By Rachel Elizabeth Stading

Introduction

Probiotics are a new trend in healthcare, but some people may not understand the role probiotics play in impacting the human body. Probiotics essentially exchange “bad” bacteria for “good” bacteria in the intestines. Human intestines contain a microenvironment of bacteria that breaks down indigestible carbohydrates, releases acids and gases, and synthesizes key vitamins. However, the overall health effects from the interactions between the bacteria living in human intestines and the rest of the body are still greatly unknown. The desire to understand more about the bacteria colonizing our intestines has recently grown due to an increased interest in probiotics in the health industry as well as groundbreaking discoveries linking intestinal bacteria to various brain conditions, such as Parkinson’s disease and Autism. Often, these bacteria play crucial roles in nutrient and drug absorption in the digestion process. In order to know more, we must investigate the microscopic world of the human intestinal tract. The question then becomes: How do we run experiments to accurately test the interactions between bacteria and the human body?

Preliminary testing of drugs and chemicals on humans is far too dangerous and variable to be a viable option for experimentation. Animal drug testing can also be inaccurate, because most animals have different microbiological systems than humans. Because of these barriers, scientists have turned to testing through in vitro models. These in vitro models allow scientists to simulate the human intestinal tract without the use of living organisms for safer and more controllable experimentation. Simply removing a piece of live human intestines for testing is dangerous and costly. The objective of our research is to create an in vitro human intestinal model that will accurately simulate the interaction between bacteria and the human intestine. Essentially, we are striving to create a human intestine in a test tube.

The human intestine is made up of several different types of cells. The goal of this experiment was to create a particular subset of human intestinal cells called an epithelial microfold (M) cell. These cells have the special characteristic of being able to transport microorganisms and macromolecules from inside the intestines to the connective tissue in the body where immune cells reside. M cells are known to be a part of the immune response, because they will often uptake antigens and present them to immune cells to react against foreign invaders and induce tolerance to beneficial bacteria.¹

IN VITRO MODELS... SIMULATE THE HUMAN INTESTINAL TRACT WITHOUT THE USE OF LIVING ORGANISMS

It was recently discovered that M-like epithelial cells could be created in vitro through the interactions between a type of human enterocyte cell-line, known as Caco-2, and a lymphocyte cell-line, known as Raji. Growing two different cell-lines together is known as a coculture. Both of these cell-lines are long-lasting and well characterized, allowing for proper experimentation. The purpose of this experiment was to determine whether M cells would become more differentiated through direct or indirect interactions between Caco-2 and Raji cells. Differentiation means that the interactions between Caco-2 and Raji cells have caused the formation of M cells.

To obtain M cells, Caco-2 and Raji cells must be cocultured in a format that allows for nutrient exchange and cell-cell interaction to promote the overall growth of the cells. Here, two coculture formats are evaluated: a direct one, in which the two cell types are allowed to be in close contact, and an indirect format, in which the two cells types are physically separated by a porous membrane that allows for nutrient exchange. After three weeks of coculture, the amount of M-cell induction was evaluated for each coculture format. It was found that direct interaction between Caco-2 and Raji cells induced Caco-2 differentiation into M-cell to a greater extent than indirect interaction. Being able to obtain M-cells using the efficient and replicable technique of direct interaction will bring the scientific community one step closer to creating an accurate replica of the human intestine. Overall, an in vitro model promises to be an important tool in studying phenomena like bacterial invasion and immune activation in the human intestine.

Methods

Caco-2 and Raji cells were routinely grown separately following protocols from the American Type Culture Collection (ATCC). For the direct coculture model, Caco-2 and Raji cells were well mixed at a 1:1 ratio and allowed to settle for 2 hours at 37°C.² Then, the cell suspension was placed onto a permeable polyester membrane as shown in Figure 1. For the indirect coculture model, the Caco-2 cells were seeded onto a membrane and were allowed to propagate for 2 weeks.³ Then, Raji cells were placed on the opposite side of the membrane to allow only for indirect interaction via chemical signals, which can also be seen in Figure 1. The control for the experiment was a culture of Caco-2 cells (monoculture) seeded onto the top side of a membrane, which was not expected to differentiate into M cells. All seeded membranes were incubated at 37°C with growth nutrient replacement every other day. Experiments were performed in quadruplicate. After incubating three weeks, the cultures were evaluated for M cell differentiation.

In order to evaluate the presence of M cells, three separate tests were performed.⁴ The first test was a transepithelial electrical resistance (TEER) test. A TEER value measures the electrical resistance across the layer formed by the cells on the membrane. The more cells on the membrane means the higher the resistance measured. However, because M cells transport particles across the layer of cells and increase its permeability, more M cells would be expected to cause a lower resistance across the membrane. For this experiment, the TEER values were measured for the monoculture, direct coculture, and indirect coculture models in order to gauge which of the three had more M cells.

Figure 1. Experimental setup of the monoculture, direct coculture, and indirect coculture.

The second test performed was a nanoparticle transport study. For this test, fluorescent nanoparticles were placed on the top side of each membrane. Because M cells transport particles like bacteria, a higher number of M cells would result in greater transportation of the fluorescent nanoparticles across the membrane. The number of fluorescent nanoparticles transported from the top to the bottom side of the membrane was measured by spectrophotometry, which measures the amount of light detected from the fluorescent nanoparticles.

