial phenotype. Immunofluorescence imaging revealed strong elongation and alignment of hiPSC-ECs parallel to the direction of flow, whereas cells cultured under static conditions or subjected to bidirectional flow on a rocking platform retained a morphology with random orientation (Figure 2).

Beyond changes in cell morphology, unidirectional flow induced collective polarization of the Golgi–nucleus axis, a well-established hallmark of endothelial mechanosensing and functional maturation (5). Under unidirectional shear, the Golgi apparatus consistently localized upstream of the nucleus, oriented opposite to the direction of flow, reflecting coordinated planar cell polarity across the endothelial monolayer. This polarization is mechanistically linked to shear-mediated cytoskeletal remodeling and spatial organization of signaling pathways involved in junction stabilization, barrier function, and anti-inflammatory endothelial states (6).  

In contrast, hiPSC-derived endothelial cells maintained under static or bidirectional flow conditions showed no preferential Golgi–nucleus orientation, indicating a failure to establish sustained polarity. Quantitative analysis using PolarityJam (7) confirmed significantly higher alignment and polarity indices under unidirectional flow compared to both control conditions. Notably, bidirectional flow—despite generating shear stress—did not induce polarization, underscoring that flow directionality, rather than shear magnitude alone, is a decisive determinant of endothelial organization and phenotype in hiPSC-derived Vascular Organ-on-Chip Model. 

Full analysed data and methods are provided in the complete application note.

Related Webinars

• 

  Webinar – Liver–Kidney OOC Model to Investigate Drug Disposition

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• 

  Webinar: Importance of Flow in Organ-on-a-Chip, featuring the Gut-on-a-Chip Model

  Watch the Recording

Acknowledgements 

The work in Leiden OoC Center and Dr. Orlova is supported by the Novo Nordisk Foundation Center for Stem Cell Medicine that is supported by a Novo Nordisk Foundation grant (NNF21CC0073729) and the LymphChip project with project number NWA-ORC 2019 1292.19.019 of the NWA research program ‘‘Research on Routes by Consortia (ORC),’’ which is funded by the Netherlands Organisation for Scientific Research (NWO). 

References 

 1. Ingber DE. Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat Rev Genet. 2022 Aug;23(8):467–91.  

2. Shakeri A, Wang Y, Zhao Y, Landau S, Perera K, Lee J, et al. Engineering Organ-on-a-Chip Systems for Vascular Diseases. Arterioscler Thromb Vasc Biol. 2023 Dec;43(12):2241–55.  

3. Jang S, Collin de l’Hortet A, Soto-Gutierrez A. Induced Pluripotent Stem Cell-Derived Endothelial Cells: Overview, Current Advances, Applications, and Future Directions. Am J Pathol. 2019 Mar;189(3):502–12.  

4. Orlova VV, van den Hil FE, Petrus-Reurer S, Drabsch Y, Ten Dijke P, Mummery CL. Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat Protoc. 2014;9(6):1514–31.  

5. Tkachenko E, Gutierrez E, Saikin SK, Fogelstrand P, Kim C, Groisman A, et al. The nucleus of endothelial cell as a sensor of blood flow direction. Biol Open. 2013 Aug 14;2(10):1007–12.  

6. Dorland YL, Huveneers S. Cell-cell junctional mechanotransduction in endothelial remodeling. Cell Mol Life Sci CMLS. 2017 Jan;74(2):279–92.  

7. Giese W, Albrecht JP, Oppenheim O, Akmeriç EB, Kraxner J, Schmidt D, et al. Polarity-JaM: an image analysis toolbox for cell polarity, junction and morphology quantification. Nat Commun. 2025 Feb 8;16(1):1474.