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  • About Mimetas
  • Supporting up to 96 tissue models on a single plate. Its unique PhaseGuide™ technology enables cells to interact and migrate freely between the two channels of each chip.

  • Supporting up to 40 tissue models on a single plate. Its unique PhaseGuide™ technology enables cells to interact and migrate freely between the three channels of each chip.
  • Supporting up to 64 tissue models on a single plate. Specially designed for automated workflows. Its unique PhaseGuide™ technology enables cells to interact and migrate freely between the three channels of each chip.
  • The first in vitro tissue culture platform that allows co-culture of spheroids, organoids, and tumors with a perfused microvascular bed and vascularization of 3D tissues.

  • Offering 38 ready-to-use Caco-2 tubules for drug exposure, transport, and permeability studies.
  • Offering 38 ready-to-use HUVEC tubules for drug exposure, transport, and permeability studies.
  • Offering 64 ready-to-sprout HUVEC tubules.
  • Driving precisely-controlled perfusion flow in the OrganoPlate® platform
  • Enabling fast, automated, and impedance-based TEER measurements in OrganoPlate®. Assay 40 tissue culture chips with a few clicks, in less than one minute.
  • Kickstart your experiments with the most sophisticated 3D tissue culture platform. Find out which training fits your 3D tissue modeling needs best.
  • OrganoPlate® is the solution for all in vitro tissue culture applications. Explore the entire OrganoPlate® family and its dedicated instruments here.

  • Start with the most sophisticated 3D tissue culture platform today.

  • Thinking of using OrganoPlate for your research? Request a quote for the product(s) of your interest.

  • Get robust compound data in human tissue models through our OrganoServices. With proven pheno­typic assays in the OrganoPlate® platform, we support your drug discovery and development needs.
  • We create novel human tissue and disease models in the OrganoPlate® platform. Our experts are looking forward to developing assays according to your specifications.
  • Expand your drug discovery capacity, shoulder to shoulder with our scientific team. Proprietary human disease biology in the OrganoPlate® platform. Together we make the therapeutics of tomorrow.
  • We offer several services to support your drug discovery and development needs. Find the overview here.
  • Layered tissues with perfused tubules in the absence of artificial membranes form the heart of our permeability and transport science. Study cell interactions, permeability, absorption, transport, and transcytosis without physical barriers.

  • Co-culture layered & structured tissues without artificial membranes with perfect imaging, to study barrier-free cellular interactions, cell-cell signaling, and migration.

  • Evaluate the effect of chemotactic triggers or cells on the migration of cells through an extracellular matrix.

  • Membrane-free microvascular formation and growth through an extracellular matrix (ECM).

  • The missing link in tissue culture: add perfusable human vasculature to your tissue models, and recreate sophisticated microenvironments with OrganoPlate® Graft.

  • OrganoPlate® enables you to study relevant 3D tissue biology by incorporating perfused tubules, co-culture, and full control over the tissue microenvironment. Find the overview of applications here.

  • Visit our Knowledge Center to get up to speed with 3D tissue culture and to learn how OrganoPlate® supports your research needs.

    Read our publications, application notes, watch our webinars, or check out the supporting protocols and brochures. All compiled for you, by our scientists.

  • Get inspired by peer-reviewed publications of our scientists, partners, and customers around the globe.

  • Get inspired by research done by our scientists, partners, and customers around the globe.

  • Giving you some food for thought. Read our blogs to learn more about 3D tissue culture, research backgrounds, developments, and its future outlook.
  • Kickstart your experiments with the most sophisticated 3D tissue culture platform. Find out which training fits your 3D tissue modeling needs best.
  • Any support questions about purchasing, products, or 3D tissue culture and analysis? Get in touch with our experts.
  • Thinking of using OrganoPlate for your research? Request a quote for the product(s) of your interest.

  • Frequently asked questions answered by our experts. Find here the answer you're looking for.
  • Learn about our mission, vision, the history of the company, and find out what we mean with MIMETAS-do.
ENGLISH

7 Ways Caco-2 Cells Can Make or Break Your Intestinal Permeability Models

Increased intestinal permeability has been described in a numberCaco-2 cells of diseases, including inflammatory bowel disorders (IBD)1, celiac disease2, and in conditions not primarily associated with the intestines.3 Even though the etiology of these diseases is far from clear, it is now widely accepted that the gut barrier plays an important role in health and disease.4 Studying intestinal permeability is critical to the development of therapeutics targeted at alleviating and avoiding the crippling symptoms associated with gut barrier disruption.

Mounting data from in vivo and in vitro studies show that intestinal permeability is influenced by stress, diet, microbial changes, and underlying genetic susceptibility. The complexity of intestinal permeability-related diseases makes it difficult to find suitable research models, let alone models that can be scaled up for high-throughput target validation and drug discovery purposes.5 While animal models have helped advance our understanding of IBD mechanisms, manipulating individual parameters such as epithelial barrier function and cell-type specific activation is challenging.5

When grown on permeable membranes, human colon adenocarcinoma (Caco-2) cells differentiate to form confluent monolayers, which display functional and structural characteristics of enterocytes (the cells that line the small and large intestine).6–8 Caco-2 cells can be used to study innumerable topics, including inflammatory triggers, bioavailability, permeability, and toxicity.9 Caco-2 cells are also considered to be the gold standard for in vitro prediction of intestinal drug permeability and oral drug absorption. However, creating intestinal permeability models with Caco-2 cells isn’t always as smooth-sailing as you might hope.

