Gene expression analysis of HUVECs cultured on uSiM (static vs flow)

Introduction

Studies have shown that the shear-stimulation of endothelial cells results in the upregulation of the shear-sensitive transcription factor Kruppel-like factor 2 (KLF2). KLF2 plays a critical role in the regulation of endothelial activity such as the regulation of vascular tone, anti-inflammatory responses, and antithrombotic functions. KLF2 is also responsible for the upregulation of endothelial nitric oxide synthase (eNOS) that promotes nitric oxide production and plays an atheroprotective function in healthy blood vessels. To demonstrate that HUVECs exhibit expected behaviors in our system, we evaluated differences in KLF2 and eNOS gene expression in shear-stimulated and static conditions.

Method

ٍHUVECs were cultured in the open-well format for 24 hours to establish a confluent monolayer. For the flow condition, the device was reconfigured and cells were stimulated with flow for another 24 hours while cells in the static condition were maintained in the open-well format. Then flow devices were reconfigured back to the open-well format and RNA extraction was carried out using standard open-well protocols (Video. 1).

Membranes were washed twice with PBS. To lyse the cells, 100 μL of TRI-Reagent (ThermoFisher Scientific, USA) was added to each device well and incubated for 5 min at room temperature. The lysate was then transferred into 1.5 mL RNase-free tubes and 1 mL of chloroform was added to each tube. After 5 min of incubation at room temperature with periodic mixing/shaking, the tubes were centrifuged at 16 000 × g and 4 °C for 15 min. The upper aqueous phase was collected and transferred into a new tube, and an equal volume of ice-cold isopropanol was added. The solutions were precipitated at −20 °C for 30 min and were then centrifuged at 16 000 × g and 4 °C for 30 min. RNA pellets were collected and washed sequentially with 70% and 100% ethanol. The solutions were air-dried for 5 min and resuspended in 11 μL of RNase-free water. RNA yield was measured using NanoPhotometer N60/N50 (Implen, Germany). High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, USA) was used to reverse transcribe 1 μg of RNA into Complementary DNA (cDNA) in a 20 μL volume reaction. Quantitative PCR with 10 ng cDNA per reaction was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems, USA). The reaction was run in a QuantStudio 3 RT-qPCR system (ThermoFisher Scientific, USA) to evaluate changes in the expression of the following genes: eNOS, KLF2, and GAPDH as the housekeeping gene (Hs01574659_m1, Hs00360439_g1, Hs02786624_g1, ThermoFisher Scientific, USA).

The following amplification conditions were used: 2 min at 50 °C, followed by 2 min at 95 °C, then 40 cycles of 95 °C for 1 s and 60 °C for 20 s. The whole experiment was conducted three times independently. Two technical replicates were used for each sample and the ΔΔCT method was used to calculate the relative expression level considering GAPDH as a reference gene.

Results

Due to the low number of cells in the uSiM (especially in the flow condition), RNA extraction yield is low; thus, we suggest combining lysate from three devices in each run and considering them as one sample.

Our results indicated that flow-stimulation of HUVECs for 24 hours under 10.7 dyne/cm2 shear stress results in 6.7× and 3.4× higher expression of KLF2 and eNOS, respectively (Figure 1). These readouts emphasize the importance of incorporating flow to mimic physiological conditions in barrier tissue models. This analysis also demonstrates the versatility of our platform for conducting downstream analysis using standard protocols designed for open-well devices.

Figure 1. Comparison of the relative expression of KLF2 and eNOS between cells cultured under flow and cells cultured in open-well m-μSiM under static condition (**p < 0.01, n = 3).

Reference

[1] Mehran Mansouri, Adeel Ahmed, S. Danial Ahmad, Molly C. McCloskey, Indranil M. Joshi, Thomas R. Gaborski, Richard E. Waugh, James L. McGrath, Steven W. Day, and Vinay V. Abhyankar. “The modular μSiM reconfigured: integration of microfluidic capabilities to study in vitro barrier tissue models under flow”, Advanced Healthcare Materials (2022)