Summary

Researchers from the University of Castilla la Mancha and University of Zaragoza have optimized a microfluidic system under flow conditions to reduce the adsorption of graphene materials on components such as tubing and microfluidic devices, as well as their 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 (rsc.org)

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, such as shear stress, which 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, 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

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.

The kidney-on-a-chip was developed by seeding a commercial line of human renal proximal tubular cells (RPTEC/TERT1), as proximal tubular cells play a crucial role in xenobiotic transport, which is highly relevant in toxicity testing. 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.

The researchers chose the peristaltic pump perfusion system due to its advantages in 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 peristaltic pump setup in a recirculation system. The pump rollers drive the culture medium through PVC tubing, which is 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

Cells were exposed to a physiological shear stress of 0.25 dyne/cm2 with a fluid flow of 75 µL/min and 0.48 dyne/cm2 with 140 µL/min 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), viability assays were conducted on RPTEC/TERT1 cells exposed to low flow and no flow, using the MTT kit (Sigma-Aldrich, TOX1) in accordance with the manufacturer’s recommendations. For high flow conditions, the Cell Counting Kit-8 (Dojindo, CK04-11) was utilized. This cell viability assay was chosen to minimize sample handling compared to the MTT kit.

The tight junction zonula occludens 1 (ZO-1) and cytoskeletal organization protein α-tubulin were fluorescently stained in the cell monolayer within the microfluidic device. To see a more detailed protocol of fixation and immunofluorescent staining visit 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 have optimized and characterized the first kidney physiological system to study the graphene aggregates under flow as an accurate system for evaluating the cytotoxic effects of 2D materials in suspension.

The researchers analyzed the amount of graphene particles and aggregates deposited on potential materials for microfluidic device fabrication (PDMS and COP) and on the tubing used in the perfusion system (PTFE and PVC). They observed that graphene aggregates were significantly higher on PDMS compared to COP. Consequently, they proceeded with experiments using commercial COP-based devices (Be-Flow from Beonchip) to ensure that the cells were exposed to a consistent concentration of the nanomaterial.

The kidney-on-a-chip created represents a more realistic alternative to conventional in vitro assays, representing a paradigm shift in experimental approaches.

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).