{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T07:44:01Z","timestamp":1793087041613,"version":"4.5.0"},"reference-count":111,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,24]],"date-time":"2025-04-24T00:00:00Z","timestamp":1745452800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p><jats:italic>Cryptosporidium hominis<\/jats:italic> is the dominant <jats:italic>Cryptosporidium<\/jats:italic> species infecting humans, but most advances in developing robust <jats:italic>in vitro<\/jats:italic> culturing platforms for <jats:italic>Cryptosporidium<\/jats:italic> have utilised <jats:italic>C. parvum<\/jats:italic>. Consequently, there is relatively little available information specific to the biology and life cycle of <jats:italic>C. hominis<\/jats:italic>. The present study utilised a pumpless and tubeless gut-on-chip to generate a physiologically relevant <jats:italic>in vitro<\/jats:italic> environment by applying a constant fluid shear stress of 0.02 dyn cm<jats:sup>-2<\/jats:sup> to HCT-8 cells. <\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Gut-on-chips were fabricated using standard soft lithography. <jats:italic>C. hominis<\/jats:italic> oocysts isolated from human pathology samples were used to infect the human ileocecal colorectal adenocarcinoma (HCT-8) cell line under a constant fluid shear stress of 0.02 dyn cm<jats:sup>-2<\/jats:sup>. Parasite growth was assessed using a <jats:italic>C. hominis<\/jats:italic>-specific quantitative PCR, a <jats:italic>Cryptosporidium<\/jats:italic> genus-specific immunofluorescence assay, and scanning electron microscopy. Differences in the HCT-8 transcriptome with and without fluid shear stress, and the host-parasite interaction, were both assessed using bulk transcriptomics.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Transcriptomic analysis of the HCT-8 cell line cultured within the gut-on-chip demonstrated a metabolic shift towards oxidative phosphorylation when compared to the same cell line cultured under static conditions. Extended <jats:italic>C. hominis<\/jats:italic> (subtype IdA15G1) cultures were sustained for up to 10 days within the gut-on-chip as shown by a <jats:italic>C. hominis<\/jats:italic>-specific qPCR and a <jats:italic>Cryptosporidium<\/jats:italic> genus-specific immunofluorescence assay, which demonstrated ~30-fold amplification in the gut-on-chip over the duration of the experiment. Scanning electron microscopy of infected monolayers identified trophozoites, meronts, merozoites, macrogamonts, microgamonts, and possible gamont-like stages at 48 h post-infection. The potential role of gamonts in the <jats:italic>Cryptosporidium<\/jats:italic> life cycle remains unclear and warrants further investigation. Transcriptomes of HCT-8 cells infected with <jats:italic>C hominis<\/jats:italic> revealed upregulation of biological processes associated with cell cycle regulation and cell signalling in <jats:italic>C. hominis<\/jats:italic>-infected cells under fluid shear stress compared to static culture. <\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>These data demonstrate that bioengineered gut-on-chip models support extended <jats:italic>C. hominis<\/jats:italic> growth and can be used to interrogate responses of host cells to infection. Owing to its relative simplicity, the pumpless and tubeless gut-on-chip can be accessible to most laboratories with established HCT-8 infection models for <jats:italic>Cryptosporidium<\/jats:italic> culture.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2025.1564806","type":"journal-article","created":{"date-parts":[[2025,4,24]],"date-time":"2025-04-24T05:29:21Z","timestamp":1745472561000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Microphysiological gut-on-chip enables extended in vitro development of Cryptosporidium hominis"],"prefix":"10.3389","volume":"15","author":[{"given":"Samantha","family":"Gunasekera","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Thierry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brendon","family":"King","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Monis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jillian M.","family":"Carr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abha","family":"Chopra","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"Watson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"O\u2019Dea","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edward","family":"Cheah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ramesh","family":"Ram","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peta L.","family":"Clode","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nawal","family":"Hijjawi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Una","family":"Ryan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,24]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.001693","article-title":"Impact of the COVID-19 restrictions on the epidemiology of Cryptosporidium spp. in England and Wales 2015\u20132021: a time series analysis","volume":"72","author":"Adamson","year":"2023","journal-title":"J. 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