Volume 50, June 2023, 102680

Introduction

Plastics are some of the most ubiquitous manmade materials and they have pervaded nearly every aspect of human life. It is estimated that 8.3 billion metric tons of virgin plastic has been generated as of 2017, and this number is expected to more than double by 2050.¹ Most plastics can break down into very small particles. Particles between 1 and 1000 μm in diameter are considered microplastics (MPs); while plastic particles between 1 and 100 nm (or up to 500 nm in some instances) are known as nanoplastics (NPs).² MPs/NPs have been consistently detected in seafood, various convenience foods and beverages, and in tap/bottle water.³ MPs/NPs are also distributed in and accumulate within living organisms in different tissues, including the digestive tract, blood, liver, pancreas, heart and, notably, the brain.⁴ In 2018, microplastics were first reported in human feces, providing evidence of accumulation, ingestion and excretion of these particles in humans and indicating exposure through processed or packaged food/water consumption.⁵ As human exposure to MPs and NPs continues to grow, MPs and NPs have been recognized as emerging contaminants within the last decade.

Ingested MPs/NPs first encounter the intestinal epithelium, the innermost layer of the mucosa, where most digestive, absorptive and secretory processes occur. This makes the intestine the primary exposure site for these particles and particularly important in terms of assessments of the impact of plastics on physiological functions.³ Investigations of the impact of MPs/NPs on organism health after oral uptake have been widely conducted in mammalian (rodent) models and reviewed. 6., 7., 8. In most of the cases, the effects in the gut correlated with size and morphology of the plastic particles, exposure concentration, exposure time, and tissue uptake and accumulation. However, there have also been studies that failed to demonstrate that MPs/NPs had a significant effect on animals. For example, a rat model showed a very low number of MPs taken up by intestinal tissue and the authors did not detect any histological lesions or significant inflammatory responses in the gut of the animals.¹⁰ Another study using rats to analyze potential neurobehavioral effects of PS NPs did not identify statistically significant behavioral changes or abnormalities.¹¹ These results represent an important clue to the accumulation and release of MPs/NPs following exposure to mammals, with remaining controversy regarding biological impact.

Despite the many studies on animals, toxicological evaluations of MPs/NPs on the human intestine in vivo are still lacking. As mentioned above, the intestine is considered the major route for particle exposure. The makeup of the native intestinal epithelium is diverse and populated with multiple differentiated epithelial cells, including enterocytes (absorption), goblet cells (mucus secretion), enteroendocrine cells (peptide and hormone production) (EECs), Paneth cells (antimicrobial protein secretion), and microfold cells (M cells, immune sensing and uptake of particulate microbial antigen).¹² In addition to its primary function of nutrient digestion and absorption, the intestinal epithelium also provides a protective barrier via apical intercellular junctions which protect against physical, chemical, and biological damages.¹³ Thus far, studies exploring the interactions between MPs/NPs and human intestinal cells have been mainly performed with monocultures of human colorectal adenocarcinoma epithelial cell-lines, Caco-2 (enterocyte-like cells) or HT29-MTX (goblet-like cells).14., 15. To achieve slightly more physiological conditions, many researchers have employed a Caco-2/HT29-MTX coculture system as an in vitro experimental model.¹⁶ To further improve upon this system, some researchers also include lymphoblast-like Raji-B cells to induce Caco-2 cells into microfold-like cells.¹⁷ Most of these studies, while revealing some level of cellular uptake and epithelial transport of MPs/NPs, showed either low or insignificant cytotoxic effects, except at very high concentrations of particles. Overall, these prior studies have not identified severe acute cytotoxic effects but have demonstrated the potential for low to moderate damage of the cells and tissues depending on the size and concentration of MPs/NPs. The degree of cellular uptake of the particles was somewhat consistent with the observations in animals. Of note, these in vitro findings were all from short-term studies (up to 48 h of exposure) with MPs/NPs exposed to human intestinal epithelial cell lines, whereas it can take up to 14 days for microplastics to pass through marine animals (compared to a normal digestion period of 2 days).¹⁸ Moreover, the intestinal epithelial cell cultures used for cytotoxicity studies were based on cancerous cell lines and were missing other important intestinal epithelial cell types. Consequently, these studies do not fully capture the cellular complexity of the native human intestine, thus, fall short of biological processes in vivo. Therefore, knowledge gaps still exist on the mechanisms and predictions regarding acute and chronic effects caused by these plastics in humans.

