Evaluation of Morphology and Viability of Spheroid Derived from Insulin-GLase Cell Line: a Model System to Understand Type 2 Diabetes Mellitus

Evaluation of Morphology and Viability of Spheroid Derived from Insulin-GLase Cell Line: a Model System to Understand Type 2 Diabetes Mellitus

Type 2 Diabetes Mellitus (T2DM) is one of the major health issues in the world. The cellular mechanism of T2DM is still not fully understood. It could be studied by using spheroid three-dimensional (3D) culture which is considered representative of the in vivo conditions. Several types of pancreatic β cell lines have been used, one of which is the insulin-GLase (iGL) cell line. This study aims to evaluate the effect of cell density and incubation time on spheroid morphology and cell viability in order to understand which one can be considered as the best option in studying T2DM using iGL cell. Spheroid was made by using the Hanging drop method. The variations of initial seeding cells were 50, 100, 200, and 400 cells/µL then incubated for 1, 2, 3, and 4 days. The evaluated parameters in this study are spheroid morphology and cell viability. Spheroid morphology was observed by using inverted phase contrast microscope integrated with camera (Nikon) and NIS-Elements Analysis D software. Cell viability was determined by using LUNA-II™ Automated Cell Counter (Logos Biosystem). The result of this study showed that spheroid in all of the group cell concentration have formed since the first day and its diameter was significantly increased on the following days (p

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  • Baidwan, S., Chekuri, A., Hynds, D. A. L., & Kowluru, A. Glucotoxicity promotes aberrant ac tivation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet β-cells: reversal of such metabolic defects by metformin. Apoptosis. 2017; 22(11); 1380–1393.
  • Bartosh, T. J., & Ylostalo, J. H. Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique. Current Protocols in Stem Cell Biology. 2014; 1(SUPPL.28)
  • Bresciani, G., Hofland, L. J., Dogan, F., Giamas, G., Gagliano, T., & Zatelli, M. C. Evaluation of Spheroid 3D Culture Methods to Study a Pancreatic Neuroendocrine Neoplasm Cell Line. Frontiers in Endocrinology. 2019; 10: 1–10.
  • Esguerra, J. L. S., Ofori, J. K., Nagao, M., Shuto, Y., Karagiannopoulos, A., Fadista, J., Sugihara, H., Groop, L., & Eliasson, L. Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA. Molecular Metabolism 2020; 32 ; 160–167.
  • Flampouri, E., Imar, S., Oconnell, K., & Singh, B. Spheroid-3D and Monolayer-2D Intestinal Electrochemical Biosensor for Toxicity/Viability Testing: Applications in Drug Screening, Food Safety, and Environmental Pollutant Analysis [Research-article]. ACS Sensors. 2019; 4(3): 660–669.
  • Gong, X., Lin, C., Cheng, J., Su, J., Zhao, H., Liu, T., Wen, X., & Zhao, P. (2015). Generation of multicellular tumor spheroids with microwell-based agarose scaffolds for drug testing. PLoS ONE. 2015; 10(6): 1–18.
  • IDF Diabetes Atlas 9th edition. 2019. IDF Diabetes Atlas 9th edition 2019. In International Diabetes Federation Diabetes Atlas, Ninth Edition.
  • Kosheleva, N. V., Efremov, Y. M., Shavkuta, B. S., Zurina, I. M., Zhang, D., Zhang, Y., Minaev, N. V., Gorkun, A. A., Wei, S., Shpichka, A. A., Saburina, I. N., & Timashev, P. S. Cell spheroid fusion: beyond liquid drops model. Scientific Reports. 2020; 10(1): 1–15.
  • Lai, X., Kang, X., Zeng, L., Li, J., Yang, Y., & Liu, D. The protective effects and genetic pathways of thorn grape seeds oil against high glucose-induced apoptosis in pancreatic β-cells. BMC Complementary and Alternative Medicine. 2014: 14: 1–7.
  • Lee, D., Pathak, S., & Jeong, J. H. Design and manufacture of 3D cell culture plate for mass production of cell-spheroids. Scientific Reports. 2019; 9(1): 1–8. Pereira, P. M. R., Berisha, N., Bhupathiraju, N. V. S. D. K., Fernandes, R., Tomé, J. P. C., & Drain, C. M. Cancer cell spheroids are a better screen for the photodynamic efficiency of glycosylated photosensitizers. PLoS ONE. 2017; 12(5): 1–21.
  • Repin, V. S., Saburina, I. N., Kosheleva, N. V., Gorkun, A. A., Zurina, I. M., & Kubatiev, A. A. 3D-Technology of the Formation and Maintenance of Single Dormant Microspheres from 2000 Human Somatic Cells and Their Reactivation In Vitro. Bulletin of Experimental Biology and Medicine. 2014; 158(1): 137–144.
  • Ryu, N. E., Lee, S. H., & Park, H. Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells. Cells. 2019; 8(12): 1–13.
  • Shi, W., Kwon, J., Huang, Y., Tan, J., Uhl, C. G., He, R., Zhou, C., & Liu, Y. Facile Tumor Spheroids Formation in Large Quantity with Controllable Size and High Uniformity. Scientific Reports. 2018; 8(1): 1–9.
  • Suzuki, T., Kanamori, T., & Inouye, S. Quantitative visualization of synchronized insulin secretion from 3D-cultured cells. Biochemical and Biophysical Research Communications. 2017; 486(4): 886–892.
  • Zanoni, M., Piccinini, F., Arienti, C., Zamagni, A., Santi, S., Polico, R., Bevilacqua, A., & Tesei, A. 3D tumor spheroid models for in vitro therapeutic screening: A systematic approach to enhance the biological relevance of data obtained. Scientific Reports. 2016; 6: 1–11.
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  • Yayın Aralığı: Yılda 4 Sayı
  • Başlangıç: 1980
  • Yayıncı: Ondokuz mayıs Üniversitesi Tıp Fakültesi