FIRST EXPERIENCE OF CULTIVATION OF BREAST CANCER CELLS
DOI: 10.22138/2500-0918-2018-15-6-860-867
S.V. Sazonov, A.A. Brilliant, F.A.
Fadeev, S.M. Demidov, S.L. Leontiev
Institute of Medical Cell Technologies, Yekaterinburg, Russian Federation;
Ural State Medical University, Yekaterinburg, Russian Federation
Abstract. Objective. To assess the changes in the receptor apparatus of breast cancer cells when creating a cell culture of the Luminal B subtype. Materials and methods. Histological specimens were prepared from the tumor, immunohistochemical studies were carried out according to standard methods to establish the cancer subtype. Immunohistochemical reactions were performed at Ventana, the United States. Part of the tumor was dissociated, the tumor cells were isolated and cultured for 5 passages. Cells were isolated using trypsin with EDTA (0.25%), dyspase, and 200 μl DNase I (concentration 1 mg / ml). To obtain the culture of mammospheres, the cell pellet was diluted in complete Mammocult medium, and transferred to a non-adhesive plate. Seeding density was no more than 4x103 cells / cm2. To obtain a culture of tumor cells of epithelial nature, the latter were planted on tissue culture plastic, covered with a thin layer of collagen. The cell pellet was diluted in complete Epi- Cult-B medium supplemented with 5% FBS. The seeding density was 1-5x104 cells / cm2. The culture was incubated for 24 hours at 37°C and 5% CO2, then the medium was replaced with serum-free medium. Cells for immunocytochemical studies were deposited on positively charged adhesive glasses, Superfrost Plus (Thermo scientific, Germany), fixed and stained in the usual way using an autostainer. Results. The cytological picture of the cells obtained was similar to the cells of the original breast cancer. The cells had a nuclear-cytoplasmic ratio. The shape of the obtained single cells resembled fibroblasts and did not differ at all stages of cultivation. To confirm the epithelial nature of the cultured cells, each passage was tested for cytokeratin expression. In all five passages, cytokeratin expression was detected in most cultured cells. In the 4th passage, the cell culture was the most dense, the cells had a rounded shape, the nuclei differed in size and shape, the amount of cytoplasm varied. There were several monolayer clusters, the cell morphology corresponded to breast cancer cells. In the course of the work, cell cultures were obtained, which differed significantly in receptor apparatus, and therefore, belonged to different immunohistochemical subtypes. Only as a result of the third passage, an immunohistochemical subtype corresponding to the primary tumor was obtained. The first, second and third passages gave a cell culture, with a phenotype characteristic of triple negative breast cancer, differing from the primary tumor by high proliferation and the absence of receptors. Thus, as a result of the study, it was revealed that the receptor apparatus of cultured breast cancer cells of the Luminale B subtype may change after each passage.
Keywords: breast cancer, receptor apparatus, cell cultivation, molecular biological subtype, Luminal B subtype
There is no conflict of interest.
Contact details of the corresponding author:
Sergey V. Sazonov
prof-SSazonov@yandex.ru
Received 27.11.2018
For citation:
Sazonov S.V., Brilliant A.A., Fadeev F.A., Demidov S.M., Leontiev S.L. First experience of cultivation of breast cancer cells. Vestn. Ural. Med. Akad. Nauki. = Journal of Ural Medical Academic Science. 2018, Vol. 15, no. 6, pp. 860–867. DOI: 10.22138/2500-0918-2018-15-6-860-867 (In Russ)
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Authors:
Sergey V. Sazonov
Dr. Sci. (Med.), Professor
Head of Department
Department of Histology, Cytology and Embryology,
Ural State Medical University
Head of Laboratory, Department Pathologist
Institute of Medical Cell Technologies
Vilonova str. 76a, Yekaterinburg, Russian Federation, 620137
imct@celltechnologies.ru
Aleksandr A. Brilliant
Institute of Medical Cell Technologies
Cand. Sci. (Bio), Senior Reseacher
Vilonova str. 76a, Yekaterinburg, Russian Federation
620137
alex_brilliant@mail.ru
Feodor A. Fadeev
Cand. Sci. (Bio), Head of Cell Culture Laboratory
Institute of Medical Cell Technologies
Vilonova str. 76a, Yekaterinburg, Russian Federation, 620137
imct@celltechnologies.ru
Sergey M. Demidov
Dr. Sci. (Med.), Senior Researcher
Institute of Medical Cell Technologies
Vilonova str. 76a, Yekaterinburg, Russian Federation, 620137
imct@celltechnologies.ru
Sergey L. Leontyev
Dr. Sci. (Med.), Professor, Head of Institute
Institute of Medical Cell Technologies
Vilonova str. 76a, Yekaterinburg, Russian Federation, 620137
imct@celltechnologies.ru
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)