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Barreto et al. | Journal of Clinical and Translational Research 2024; 10(6): 325-333 327
establishment. Large quantities of the three cell lines were then 2.6. Tumor histopathology
routinely frozen at −80°C in FBS supplemented with 10% (v/v)
dimethyl sulfoxide (DMSO; #D2650; Sigma-Aldrich, USA) and Tumor pieces designated for tissue analysis were immersed
stored long-term in a cryogenic freezer within liquid nitrogen in PFA for 24 h. Thereafter, the tissue pieces were washed with
distilled water 3 times (10 min each) to remove any residual
vapor (biobank). For the immunofluorescence assay, cells were PFA. The samples were then dehydrated through an ethanol
thawed, cultured, and allowed to reach 90% confluence before
being carefully scraped into 48-well plates (Corning Costar , gradient and embedded in Paraplast (#P3558; Sigma-Aldrich,
®
USA). In addition, 3 × 10 cells were also obtained for the USA). Sections of 5 µm were obtained and stained with H&E
5
before evaluation under a microscope.
tumorigenicity assay as described below.
2.7. Morphological evaluation
2.4. Immunofluorescence
For evaluation of morphology, the three established
Characterization and authentication of the three established
cell lines were assessed using immunostaining for glial fibrillary cell lines (obtained from patients) and those acquired after
tumor excision from mice were seeded in 6-well plates
acidic protein (GFAP), an astrocyte marker. Cell morphology (2 × 10 cells/well) and incubated at 37°C, 5% CO for
5
was also assessed using phalloidin to selectively label actin 48 h. The cells were then photographed under an inverted
2
filaments. This assay was based on a standardized protocol in microscope (Nikon Eclipse Ts2; Nikon, Japan) and the
our laboratory, which was described in a previous study [13]. NIS-Elements D software to analyze their general morphology.
Cells were initially seeded in 48-well plates (2 × 10 cells/well) The entire methodology used in this work is summarized and
5
with a complete medium. At 90% confluence, the medium was exemplified in Figures 1A and B.
removed from the wells and replaced with fresh medium. Cells
were then fixed with 4% paraformaldehyde (PFA) (15 min), 3. Results
washed three times with phosphate-buffered saline (PBS)
(pH 7.4), and incubated with a permeabilization solution (0.1% 3.1. Establishment and biobanking of the three high-grade
Triton X-100 in PBS) for 10 min at room temperature. After, the glioma cell lines from patient tumor tissues and morphological
wells were washed with PBS and then treated with a blocking evaluation
solution (1% bovine serum albumin [BSA] 467 supplemented A tumor mass was observed in the MRI of the patient before
0.2% Tween 20 in PBS) for 1 h. Subsequently, the cells were surgery (Figures 2A, 3A, and 4A). Using in vitro patient-
incubated with a Phalloidin probe (1:200, P5282; Sigma- derived specimens, the three HGG cell lines (C03, N07, and
Aldrich, USA) diluted in 0.3% BSA solution (supplemented with L09) were successfully established. From the primary culture,
0.1% Tween 20 in PBS) for 2 h at room temperature. Following it was possible to freeze, thaw, and subculture cells, allowing
this, the wells were washed with PBS and incubated overnight the creation of a biobank and the performance of in vitro
with anti-GFAP (1:500; #16825-I-AP; Proteintech, USA) in the characterization (GFAP labeling), as described below. During
same dilution solution. A negative control group without anti- the first 2 weeks of culture, the GB (N07) cell line grew faster
GFAP incubation was also prepared. The next day, cells were than the astrocytoma (C03 and L09) cell lines. Later, growth
washed again with PBS and incubated with rhodamine (TRITC) was similar for all three HGG cell lines. All of them reached the
– conjugated goat anti-rabbit IgG (1:100; #SA00007-2; tenth passage in about 2 months. Under light microscopy, all
Proteintech, USA) for 1 h, and then counterstained with a drop three cell lines grew in monolayer and most of them exhibited
of the nucleus dye DAPI (#P36971; ThermoFisher, USA). Cell spindle-shaped and polygonal morphologies; some of them
imaging was performed using Apotome.2 (Zeiss, Germany) and exhibited long protrusions (Figures 2B, 3B, and 4B).
analyzed with Zen 2.6 and Image J software.
3.2. Tumorigenicity of the three high-grade glioma cell lines in
2.5. Tumorigenicity in RAG black mice C57BL/6 RAG −/− mice and morphological evaluation
−/−
To investigate tumorigenicity, 3 × 10 cells from each of the To assess whether the three cell lines retained their
5
three cell lines were suspended in 0.1 mL PBS and injected tumor-forming ability, a tumorigenicity assay was
subcutaneously into the dorsal flank of adult immunodeficient conducted. In vivo, xenograft tumors were developed after
C57BL/6 RAG female or male mice (12 months old). The subcutaneous implantation of all three established cell lines
−/−
animals were monitored weekly during tumor development. (Figures 2C and D; 3C and D; and 4C; and D). After 22 days
After 22 days of cell implantation, the mice were anesthetized of inoculation, the animals were anesthetized and the tumor
with ketamine (80 mg/kg) and xylazine (10 mg/kg) and masses were excised. The tumors reached a volume of
euthanized. Tumors were excised and divided into two parts for 2.5, 0.7, and 2.02 cm , with weights of 1.6, 0.8, and 1.5 g,
3
different processing methods. One part was fixed in 4% PFA respectively (for C03, N07, and L09). The morphology of the
and embedded in paraffin for tissue analysis by hematoxylin and xenograft tumors during subculture resembled the cell line
eosin (H&E) staining. The other part was placed in a falcon tube characteristics of the three cell lines before implantation,
containing a complete medium for subculture, morphological displaying spindle-shaped and polygonal morphologies
analysis, and creation of a biobank. (Figures 2E, 3E, and 4E).

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