New Gut-on-a-Chip System

Everybody’s human, but there are innumerable nuances that make up every single person. Now, where this is glaringly obvious is in medicine. Sure, people behave generally the same way. But disease presentation can vary from person to person. And most treatments need to be further tailored to the individual. Inflammatory Bowel Disease (IBD) is something that affects over 7 million individuals around the world. And IBD does not look the same for all people at any given time, and gives those affected many serious difficulties. Therefore, having easily producible ways to study the disease at the level of the individual would be a tremendous step in managing this disease, and hopefully finding a cure for it. So what is being done to make this possibility a reality?

Hi folks, my name is Cole and I have a Master’s of Immunology. Today on Investigate Explore Discover, we’re going to be looking at a new gut-on-a-chip system. So hang around with me throughout this whole presentation to get all of the relevant background information so we can dive into some exciting experimental results. Plus, there’s more information for you in the description below.

Now, the gut and the microbes that inhabit it is a complex place that interacts with many different parts of our bodies. You name an organ, and it’s likely that the gut has an influence on it because the billions of microbes in our gut are all important. They do things like training our immune system, synthesizing digestible nutrients, and protecting us from harmful pathogens. In our guts, there are many different types of bacteria that all play different roles. And as it stands, we are just starting to scratch the surface of how all of them interact. What we do know is that an imbalance between the composition of the gut microbiome is associated with diseases like IBD, diabetes, cancer and neurodegenerative diseases. But I’m getting carried away here. To be better able to study the gut, we need to have an accurate model system. And to create those systems, we need to understand what we’re trying to model.

In the gut, there are many mechanical forces at work, like how we move our bodies and the fluids or semi fluids that move through it. Like the rest of our body, our gut is made up of specific cells. Starting from the inside out, there are Endothelial cells, which line our blood vessels and interact with our bloodstream. There is then a space where other transient cells reside. And there are our Epithelial cells, which line our gut and interact with the material moving there. Now, inside of the cells, there are multiple proteins like Actin, which helps the cells to hold their shape. On the apical gut facing side of the Epithelium, there are structures called Microvilli, which increase the surface area for nutrient absorption. And the longer the Microvilli and Epithelial cells, the more healthy they are. There are also proteins like Mucin and Glycocalyx, which act as scaffolding and barriers against harmful bacteria.

Between the column-like Epithelial cells, there are tight junctions, which prevent bacteria or other unwanted materials from crossing this tissue and getting into our body. The cellular integrity of these junctions can be measured using electrical resistance. The healthier the cells are, the more they prevent harmful things from entering our bodies and thus the greater resistance they have. Now when our guts are damaged, the Epithelial cells become shorter. This can be caused by inflammation of the cells due to LPS, which is derived from pathogenic bacteria. To date, we utilize many model systems to better understand the interactions between the gut microbiomes and the host.

Animal models give us robust information about how the gut functions, but the experiments are slow, they’re expensive, and are not fully scalable to human physiological responses. Thus, there have been great efforts to create human-like environments using in vitro models, static cell cultures and 3D cellular organoids, which are super cool in their own right are useful, but to a degree. They usually take a long time to grow and do not fully encompass the complexity of the gut microenvironment, thus decreasing their physiological relevance. Organs-on-a-chip helped to solve this issue by creating dynamic environments that better replicate what actually happens in the gut. They can alter multiple parameters like fluid flow, mechanical forces, oxygen content, and the growth of microbes, which give us a better understanding of what is really happening. Now, other chips that have already been developed are complicated to manufacture, deal with limited cell types and can take weeks for the cells to be ready for experimentation. Thus, why there is a need to have a gut-on-a-chip system that is easy to manufacture, quickly establishes cell culture and mimics the environmental complexity of the gut using multiple cell lines.

All of that being said, I want to take a moment and really stress the importance of creating new organ-on-a-chip systems. Gut-on-a-chip systems are important because they help to recreate the environment of the gut without using animals. In doing so, this allows increased testing throughput, which broadens our ability to understand how the gut functions. This fosters faster analysis and development of therapeutics, paving the way for more personalized medicine that would benefit you and me.

This brings us to the paper that we’re focusing on today. This paper is called Contributions of the microbiome to intestinal inflammation in a gut-on-a-chip by Jeon et al. from Sogang University in Seoul, South Korea. And in this paper, the authors optimized a physiologically relevant gut-on-a-chip system that allows them to look at the impact of adding microbes to it. To set the stage for this, the authors embedded a glass chip with microelectrode arrays, which was pretty simple to make. And in doing so, they were able to control osmotic pressure and fluid flow, mimicking the internal environment of the gut lumen. Now this chip was also pretty neat because it had three parallel micro channels. As you can see here, this was beneficial because it allowed for the growing of Epithelial and Endothelial cells together, but not touching, like in the gut. However, before the authors could actually use this chip, they needed to optimize growth conditions. Instead of doing many experiments to figure this out, they used a computational fluid dynamics simulation, which was used with the Navier-Stokes and Continuity equations. This allowed them to assess the correlation between wall shear stress and the dimensions of the fluidic channel.

All of these associated factors are shown here, for those who are really interested. But I’m not an expert in the math going on here. The takeaway message is that by adjusting the height, width and micro pillar distance, correlations between the shear stress that would be applied to the cells were identified, because you don’t want too much. This allowed optimizations of the parameters, which would be optimal for cell growth. They found that by decreasing the height, increasing the width, and increasing the micro pillar distance, all increase the shear stress on the cells. Now to get a more complete understanding of how the device would function, they also found that the more viscous the liquid was, and the faster it moved, the more it caused shear stress. Taking everything that they learned through modeling, they came up with the optimal growth parameters here.

