The impact of cell density variations on nanoparticle uptake across bioprinted A549 gradients
DOKPE
- Di Stolfo, Luigi ORCID University of Fribourg
- Lee, Wang Sik ORCID University of Fribourg
- Vanhecke, Dimitri ORCID University of Fribourg
- Balog, Sandor ORCID University of Fribourg
- Taladriz-Blanco, Patricia ORCID University of Fribourg
- Petri-Fink, Alke ORCID University of Fribourg
- Rothen-Rutishauser, Barbara ORCID University of Fribourg
- 2025
Published in:
- Frontiers in Bioengineering and Biotechnology. - Frontiers Media SA. - 2025, vol. 13
cell density
bioprinter
cell density gradients
SiO2 nanoparticles
nanoparticle uptake
advanced in-vitro model
nanoparticle-cell membrane interaction
3D image analysis
English
Introduction: The safe-by-design of engineered nanoparticles (NPs) for any
application requires a detailed understanding of how the particles interact with
single cells. Most studies are based on two-dimensional, uniformly dense cell
cultures, which do not represent the diverse and inhomogeneous cell
environments found in situ. In-vitro models that accurately represent tissue
complexity, including realistic cell densities, are essential to increase the predictive
accuracy of studies on cell-NP interactions. This study uses a bioprinted cell gradient
model to examine the relation between cell density and NP uptake in one dish.
Method: A549 lung epithelial cell density gradients within single inserts were
produced with a bioprinter by modulating inter-droplet distances. After two days
in culture, cells were exposed to Cy5-labeled silica NPs (SiO2 NPs, ~112 nm,
20 μg/mL) for up to 48 h. Confocal fluorescence microscopy and 3D image
analysis were used to quantify NP uptake, cell surface area, and cell volume. The
relationship between NP uptake and the other parameters was then investigated
statistically.
Results: Bioprinting enabled the creation of reproducible linear cell density
gradients, allowing controlled modeling of density variations while preserving
cell viability throughout the experiment. Increasing inter-droplet distances, from
0.1 mm to 0.6 mm, were used to achieve uniformly decreasing cell densities. SiO2
NP uptake per cell was around 50% higher in low-density regions compared to
high-density areas across all time points, i.e., 6, 24, and 48 h post-exposure. This
inverse relationship correlated with greater average cell surface area in lowerdensity regions, while differences in the proliferation rates of the A549 cells at
varying densities did not significantly impact uptake, did not significantly
impact uptake.
Conclusion: SiO2 NP uptake is significantly enhanced at lower cell densities,
mainly due to the increased available surface area, revealing potential cell-NP
interaction differences in tissues that present cell density variability. Our drop-on-demand bioprinting gradient model successfully supports the implementation of
cell density gradients in in-vitro models to increase their relevance as new
approach methodologies (NAMs) for next-generation risk assessment strategies
application requires a detailed understanding of how the particles interact with
single cells. Most studies are based on two-dimensional, uniformly dense cell
cultures, which do not represent the diverse and inhomogeneous cell
environments found in situ. In-vitro models that accurately represent tissue
complexity, including realistic cell densities, are essential to increase the predictive
accuracy of studies on cell-NP interactions. This study uses a bioprinted cell gradient
model to examine the relation between cell density and NP uptake in one dish.
Method: A549 lung epithelial cell density gradients within single inserts were
produced with a bioprinter by modulating inter-droplet distances. After two days
in culture, cells were exposed to Cy5-labeled silica NPs (SiO2 NPs, ~112 nm,
20 μg/mL) for up to 48 h. Confocal fluorescence microscopy and 3D image
analysis were used to quantify NP uptake, cell surface area, and cell volume. The
relationship between NP uptake and the other parameters was then investigated
statistically.
Results: Bioprinting enabled the creation of reproducible linear cell density
gradients, allowing controlled modeling of density variations while preserving
cell viability throughout the experiment. Increasing inter-droplet distances, from
0.1 mm to 0.6 mm, were used to achieve uniformly decreasing cell densities. SiO2
NP uptake per cell was around 50% higher in low-density regions compared to
high-density areas across all time points, i.e., 6, 24, and 48 h post-exposure. This
inverse relationship correlated with greater average cell surface area in lowerdensity regions, while differences in the proliferation rates of the A549 cells at
varying densities did not significantly impact uptake, did not significantly
impact uptake.
Conclusion: SiO2 NP uptake is significantly enhanced at lower cell densities,
mainly due to the increased available surface area, revealing potential cell-NP
interaction differences in tissues that present cell density variability. Our drop-on-demand bioprinting gradient model successfully supports the implementation of
cell density gradients in in-vitro models to increase their relevance as new
approach methodologies (NAMs) for next-generation risk assessment strategies
- Faculty
- Faculté des sciences et de médecine
- Department
- AMI - Bio-Nanomatériaux
- Language
-
- English
- Classification
- Medicine
- Other electronic version
- License
-
CC BY
- Open access status
- gold
- Identifiers
-
- DOI 10.3389/fbioe.2025.1584635
- ISSN 2296-4185
- Persistent URL
- https://folia.unifr.ch/unifr/documents/333672
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