Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Abstract

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow. Video Link The video component of this article can be found at https://www.jove.com/video/58678/ This protocol is useful for the assessment of inflammatory responses in hiPSC-ECs in drug screening, disease modeling and personalized regenerative medicine.

Endothelial Cells as Tools to Model Tissue Microenvironment in Hypoxia-Dependent Pathologies

“…Therefore, in in vitro conditions the degree of activation of adhesion molecules after cytokine stimulation can be assessed, such as E-Selectin expression after TNFα or IL-1 stimulation [ 16 ]. Functional studies include a simple test for the adhesion of immune cells to a monolayer of endothelial cells [ 56 , 57 ] or a more advanced microfluid test, where the rolling and adhesion of leukocytes can be observed in real time [ 20 , 58 ]. Bielawska-Pohl et al showed that interaction between immune cells and organospecific endothelial cells can be a potential target to block vascular injury [ 59 ].…”

Generation and Functional Characterization of Monocytes and Macrophages Derived from Human Induced Pluripotent Stem Cells

“…Therefore, leukocyte adhesion in a microfluidic flow assay is a more physiologically relevant way of assessing functionality of hiPSC‐mono and allows cross comparison of them with primary cells. Previously we described a detailed protocol to study adhesion of monocytes to hiPSC‐ECs in a microfluidic chip (Halaidych et al., 2018b). The protocol was applied to study adhesion of human monocytic cell line (THP1; Halaidych et al., 2018a), blood‐mono, and hiPSC‐mono (Cao et al., 2019).…”

“…2B-D). The flow assay had been described in detail in our earlier publication (Halaidych, van den Hil, Mummery, & Orlova, 2018b) and is only briefly introduced here. In this assay, hiPSC-mono should be highly adhesive to both hiPSC-ECs and HUVECs that are stimulated with tumor…”

Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium

“…To improve the lead time and simplicity of biochip production, we followed a modified protocol for endothelial cell priming and surface endothelialisation, which does not require an overnight cell culture perfusion system [21]. Immunostaining showed a confluent endothelial surface (Figure 2).…”

“…Perfusion of whole blood in endothelialised chips has been used predominantly to study platelet adhesion and thrombus formation [20]. The study of whole blood neutrophil adhesion in the context of cardiovascular thrombosis is limited with chips utilising isolated neutrophils or cell lines (e.g., THP-1) [21,22]. A summary of the published endothelialised microfluidic chips assessing thromboinflammation to date is provided in Table 1.…”