Lung organoids and microplastic fibers: a new exposure model for emerging contaminants

Background Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but they have been applied only once to evaluate the biological effects associated with microplastic exposure. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is still poorly understood. Objectives In this study, we aimed to establish an in vitro organoid model of human lung epithelial cells to evaluate its suitability for studying the effects of airborne microplastic contamination on humans. Furthermore, we aimed to characterize the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and to test their interactions and inflammatory effects on the established lung organoids. Methods The polyester fibers emitted from the drying of synthetic fabrics were collected. Morphological characterization of the fibers released into the air filter was performed by optical microscopy and scanning electron microscopy (SEM)/energy dispersive x-ray spectroscopy (EDS). The organoids were exposed to various MPF concentrations (1, 10, and 50 mg L−1) and analyzed by optical microscopy, SEM, and confocal microscopy. Gene expression analysis of lung-specific genes, inflammatory cytokines, and oxidative stress-related genes was achieved by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Results We successfully cultured organoids with lung-specific genes. The presence of MPFs did not inhibit organoid growth, but polarized cell growth was observed along the fibers. Moreover, the MPFs did not cause inflammation or oxidative stress. Interestingly, the MPFs were coated with a cellular layer, resulting in the inclusion of fibers in the organoid. Discussion This work could have potential long-term implications regarding lung epithelial cells undergoing repair. This preliminary exposure study using human lung organoids could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.


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The human health effects of MPs and nanoplastics (NPs) are usually deduced from in vivo exposure  Total RNA from organoids was isolated using TRIzol reagent, according to the manufacturer's instructions.

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The RNA concentration and purity were verified using a NanoDrop ND-100 spectrophotometer (NanoDrop 1 4 5 Technologies). For the qRT-PCR assay, cDNA was synthesised from 200 ng of total RNA with SuperScript 1 4 6 IV VILO. The cDNA was diluted 10-fold, and 1 μ L of the sample was used as a template for qRT-PCR 1 4 7 analysis with SYBR Select Master Mix on a CFX96 thermal cycler (Bio-Rad). The relative expression levels 1 4 8 of the selected target genes were determined using the Δ Δ C t method and normalized to the geometric mean 1 4 9 of the ACTB and TBP mRNA levels using the primers listed in Table S2.

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To characterize the hAO, gene expression analysis by qRT-PCR was performed after 10-14 days of 1 5 1 standard culture and again after MPF exposure to investigate possible changes in the lung cell To generate environmentally relevant MPFs for the human organoid exposure experiment, polyester fibers 1 5 6 emitted from the drying of synthetic clothes and fabrics were collected. Specifically, one polyester t-shirt, six 1 5 7 sweatshirts, and two blankets of different colors (dry weight: 5,427 g) were washed in a washing machine 1 5 8 and subsequently dried in a common domestic tumble dryer. The dryer filter was previously cleaned by a 1 5 9 vacuum cleaner, and all fibers derived from the first drying cycle were discarded. The same items were then 1 6 0 washed again, and the synthetic fibers from the filtered exhaust air of the tumble dryer were collected ( Figure   1 6 1 S1), wrapped in aluminum foil, and transported to the laboratory for subsequent analyses.

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The MPFs were first morphologically analyzed with a Leica EZ4D stereomicroscope and then with a 1 6 3 Leica DMRA2 light microscope equipped with a Leica DC300 F digital camera. The detailed morphology and 1 6 4 the elemental composition of the fibers were studied with a Zeiss LEO 1430 scanning electron microscope 1 6 5 (SEM) coupled with a Centaurus detector for energy dispersive x-ray spectroscopy (EDS) analysis. A The MPFs collected from the filter of the dryer machine were resuspended at a final concentration of 500 mg 1 7 2 L -1 in AdDMEM/F12 supplemented with 10 mM Hepes, 1× penicillin/streptomycin, and 1× Glutamax; then the 1 7 3 sample was sonicated at a high intensity (3 × 5 min) using a Bioruptor sonicator (Diagenode). To obtain 1 7 4 organoid-MPF cocultures, the hAOs were split as described above, and then the organoid fragments were  To study the effects of MPFs on organoid growth, the control and exposed samples (1, 10, and 50 mg L -1 ) 1 8 8 were fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate-buffered 1 8 9 solution at pH 7.4. After several washes in the same buffer, the samples were post-fixed in 1% OsO 4 for 1.5 1 9 0 h at 4°C and then dehydrated in a graded ethanol series. As a final step, the organoids were treated with 1 9 1 hexamethyldisilazane for complete chemical dehydration. All samples were mounted onto standard 1 9 2 aluminum stubs, gold sputtered, and analyzed under a Zeiss LEO 1430 SEM at 20 kV. To investigate the possible changes in the inflammatory cytokine expression in hAOs after MPF exposure, 2 0 8 gene expression analysis by qRT-PCR was performed as described above at the end of the coculture with 2 0 9 the highest MPF concentration using the primers listed in Table S2. Considering that the exposure of MPFs 2 1 0 could induce oxidative stress in human tissues (Hu and Palić 2020), we analyzed the expression of genes 2 1 1 involved in oxidative stress pathways reported in relation to MPF exposure, including superoxide dismutase 2 1 2 family genes (SOD1 and SOD2), glutathione detox-related genes (GSTA1 and GPX1), catalase (CAT), and 2 1 3 ROS-controlling genes (NOX2, COX1, and ND1). In addition, the capacity of our 3D structures to respond to 2 1 4 standard inflammatory stimuli was evaluated after treatment with poly(I:C) (50 µg mL -1 ) as a positive control. For statistical analysis of the gene expression data, a two-way analysis of variance followed by Bonferroni's 2 1 8 multiple comparison test (α = 0.05) was performed. Photoshop was used to export the image graphs. similar gene expression profiles (compared to human pancreas organoids as a control).

