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

2. Materials and methods The ceramic particles were composed of zirconia-
toughened alumina (ZTA; Inframat Advanced Materials,
2.1. Cell culture preparation USA), while the metallic particles were made from a cobalt–
2.1.1. Neural cell lines and cell culture media chromium–molybdenum (CoCrMo) alloy (American
C6 astrocyte-like (rat glioma) and NG108-15 (derived Elements, USA). These materials were selected to represent
from mouse neuroblastoma fused with rat glioma) cell common biomaterials used in spinal implants, providing
lines were obtained from the American Tissue Culture a comprehensive analysis of the biological impact of wear
Collection (ATCC) and the European Collection of Cell debris from diverse material types.
Culture (ECACC), respectively. The cell culture medium 2.2.2. Particle preparation and separation
for C6 astrocyte-like cells consisted of Ham’s F-12 nutrient Sequential filtration was performed to separate particles
mix (Sigma-Aldrich, Germany) supplemented with 10% by size using 25 mm polycarbonate membrane filters
(v/v) fetal bovine serum (FBS) (Bovogen, Australia). (8, 0.8, and 0.1 μm pore sizes; Whatman). Membrane filters
For NG108-15 cells, the cryopreservation medium were sterilized by washing with 70% (v/v) ethanol, rinsing
comprised Dulbecco’s Modified Eagle’s Medium (DMEM; with deionized water, air-drying at room temperature, and
Gibco, USA) supplemented with 20% (v/v) FBS and 10% exposing them to ultraviolet (UV) light for 30 min. A 100 mg
(v/v) dimethyl sulfoxide (DMSO; Sigma-Aldrich). For particle sample was suspended in 20 mL of distilled water
cell culture, the cryopreservation medium was further in a 50 mL centrifuge tube and then sonicated (Unisonics
supplemented with 100 U/mL penicillin (Gibco) and 100 Australia) for 40 min to ensure uniform dispersion
μg/mL streptomycin (Gibco). (5 g/L concentration). Immediately after sonication, the
suspension was filtered through the membranes. The
2.1.2. Cell culture filters were carefully transferred to sterile Petri dishes using
Cell passaging was performed when cultures reached tweezers and dried in an oven (Skope, New Zealand) at 50°C
approximately 70–80% confluence. Before passaging, a for at least 30 min. The size-separated particles were then
complete cell culture medium and trypsin (Gibco) were subjected to scanning electron microscopy (SEM; Zeiss;
pre-warmed to 37°C in a water bath (Thermoline Scientific, EVO LS 15) characterization and cell culture experiments.
Australia). The existing medium in the culture flask was
aspirated, and the cells were washed with Dulbecco’s 2.2.3. Scanning electron microscopy imaging
Phosphate Buffered Saline solution (PBS; Sigma-Aldrich). Dried filters were directly coated with a 3 nm layer of gold/
Subsequently, 0.25% (v/v) trypsin-EDTA (Gibco) was palladium or carbon using a sputter coater before SEM
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added (1 mL for a T25 flask and 3 mL for a T75 flask), and imaging. PEEK-OPTIMA™, Ceridust 3615, and ZTA
the flasks were incubated at 37°C with 5% CO₂ for 3 min. samples were coated with gold/palladium, while CoCrMo
An equal volume of complete medium was then added to samples were coated with carbon. Imaging was performed
neutralize the trypsin. The cell suspension was transferred at 5 kV or 10 kV with a working distance of 4.9–21 mm.
to a 15 mL centrifuge tube and centrifuged (Thermoline Four magnifications (100×, 400×, 700×, and 1500×) were
Scientific) at 300 × g for 5 min. Following centrifugation, used, with at least three images captured per magnification
the supernatant was aspirated and the cells were then for each filter and material.
transferred into new culture flasks containing fresh
medium and maintained at 37°C with 5% CO₂ (Heracell™ 2.2.4. Particle characterization
The SEM images were analyzed using ImageJ open-
VIOS 160i, Thermo Fisher Scientific, USA). Cell culture source software (Open-source image processing software
was conducted both in isolation and co-culture conditions. developed by the National Institutes of Health [NIH]
When required, cell counting was performed using a in the USA) to measure particle size, area distribution,
hemocytometer and the trypan blue (Sigma-Aldrich) and morphology. Measurements included particle area,
exclusion assay. perimeter, length (Feret’s diameter along the major axis),

2.2. Particles and width (Feret’s diameter along the minor axis). Particles
were categorized into size ranges: <0.1, 0.1–0.8, 1.0–8,
2.2.1. Particle types and >8 μm. Additionally, the particle count per image
This study utilized a variety of particulate materials, area (N/A) and the average particle area within each size
encompassing polymers, metals, and ceramics. The range (P/N) were calculated. A minimum of 100 particles
polymer particles included polyetheretherketone (PEEK- per material were manually measured by outlining
OPTIMA™; from Invibio Biomaterial Solutions, UK) and each particle. To minimize human error, overlapping or
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polyethylene (Ceridust 3615; from Hoechst, Germany). agglomerated particles were excluded from the analysis.

Volume 11 Issue 4 (2025) 258 doi: 10.36922/IJB025180174