Page 268 - v11i4

International Journal of Bioprinting 3D cell culture model for neural cell analysis

disruption and ATP stabilization. The plate was then for approximately 45 min. After incubation, the droplets
shaken for 5 min at 50 × g. Following cell lysis, 50 µL of were washed again with PBS, and 50 μL of a fluorescently
luminescent substrate solution was added to each well. labeled secondary antibody was added, followed by a 25-
The plate was shaken again under the same conditions min incubation under the same conditions. After a final
for an additional 5 min to ensure proper mixing and PBS wash, mounting medium containing Hoechst dye was
reaction initiation. Subsequently, the plate was incubated applied to stain cell nuclei. The droplets were then imaged
in the dark at room temperature for 10 min to allow the using a fluorescence microscope to quantify γ-H2AX
luminescent signal to stabilize. Luminescence, which is foci formation.
directly proportional to the intracellular ATP content
and hence viable cell number, was then measured using a 2.5. Biological assessments in 2D
microplate reader (Varioskan LUX, Thermo Scientific). For To assess cell viability in 2D culture, a luminescent ATP
qualitative analysis, GelMA droplet hydrogels were imaged assay kit (Abcam) was used. Briefly, before conducting
immediately using a confocal EVOS M5000 microscope at the luminescent ATP detection assay, cells were seeded
into 96-well plates. Fifty microliter of lysis buffer was
10× magnification with green fluorescent protein (GFP; added to each well to facilitate cell membrane disruption
emission: 525/50 nm), red fluorescent protein (RFP;
emission: 593/40 nm), and 4’,6-diamidino-2-phenylindole and ATP stabilization. The plate was then shaken for 5
(DAPI; emission: 447/60 nm) emission filters for calcein min at 50 × g. Following cell lysis, 50 µL of luminescent
AM, ethidium homodimer-1, and Hoechst 33342 stains, substrate solution was added to each well. The plate was
respectively. Three replicates of GelMA droplets were shaken again under the same conditions for an additional
5 min to ensure proper mixing and reaction initiation.
bioprinted for each particle type and a total of three images Subsequently, the plate was incubated in the dark at room
were taken for each droplet. In addition, a negative control temperature for 10 min to allow the luminescent signal to
of cell only and a positive control of 5% (v/v) DMSO stabilize. Luminescence, which is directly proportional to
was utilized, and a particle-only control was also used to the intracellular ATP content and hence viable cell number,
ensure that particles did not interfere with the luminescent was then measured using a microplate reader (Days 1,
readings for this assay. 3, and 7). Fluorescence microscopy (DP80 and Nikon
2.4.2. Reactive oxygen species detection assay ECLIPSE Ti2, Japan) was utilized for imaging fluorescently
The DCFDA - Cellular ROS Assay Kit (Abcam, UK) was stained cells for qualitative analysis of cell viability. An
used to measure the production of reactive oxygen species inverted microscope (Olympus CKX53, Japan) was used
(ROS) by cells in different particle model bioprinted for bright-field microscopy.
droplets. The cell culture media was aspirated from each 2.6. Statistical analysis
well, and bioprinted droplets were washed with ROS assay Statistical analysis was performed using the SPSS software,
buffer. The cells were stained with 100 μL diluted DCFDA v22.0 (IBM Corp., Armonk, NY, USA). Two-way analysis
(2ʹ,7ʹ-dichlorofluorescin diacetate; a cell-permeant of variance (ANOVA) was utilized to assess cell viability
reagent) solution and incubated at 37°C and 5% (v/v) CO and ROS levels between 3D model particles. Fixed factors
2
for 45 min. The positive control was established by using included cell culture time points and biomaterial types,
100 μL of 200 μM hydrogen peroxide, followed by a 2-h analyzed with statistical significance of 0.05. Post-hoc
incubation (96 well plate). Subsequently, fluorescence was multiple comparisons were conducted using a Bonferroni
measured with a plate reader at excitation and emission of adjustment. All experiments included at least three
485 and 535 nm, respectively. biological replicates. In addition, statistical significance

2.4.3. DNA damage was analyzed within 3D model particles using one-
To evaluate the impact of model wear particles on way ANOVA tests. For proportional or percentage data
DNA integrity in C6 astrocyte-like cells, γ-H2AX foci that does not follow a binomial distribution, an arcsine
formation—a marker of DNA double-strand breaks— transformation was used to help normalize the distribution
was assessed at multiple time points (1, 2, and 4 h post- of data.
exposure). For immunofluorescent detection, bioprinted
droplets were first washed with PBS and permeabilized 3. Results
with 100 μL of 0.5% Triton X-100 solution for 3 min at room 3.1. Particle characterization
temperature. The droplets were then washed twice with This study utilized particles from various materials,
®
PBS, followed by the addition of 50 μL of primary antibody including PEEK-OPTIMA™, Ceridust 3615, ZTA, and
solution targeting γ-H2AX (Abcam). Samples were CoCrMo alloy. Particle characterization involved size
incubated at 37°C in a humidified atmosphere with 5% CO₂ distribution analysis and morphological assessment

Volume 11 Issue 4 (2025) 260 doi: 10.36922/IJB025180174