Abstract
The aim of this study is the comparison of the morphological parameters of the shape of various types of human dermal fibroblasts, cultured in Laboratory of Tissue Engineering. This investigation compares three groups of fibroblasts: control group and with two kinds of genetic modifications. Those are: fibroblasts isolated from human skin (control group), after transduction with lentivirus bearing EGFP fluorescent marker and transduction with lentivirus bearing DsRed2 fluorescent markers. The experimental sequences of images of a monitored cells’ culture have been analysed using image processing methods, such as image segmentation and morphological feature quantification of cells. The following features: area, perimeter, eccentricity, roundness, elongation ratio, length of the major axis, roundness and Hu and Zernike moments have been analyzed in two types of cells models: lamina model and wire model. It appears that the differences in eccentricity, roundness, elongation ratio between modified human skin fibroblasts and their reference culture are not statistically significant. However, the differences in the area, the length of the major axis and the length of perimeter between the control group of fibroblasts and both groups of transduced fibroblasts are statistically significant. The next feature – roundness of fibroblasts – presents statistically significant difference for both transdused groups and fibroblast control group. There is no statistically significant difference between fibroblasts transduced with EGFP marker and those transdused with DsRed2 marker. Genetically modified fibroblasts after extra gene transduction occupy bigger area and assume more elongated shapes. Their compactness is not affected and seems to be similar in all groups.
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References
Telford, W.G., Hawley, T., Subach, F., Verkhusha, V., Hawley, R.G.: Flow cytometry of fluorescent proteins. Methods Elsevier BV 57(3), 318–330 (2012)
Baens, M., Noels, H., Broeckx, V., Hagens, S., Fevery, S., Billiau, A.D., Vankelecom, H., Marynen, P.: The dark side of EGFP: defective polyubiquitination. PLoS ONE 1(1), e54 (2006)
Tao, W., Evans, B.-G., Yao, J., Cooper, S., Cornetta, K., Ballas, C.B., Hangoc, G., Broxmeyer, H.E.: Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not. Stem Cells 25(3), 670–678 (2006)
Zhou, J., Lin, J., Zhou, C., Deng, X., Xia, B.: Cytotoxicity of red fluorescent protein DsRed is associated with the suppression of Bcl-xL translation. FEBS Lett. 585(5), 821–827 (2011)
Liu, H.-S., Jan, M.-S., Chou, C.-K., Chen, P.-H., Ke, N.-J.: Is green fluorescent protein toxic to the living cells? Biochem. Biophys. Res. Commun. 260(3), 712–717 (1999)
Pluta, K., Kacprzak, M.: Use of HIV as a gene transfer vector. Acta Biochim. Pol. 56(4), 531–595 (2009)
Pluta, K., Diehl, W., Zhang, X.Y., Kutner, R., Bialkowska, A., Reiser, J.: Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study. BMC Biotechnol. 7(1), 41 (2007)
Zakrzewska, K., Samluk, A., Pluta, K., Pijanowska, D.: Evaluation of the effects of antibiotics on cytotoxicity of EGFP and DsRed2 fluorescent proteins used for stable cell labelling. Acta Biochim. Pol. 61, 809–813 (2014)
Samluk, A., Zakrzewska, K.E., Pluta, K.D.: Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models. Artif. Organs 37(7), E123–E130 (2013)
Zakrzewska, K.E., Samluk, A., Wierzbicki, M., Jaworski, S., Kutwin, M., Sawosz, E., Chwalibog, A., Pijanowska, D.G., Pluta, K.D.: Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines. PLoS ONE 10(3), e0122579 (2015)
Gonzalez, R.C., Woods, R.E.: Digital Image Processing. Prentice Hall, Upper Saddle River (2007)
Nieniewski, M.: Mathematical morphology in image processing (in Polish). In: AOW PLJ (1998)
Iwanowski, M., Korzynska, A.: Detection of the area covered by neural stem cells in cultures using textural segmentation and morphological watershed. In: Kurzynski M., Wozniak M. (eds.) Computer Recognition Systems 3. Advances in Intelligent and Soft Computing, vol. 57, pp. 543–557. Springer, Heidelberg (2009). doi:10.1007/978-3-540-93905-4_64
