Page 14 - IJB-9-4

International Journal of Bioprinting Biomimetic 3D printed glioma model

image of Ca was examined at excitation wavelength only spherical in the Far area in G/N and in the whole area
2+
of 488 nm under a laser scanning confocal microscope in G. In order to quantitatively analyze the distribution
(LSCM) (A1, Nikon, Japan). ImageJ software was used of GL261 cells in different models, the number of GL261
for the quantification of fluorescence intensities. Each cells in different areas and models was statistically counted
experiment was repeated three times (n = 3). by ImageJ software (Figure 4D). It was found that the
amount of GL261 cells in the Near area (42.33 ± 4.72) were
2.6.3. Live/dead cell assay significantly higher than those in the Far area (21.33 ±
LIVE/DEAD Viability/Cytotoxicity Kit was used to label 2.01) in G/N. In addition, the amount of GL261 cells in
both live and dead cells in the printed structure. On days both the Far and Near area in G/N were superior to that
1, 3, and 5 of tissue culture, specimens were harvested observed in the whole area in G.
from incubator and washed three times in PBS. Later,
live/dead working solutions were applied simultaneously 3.1.2. Intercellular Ca2+ concentration of GL261 cells
to the specimens, and the specimens were then kept in a After 5 days of culture, the intracellular Ca in the
2+
dark environment. Specimens were washed once before specimens was stained with cell calcium staining agent
imaged. Exciting light waves were of 515 nm and 580 nm and imaged by LSCM (Figure 5A and B). The fluorescence
for live and dead cells imaging, respectively. The live cells intensity of the images was carried out with ImageJ
were in green whilst the dead cells were in red. Z-stacks of software to analyze the intracellular Ca concentration
2+
50–90 µm in height were flattened, and the green and red quantitatively (Figure 5C). Similar to cell morphology and
channels were analyzed separately to determine numbers distribution, intracellular Ca concentration of GL261
2+
of live and dead cells. Live and dead cells numbers were cells in G/N also showed differences in the Far and Near
counted using ImageJ, and the percentage of viable cells areas. Compared with the GL261 cells in the Far area in
was quantified as the number of live cells divided by the G/N, those in the Near area in G/N significantly expressed
total number of cells. Each experiment was repeated three higher fluorescence intensity of Ca , which is not found in
2+
times (n = 3). G. In addition, the intracellular Ca concentration in both
2+
the Far and Near areas in G/N is higher than that observed
2.7. Statistical analysis in the whole area in G.
Statistical analysis was applied to evaluate significant
differences among experimental data sets. To obtain 3.2. Effects of GL261 cells on neurons in the
statistical results, the number (n) of data sets should bioprinted glioma in vitro 3D model
be more than 3, or n ≥3. Two-way analysis of variance 3.2.1. Survival rate of neurons
(ANOVA) was used to investigate the trend of a series of After 1, 3, and 5 days of culture, a live/dead assay was
data sets, whereas t-test was used to compare two data carried out, images of different bioprinted 3D models
sets based on the difference of their characteristics. ∗p, were obtained by LSCM (Figure 6A). The survival rate
∗∗p, ∗∗∗p, and ∗∗∗∗p were used to measure the degree of of neurons was analyzed quantitatively (Figure 6B). On
significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p day 1, it was shown that the neuron survival rate in G/N
< 0.0001. was 70.24 ± 2.78%, which was 60.00 ± 2.76% in N. After
3 days, the survival rate of neurons in the two models was
3. Results approximately equal (87.71 ± 1.23% in G/N and 87.36 ±
1.19% in N) and much higher than that observed on day 1.
3.1. Effects of neurons on GL261 cells in the After 5 days of culture, the survival rate of neurons began
bioprinted glioma in vitro 3D model to decline in both models, which was 58.31 ± 1.09% in G/N
3.1.1. Cell morphology and distribution of GL261 cells and 54.57 ± 1.44% in N. From day 1 to day 5, neurons in
After 5 days of culture, the cell morphology and G/N consistently exhibited equal or higher survival rates
distribution of GL261 cells in the different in vitro 3D than that observed in N.
models was observed by LSCM (Figure 4A and B).
Visual inspection demonstrated that the morphology and 3.2.2. Neurite characteristics of neurons
distribution of GL261 cells co-cultured with neurons in As shown in Figure 6A, a superior proportion of neurons
G/N specimens showed difference in the area far away in G/N outgrew neurites and formed connections with
from neurons and in the area close to neurons. Thus, the each other than those in N after 1 day and 3 days of culture.
observation area in specimens were divided into Far area The neurite characteristics of neurons in the bioprinted
(the area away from neurons) and Near area (the area near 3D models were analyzed using Image J software, and the
neurons) (Figure 4C). GL261 cell morphology was found results obtained are shown in Figure 7. During the 5 days of
diverse, i.e., some cells were spherical, and the other were culturing, the neurite number of individual neurons in both
elongated with filopodium in the Near area in G/N, but G and N models showed a slight decrease. The neurons in

Volume 9 Issue 4 (2023) 6 https://doi.org/10.18063/ijb.715