The third test performed was a microvillus staining test. Microvilli are hair-like structures that project from many cells in the human intestine to aid in nutrient absorption. M cells, however, do not have microvilli. This test stains any cells that have microvilli; therefore, more M cells on the membrane would yield a greater number of unstained areas (e.g., dark spots) present on an image of the layer of cells. Statistical significance of the data obtained was evaluated with a significance of 0.05.

Results

The TEER values for the control monoculture, direct coculture, and indirect coculture can be seen in Figure 2. According to Figure 2, the monoculture had the highest TEER values. This result is expected, because the monoculture cannot differentiate into M cells. The indirect coculture had significantly lower TEER values, suggesting that M cells were present on the membrane, causing less resistance between the top and bottom sides of the membranes. The direct coculture showed the least resistance across the membrane, suggesting the presence of the greatest portion of M cells out of the three cultures.

Figure 2. TEER values for the control, indirect coculture, and direct coculture membranes with respect to the control. *denotes significant difference with respect to control (p-value < 0.05).

Figure 3. Results for the nanoparticle transport study of the monoculture, indirect coculture, and direct coculture.

The nanoparticle transport study measured the amount of fluorescence transported across the membrane. The results of this study can be seen in Figure 3. Based on these values, the monoculture and indirect coculture did not show a significant difference in the number of fluorescent nanoparticles transported across the membrane. However, the direct coculture showed a much higher number of nanoparticles transported than either the monoculture or the indirect coculture, suggesting a much higher presence of M cells.

The microvilli staining experiment tested to see if there were any cells that did not contain microvilli (a characteristic of M cells). Figure 4 displays the results from this staining experiment. As seen in these images, the monoculture is almost completely stained with very few dark spots. The indirect coculture contains a few more unstained regions. The direct coculture shows a large amount of very dark spots where the stain did notadhere. Therefore, the staining images suggest that the direct coculture contains the greatest amount of M cells without microvilli because the stain did not adhere.

Figure 4. The microvilli staining images of the monoculture, indirect coculture, and direct coculture. Light coloring denotes adherence of stain and dark coloring denotes lack of stain. Images are each 1200x900 mm.

THIS EXPERIMENT... BRINGS US CLOSER TO CREATING AN ACCURATE REPLICA OF THE HUMAN INTESTINE

Conclusion

The results from the three tests confirm that the coculture of Caco-2 and Raji cells directly interacting with one another had a higher differentiation of M cells than the coculture of indirect interaction. The direct coculture had the lowest TEER values, the highest number of fluorescent nanoparticle transportation, and the greatest number of unstained regions for the microvilli staining. All of these results indicate the largest presence of M cells.

Based on the experimental results, a direct coculture model should be chosen over indirect coculture to differentiate M cells from interacting Caco-2 and Raji cells. Increasing the number of differentiated M cells allows for a more accurate creation of an in vitro human intestinal environment. The efficiency and simplicity of using the direct coculture model instead of the indirect coculture model will save time and energy for future scientists when obtaining M cells—an important subset of cells in the human intestines—for experimentation. This experiment signifies that direct interaction is a proven in vitro method for procuring M cells, which brings us closer to creating an accurate replica of the human intestine in a test tube.

Because of the success of this experiment, the next step for this research is to add other key human intestinal cells to the in vitro environment in order to build a more representative model of the human intestinal tract. Furthermore, we plan to run experiments with M cells that incorporate different types of immune cells to study their joint immune response when presented with harmful bacteria and pathogens.

Creating an accurate and controllable in vitro model of the human intestinal tract is essential for exploring the relationship between the bacteria in human intestines and the overall health of the human body. With this more comprehensive in vitro model, intestinal bacteria’s effect on infections, brain conditions, drug administration, and nutrient absorption can be tested and better understood by the medical community. The ultimate goal of this research would be that probiotics could one day be specifically designed to cater to the specialized needs of the individual.

Acknowledgments

I would like to thank my research advisor, Dr. Arul Jayaraman, for his guidance and support throughout my research experience. I would also like to thank Dr. Nitesh Sule who trained me when I first began my research, and who encouraged me to apply for this publication. Finally, I would like to thank my direct mentor, Daniel Penarete Acosta, who is a doctoral candidate. Daniel has spent countless hours training me, guiding my research, and improving my research techniques. Daniel also partnered with me for much of this experiment and was instrumental in obtaining key results. I am tremendously grateful for his mentorship and constant support.

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References

1. A. Gebert, H. J. Rothkotter, and R. Pabst, “M cells in Peyer’s patches of the intestine,” International Review of Cytology 167, (1996): 91–159.

2. P. Chaikhumwang, D. Nilubol, A. Tantituvanont, and P. Chanvorachote, “A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor,” European Journal of Pharmaceutical Sciences 106 (2017): 49–61.

3. A. des Rieux, E. Ragnarsson, E. Gullberg, V. Préat, Y. J. Schneider, and P. Artursson, “Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium,” European Journal of Pharmaceutical Sciences 25, no.4-5 (2005): 455–65.

4. A. Beloqui, D. J. Brayden, P. Artursson, V. Préat, and A. des Rieux, “A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation,” Nature Protocols 12 (2017): 1387–99.

Rachel Elizabeth Stading ‘19

Rachel Elizabeth Stading ‘19 is a chemical engineering major from Katy, Texas. Rachel began her research in order to explore the unknown questions pertaining to the human body. Rachel hopes to attend medical school to become a doctor so that she may pursue a life-long passion of exploring and studying the human body and helping others.

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