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326 Barreto et al. | Journal of Clinical and Translational Research 2024; 10(6): 325-333
brain tumors in adults [1]. According to the 2021 World Health and opens perspectives for studies aimed at developing new
Organization (WHO) Classification of Tumors of the Central treatments against these tumors.
Nervous System (WHO CNS5), adult diffuse gliomas are
classified into: (i) Astrocytoma, IDH-mutant (grades 2, 3, or 4), 2. Methods
(ii) oligodendroglioma, IDH-mutant, and 1p/19q-codeleted 2.1. Ethical considerations
(grades 2 or 3); and (iii) glioblastoma (GB), IDH-wildtype
(grade 4) [2]. CNS WHO Grades 1 – 4 are related to the degree Tissue samples were obtained from the department of
of malignancy, with a higher grade corresponding to a poorer neurosurgery and imaging examinations were acquired from
prognosis [2-4]. Standard treatments for HGGs involve a the department of radiology, both at the Central Hospital of
combination of maximal surgical resection, radiotherapy, and Santa Casa (Brazil) after informed patient consent (Termo de
chemotherapy [5,6]. However, these treatments have limited Consentimento Livre e Esclarecido [TCLE]). Tumor samples
efficacy in preventing glioma progression. GB is the most were acquired following the ethical guidelines of the Research
common and highly malignant type of brain tumor in adults, Ethics Committee of the University of Campinas (UNICAMP,
with a median survival of <2 years for most patients [7,8]. Brazil; CAAE: 15215219.5.0000.5404) and the Central Hospital
Given the poor outcomes associated with HGGs, there is a need of Santa Casa (Brazil; CAAE: 15215219.5.3001.5479). Animal
to explore new therapeutic approaches or even improve the experiments were performed in accordance with the Ethical
efficacy of conventional therapies against these tumors. Principles in Animal Research, adopted by the Brazilian College of
Patient-derived cancer cell lines are the most commonly used Animal Experimentation (Colégio Brasileiro de Experimentação
models to study tumor biology and discover novel therapeutics, Animal [COBEA]); all procedures were previously approved by
not only in vitro but also in murine in vivo assays [9,10]. Once the Animal Use Ethics Committee (CEUA/UNICAMP; 6200-
established, malignant cell cultures provide excellent and 1/2023). The animals were carefully monitored and cared for
permanent materials for studying tumor biology and behavior. daily to ensure their well-being. They were kept in standard
They facilitate the analysis of bioactive components produced by filter-top cages with unrestricted access to sterile water and food
tumors, the determination of cell viability and proliferation, and in the Animal Facility of the Institute of Biology, Department of
the assessment of cell migration and invasion under treatment Functional and Structural Biology (UNICAMP).
conditions [11]. Therefore, in vitro assays are a fundamental 2.2. Specimen collection
step in drug development, as they permit screening for agents
that selectively target tumors. For example, the successful After surgical resection, tissue samples were obtained from
establishment of a human GB cell line (named NG97) [11,12] three different patients: a 55-year-old male (specimen designated
allowed our research group to identify spider venom molecules C03), a 56-year-old male (N07), and a 36-year-old male (L09).
with antitumor effects [3,13-16]. These studies emphasize the Under aseptic conditions, a portion of the samples from each
importance of establishing cancer cell lines. However, their patient was placed in separate falcon tubes containing Iscove’s
focus primarily remained on one glioma cell type (GB-NG97). Modified Dulbecco´s Medium (IMDM; #l7633; Sigma-Aldrich,
Considering the heterogeneity of gliomas, trials involving United States of America [USA]) supplemented with 10% fetal
multiple cell types from different patients could provide a more bovine serum (FBS) and penicillin-streptomycin-amphotericin B
comprehensive understanding of the effects of animal venom solution (10.000 U/mL; #A5955; Sigma-Aldrich, USA) (pH 7.4)
molecules on these tumors. (referred to as complete medium). These samples were intended
Despite being an excellent model for cancer research and for the preparation of primary cell cultures. The remaining portions
drug development, establishing cell lines from fresh tissue were placed in other falcon tubes containing 10% formaldehyde
obtained from patients is time-consuming and challenging. In and sent to a histopathology laboratory, where the tumors were
contrast to established cell lines, primary cells are sensitive characterized immunohistochemically and molecularly according
to new environments and more susceptible to contamination. to WHO CNS5 2021, as follows: astrocytoma IDH-mutant,
Bian et al. [17] reported that the success rate of cell line WHO Grade 3 (C03); GB IDH-wildtype, WHO grade 4 (N07);
establishment from fresh tumor tissues is around 10%. In the and astrocytoma IDH-mutant, WHO Grade 3 (L09).
present study, we aimed to establish and characterize three new 2.3. Cell lines establishment
human cell lines derived from fresh tissue samples of three male
patients diagnosed with HGGs. Through a simple yet carefully Tumor specimens were maintained in a complete medium,
optimized process, we aimed to establish the cell lines after only minced into 1 – 2 mm fragments, and centrifuged (1500 rpm,
3
a few passages, ensuring they became stable while remaining 4°C, 5 min) to remove debris. The supernatant was then
as close as possible to the original tumor tissue. These newly discarded, and the cells (pellet) were seeded into 25 cm culture
2
established cell lines will facilitate future studies related to bottles. Primary cells were cultured (IMDM) and maintained
the understanding of glioma biology, especially due to their at 37°C in a 5% CO humidified atmosphere until they reached
2
distinct histological and molecular characteristics. These cell the tenth passage. Each passage involves releasing the cells
lines will also enable our research group to conduct new tests from the bottle using a cell scraper and transferring them to a
investigating the effects of animal venom molecules. A well- larger bottle or two other bottles. By the tenth passage, the cells
established tumor cell bank provides heterogeneous cell lines were observed to be stable after thawing, indicating successful

DOI: http://doi.org/10.36922/jctr.24.00028