Now, computer simulations are great, but they need to be able to hold up experimentally. So to confirm their simulations, the author started by culturing just epithelial cells, with or without luminal flow. After five days, the epithelial cells were able to grow under both static and fluid conditions on the chip. However, there were many more cells under the fluid condition. In fact, in the fluid condition, the Actin Cytoskeleton in the cells formed a continuous ring, and they were able to form more tight junctions along the edges of the cell. These data suggest that the Epithelial cells under fluid forces look more like in vivo gut cells.

However, as Epithelial cells grow with Endothelial cells in the gut environment, the authors then cultured them together and found that both cell types were able to grow in the same device, which just looks amazing. The authors next wanted to see what effect co-culture of the two cells has on Epithelial cells. As controls, they showed that both cell types are able to grow and you can see that under luminal conditions, the Epithelial cells look more organized. Notably, co-cultured Epithelial cells have strong actin staining and exhibited polarized column like characteristics. They were also able to produce more of the structural protein Mucin at the tips, which helps protect them from too much mechanical stress.

Furthermore, the Epithelial cells also produce the defense protein Glycocalyx, which protects the cells from pathogens. Another important factor to assess is the cell morphology and how closely that resembles life conditions. These are some of the pictures of how the cells looked under static or flow conditions. And it looks like there are more cells growing under flow conditions because of the increase in blue staining. But also when quantifying the Epithelial cell height, the co-cultured cells in the flow condition were almost twice as tall.

So taken together, these results suggest that the mechanical factors of the fluid flow and cellular components are the crucial microenvironmental cues that allow intestinal epithelial cells to mimic those found in human guts. Impedance spectroscopy is a non-invasive method used on living cells that measure cell properties like electrical resistance, and thus cellular integrity in real time. Over the 5 days studied, the authors measured cellular resistance and found that it continued to increase as time progressed. This value was greatest when the two cell types were co-cultured together in flowing conditions, indicating that these gut-on-a-chip culturing conditions recapitulate key cellular factors present in in vivo cells in a short timeframe.

Now the establishment of symbiotic bacteria in the gut is crucial for immune system training and intestinal protection. There are many beneficial bacteria that helped to do this. In fact, we can influence our own microbiota composition through consuming choice probiotics. To make this gut-on-a-chip system mimic the real life situation in the gut, the authors next sought to identify which probiotic bacterial strains would actually establish in their system. This was measured by bacterial adherence to the Epithelial cells in fluidic conditions. They use a control L. plantarum strain and two probiotics, HY7715 and HY8002. And all of the bacteria that they use fluoresce green. So as you can see, the control bacteria was able to grow up until day 3 where it starts to get washed out. What is noticeable about the probiotics, though, is that HY7715 adhere to well into day 5 of the experiment. Although the procedure does need to be more fully optimized for attachment of these bacteria to the Epithelial cells because damaged and inflamed guts are a hallmark of many diseases like IBD.

The authors also wanted to further explore how their gut-on-a-chip system would mimic intestinal inflammation. To do this, after the epithelial cells had time to establish themselves, they were exposed to LPS, which is known to induce intestinal inflammation. The LPS causes disorganization and fainter staining of the cell Cytoskeleton and disrupted organization of the tight junctions between the cells. Furthermore, the addition of LPS caused a decrease in the Microvilli length by about 1/3, which is indicative of further intestinal damage. The paracellular permeability was also decreased as measured through resistance. But when the probiotic HY7715 was added, the cellular resistance of the cells continued to increase after three days. These results indicate that certain bacteria can be used as a tool on these chips to better mimic in vivo conditions.

Now, to quickly summarize everything altogether, the authors created a three-channel microfluidic system that allowed for the culturing of Epithelial and Endothelial cells together without contact. This caused increased Epithelial cell growth and a new structural characteristics that more closely mimic the cells in the gut. Extracellular proteins that would help protect the cells from bacterial injury were also increased under these conditions. Furthermore, the authors found that they could culture a known probiotic bacterial strain on their cells, and these bacteria helped to reduce inflammation induced by pathogenic bacterial products.

Ultimately, though, there’s more work to be done. But this is a promising model that can be used for further applications. Not only do I think these new developments are exciting to investigate and learn about, they are also significant in a broader context. This information is significant because this model showed that they were capable of culturing human Epithelial cells in a small amount of time, with gut-like forces being applied to them. These epithelial cells, when co cultured with non-touching Endothelial cells, better mimicked in vivo cells in the gut environment. This model can also harbor probiotic growth, allowing for additional microbiome studies.

Now, all science is basically a stepping stone for new knowledge. And these steps are driven by questions. And I had a few questions myself after reviewing this information. The first question that I had was more from a technical standpoint. It was interesting to see that the mere presence of Endothelial cells help to differentiate the Epithelial cells. But why was that? What about the Endothelial cells is causing this change? I was also curious whether this chip could push experimental limits even further. Could this gut-on-a-chip be modified so that it could incorporate immune cells, thus further mimicking the environment that is actually going on in our own guts? Speaking of our own guts, to even better mimic the gut microenvironment, is it possible to culture multiple bacterial strains using this system? Perhaps by using stool samples to even better recapitulate what is happening at the level of the individual?

As always though, my final question revolves around you. What sort of ideas or questions popped into your head when hearing about this information? I would love to hear about them in the comment section below. Also, let me know if there are any topics that you’d like to hear about in the future. Ultimately, I hope that you learned something. But more importantly, I hope that you enjoyed your time doing so. Well, that’s everything for today. Thank you for watching, and I’ll see you next time.

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