scholarly journals Preparation of primary glial tumor cell lines

2021 ◽  
Vol 37 (5) ◽  
pp. 79-89
Author(s):  
T. V. Shamova ◽  
A. O. Sitkovskaya ◽  
E. E. Rostorguev ◽  
N. S. Kuznetsova ◽  
S. E. Kavitsky
Keyword(s):  
Cell Lines ◽  
Anaplastic Astrocytoma ◽  
Malignant Tumors ◽  
Anaplastic Oligodendroglioma ◽  
Diffuse Astrocytoma ◽  
Glial Tumor ◽  
Glial Tumors ◽  
Viable Cells ◽  
Primary Cell Lines ◽  
Tumor Material

Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method. Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed. Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes. At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days. Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.

Formation of primary cell line collections of glial tumors for cancer research.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14030-e14030
Author(s):  
Tatiana V. Shamova ◽  
Irina V. Mezhevova ◽  
Anastasia O. Sitkovskaya ◽  
Svetlana Yu. Filippova ◽  
Sofia V. Timofeeva ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cell Cultures ◽  
Anaplastic Astrocytoma ◽  
Primary Cell ◽  
Anaplastic Oligodendroglioma ◽  
Diffuse Astrocytoma ◽  
First Passage ◽  
Glial Tumors ◽  
Primary Cell Lines

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.

scholarly journals FEATURES OF EXPRESSION OF OCT4, NANOG AND ENDOGLIN IN RENAL CELL CARCINOMAS

2021 ◽  
Vol 10 (3) ◽  
pp. 39-42
Author(s):  
A. O. Sitkovskaya ◽  
E. E. Rostorguev ◽  
S. V. Timofeeva ◽  
I. V. Mezhevova ◽  
S. Yu. Filippova ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Cell Lines ◽  
Anaplastic Astrocytoma ◽  
Primary Cultures ◽  
Microsatellite Analysis ◽  
Primary Cell ◽  
Cellular Heterogeneity ◽  
Malignant Neoplasms ◽  
Glial Tumors ◽  
Primary Cell Lines

Glial tumors, in particular those composed of astrocytes, are the most common group of primary brain tumors. The most common glial tumor is astrocytoma. The biology of glial tumors was routinely studied on immortalized cell lines. However, multiple passages result in a loss of cellular heterogeneity. Therefore, more and more scientific laboratories in their research are beginning to use primary cell lines obtained directly from the patient’s native postoperative material. The question of the timing of the genetic stability of primary cell lines remains open. A special type of genetic instability is microsatellite instability, which affects microsatellites found throughout the genome. Microsatellites have several alleles, which are determined by changes in the number of repetitions of a motif unit. Microsatellite instability is of great importance in the oncogenesis of malignant neoplasms. The aim of this work was to study the microsatellite instability of primary cell lines of poorly differentiated glial tumors at different passages in comparison with the patient’s primary tumor material. Tumor cells of primary cultures of anaplastic astrocytoma and glioblastoma at different passages were used as material for the study. Formalin-fixed paraffin wax (FFPE) slides were used as a microsatellite control. Microsatellite analysis was performed on primary cultures of anaplastic astrocytoma and glioblastoma using the following markers: D17S250, D2S123, D5S346, NR21, NR24, NR27, BAT25, BAT26 by PCR. Microsatellite analysis has shown that primary glioblastoma cell lines are genetically more stable than primary anaplastic astrocytoma cell lines.

Frequency and Topography of AstrocyticTumor: Experience of 567 Cases at Referral Neuroscience Hospital in Bangladesh

2020 ◽  
Vol 6 (2) ◽  
pp. 91-95
Author(s):  
Md Nowfel Islam ◽  
Naila Haq ◽  
Md Badius Salam ◽  
Monsur Ahmed ◽  
Sk Muhammad Ekramullah ◽  
...  
Keyword(s):  
Pilocytic Astrocytoma ◽  
Anaplastic Astrocytoma ◽  
Pleomorphic Xanthoastrocytoma ◽  
Subependymal Giant Cell Astrocytoma ◽  
Diffuse Astrocytoma ◽  
Female Ratio ◽  
Glial Tumors ◽  
The Mean ◽  
Male To Female ◽  
Good Number

Background: Glioma is the most commonly occurring malignant brain tumor that varies by age, sex, race or ethnicity. A very few number of records on CNS tumors are available in Bangladesh. National Institute of Neurosciences and Hospital (NINS), Dhaka has a good number of CNS surgeries. Regularly both tumorous and non-tumorous ICSOL samples are examined here. Objective: The aim of the study was to see the subtypes, frequency and topography of Astrocytic tumors at NINS setting. Methodology: Data from the department of Neuropathology department of NINS since January 2013 to June 2019 were evaluated. Tissue were fixed in formalin, paraffin embedded, stained with H&E. Histomorphology and WHO 2007 CNS tumor classification were used. Result: From 3945 routine sample 567 cases were sorted out as Astrocytic tumor. Total male were 61% (346) and female 39% (221), male to female ratio was 1.6:1. The mean age was 32.64 and ranged from 1 to 80 years. Sixty six percent (66%) tumors were in supratentorial compartment, 15% infratentorial, 6.3% spinal and 9.7% in midline areas like thalamus, hypothalamus and seller region. In this study 34.6% (196) cases were Glioblastoma, followed by Anaplastic Astrocytoma 8.3%(47), Diffuse Astrocytoma 29% (165), Pilocytic Astrocytoma 26.6% (151), Pilomyxoid Astrocytoma 0.4% (2), Subependymal giant cell Astrocytoma 0.9% (5) and Pleomorphic Xanthoastrocytoma 0.1 (0.1). Topographically 66% glial tumors are supratentorial. Among the glial tumors 34.6% is Glioblastoma, 8.3% Anaplastic Astrocytoma, 29% Diffuse Astrocytoma and 26.6% Pilocytic astrocytoma. Common age group of Glioblastoma is 41-60 (52%) years, diffuse astrocytoma is 21-40 years 60.60 and Pilocytic Astrocytoma is 1-20 years (66.88). Glioblastoma, Anaplastic Astrocytoma and Diffuse astrocytoma are more common in male than female. Conclusion: There is no gender difference in case of Pilocytic Astrocytoma. Journal of National Institute of Neurosciences Bangladesh, 2020;6(2): 91-95