Here are seven problems you might be facing when creating your intestinal permeability model with Caco-2 cells:

1. Caco-2 cell tolerance of DMSO

Dimethylsulfoxide (DMSO) is a standard solvent used forDMSO preparing stock solutions and is one of the principal additives in assay buffers.10 Although cytotoxic at high doses, it is often used in low concentrations as the control reagent during exposure, and in cryopreservation. The tolerance for DMSO varies across cells and transport proteins, and its possible effects on your results are worth bearing in mind; a recent epigenetic study revealed that low doses of DMSO induced major changes in DNA methylation in cardiac (but not hepatic) microtissues.11

Above a certain threshold, DMSO can affect cell growth, protein stability and aggregation, and binding of drug compounds.10,12 Some protocols advise running a solvent tolerance test to ensure your results are not affected by the concentration of DMSO used in your cell culture experiment.6,10

2. Caco-2 cells taking too long to grow

If you’ve ever grizzled about your Caco-2 cells takingCaco-2 Grow Slowly too long to grow, know that you are not alone. Slow-growing cells can be frustrating, especially when the problem significantly drags out your experiments, and limits the number of experiments you are able to complete.

Troubleshooting can be time-consuming as there are many variables and possible reasons for slow growth; mycoplasma contamination and an overly dense feeder layer are two major possibilities.

3. Overcrowding of Caco-2 cells

The design of transport assays is based on the assumption that cells are organized in a monolayer, mimicking enterocytes in vivo. However, dome formation of Caco-2 cells can occur when a cell layer is pushed away from the culture dish by the accumulation of fluid beneath.13 This distortion can create fluid pockets, leading to uneven treatment distribution, and inadequate oxygen supply.14 These factors can compromise your transport assay results. Overcrowding can also occur when cells become too confluent, and differentiate before you are ready to run your assay. Subculturing cells before they have reached confluence can help cells to form a more homogenous and polarized monolayer.15 One protocol suggests subculturing cells when they have reached 50% of confluence, in contrast to an existing recommendation of 80%.15

4. Stability or instability over time

The number of times a cell culture is subcultured, or transferred from one vessel to another, is known as the “passage number”. Higher passage numbers and longer culture times can compromise genome stability and alter critical cell characteristics. Caco-2 cells are no exception; gene expression, phenotype and signaling pathways have been reported to drift over time in Caco-2 cells.16 Drifting gene expression levels can affect cell function and contribute to erroneous results.

Protocols generally recommend limiting continuous cell cultures to three months, or a particular number of passages. Excessive time in culture can also increase the risk of cell culture contamination, which is another reason to keep culture times on the shorter side where possible. There is no “magic number” of acceptable passages, and constant monitoring for changes in phenotype and cell density is essential.

5. Potential scalability for drug discovery

From a drug discovery perspective, models are needed thatPills allow researchers to screen thousands of drug candidates. Conventional membrane insert-based systems are limited in their scalability on this level, and typically do not cater for 3D cellular models – an aspect that is desirable for improving physiological relevance. While you might have the ideal cell type for modeling intestinal permeability, having the potential to incorporate 3D models or other cell types would be highly beneficial.17

6. Research costs associated with compounds, media, and reagents

Whenever you are troubleshooting issues with culturing your Caco-2 cells, you could potentially be wasting huge amounts of money on expensive compounds – not to mention copious amounts of fetal bovine serum and other reagents. Any technology that reduces your reagent, cell, and time consumption can significantly reduce research and development costs.

7. The need for specialized instruments and equipment

Modern drug discovery approaches rely on high-resolution kinetic measurements and image-based readouts. However, conventional transwell models are poorly suited to the instruments and imagers required for kinetic measurements.17 Having a platform compatible with automated plate readers, microscopes, and other technologies could help researchers to extract critical information about intestinal permeability from their Caco-2 cells.

Some research groups working with Caco-2 cells might find they have a shortage of laboratory personnel with the specialized skills required for setting up desired experiments. Certain research models may require staff to be knowledgeable in microfluidics, or experienced in assessing barrier integrity. The transepithelial electrical resistance (TEER) is a measure of the integrity of a monolayer corresponding to barrier function, with differentiated polarized cells that have formed tight junctions.18 TEER is used to gauge the effect of different exposure times and concentrations. Without prior experience, navigating the literature surrounding TEER techniques19 can be challenging.

One Nature Protocols paper states that “Given the necessary care and attention, most cell lines are easy to maintain and grow”.20 Despite this, most researchers still find there are many obstacles to navigate when working with Caco-2 cells. If Caco-2 cells are critical to your work, be sure to check this blog post where we will show you how you can grow your intestinal permeability models in less than five days.

