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Investigating the intestinal transport of mercury ions with a gut-on-a-chip device

Investigating the intestinal transport of mercury ions with a gut-on-a-chip device
Gut-on-a-chip integrated with sensors. a) Device fabrication process. b) Annotated device decomposition diagram. The top and bottom microchannels were separated by the porous membrane. Simultaneous integration of three-electrode sensors and an Ag/AgCl electrode for in situ detection of Hg(II) and TEER. c) Photograph of the gut-on-a-chip integrated with sensors. Credit: Microsystems & Nanoengineering (2023). DOI: 10.1038/s41378-022-00447-2

The transport of mercury ions across intestinal epithelial cells can be studied for toxicology assessments by using animal models and static cell cultures. However, the concepts do not reliably replicate conditions of the human gut microenvironment to monitor in situ cell physiology. As a result, the mechanism of mercury transport in the human intestine is still unknown.

In a new report now published in Nature Microsystems and Nanoengineering, Li Wang and a research team in mechanical engineering and regenerative medicine in China developed a gut-on-a-chip instrument integrated with transepithelial electrical resistance (TEER) sensors and electrochemical sensors.

They proposed to explore the dynamic concept to simulate the physical intestinal barrier and mirror biological transport and adsorption mechanisms of mercury ions. The scientists recreated the cellular microenvironment by applying fluid shear stress and cyclic mechanical strain.

Wang and the team studied mercury adsorption and the physical damage caused by the toxic element on epithelial cells via the performance of electrochemical sensors after exposing them to intestinal cells growing under diverse concentrations of mercury mixed in the cell culture medium. The team noted the corresponding expression and upregulation of Piezo1 and DMT1 (divalent metal transporter), both mechanosensory ion channels and iron transporters respectively on the cell surface.

Developing an intestinal model

Mercury ions are non-biodegradable and can accumulate in the body at low concentrations to cause damage to major organs. Toxic mercury ions can interact with antioxidant components, DNA repair enzymes and proteins at the subcellular level to disrupt cell homeostasis and produce disordered cellular structure and function.

While mercury adsorption occurs predominantly in the small intestine, high levels of mercury ingestion can cause internal bleeding and perforation in a short timeframe. The long-term intake of low concentrations of mercury ions can also lead to chronic intestinal diseases. Since the intestinal epithelium provides an initial barrier to limit the penetration of ingested mercury in the blood stream and reduce its harmful effects; a reasonable intestinal model is of great significance to explore transport mechanisms of mercury in the lab. Although animal models and static cell cultures are traditionally used to study intestinal adsorption and mercury transport, these models do not efficiently recapitulate the intestinal microenvironment to emulate the living intestine.

Investigating the intestinal transport of mercury ions with a gut-on-a-chip device
Quantification of physical properties in gut-on-a-chip. a) FEA results of fluid shear stress in a microchannel. b) Calibration relationship between the actual flow rate of the culture medium into the gut-on-a-chip and the input flow by the syringe pump. Inset (I): Calibration experimental platform. Inset (II): Microscopic image of fluid flow in the gut-on-a-chip (scale bar, 200 μm). c) The FEA results of shear stress in the gut-on-a-chip were compared with the actual results (n = 3). d Quantitation of the mechanical strain produced in the adherent gut epithelial cells as a function of pressure applied by the vacuum controller. Black lines represent linear fitting lines (y = 0.099 + 0.270x; R2 = 0.988). Inset (I): Comparison of cell masses on porous membranes before and after stretching by 3% (scale bar, 20 μm). e Variation in the pore diameter of the porous film within 10 days under 1% tensile strain. RSD = 1.81% (n = 3). f) The change in Young’s modulus of the porous membrane under 1% tensile strain within 10 days. RSD = 0.07% (n = 3). Credit: Microsystems & Nanoengineering (2023). DOI: 10.1038/s41378-022-00447-2

In this work, the researchers developed a gut-on-a-chip model integrated with label-free sensors, to non-invasively monitor changes in transepithelial electrical resistance (TEER) during cellular behavior of mercury absorption, in real time. The team identified key features of the gut via electrical measurements and immunohistochemistry studies, to assess the effect on mechanosensory ion channels.

Investigating the intestinal transport of mercury ions with a gut-on-a-chip device
Characteristics of the intestinal epithelium in the gut-on-a-chip. a) Tight junctional integrity of the epithelium quantified by measuring TEER (n = 3). b) AKP activity under static (3 days, 7 days and 21 days) and dynamic (3 days and 7 days) cultures (n = 3; *P < 0.05, **P < 0.01). c) (I) Confocal fluorescence view of a tight junction protein (ZO-1; red) and brush border protein (ezrin; green) in static (3 days; 21 days) and dynamic cultures (3 days) (scale bar 20 μm). (II) Confocal fluorescence view of vertical cross section of cell monolayer in static (3 days; 21 days) and dynamic culture (3 days) (scale bar 10 μm). d) Average fluorescence intensity analysis of ZO-1 and ezrin proteins in static and dynamic cultures (3 days) (n = 3; ***P < 0.001). e) Average cell height cultured in static and dynamic cultures (n = 3; *P < 0.05). f) Microscope top-down views of intestinal villi-like structures (scale bar 100 μm). g) An SEM image of intestinal villi-like structures. h) A fluorescence microscopic view highlighting the nuclei (DAPI) of intestinal villi-like structures (scale bar 100 μm). i) A photograph showing the proposed gut-on-a-chip with its monitoring and culturing component. Credit: Microsystems & Nanoengineering (2023). DOI: 10.1038/s41378-022-00447-2

Numerical analysis of the device

The research team developed a chip that showed mechanical behavior similar to the living intestine and introduced representative fluid flow and cyclic mechanical stretching. For instance, a shear stress approximating 0.02 dyne/cm2 produced a fluid flow required for intestinal epithelial morphogenesis. Based on additional calculations on the organ chip, the team obtained a flow rate of 160 μL/hour for the corresponding dimensions of shear stress.