In Vivo-like model of glial tumors: Creation of primary cell lines.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e14515-e14515
Author(s):  
Sofia V. Timofeeva ◽  
Oleg I. Kit ◽  
Anastasia O. Sitkovskaya ◽  
Irina V. Mezhevova ◽  
Svetlana Yu. Filippova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Primary Cell Culture ◽  
Tumor Tissue ◽  
Primary Cell ◽  
Glial Tumor ◽  
Ki 67 ◽  
Glial Tumors

e14515 Background: The choice of cell source for 3D bioprinting of in vivo-like models of glial tumors is crucial and must take into account the ability to proliferation and stable metabolism. Oral administration of 5-aminolevulinic acid (5-ALA) in patients prior to surgery increases the fluorescent contrast between tumor and surrounding tissue, but the effect of contrast agents on cells in vitro is unknown. The aim of the study was obtaining viable glial tumor tissues using 5-ALA, as well as the development of a stable primary cell culture for 3D bioprinting. Methods: Tumor tissue was obtained from patients with glioblastoma during surgery under visual control using the Opmi Pentero Blue E400 microscope and 5-ALA. Material was disaggregated on a BD Machine using Medicons 50 μm (BD). Glioblastoma cells were cultured in DMEM/F12 medium with L-glutamine (Gibco) containing 10% fetal bovine serum (Biolot, Russia), 1% non-essential amino acids (NEAA, Sigma-Aldrich) and 0.5% penicillin-streptomycin (Biolot) at 37C. Glial cell lines were characterized immunohistochemically using antibodies to the glial fibrillary acidic protein (GFAP) and proliferation index (Ki-67). Microsatellite analysis was performed using three dinucleotide repeat markers D2S123, D17S250, D5S346 and five mononucleotide loci BAT25, BAT26, NR21, NR24 and NR27. Results: The positive expression of GFAP on the cell processes of the star-like shape was clearly visualized, indicating a morphological feature of glial tumors. The Ki-67 labeling index was 70%. Changes were observed at the D17S250 locus (148-148/148-152) for the glial tumor primary cells after the sixth passage. Microsatellite instability was not observed in the primary cell culture. Conclusions: The accumulation of porphyrins from 5-ALA in glial tumor cells does not prevent the in vitro creation of a cell culture from tumor tissue. Microsatellite analysis showed that the obtained glioblastoma cell lines remain stable for at least 10 passages. Material obtained during resection using 5-ALA is a reliable source of stable glial tumor cell lines.

scholarly journals Primary tumor cell cultures: сurrent methods of obtaining and subcultivation

2020 ◽  
Vol 1 (3) ◽  
pp. 36-49
Author(s):  
I. V. Mezhevova ◽  
A. O. Sitkovskaya ◽  
O. I. Kit
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Primary Cell ◽  
The Past ◽  
Primary Cell Line ◽  
Primary Cell Lines ◽  
Tumor Material ◽  
Personalized Cancer Therapy ◽  
Personalized Cancer ◽  
Selection Of

Over the past decades, transplantable cell lines have been an affordable model for studying the biology and effect of chemotherapeutic drugs on tumors. However, numerous studies have shown that these cell lines are not heterogeneous enough and cannot reflect the drug resistance of tumors that occurs in some patients. Primary cell line cultures isolated from solid tumors have become widespread in personalized cancer therapy. This review discusses the basic methods for the preparation and cultivation of primary cell lines. A brief description is given of the methods for the disaggregation of tumor material using enzymatic, chemical and mechanical dissociation. The systems of cultivation of primary cell cultures. The selection of an appropriate dissociation method and cultivation is important to preserve the benefits of primary culture in preclinical studies.

Ectodermal and ectomesenchymal marker expression in primary cell lines of complex and compound odontomas: a pilot study

2019 ◽  
Vol 68 (3) ◽  
Author(s):  
David A. Trejo-Remigio ◽  
Luis F. Jacinto-Alemán ◽  
Elba R. Leyva-Huerta ◽  
Bogdan R. Navarro-Bustos ◽  
Javier Portilla-Robertson
Keyword(s):  
Pilot Study ◽  
Cell Lines ◽  
Primary Cell ◽  
Primary Cell Lines ◽  
Marker Expression

scholarly journals PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

2021 ◽  
Vol 11 (9) ◽  
pp. 3729
Author(s):  
Katarzyna Balon ◽  
Benita Wiatrak
Keyword(s):  
Cell Culture ◽  
Dna Damage ◽  
Cell Lines ◽  
Inflammatory Factor ◽  
Research Model ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Primary Cell Lines ◽  
Neurobiological Research ◽  
The Impact

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

In vitro effects of erythromycin and florfenicol on primary cell lines of Unio crassus and Cyprinus carpio

2021 ◽  
Author(s):  
Pınar Arslan ◽  
Begum Yurdakok-Dikmen ◽  
Saniye Cevher Ozeren ◽  
Ozgur Kuzukiran ◽  
Ayhan Filazi
Keyword(s):  
Cell Lines ◽  
Cyprinus Carpio ◽  
Primary Cell ◽  
Primary Cell Lines ◽  
In Vitro Effects
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