Thanks to advances in stem cell biology, organoids have been established as new model system for biomedical research and have been extensively reviewed.¹⁹ Organoids are in vitro miniaturized organ models originating from self-organizing stem cells. Compared to conventional cancerous cell lines, organoids are capable of physiologically mimicking the in vivo structure and function of an organ. They can be produced from adult (murine and human) stem cell-containing tissue samples, organ-specific single adult stem cells (ASCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs) and expanded indefinitely with the support of a cocktail of various growth factors in their culture media. Organoids can be grown to model many human organs, including the stomach, intestine, kidneys, heart, pancreas, brain, and liver. For the human intestine, organoids can be generated from fresh or frozen patient biopsies of intestinal tissue specimens by isolating crypts, the intestinal stem cell niche.²⁰ The isolation procedure yields spheroid-shaped and budding organoids which become ever-expanding in a 3D Matrigel in the presence of Wingless-related integration site (Wnt) pathway agonist, R-spondin (Rspo), epidermal growth factor (EGF), and the BMP inhibitor, Noggin.²¹ As these organoids contain stem cells, they have the ability to differentiate toward different intestinal epithelial cell lineages. For specific (and rare) cell types, the differentiation protocols for intestinal organoids have been updated and standardized to direct stem cell maintenance or differentiation toward specific cell fates. For example, by removing the Wnt, Noggin, and R-spondin from the culture media, the organoids simultaneously differentiate into a continuous intestinal epithelium containing enterocytes, goblet cells, EECs, and Paneth cells with an abundance that is similar to what is found in vivo.22., 23. By treating the organoids with nucleic factor-kappa B ligand (RANKL), a common M cell inducer, increased M-cell differentiation can be achieved in the cultures.²⁴ Over the past decade, intestinal organoids have been derived from healthy and diseased intestinal tissues and can be used as an alternative to Caco-2 drug absorption and permeability assays for drug screening and for personalized medical decisions to better predict patients' treatment response and, importantly, any potential drug toxicity.²⁵

We have previously used human intestinal organoids from both large and small intestine for intestinal tissue engineering,²⁶ inflammatory bowel disease modeling,²⁷ and host cell-microorganism interactions.²² In the present study, we explored the application of these organoids as a platform for predicting potential risks of MP and NP exposure. Specifically, the goals of this work were: 1) to establish and characterize two organoid-derived intestinal epithelial monolayer models: monolayers without M cells (enterocytes, goblet cells, EECs, and Paneth cells) and monolayers with M cells (enterocytes, goblet cells, EECs, Paneth cells, and M cells); 2) to compare the dynamics of cellular uptake and translocation of different sizes of MPs and NPs over time in these two different tissue models; 3) to evaluate the potential dose-, size- and exposure period-dependent cytotoxic effects of the plastic particles on the intestinal barrier integrity and immune response in the tissue models; 4) to distinguish the role of M cells regarding the transport of plastic particles across the intestinal epithelium and how M cells shape specific immune responses to these particles. In this work, PS MPs and NPs were selected as the model particles for the following reasons: 1) they are commercially available with a wide range of specific sizes; 2) they are fluorescently labeled, allowing for localization and tracking in cell compartments; and 3) they are biologically stable in various cell culture media.

Section snippets

Materials and methods

In this study, a comprehensive description of the materials and methods utilized is available in the Supplementary Materials section. This section offers an in-depth account of the experimental procedures, encompassing aspects such as the sourcing and characterization of PS MPs and NPs, establishment and characterization of the cell model, administration of particles, sample collection and characterization, and statistical analysis. The detailed description ensures a thorough understanding of

Characterization of PS MPs and NPs

PS MPs and NPs in different sizes (1 μm, 500 nm, 100 nm, and 30 nm) and fluorescently labeled are commercially available from Sigma Millipore. All the plastic particles diluted in DMEM were detected using fluorescence microscopy and SEM to validate the manufacturing sizes and the fluorescence. Microscopy images showed significant fluorescence signals from different sizes of particles confirming the fluorescence labels on the particles (Fig. 1). In addition, a uniform dispersion of particles

Discussion

Plastics are extensively used in our daily lives and are increasingly contributing to both land and water pollution. Over time, these plastics degrade and generate micro- or nano-sized plastic particles (MPs or NPs). In 2018, microplastics were first reported as present in human feces, providing evidence of accumulation, ingestion, and excretion of these particles in humans and indicating exposure through processed or packaged food/water consumption.⁵ Since then, plastics have been detected in

CRediT authorship contribution statement

Ying Chen: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing – original draft, Supervision, Project administration, Funding acquisition. Ashleigh M. Williams: Methodology, Validation, Formal analysis, Investigation, Writing – review & editing. Edward B. Gordon: Methodology, Validation, Formal analysis, Investigation. Sara E. Rudolph: Investigation, Writing – review & editing. Brooke N. Longo: Methodology, Investigation. Gang Li: Methodology, Writing

Acknowledgements

This work was performed in part at the Harvard University Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI). Figs. 2A and 3A were created with BioRender.com

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