They then simulated the tensile strain/stress dynamics via finite element analysis to understand the effects of the parameters on the physical properties of the instrument including its pore diameter and their impact on the cytodifferentiation by analyzing the catalytic activity of alkaline phosphatase; typically used as a marker of bone and liver damage. The team noted greater catalytic activity among cells under fluid flow and those undergoing mechanical strain on a chip after only seven days of cell culture.

Investigating the intestinal transport of mercury ions with a gut-on-a-chip device
Damage to cells caused by Hg(II). a) Immunofluorescence of cells exposed to different concentrations of Hg(II) for 5, 12, and 24 h in static culture (red: dead cells; green: living cells). b) Changes in cell activity under the treatment of different concentrations of Hg(II). The cell activity remained above 80% within 24 h under low concentrations of Hg(II) (1, 10 μM) (n = 3). c) Epithelial cells were exposed to 100 μM Hg(II), and LDH was detected at 5, 12′ and 24 h. Within 24 h, the results of LDH detection showed that the expression of LDH in static samples was 1.3-fold higher than that in dynamic samples (n = 3; *P < 0.05). d) The change rate of TEER in static and dynamic cultures of cells exposed to 100 μM Hg(II) within 24 h. The TEER under dynamic conditions was 4.4-fold higher than that under static conditions (n = 3; *P < 0.05). e) In the absence or presence of Hg(II) (100 μM), confocal immunofluorescence of ZO-1 (red) and ezrin (green) protein was observed (scale bar 20 μm). (f, g) Analysis of the average fluorescence intensity of ZO-1 and ezrin proteins after epithelial cell injury. Compared with the control group, the fluorescence intensity of ZO-1 and ezrin decreased by 1.8-fold (n = 3; ***P < 0.001). Credit: Microsystems & Nanoengineering (2023). DOI: 10.1038/s41378-022-00447-2

The outcomes highlighted the biomimetic approach and the capacity for growth and differentiation of the cell monolayer under mechanical stimulation. The gut-on-a-chip instrument provided biomimetic intestinal villus-like structures to maintain the integrity of the tissue barrier and represented a key physiologically relevant human intestine.

Investigating the intestinal transport of mercury ions with a gut-on-a-chip device
a) The TEER value of the cell monolayer on the 7th day under different mechanical stretching conditions (1%, 3%, 5%). The TEER values were 26.65 ± 1.17 kΩ cm2, 32.21 ± 1.05 kΩ cm2 and 34.10 ± 0.93 kΩ cm2, and the cell barrier ability was increased by 20.86% and 27.95% (1% mechanical stimulation as the control group) (n = 3). b) Changes in intestinal epithelial cell monolayer absorption of Hg(II) within three hours under different mechanical stretching conditions (1%, 3%, 5%). The concentrations of Hg(II) absorbed by cells under three mechanical stretching stimuli were 1.78 ± 0.11 μM, 1.98 ± 0.03 μM, and 2.20 ± 0.14 μM (n = 3). c) The change in the Papp value under different mechanical stretching (1%, 3%, 5%). Compared with 1% mechanical stimulation, the ability of cells to absorb Hg(II) increased by 11.65% and 17.96% under 3% and 5% mechanical stimulation, respectively (n = 3). d) The expression of Piezo1 protein and DMT1 protein under different mechanical stretching conditions (Piezo1: red; DMT1: green; nucleus: blue) (scale bar 20 μm). Credit: Microsystems & Nanoengineering (2023). DOI: 10.1038/s41378-022-00447-2

Functional assays

The research team next exposed the cells to mercury under static culture conditions to understand the process of cell death. And noted an increase to the process when upon increasing the mercury concentration and culture time. They noted that the activity of lactate dehydrogenase (LDH) increased with time in the assays to show equal effects of the toxin across both cell cultures. However, the degree of injury between the two cultures differed. For instance, the expression of LDH was greater in the static culture compared to the dynamic cell cultures after mercury treatment.

The scientists used an electrochemical sensor array integrated into a gut-on-a-chip device to observe epithelial cell interactions with mercury. They explored the transport mechanism of the element relative to its absorption on epithelial cells and investigated the expression of key proteins such as Piezo1 and DMT1 relative to mechanosensory ion channels and active iron transporters. They studied the effects of different tensile strains on the cell barrier and noted that an increase in mechanical stimulation led to increased adsorption of mercury via intestinal epithelial cells, where the mechanosensory ion channels too showed a positive correlation.

Outlook

In this way, Li Wang and colleagues developed a gut-on-a-chip device integrated with transepithelial electrical resistor sensors and multiple electrochemical sensors to stimulate mercury transport in the human intestine in vitro. The chip dynamics emulated a physical intestinal barrier and microenvironment to observe the transport of mercury in real-time. The team noted the cell monolayer on the gut-on-a-chip to differentiate to form a complete cellular barrier via imaging and immunohistochemistry. The team intends to further understand additional mechanisms underlying human intestinal diseases using organ chips to promote personalized drug development.

More information: Li Wang et al, Gut-on-a-chip for exploring the transport mechanism of Hg(II), Microsystems & Nanoengineering (2023). DOI: 10.1038/s41378-022-00447-2

Sangeeta N Bhatia et al, Microfluidic organs-on-chips, Nature Biotechnology (2014). DOI: 10.1038/nbt.2989

Journal information: Nature Biotechnology 

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