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Barreto et al. | Journal of Clinical and Translational Research 2024; 10(6): 325-333 331
hinder many preclinical studies from translating into clinical culture, forming additional cellular extensions that connect to
applications. The occurrence of multiple passages makes these one another as they adapt to the new environment. Despite the
cell lines less similar to the original tissue [19,20]. This allows GB (C03) cell line growing faster at the beginning of culture,
newly established cell lines to become a better study model its morphology remained similar to the other astrocytic (N07
than commercially available cell lines. In the present study, we and L09) cell lines. In addition to morphological analyses,
were able to establish three new HGG cell lines (C03, N07, and to characterize and confirm the established cell lines as
L09) after 10 passages, obtained directly from primary tumors astrocytoma and GB, immunofluorescence staining for GFAP
of different patients. During the adaptation period, astrocytoma was performed. GFAP, a protein involved in the structure and
cell lines (C03 and L09) slowly adhered to the culture flasks function of the cytoskeleton, is commonly used as a marker of
and began to proliferate more rapidly until reaching the tenth astrocytes [21,22]. Our results indicated that the three HGGs
passage. Interestingly, the GB cell line (N07) grew faster in the were positive for GFAP, confirming their astrocytic origin.
first 2 weeks of culture than the astrocytomas. Thereafter, growth Furthermore, for the validation of the cell lines, it was also
was similar for all three HGG cell lines. These differences may important to evaluate whether the cell lines maintained the
be related to GB being a more aggressive tumor; however, after ability to form tumors. Studies report that the implantation
the 2 week, its growth remained similar to that of the other of tumor cell lines in mice does not always lead to tumor
nd
cell lines. After reaching the tenth passage (approximately development [21]. However, this is an important factor to
2 months), it was noted that they became stable after thawing. reliably attest to the tumorigenicity of new tumor cell lines. In
Compared to other cell culture models, like sphere culture, the present study, all three HGG cell lines were tumorigenic in
adherent cells are easy to manipulate and are ideal for drug RAG-/- C57BL6 mice, indicating that the cells are neoplastic
screening experiments, such as cell proliferation, viability, and and malignant. Histopathological analysis of xenograft tumors
migration assays. Although easy to manipulate, primary cultures from all three cell lines revealed increased cellularity, variation
are more susceptible to contamination. They are also more in nuclear size, and mitotic figures. These characteristics are
sensitive to microenvironmental components, making the choice in line with other studies describing malignant astrocytoma
of an appropriate culture medium essential. Cheng et al. [21] tumors, which are distinguished by high mitotic activity and
cultured 10 GB cell lines from different patients using high- increased cellularity [23,24]. The difference in size and weight
glucose DMEM + 10% FBS, but only one of them (GWH04) of the tumors obtained did not influence the histopathological
acquired unlimited proliferation capacity. In the present study, analyses, which were very similar, despite being from three
all three HGG cell lines acquired stability and high replicative different cell lines.
ability with complete medium (IMDM + 10% FBS), without
the need for additional glucose supplementation. Morphological 5. Conclusion
analysis is commonly used to assess the characteristics of cancer Three HGG cell lines (C03, N07, and L09) obtained from
cell lines. Despite being different types of gliomas, all three different patients were successfully established. Characterization
HGG cell lines grew in a monolayer and exhibited spindle- confirmed their astrocytic origin, and validation in a xenograft
shaped and polygonal morphologies. These morphological murine model demonstrated their ability to form tumors post-
findings are in agreement with studies of other established establishment. Therefore, these newly established cell lines
primary glioma cell lines. Grube et al. [20] established 16 of prove to be useful for studying cell and molecular biology, as
34 GB cell lines that exhibited a spindle-form or polygonal- well as for the development of new glioma treatments.
to-amorphous morphology and grew as a monolayer. In the
present study, we also investigated cell morphology by labeling Acknowledgments
cytoskeleton components (using the phalloidin probe). The
cells exhibited a morphology similar to those observed by light We would like to thank the support and the access to
microscopy. Interestingly, the presence of rounder rather than equipment provided by the National Institute of Science and
polygonal cells was noted. Technology on Photonics Applied to Cell Biology (INFABIC)
The differences observed in morphology may be related at the State University of Campinas. Figure 1 was created using
to the time at which the cells were imaged, as phalloidin BioRender (BioRender.com).
labeling was performed in the initial weeks of culture, Funding
whereas light microscopy was conducted after establishment.
Machado et al. [11] observed a similar pattern during GB This study was financed by the São Paulo Research Foundation
(NG97) establishment: in the first passages, GB cultures (FAPESP; #2019/10003-3 [Natália Barreto’s scholarship],
grew more slowly and presented a more rounded shape. At #2022/03543-4, and #2015/04194-0), the National Council
the 13 passage, dendritic-like cells, with more extensive for Scientific and Technological Development (Conselho
th
cytoplasmatic extensions, appeared in the culture. Subsequently, Nacional de Desenvolvimento Científico e Tecnológico
spindle-shaped cells appeared as the culture became dense. [CNPq]; 148156/2019-3 [Natália Barreto’s scholarship]), and
We hypothesize that these differences may be related to the the Education, Research, and Extension Support Fund (Fundo
heterogeneity of the tumor and the slow adaptation of the cells to de Apoio ao Ensino, à Pesquisa e à Extensão [FAEPEX/
a new environment. Cells may create more connections during UNICAMP]; #2588/20, #2124/21, #2569/21, #2389/22,

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