A low-cost device for measuring TEER at cultivation of epithelial/endothelial cells on inserts.

Abstract

Background: The barrier properties of epithelial and endothelial cells are usually studied in vitro when cells are cultured on mesh inserts in culture plates, and it is necessary to assess the state of the cell monolayer on the membrane of the inserts before the experiment. Typically, monolayer density is analyzed by passing a fluorescent label through the insert with cells or by measuring the transendothelial resistance (TEER) of the cell layer. Many studies use both methods of assessing monolayer integrity in parallel, depending on the purpose of the study. The TEER method also allows to record early changes in the monolayer state under the action of various substances.

Aim of this work is to demonstrate the possibility of TEER measurements using the proposed device (conductometer) made from freely available components using endothelial/epithelial cells as an example.

Materials and Methods. A device (conductometer) for measuring TEER, created on the basis of easily accessible components, is presented. The device was tested by culturing two cell lines – hybridoma of endothelial origin Ea.hy926 and human colon adenocarcinoma Caco-2. Caco-2 cells were cultured for 22 days and Ea.hy926 cells were cultured for 7 days. The integrity of the cell monolayer and the density of intercellular contacts were evaluated by the TEER value determined by the proposed conductometer, as well as by the fluorescein permeability of the cell monolayer.

Results: The results of TEER measurements using the proposed device and at the same time, the fluorescein permeability of the cell monolayer during the cultivation of Caco-2 and EA.hy926 cells are presented. For Caco-2 cells from the moment of 100% confluence the TEER value gradually increased reaching maximum values by 20-21 days, after which it decreased slightly. The permeability values decreased as the cells were cultured, indicating the formation of dense contacts. For EA.hy926 cells the rise in TEER values are observed on day 3  and decrease was observed on day 7. The results of TEER and permeability obtained by the proposed conductometer have a strong inverse correlation for both cell lines and are in good agreement with each other.

Conclusions. The presented device, made on the basis of simple and affordable components, is similar to commercially available devices and can be used to study the integrity and density of a monolayer in the cultivation of epithelial/endothelial cells, to study the processes of trans/paracellular transport under the action of various substances, as well as in experiments with the co-cultivation of various cell lines.

Keywords

transepithelial electrical resistance / cellular monolayer / fluorescein permeability / Caco-2 cells / EA.hy926 cells.

References

[1] EVOM™ MANUAL FOR TEER MEASUREMENT [cited 2023 Mar 3] https://www.wpiinc.com/evm-mt-03-01-evomtm-manual-for-teer-measurement.html

[2] EVOM™ MANUAL FOR TEER MEASUREMENT. [дата обращения: 3.03.2023] Доступ по ссылке: https://www.wpiinc.com/evm-mt-03-01-evomtm-manual-for-teer-measurement.html

[3] Raut, B. Chen, L.-J., Hori, T. et al.. An Open-Source Add-On EVOM® Device for Real-Time Transepithelial/Endothelial Electrical Resistance Measurements in Multiple Transwell Samples. Micromachines 2021;12: 282. doi.org/10.3390/mi12030282

[4] Raut, B. Chen, L.-J., Hori, T. et al.. An Open-Source Add-On EVOM® Device for Real-Time Transepithelial/Endothelial Electrical Resistance Measurements in Multiple Transwell Samples // Micromachines. 2021. Vol.12. P.282. doi.org/10.3390/mi12030282

[5] CELLZCOPE The Automatic Cell Monitoring System. [cited 2023 Apr 20] www.nanoanalytics.com/en/products/cellzscope.html

[6] CELLZCOPE The Automatic Cell Monitoring System. [дата обращения: 20.04.2023] Доступ по ссылке:https://www.nanoanalytics.com/en/products/cellzscope.html

[7] REMS Autosampler. [cited 2023 Jul 8] www.yumpu.com/en/document/view/ 34494981/rems-autosampler-instruction-manual-world-precision-instruments/48

