Summary

Researchers from the University of Castilla la Mancha and University of Zaragoza have optimized a kidney-on-chip for graphene particle testing. The microfluidic system was placed under flow conditions to reduce the adsorption of graphene materials on the system components. Also, graphene deposition on cells. The researchers evaluated the response of a kidney cell line under both static and flow conditions, validating the effectiveness of their approach. They also created a kidney-on-a-chip model that mimics the physiological shear stress experienced by cells. This research paves the way for the creation of other microphysiological systems that simulate other tissues, whose functions can be influenced by interactions with 2D materials.

Read the full article here: A microphysiological system for handling graphene-related materials under flow conditions.

Introduction

Graphene, a single layer of carbon atoms arranged in a two-dimensional honeycomb lattice, has garnered significant attention for its potential applications in biological tissues due to its unique thermal, electrical, and mechanical properties. These properties open up a wide range of applications in biological tissues, from drug delivery and biosensing to tissue engineering and cancer treatment. Its versatility and potential for functionalization make it a highly promising material in the field of biomedicine [1].

The study of graphene has traditionally been conducted using static in vitro models or animal models. Static in vitro models offer an overly simplified approach that does not account for mechanical stimuli present in physiological tissues. For example, shear stress is especially relevant in systems like renal or vascular environments. On the other hand, animal models fail to accurately replicate human physiology due to interspecies differences, and they also raise significant ethical concerns [2].

The expanding biomedical applications of graphene-based materials have sparked concerns regarding their short- and long-term (cyto)toxicity. Graphene, due to its unique properties such as high surface area, electrical conductivity, and mechanical strength. Due to them, it holds immense potential for use in drug delivery systems, biosensors, tissue engineering, and other medical applications. However, these same properties that make graphene so versatile also raise questions about its safety [3].

This study utilizes microfluidic devices to examine the interaction and cytotoxic effects of graphene-based nanomaterials (GRMs) on a kidney model under physiologically relevant fluid shear stress conditions.

Methods

Be-Flow device

COP-based devices like Be-Flow have a very low capacity for molecule retention due to their low porosity. Additionally, their optical properties, including low autofluorescence and high transparency, enable high-quality monitoring through brightfield and confocal microscopy.

Cell culture

Researchers developed the kidney-on-a-chip by seeding a commercial line of human renal proximal tubular cells (RPTEC/TERT1). This approach is particularly effective because proximal tubular cells play a crucial role in xenobiotic transport. In fact, it is a function that is highly relevant for toxicity testing. Consequently, the kidney-on-a-chip offers a valuable platform for advancing toxicity research and understanding drug interactions. To see more about how to perform an ECM coating or seeding cells in the Be-Flow, check our guide and take a look on our how-to-do video.

Perfusion conditions

The researchers chose the peristaltic pump perfusion system due to its advantages, Namely, recirculating the culture medium while maintaining unidirectional flow, and its ability to use small volumes of medium during a 72-hour experiment.

Figure 1. Diagram of kidney-on-chip for graphene particle testing setup in a recirculation system. The pump rollers drive the culture medium through PVC tubing, connected to PTFE tubing that links the microfluidic device to the medium reservoir. A magnetic stirrer is used in the reservoir to prevent graphene deposition.

The Hage-Poiseuille formula was used to calculate the shear stress (T), where Q represents the flow rate, ɳ is the medium viscosity, w is the channel width, and h is the channel height [4].

T =6ɳQ/wh2

Researchers exposed cells to physiological shear stress by maintaining a fluid flow of 75 µL/min (0.25 dyne/cm²) and 140 µL/min (0.48 dyne/cm²) using a peristaltic pump (Reglo Digital Pump, 4-Channel 12-Roller, Masterflex Ismatec) in a closed circular system. After 72 hours of exposure to various graphene conditions (Figure 2), they conducted viability assays on RPTEC/TERT1 cells. For low-flow and no-flow conditions, they utilized the MTT kit (Sigma-Aldrich, TOX1) following the manufacturer’s recommendations. Meanwhile, for high-flow conditions, they employed the Cell Counting Kit-8 (Dojindo, CK04-11), choosing this assay to minimize sample handling compared to the MTT kit.

In parallel, researchers fluorescently stained the cell monolayer within the microfluidic device to analyze the tight junction protein zonula occludens 1 (ZO-1) and the cytoskeletal protein α-tubulin. For more details on the fixation and immunofluorescent staining protocol, refer to our technical note.

Figure 2. Confocal microscopy images of RPTEC/TERT1 cells under 140 µL/min fluid flow conditions (0.48 dyne/cm2 shear stress) for 72 hours in control conditions and with perfusion of GO 10 µg/mL and FLG 10 µg/mL. Scale bar 20 µm.

Relevant conclusions

The authors optimized and characterized the first kidney physiological system to study graphene aggregates under flow, establishing an accurate model for evaluating the cytotoxic effects of 2D materials in suspension.

To refine the system, the researchers analyzed the amount of graphene particles and aggregates deposited on materials commonly used in microfluidic device fabrication, such as PDMS and COP, as well as on tubing materials in the perfusion system, including PTFE and PVC. Through their analysis, they observed that graphene aggregates were significantly higher on PDMS compared to COP. Therefore, they decided to conduct experiments using commercial COP-based devices (Be-Flow from Beonchip) to ensure a consistent concentration of the nanomaterial during cell exposure.

By creating this kidney-on-chip for graphene particle testing, the researchers introduced a more realistic alternative to conventional in vitro assays, marking a paradigm shift in experimental approaches for studying cytotoxicity.

Read the full article here.

More information about the Be-Flow device and how to use it in our website.

Bibliography

1. Tadyszak, K., Wychowaniec, J. K. & Litowczenko, J. Biomedical applications of graphene-based structures. Nanomaterials 8, 1–20 (2018).
2. Denayer, T., Stöhrn, T. & Van Roy, M. Animal models in translational medicine: Validation and prediction. New Horizons Transl. Med. 2, 5–11 (2014).
3. Chen, Y. et al. Two-Dimensional Metal Nanomaterials: Synthesis, Properties, and Applications. Chem. Rev. 118, 6409–6455 (2018).
4. Meng, F. et al. In vitro fluidic systems: Applying shear stress on endothelial cells. Med. Nov. Technol. Devices 15, 100143 (2022).