Learn more about MIMETAS organ-on-a-chip technology >>

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References

  1. Vivinus-Nébot, M., Frin-Mathy, G., Bzioueche, H., Dainese, R., Bernard, G., Anty, R., Filippi, J., Saint-Paul, M. C., Tulic, M. K., Verhasselt, V., Hébuterne, X., & Piche, T. (2014). Functional bowel symptoms in quiescent inflammatory bowel diseases: Role of epithelial barrier disruption and low-grade inflammation. Gut, 63(5), 744–752.
  2. Heyman, M., Abed, J., Lebreton, C., & Cerf-Bensussan, N. (2012). Intestinal permeability in coeliac disease: Insight into mechanisms and relevance to pathogenesis. Gut, 61(9), 1355–1364.
  3. González-González, M., Díaz-Zepeda, C., Eyzaguirre-Velásquez, J., González-Arancibia, C., Bravo, J. A., & Julio-Pieper, M. (2019). Investigating Gut Permeability in Animal Models of Disease. Frontiers in Physiology, 9, 1962.
  4. Vancamelbeke, M., & Vermeire, S. (2017). The intestinal barrier: A fundamental role in health and disease. Expert Review of Gastroenterology & Hepatology, 11(9), 821–834.
  5. Beaurivage, C., Naumovska, E., Chang, Y., Elstak, E., Nicolas, A., Wouters, H., van Moolenbroek, G., Lanz, H., Trietsch, S., Joore, J., Vulto, P., Janssen, R., Erdmann, K., Stallen, J., & Kurek, D. (2019). Development of a Gut-on-a-Chip Model for HighThroughput Disease Modeling and Drug Discovery. International Journal of Molecular Sciences, 20(22), 5661.
  6. Hubatsch, I., Ragnarsson, E. G. E., & Artursson, P. (2007). Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols, 2(9), 2111–2119.
  7. Hilgers, A., Conradi, R., & Burton, P. (1990). Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharmaceutical Research, 7(9), 902–910.
  8. Jumarie, C., & Malo, C. (1991). Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro. Journal of Cellular Physiology, 149(1), 24–33.
  9. Maxnah, I. Jr. (1999). The use of Caco-2 cells as an in vitro method to study bioavailability of iron. Malaysian Journal of Nutrition, 5(1), 31–45.
  10. Tjernberg, A., Markova, N., Griffiths, W. J., & Hallén, D. (2006). DMSO-Related Effects in Protein Characterization. Journal of Biomolecular Screening, 11(2), 131–137.
  11. Verheijen, M., Lienhard, M., Schrooders, Y., Clayton, O., Nudischer, R., Boerno, S., Timmermann, B., Selevsek, N., Schlapbach, R., Gmuender, H., Gotta, S., Geraedts, J., Herwig, R., Kleinjans, J., & Caiment, F. (2019). DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Scientific Reports, 9(1), 4641.
  12. Rodríguez-Burford, C., Oelschlager, D. K., Talley, L. I., Barnes, M. N., Partridge, E. E., & Grizzle, W. E. (2003). The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines. Biotechnic & Histochemistry, 78(1), 17–21.
  13. Misfeldt, D., Hamamoto, S., & Pitelka, D. (1976). Transepithelial transport in cell culture. Proceedings of the National Academy of Sciences, 74(3), 1212–1216.
  14. von Köckritz-Blickwede, M., Zeitouni, N., Fandrey, J., & Naim, H. Y. (2015). Measuring oxygen levels in Caco-2 cultures. Hypoxia, 53.
  15. Natoli, M., Leoni, B. D., D’Agnano, I., D’Onofrio, M., Brandi, R., Arisi, I., Zucco, F., & Felsani, A. (2011). Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. Journal of Cellular Physiology, 226(6), 1531–1543.
  16. Briske-Anderson, M. J., Finley, J. W., & Newman, S. M. (1997). The Influence of Culture Time and Passage Number on the Morphological and Physiological Development of Caco-2 Cells. Experimental Biology and Medicine, 214(3), 248–257.
  17. Trietsch, S. J., Naumovska, E., Kurek, D., Setyawati, M. C., Vormann, M. K., Wilschut, K. J., Lanz, H. L., Nicolas, A., Ng, C. P., Joore, J., Kustermann, S., Roth, A., Hankemeier, T., Moisan, A., & Vulto, P. (2017). Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes. Nature Communications, 8(1), 262.
  18. Sakharov, D., Maltseva, D., Knyazev, E., Nikulin, S., Poloznikov, A., Shilin, S., Baranova, A., Tsypina, I., & Tonevitsky, A. (2019). Towards embedding Caco-2 model of gut interface in a microfluidic device to enable multi-organ models for systems biology. BMC Systems Biology, 13(S1), 19.
  19. Srinivasan, B., Kolli, A. R., Esch, M. B., Abaci, H. E., Shuler, M. L., & Hickman, J. J. (2015). TEER Measurement Techniques for In Vitro Barrier Model Systems. Journal of Laboratory Automation, 20(2), 107–126.
  20. Masters, J. R., & Stacey, G. N. (2007). Changing medium and passaging cell lines. Nature Protocols, 2(9), 2276–2284

Blog post written by Michele Wilson at choicesciencewriting.com

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