Formation of primary cell line collections of glial tumors for cancer research.
Authors: Shamova Tatiana V. , Mezhevova Irina V. , Sitkovskaya Anastasia O. , Filippova Svetlana Yu. , Timofeeva Sofia V. , Rostorguev Eduard E. , Kit Oleg I. 
Published: June 2021

e14030

Background: Gliomas are the most common primary intracranial tumors. Primary cell cultures are obtained directly from patient’s tumor after surgery, so they display tumor properties in vivo much better than established cell lines. This advantage makes it possible to use primary cell cultures in development of individualized cancer treatment. The aim of the study was to obtain primary cell lines of malignant brain tumors using the explant method. Methods: Twenty-six patients of both sexes aged 22 to 66 years with such diagnoses as glioblastoma, glioblastoma with anaplastic astrocytoma, anaplastic oligodendroglioma, diffuse astrocytoma, anaplastic astrocytoma, oligoastrocytoma and diffuse protoplasmic astrocytoma were recruited for the study. Tumor samples obtained during surgery was disintegrated with sterile scalpels in a Petri dish with 5 ml of DMEM. Further cultivation of fragmented tumor tissue was in a culture flask with 5 ml of complete media contained DMEM (Gibco, USA), 10% FBS (HyClone, USA), 1% penicillin/streptomycin (BioLot, Russia), 1% NEAA (Sigma-Aldrich, USA), 1 ng/ml FGF (Sigma-Aldrich, USA). Microscopic investigation and media replacement were carried out once in 3-5 days. Cell counting was executed with automatic cell counter EVE (NanoEnTek, Korea). Results: At the zero passage, the rate of a 100% confluence monolayer formation varied from 22 to 85 days (46.3 days in average). At the zero passage, glioblastoma lines formed a 100% confluence monolayer longer than all the other lines, with an average of 57.1 days. Diffuse astrocytoma, anaplastic oligodendroglioma, and glioblastoma with anaplastic astrocytoma lines took the shortest time to reach a 100% confluence - 22-24 days. However, at the first passage, the cells reached a full monolayer within 4 to 25 days (7.1 days in average). It demonstrates a tendency in increase of the cell proliferation rate by 6.5 times at the first passage which indirectly indicates the effectiveness of the used cultivation conditions. Success in the cell cultivation is particularly important for creating a bank of primary cell lines for further screening of anticancer therapeutic agents. Glioblastoma samples had a tendency for more active cell proliferation at the first passage compared with other glial tumors: 5.7 days against 8.5 days in average. At the same time, glioblastoma with anaplastic astrocytoma lines showed a tendency for 2-4 times decrease in the rate of monolayer formation compared with glioblastoma samples: 19.5 days against 5.7 days in average. Conclusions: Twenty-six primary human brain cancer cell lines were obtained: four diffuse astrocytoma lines, five anaplastic astrocytoma lines, anaplastic oligodendroglioma line, thirteen glioblastoma lines, two glioblastoma with anaplastic astrocytoma lines, oligoastrocytoma line. The explant method was shown to be more effective for obtaining of glioblastoma cell lines in comparison with other glial tumors.