[8] REMS Autosampler [дата обращения: 08.07.2023] Доступ по ссылке: https://www.yumpu.com/en/document/view/34494981/rems-autosampler-instruction-manual-world-precision-instruments/48

[9] Theile, M., Wiora, L., Russ, D., Reuter, et al., A simple approach to perform TEER measurements using a self-made volt-amperemeter with programmable output frequency. JoVE (Journal of Visualized Experiments), 2019: 152. doi:10.3791/60087

[10] Theile, M., Wiora, L., Russ, D., Reuter, et al., A simple approach to perform TEER measurements using a self-made volt-amperemeter with programmable output frequency. JoVE (Journal of Visualized Experiments). 2019: 152, e60087. doi:10.3791/60087

[11] Rajabzadeh, M., Ungethum, J., Herkle, A., et al. A PCB-Based 24-Ch. MEA-EIS Allowing Fast Measurement of TEER. IEEE Sensors Journal, 2021:21(12): 13048–13059. doi:10.1109/jsen.2021.3067823

[12] Rajabzadeh, M., Ungethum, J., Herkle, A., et al. A PCB-Based 24-Ch. MEA-EIS Allowing Fast Measurement of TEER. IEEE Sensors Journal. 2021. Vol. 21, N 12. P. 13048–13059. doi:10.1109/jsen.2021.3067823

[13] D. Bouis, G.A. Hospers, C. Meijer, et al. Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis, 2001:4 (2): 91-102. doi.org/10.1023/A:1012259529167

[14] D. Bouis, G.A. Hospers, C. Meijer, et al. Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis. 2001.Vol.4 , N 2. P. 91-102.

[15] Lisec, B. , Bozic, T. , Santek, I. Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles. Cell Communication and Signaling . 2024: 22 (1):118. doi:10.1186/s12964-024-01503-2

[16] Lisec, B. , Bozic, T. , Santek, I. Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles. Cell Communication and Signaling . 2024. Vol. 22, N 1. P. 118. doi:10.1186/s12964-024-01503-2

[17] Tranova Yu.S., Slepnev A.A., Chernykh I.V., Shchulkin A.V., Mylnikov P.Yu., Popova N.M., Povetko M.I., Yakusheva E.N. Method for Testing of Drugs Belonging to Substrates and Inhibitors of the Transporter Protein BCRP on Caco-2 Cells. Drug development & registration. 2023;12(2):87-94. (In Russ.) doi.org/10.33380/2305-2066-2023-12-2-87-94

[18] Транова Ю. С., Слепнев А. А., Черных И. В., и др. Методика тестирования лекарственных средств на принадлежность к субстратам и ингибиторам белка-транспортера BCRP на клетках линии Caco-2. Разработка и регистрация лекарственных средств. 2023. Т. 12, № 2. С. 87-94. doi.org/10.33380/2305-2066-2023-12-2-87-94.

[19] Yutaka KONISHI, Keiko HAGIWARA, Makoto SHIMIZU. Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers and Use of Such Transport in In Vitro Evaluation of Phenolic Acid Availability. Bioscience, Biotechnology, and Biochemistry. 2002;66(11): 2449-2457. doi: 10.1271/bbb.66.2449.

[20] Yutaka Konishi, Keiko Hagiwara, Makoto Shimizu. Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers and Use of Such Transport in In Vitro Evaluation of Phenolic Acid Availability. Bioscience, Biotechnology, and Biochemistry. 2002. Vol. 66, N 11. P. 2449-2457. doi: 10.1271/bbb.66.2449.

[21] Prachi Shekhawat, Milind Bagul, Diptee Edwankar, et al. Enhanced dissolution/caco-2 permeability, pharmacokinetic and pharmacodynamic performance of re-dispersible eprosartan mesylate nanopowder. European Journal of Pharmaceutical Sciences. 2019; 132: 72-85, doi.org/10.1016/j.ejps.2019.02.021.