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The organoids were analyzed by optical microscopy, confocal 3D construction, and SEM imaging.

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The normal organoids (control group) exhibited a spherical shape with a typical diameter of 200-300 µm and 2 2 7 an inner cavity (Figure 2A  The total weight of the MPFs removed from the air filter of a dryer machine was 2.3 g ( Figure S1), which was

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Interestingly, the shape of these MPFs differed at the transverse surface; no MPFs had a round profile along 2 4 7 the entire length, but they exhibited a varying profile from flat and twisted to tattered ( Figure 3C-E). Flat

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MPFs exhibited maximum widths of 21-24 µm along their widest dimension and a minimum height of 1-3 2 4 9 µm along their thinnest dimension. Furthermore, we observed that some fibers showed a rough surface, 2 5 0 where small pieces of <10 µm flaked off from the fiber ( Figure 3D) or a chapped ending was revealed ( Figure   2 5 1 3E), which could lead to the release of very small MPs from the fibers.

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Considering the average dimensions of the fibers described by SEM analyses, we obtained a fiber 2 5 3 volume of 28,485 µm 3 fiber -1 (633 µm × 22.5 µm × 2 µm) or 2.8 × 10 -5 mm 3 fiber -1 , indicating that the 2.3 g of 2 5 4 polyester fibers collected in the filter of a dryer machine contained 59 × 10 6 fibers (considering that the 2 5 5 density of polyester is 1.38 mg mm -3 ). Elemental analysis showed a C:O ratio of 74:26, which corresponds to 2 5 6 that of polyester (see Figure S2 for the EDS spectrum), thus confirming that the fibers were made of 2 5 7 polyester.
2 5 8 2 5 9 Effects of MPFs on hAOs 2 6 0 The hAOs exposed to MPFs were affected by all concentrations of MPFs (1, 10, and 50 mg L -1 ). Optical 2 6 1 microscopy, confocal 3D construction, and SEM image analyses revealed that the organoids exposed to (ciliated cells), and KRT5 (basal cells); no significant differences were observed between the organoids 2 7 3 exposed to MPFs and the control organoids ( Figure 5B). The only notable observation was a higher density 2 7 4 of polarization of the cytoskeleton near the fiber contact site, visible as an intensification of the red color in 2 7 5 Figure 6B. Owing to an organoid that had broken apart (likely during the transfer of the dehydrated organoid 2 7 6 onto the aluminum stub), we were able to take SEM images of the inner surface of the organoids and could 2 7 7 observe the same cellular differentiation (multiciliated and nonciliated cells) as the outer surface ( Figure 6E).

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Rather than depending on the MPF concentration, the observed effects varied with the degree of MPF-2 7 9 organoid contact, which presumably depended on the development phase of the organoids. The organoids 2 8 0 that made contact with fibers during maturation grew around the fibers and integrated them into their body 2 8 1 ( Figure 6F-H).

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Interestingly, the organoids closely bonded to an MPF formed an organoid-fiber interlacement or fully 2 8 3 encompassed the MPF. We further observed that some organoids grew polarized around the fibers. For covering a fiber with a thin layer stands in contrast to the images depicted in Figure 6F-H showing that cells 2 9 0 did not cover the fibers once they left the organoid. We also noticed that organoids in suspension with MPFs 2 9 1 can be damaged by fibers piercing the cell tissue ( Figure 7C). ( Figure 7A).

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This study presents an attempt to use human lung organoids for toxicological assessment of the total amount of MPFs released in the air flux can be even higher.

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The effects of MPs/NPs on human health have been tested previously using a variety of 2D cell 3 3 2 lines. The observed effects were very diverse and depended on the properties of the particle (e.g., size and person's lungs could be exposed to 26-130 airborne MPs a day. In addition, Vianello et al. (2019)

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Microplastic fibres from synthetic textiles: Environmental degradation and additive chemical content.   5  7  3   T  h  e  i  m  a  g  e  c  a  n  n  o  t  b  e  d  i  s  p  l  a  y  e  d  .  Y  o  u  r  c  o  m  p  u  t  e  r  m  a  y  n  o  t  h  a  v  e  e  n  o  u  g  h  m  e  m  o  r  y  t  o  o  p  e  n  t  h  e  i  m  a  g  e  ,  o  r  t  h  e  i  m  a  g  e  m  a  y  h  a  v  e  b  e  e  n  c  o  r  r  u  p  t  e  d  .  R  e  s  t  a  r  t  y  o  u  r  c  o  m  p  u  t  e  r  ,  a  n  d  t  h  e  n  o  p  e  n  t  h  e  f  i  l  e  a  g  a  i  n  .  I  f  t  h  e  r  e  d  x  s  t  i  l  l  a  p  p  e  a  r  s  ,  y  o  u  m  a  y  h  a  v  e  t  o  d  e  l  e  t  e  t  h  e  i  m  a  g  e  a  n  d  t  h  e  n  i  n  s  e  r  t  i  t  a  g  a  i  n .