A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations

Our plain-English summary of A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations, by P.K. Kabadi, A.L. Rodd, A.E. Simmons, et al (Particle and Fibre Toxicology, 2019).

The study

• Authors: P.K. Kabadi, A.L. Rodd, A.E. Simmons, N.J. Messier, R.H. Hurt, A.B. Kane
• Journal: Particle and Fibre Toxicology
• Published: 2019;16(1):15
• Cited by: 33 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm.

Results: As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins.

Conclusion: 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

In plain English

This was a laboratory study of cultured cells, not of people or animals. The authors built tiny three-dimensional clusters of human lung cells, called microtissues, of about 316 cells each. Each one combined lung bronchial epithelial cells (BEAS-2B), lung fibroblasts (IMR-90) and THP-1 cells turned into macrophages. The microtissues were exposed for 4 or 7 days to multi-walled carbon nanotubes, M120 carbon black nanoparticles, or crocidolite asbestos fibers, at 0.5 to 10 micrograms per mL. By day 7, cell survival fell as dose rose, and the nanotubes caused a drop of more than 40% at the highest dose. Cells touching asbestos fibers showed patches of cell death, but overall toxicity was lower than with nanotubes. Nanotubes and asbestos raised several of the same inflammation and matrix-remodeling genes, which carbon black did not raise. In four-day follow-up gene tests (qRT-PCR), increases in two such genes, MMP1 and MMP3, were statistically significant only for nanotubes. The authors concluded the model could help screen nanomaterials for possible fibrosis-causing effects. They noted it lacks some lung and immune cell types, links to other organs, and particle clearance.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study developing and testing a three-dimensional human lung cell culture model (microtissues) made from three human cell lines
• Who was studied: Human cell lines grown at Brown University: BEAS-2B lung bronchial epithelial cells, IMR-90 lung fibroblasts and THP-1 monocytic cells differentiated into macrophages. They were combined at a 4:1:1 ratio of macrophages to epithelial cells to fibroblasts, about 316 cells per microtissue.
• Exposure: Multi-walled carbon nanotubes (MWCNT-7, Mitsui), M120 carbon black nanoparticles (a non-fibrous reference material) and crocidolite asbestos fibers, at doses of 0.5 to 10 micrograms per mL, for 4 or 7 days. Gene and protein tests used the 10 micrograms per mL dose.
• Main results:
  • There was no significant decrease in microtissue viability on day 4. By day 7, toxicity rose with dose, and MWCNTs caused the greatest toxicity: a more than 40% decrease in viability at the highest dose.
  • In asbestos-exposed microtissues, focal cell death was seen in cells in direct contact with the fibers. Overall toxicity was lower than in MWCNT-exposed microtissues.
  • After 4 days at 10 micrograms per mL, collagen type 3 (COL3A1) gene expression, measured as mean fold change relative to untreated microtissues, was 6.33 plus or minus 0.65 with carbon nanotubes (p less than 0.001). It was 2.57 plus or minus 0.25 with asbestos (p = 0.002) and 1.33 plus or minus 0.15 with carbon black (p = 0.555).
  • Decorin gene expression (mean fold change) was 10.97 plus or minus 1.58 with carbon nanotubes (p less than 0.001) and 3.53 plus or minus 1.69 with asbestos (p = 0.065, not statistically significant). Collagen type I (COL1A1) was 3.64 plus or minus 0.82 with carbon nanotubes (p = 0.005) and 2.12 plus or minus 0.51 with asbestos (p = 0.062).
  • In the PCR array analysis, several highly upregulated genes, including MMP1, MMP3 and decorin, were induced by both asbestos and MWCNT exposure.
  • In the 4-day qRT-PCR dose-response tests, MMP1 and MMP3 gene expression increased with dose in asbestos-exposed microtissues, but statistically significant increases were seen only after exposure to carbon nanotubes.
  • In protein measurements after 4 days at 10 micrograms per mL, based on duplicate experiments, increased levels of IL-1 beta, TNF-alpha and IL-6 were detected in microtissues exposed to carbon nanotubes or asbestos fibers.
  • The genes induced by both MWCNTs and asbestos were not induced in microtissues exposed to carbon black.
• The authors' conclusion: The authors concluded that the 3D human lung microtissue model has potential use in a tiered testing strategy to detect potentially fibrosis-causing nanomaterials, and may reduce the need for expensive rodent inhalation studies.
• Limitations the authors state:
  • Some cell types, such as type I and II alveolar cells and other immune cells, are not included in the model.
  • Like any in vitro model, the microtissues lack cross-talk with other organ systems that contribute to whole-body inflammatory responses.
  • The microtissues lack the particle clearance mechanisms of a living lung, which may increase the model's sensitivity to biopersistent materials.
  • At the 4- and 7-day time points, the authors could not detect mature, cross-linked collagen. They noted that longer exposures, with ascorbic acid added, may be required.
  • The complexity of the model makes it harder to test many materials quickly.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Fibroblast, Macrophage, Crocidolite, Inflammation.

Related studies

• Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation — H. Barosova, S. Chortarea, et al. (2018)
• A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials — V.C. Sanchez, P. Weston, et al. (2011)
• Progressive lung cell reactions and extracellular matrix production after a brief exposure to asbestos — L.Y. Chang, L.H. Overby, et al. (1988)
• SWCNT suppress inflammatory mediator responses in human lung epithelium in vitro — E. Herzog, H.J. Byrne, et al. (2009)
• Cell and extracellular matrix interaction models in benign mesothelial and malignant pleural mesothelioma cells in 2D and 3D in-vitro — R.M. Jagirdar, E.D. Papazoglou, et al. (2021)
• Determination of cytotoxicity attributed to multiwall carbon nanotubes (MWCNT) in normal human embryonic lung cell (WI-38) line — J.S. Kim, K.S. Song, et al. (2010)

Browse more research in Particle and Fibre Toxicology, or return to medical research.