[22] Prachi Shekhawat, Milind Bagul, Diptee Edwankar, et al. Enhanced dissolution/caco-2 permeability, pharmacokinetic and pharmacodynamic performance of re-dispersible eprosartan mesylate nanopowder. European Journal of Pharmaceutical Sciences. 2019. Vol. 132, P. 72-85. doi.org/10.1016/j.ejps.2019.02.021.

[23] Shchulkin A.V., Tranova Yu.S., Abalenikhina Yu.V., et al. Cells of the Caco-2 line as a model for studying the absorption of medicinal substances. Experimental and Clinical Gastroenterology. 2022:10:63-69. (In Russ.) doi.org/10.31146/1682-8658-ecg-206-10-63-69

[24] Щулькин АВ, Транова ЮС, Абаленихина ЮВ, и др. Клетки линии Сaco-2 как модель для изучения абсорбции лекарственных веществ. Экспериментальная и клиническая гастроэнтерология. 2022. Т. 10. С. 63-69. doi.org/10.31146/1682-8658-ecg-206-10-63-69.

[25] Hubatsch, I., Ragnarsson, E. G. E., Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols, 2007:2(9): 2111–2119. doi:10.1038/nprot.2007.303.

[26] Hubatsch, I., Ragnarsson, E. G. E., Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols. 2007. Vol.2, N 9. P. 2111–2119. doi:10.1038/nprot.2007.303.

[27] Poenar, D. P., Yang, G., Wan, W. K. & Feng, S.: Low-Cost Method and Biochip for Measuring the Trans-Epithelial Electrical Resistance (TEER) of Esophageal Epithelium. Materials (Basel) 2020; 13(10): 2354. doi:10.3390/ma13102354

[28] Poenar, D. P., Yang, G., Wan, W. K. & Feng, S.: Low-Cost Method and Biochip for Measuring the Trans-Epithelial Electrical Resistance (TEER) of Esophageal Epithelium. Materials (Basel). 2020. Vol. 13, N 10. P. 2354. doi:10.3390/ma13102354

[29] Srinivasan, B. Kolli, A. R., Esch, M. B., et al. TEER measurement techniques for in vitro barrier model systems. Journal of Laboratory Automation. 2015: 107–126. doi:10.1177/2211068214561025.

[30] Srinivasan, B. Kolli, A. R., Esch, M. B., et al. TEER measurement techniques for in vitro barrier model systems. Journal of Laboratory Automation. 2015. Vol. 20, N 2. P. 107–126. doi:10.1177/2211068214561025.

[31] Wuttimongkolchai, N., Kanlaya, R., Nanthawuttiphan, S., et al. 2022. Chlorogenic acid enhances endothelial barrier function and promotes endothelial tube formation: a proteomics approach and functional validation. Biomedicine & Pharmacotherapy. 2022; 153: 113471. doi.org/10.1016/ j.biopha.2022.113471

[32] Wuttimongkolchai, N., Kanlaya, R., Nanthawuttiphan, S., et al. Chlorogenic acid enhances endothelial barrier function and promotes endothelial tube formation: a proteomics approach and functional validation. Biomedicine & Pharmacotherapy. 2022. N 153, P. 113471. doi.org/10.1016/j.biopha.2022.113471

[33] Velandia-Romero, M. L., Calderón-Peláez, M. A., Balbás-Tepedino, A., et al. Extracellular vesicles of U937 macrophage cell line infected with DENV-2 induce activation in endothelial cells EA.hy926. 2020. PLoS One. 15(1): e0227030.

[34] Velandia-Romero, M. L., Calderón-Peláez, M. A., Balbás-Tepedino, A., et al. Extracellular vesicles of U937 macrophage cell line infected with DENV-2 induce activation in endothelial cells EA.hy926. PLoS One, 2020. Vol. 15, N 1. P. e0227030.

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