Study population
This study was approved by the Institutional Review Board of Severance Hospital (IRB No. 2023-0425-001), and informed consent was obtained from all participants.
Endometrial Stromal Cell Isolation and Culture
Endometrial stromal cells (ESCs) were isolated from healthy human eutopic endometrial tissue. Tissue samples were minced and incubated in PBS containing 2.0 mg/mL collagenase type I (Gibco, Waltham, MA, USA) for 2 h at 37°C in a 5% CO2 incubator. The cells were then filtered through a 40-µm cell strainer (BD Biosciences, San Jose, CA, USA) and collected. The supernatant was discarded, and the cells were resuspended in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 2% penicillin-streptomycin (P/S; Cytiva, Marlborough, MA, USA) at 37°C in a 5% CO2 atmosphere. For passage, cells at 80–90% confluence were treated with 2 mL of 0.25% trypsin-EDTA (Gibco, Waltham, MA, USA) for 5 min in an incubator, and the reaction was stopped by adding 1 mL of culture medium. Cells from passages 3 to 6 were used for subsequent experiments.
Treatment of plastics on cells
ESCs were seeded at 3 × 105 cells/well in a 6-well plate and cultured to approximately 60% confluence. The medium was then exchanged to DMEM/F12 containing 100 nm NPs, 1 µm or 5 µm MPs (all from Merck, Darmstadt, Germany) for 24 h at 37°C in a 5% CO2 incubator.
Cell Proliferation Assay
ESCs were seeded at 6 × 103 cells/well in 96-well plates. After 24 h, the culture medium was replaced with DMEM/F12 containing increasing concentrations (from 0 to 10,000 µg/mL) of 100 nm NPs, 1 µm or 5 µm MPs (Merck, Darmstadt, Germany). Cell proliferation assays were performed by dropping 20 µL of Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) into each well, and incubating at 37°C for 2 h. The supernatants measured at an optical density (OD) of 450 nm using a VersaMax microplate reader (Molecular Devices, San Jose, CA USA).
Characterization of PS Particles
Green-fluorescent unmodified nanoplastics beads of 100 nm (1.05 g/mL solution) and microplastic beads of 1 µm (1.05 g/mL solution) were acquired from CD Bioparticles – Drug Delivery Research (NY, USA) for live-cell and confocal fluorescence microscopy imaging. The sizes, colors, and shapes of polystyrene (PS) particles were characterized using a scanning confocal microscope (LSM 980, Carl Zeiss, Oberkochen, Germany).
Live-Cell Imaging Assay
The IncuCyte Live-Cell Analysis System (Sartorius, Goettingen, Germany) was used to capture images of cells at set time points in real-time over long periods of time, and to perform analyses based on those images, such as creating videos. ESCs were seeded on poly-L-lysine (Merck, Darmstadt, Germany) -coated coverslips. when reached ~ 60% confluency, ESCs were treated with 100 µg/mL of 100 nm green-fluorescent NPs (CD Bioparticles, NY, USA) or 1 µm live MPs (CD Bioparticles, NY, USA), and then cells were loaded with a final concentration of 1 µM of NucSpot Live 650 Nuclear Stain (Biotium, San Francisco, USA) to stain nucleus for 10 min at 37°C in a 5% CO2 incubator. After that, the sample was incubated in an incubator equipped with an IncuCyte Live-Cell Analysis system for live-cell imaging.
Confocal Fluorescence microscopy for fixed cell imaging
After live-cell imaging, the media was removed, and cells were fixed with 4% paraformaldehyde (Forbio, Seoul, Korea) on a shaker (Daihan Scientific, Wonju, Korea) at 40 rpm for 15 min at room temperature. The cells were mounted with mounting medium (Dako, Seongnam, Korea) and examined with a confocal microscope (LSM 980, Carl Zeiss, Oberkochen, Germany) with an oil immersion objective lens (Plan-Apochromat 40x/1.4 oil DIC M27; Carl Zeiss Microscopy, GmbH).
Quantification of Polystyrene accumulation
To quantify the degree of plastic accumulation within the cells, fixed ESCs treated with nano- and microplastics were subjected to nuclear staining with NucSpot Live 650 Nuclear Stain (Biotium, San Francisco, USA) and mounted with mounting medium (Dako, Seongnam, Korea). Subsequently, the number of 1 µm MPs (green) (CD Bioparticles, NY, USA)- Nucspot650 (red) stained cells was counted and averaged from four randomly selected microscopic fields at 100x magnification observed with a confocal microscope (LSM 980, Carl Zeiss, Oberkochen, Germany). Due to the small size of 100 nm NPs, the number of 100 nm NPs (green) (CD Bioparticles, NY, USA)- Nucspot650 (red) stained cells was counted and averaged from eight randomly selected microscopic fields at 630x magnification observed with a confocal microscope (LSM 980, Carl Zeiss, Oberkochen, Germany).
Statistical analysis
GraphPad Prism program (GraphPad Software Inc, San Diego, CA, USA) was used to generate graphs and conduct statistical analyses. Data collected from at least three independent replicates were analyzed and expressed as means ± S.E.M. In vitro, differences between the control group and each NPs or MPs-treated group were analyzed using one-way ANOVA. Differences with a P-values less than 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001