Wu, Q., Merchant, F., Castleman, K.: Microscope Image Processing. Academic Press, Boston (2008)
Niblack, W.: Introduction to Digital Image Processing. Pearson Education Limited, Singapore (1967)
Moczko, J., Brȩborowicz, G., Tadeusiewicz, R.: Statistics in Medical Research. Springer PWN (1998)
Ming-Kuei, H.: Visual pattern recognition by moment invariants. IEEE Trans. Inf. Theor. 8(2), 179–187 (1962)
Zhenjiang, M.: Zernike moment-based image shape analysis and its application. Pattern Recogn. Lett. 21(2), 169–177 (2000)
Zhang, D., Guojun, L.: Review of shape representation and description techniques. Pattern Recogn. 37(1), 1–19 (2004)
Korzyńska, A.: Neutrophils movement in vitro. Ann. New York Acad. Sci. 972(1), 139–143 (2002)
Korzyńska, A., Iwanowski, M.: Multistage morphological segmentation of bright-field and fluorescent microscopy images. Opto-Electron. Rev. 20(2), 174–186 (2012)
Korzyńska, A., Zychowicz, M.: A method of estimation of the cell doubling time on basis of the cell culture monitoring data. Biocybern. Biomed. Eng. 28(4), 75–82 (2008)
Korzynska, A., Strojny, W., Hoppe, A., Wertheim, D., Hoser, P.: Segmentation of microscopic images of living cells. Pattern Anal. Appl. 10(4), 301–319 (2007)
Roszkowiak, L., Korzynska, A., Pijanowska, D.: Segmentation of immunonegative cell nuclei from images of immunohistochemichal slides of breast cancer, stained with dab&h - an introductory analysis. In: Abstracts of XIX KKBiIB (2015)
Osher, S., Paragios, N. (eds.): Geometric Level Set Methods in Imaging, Vision, and Graphics. Springer, New York (2007)
Li, C., Xu, C., Gui, C., Fox, M.D.: Level set evolution without re-initialization: a new variational formulation. In: 2005 IEEE Computer Society Conference on Computer Vision and Pattern Recognition (CVPR05). IEEE
Wang, Y., Jeong, Y., Jhiang, S.M., Yu, L., Menq, C.-H.: Quantitative characterization of cell behaviors through cell cycle progression via automated cell tracking. PLoS ONE 9(6), e98762 (2014)
Jiang, K., Liao, Q.-M., Dai, S.-Y.: A novel white blood cell segmentation scheme using scale-space filtering and watershed clustering. In: Proceedings of the 2003 International Conference on Machine Learning and Cybernetics (IEEE Cat. No. 03EX693). IEEE
Iwanowski, M., Korzyńska, A.: Segmentation of moving cells in bright field and epi-fluorescent microscopic image sequences. In: Computer Vision and Graphics, pp. 401–410. Springer, Heidelberg (2010)
Calabuig, A., Mugnano, M., Miccio, L., Grilli, S., Ferraro, P.: Investigating fibroblast cells under “safe" and “injurious" blue-light exposure by holographic microscopy. J. Biophotonics 10(6–7), 919–927 (2016)
Estévez, M., Martínez, E., Yarwood, S.J., Dalby, M.J., Samitier, J.: Adhesion and migration of cells responding to microtopography. J. Biomed. Mater. Res., Part A 103(5), 1659–1668 (2014)
Sugimoto, H., Mundel, T.M., Kieran, M.W., Kalluri, R.: Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol. Ther. 5(12), 1640–1646 (2006)
Acknowledgements
This study was supported by statutory funds of Nalecz Institute of Biocybernetics and Biomedical Engineering Polish Academy of Sciences.
Authors are very grateful to Malgorzata Calka and Natalia Nowak from Nencki Institute of Experimental Biology for their help in microscopic adjustment.
Karolina Zakrzewska acquired the results during her work in Nalecz Institute of Biocybernetics and Biomedical Engineering but now she is working in the Institute of Fundamental Technological Research.
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Korzynska, A. et al. (2018). The Analysis of the Shape of the Genetically Modified Human Skin Fibroblasts in Culture. In: Augustyniak, P., Maniewski, R., Tadeusiewicz, R. (eds) Recent Developments and Achievements in Biocybernetics and Biomedical Engineering. PCBBE 2017. Advances in Intelligent Systems and Computing, vol 647. Springer, Cham. https://doi.org/10.1007/978-3-319-66905-2_8
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DOI: https://doi.org/10.1007/978-3-319-66905-2_8
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