Testing in vitro toxicity of nanoparticles in 3D cell culture with various extracellular matrix scaffold

Nanomaterials are used in a variety of fields and toxicity assessment is paramount for their development and application. Although most toxicity assessments have been performed in 2D (2-Dimensional) cell culture, the inability to adequately replicate the in vivo environment and toxicity is a limitation. To overcome the limitation, a 3D (3-Dimensional) cell culture method has been developed to make an environment closer to an in vivo system. In this study, 20 nm SiO2 nanoparticles were dispersed in serum-containing (SC) and serum-free (SF) media to compare 2D cell culture and 3D cell culture toxicity. The cells were subjected to a 3D cell culture method in which HepG2, a human-derived liver cancer cell line, was mixed on a scaffold. We found that nanoparticles induced toxicity in 2D cell culture, but toxicity was not observed in 3D cell culture similar to in vivo environment. However, differences in toxicity were observed between the three types of scaffolds in the absence of serum as the number of cells decreased.


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Nanomaterials can be applied to diverse fields in a novel way or to provide enhanced 38 functionalities depending on their particle size and surface modifications and therefore have 39 been the subject of intense research in a wide range of fields, such as medicine, chemistry, 40 biotechnology, foods, and electronics. As it has become possible to apply nanomaterials to 41 various products and fields, many studies are underway investigating the effects that 42 nanoparticle exposure may have on the body. In particular, silica nanoparticles are incorporated 43 into various products, including drugs, cosmetics, food additives, and coatings, and are 44 extensively studied in the biochemical field regarding their application as drug carriers and/or 3 45 biomarkers [1,2]. Cell cultures are used for such in vitro biochemical evaluation, and a variety 46 of cell culture models and platforms have been developed to study investigational products in 47 an environment that more closely mimic an in vivo system [3,4]. Cell cultures are used in a 48 wide range of fields to study various biochemical and physiological events that may occur in 49 vivo, including the effects of drugs or toxic compounds on cells and development of mutations. such as collagen, enzymes, and glycoproteins and is known to support 3D cell growth and act 69 as mediator for cell growth, migration, differentiation, survival, homeostasis, and 70 morphogenesis [14-16]. In addition, moving away from 2D cell culture technology that uses 71 culture dishes for cell culture, allows cells cultured in 3D, using various newly developed 72 platforms, to be effectively applied to research. In this study, we compared the toxicity of silica 73 nanoparticles in two models, using a widely used 2D cell culture model and a newly developed 74 3D cell culture model. Fig 1 shows a  assessments were performed on cultured cells (1 x 10 3 cells, 5 x 10 3 cells, and 1 x 10 4 cells) to 81 assess differences in toxicity by ECM type.     x 10 3 also. In this study, three types of ECM (alginate, Matrigel, and collagen) were used as 129 scaffolds for 3D cell growth. The first type of scaffold is 3% alginate, which is liquid at 4 °C,   which was diluted to a final concentration of 150 μM and then further diluted in two-folds to 160 prepare various concentrations mentioned above. In this study, cytotoxicity was confirmed 161 using a toxicity test method based on absorbance measurement.  with the standard error of the mean (SEM) of three or more independent experiments (n = 3).

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The t-test was performed by dividing the equal variance and this variance through the F-test 183 using Excel, and the p-value was statistically processed as a value expressed in both directions.

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In the 2D cell culture statistical comparisons were performed between two groups, and in 3D

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Size changes in 20 nm SiO 2 with or without serum 192 The particle size distribution is plotted in Fig 2 (a)   To determine the cytotoxicity of the 20 nm SiO 2 nanoparticles, they were mixed with 210 culture media to expose cells to the nanoparticles. In addition, it has been reported that proteins 211 or polymers present in culture media get adsorbed to the nanoparticles and form protein corona 212 that can alter the size of the particles in an aqueous solution [19,20]. Therefore, prior to 213 experimentation, particle size in the culture media with or without serum, in which the cells 214 were exposed to the nanoparticles, was determined using DLS. As shown in Fig 3, we were 215 able to confirm that the size of 20 nm SiO 2 nanoparticles dispersed in SC media increased to 216 as high as 158 nm because the serum led to the formation of protein corona around the 217 nanoparticles. In contrast, we found that the size of 20 nm SiO 2 nanoparticles dispersed in SF 218 media was 23 nm, which was similar to the original size.   However, collagen appears to constitute a spheroid in which cells are completely coagulated.

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MTS analysis showed that cell growth was promoted by collagen rather than alginate and 239 Matrigel. These results were similar to those reported previously, stating that collagen     To overcome the limited ability of 2D cell culture to adequately express the in vivo 331 environment, this study aimed to determine the toxicity of nanoparticles using 3D cell culture 332 technology. Among various methods for culturing cells in 3D, a method of distributing cells 333 mixed with ECM acting as a scaffold in a column was used in this study. The toxicity of 334 nanoparticles in 3D cell culture method used in this study was compared to 2D cell culture. 335 The results showed that the HepG2 cells grown in 3D are less susceptible to toxicity regardless 336 of protein-corona formation. In addition, it was found that there were differences in toxicity 337 according to the scaffold (ECM) type and cell number suggesting that 3D cell culture needs 338 more research and development. Cells grown in 2D are designed to mimic in vivo conditions. 339 However, the environment is significantly different with regard to morphology, exposed