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Hoʻokumu ka morphogenesis ʻōpū kanaka i nā hiʻohiʻona crypt-villus o ka microarchitecture epithelial 3D a me ka hoʻonohonoho spatial. Pono kēia ʻano kū hoʻokahi e mālama i ka homeostasis ʻōpū ma ka pale ʻana i ka niche cell stem i loko o ka crypt basal mai nā antigens microbial exogenous a me kā lākou metabolites. Eia kekahi, hōʻike ka villi ʻōpū a me ka mucus huna i nā cell epithelial i hoʻokaʻawale ʻia me kahi pale pale ma ka ʻili mucosal ʻōpū. No laila, he mea koʻikoʻi ka hana hou ʻana i nā ʻano epithelial 3D no ke kūkulu ʻana i nā hiʻohiʻona ʻōpū in vitro. ʻIke ʻia, hiki i ka gut-on-a-chip mimetic organic ke hoʻoulu i ka morphogenesis 3D spontaneous o ka epithelium ʻōpū me nā hana physiological i hoʻonui ʻia a me nā biomechanics. Maanei, hāʻawi mākou i kahi protocol reproducible e hoʻoulu ikaika i ka morphogenesis ʻōpū i loko o ka ʻōpū ma kahi chip microfluidic a me kahi chip hybrid i hoʻokomo ʻia ʻo Transwell. Hōʻike mākou i nā ʻano kikoʻī no ka hana ʻana i nā mea hana, ka mahi ʻana o Caco-2 a i ʻole nā ​​​​cell epithelial organoid intestinal i nā hoʻonohonoho maʻamau a ma kahi kahua microfluidic, ka hoʻokomo ʻana o 3D morphogenesis, a me ke ʻano o ka epithelia 3D i hoʻokumu ʻia me ka hoʻohana ʻana i nā modalities kiʻi he nui. ʻO kēia protocol hoʻokō i ka hana hou ʻana o ka microarchitecture gut hana ma o ka kaohi ʻana i ke kahe wai basolateral no 5 d. Hoʻohana kā mākou ʻano morphogenesis in vitro i ke koʻikoʻi shear pili physiologically a me ka neʻe mechanical a ʻaʻole koi i ka ʻenekinia cell paʻakikī a i ʻole ka manipulation, kahi e ʻoi aku ka maikaʻi o nā ʻenehana ʻē aʻe. Ke manaʻo nei mākou e hiki i kā mākou protocol i manaʻo ʻia ke loaʻa nā hopena ākea no ke kaiāulu noiʻi biomedical, e hāʻawi ana i kahi ʻano e hana hou ai i nā papa epithelial intestinal 3D in vitro no nā noi biomedical, clinical, a me nā lāʻau lapaʻau.
Hōʻike nā hoʻokolohua e hiki i nā pūnaewele epithelial Caco-2 intestinal i mahi ʻia i loko o nā gut-on-a-chip1,2,3,4,5 a i ʻole nā ​​​​​​mea hana microfluidic bilayer6,7 ke hana i ka morphogenesis 3D spontaneous in vitro me ka ʻole o ka hoʻomaopopo maopopo ʻana i ke ʻano kumu. I kā mākou noiʻi hou, ua ʻike mākou he mea pono ka wehe ʻana i nā antagonists morphogen i hūnā ʻia e basolaterally mai nā mea hana moʻomeheu a lawa ia e hoʻoulu ai i ka morphogenesis epithelial 3D in vitro, ka mea i hōʻike ʻia e Caco-2 a me nā organoids intestinal i loaʻa mai ka mea maʻi. Ua hōʻoia ʻia nā pūnaewele epithelial. Ma kēia haʻawina, ua kālele pono mākou i ka hana ʻana o nā cell a me ka hoʻolaha ʻana o ka nui o kahi antagonist Wnt ikaika, ʻo Dickkopf-1 (DKK-1), i loko o nā mea hana microfluidic gut-on-a-chip a me nā mea i hoʻololi ʻia me nā mea hoʻokomo Transwell, i kapa ʻia ʻo "Hybrid Chip". Hōʻike mākou i ka hoʻohui ʻana o nā antagonists Wnt exogenous (e like me DKK-1, Wnt repressor 1, huna ʻia ka protein frizzled-related 1, a i ʻole Soggy-1) i ka ʻōpū on-chip e kāohi ana i ka morphogenesis a hoʻopilikia paha i ka papa epithelial 3D i hoʻonohonoho mua ʻia, e hōʻike ana i ke kaumaha antagonistic i ka wā o ka moʻomeheu ke kuleana no ka morphogenesis ʻōpū in vitro. No laila, ʻo kahi ala kūpono e hoʻokō ai i ka morphogenesis ikaika ma ka interface epithelial, ʻo ia ka wehe ʻana a i ʻole ka mālama liʻiliʻi ʻana i nā pae o nā antagonists Wnt i loko o ka ʻāpana basolateral ma o ka flushing hana (e laʻa, i loko o nā kahua gut-on-a-chip a i ʻole hybrid-on-a-chip) a i ʻole ka diffusion. Nā media Basolateral (e laʻa, mai nā mea hoʻokomo Transwell i loko o nā waihona basolateral nui i loko o nā luawai).
Ma kēia protocol, hāʻawi mākou i kahi ʻano kikoʻī no ka hana ʻana i nā microdevices gut-on-a-chip a me nā ʻāpana hybrid Transwell-insertable (nā ʻanuʻu 1-5) e hoʻoulu i nā cell epithelial intestinal ma nā membrane porous polydimethylsiloxane (PDMS) (nā ʻanuʻu 6A, 7A, 8, 9) a i ʻole nā ​​membrane polyester o nā mea hoʻokomo Transwell (nā ʻanuʻu 6B, 7B, 8, 9) a hoʻoulu i ka morphogenesis 3D ma vitro (kaʻanuʻu 10). Ua ʻike pū mākou i nā hiʻohiʻona cellular a me nā molekala e hōʻike ana i ka histogenesis kikoʻī a me ka hoʻokaʻawale ʻana o ke kelepona ma muli o ka lineage ma o ka hoʻopili ʻana i nā modalities kiʻi he nui (nā ʻanuʻu 11-24). Hoʻoulu mākou i ka morphogenesis me ka hoʻohana ʻana i nā cell epithelial intestinal kanaka, e like me Caco-2 a i ʻole nā ​​​​organoids intestinal, ma nā ʻano moʻomeheu ʻelua me nā kikoʻī loea e pili ana i ka hoʻololi ʻana o ka ʻili o nā membrane porous, ka hana ʻana o nā monolayers 2D, a me ka biochemical intestinal a me ka hana hou ʻana o ka microenvironment biomechanical. ma vitro. No ka hoʻoulu ʻana i ka morphogenesis 3D mai nā monolayers epithelial 2D, ua wehe mākou i nā antagonists morphogen ma nā ʻano i hoʻoulu ʻia ma ke kahe ʻana. ʻO ka mea hope loa, hāʻawi mākou i kahi hōʻike o ka pono o kahi papa epithelial 3D regenable hiki ke hoʻohana ʻia e hoʻohālike i ka ulu ʻana o ka epithelial morphogen-dependent, longitudinal host-microbiome co-cultures, pathogen infection, inflammatory injury, epithelial barrier dysfunction, a me nā probiotic-based therapies Example.influences.
Hiki i kā mākou protocol ke lilo i mea pono i kahi ākea o nā kānaka ʻepekema ma ke kumu (e laʻa, ka biology mucosal intestinal, ka biology cell stem, a me ka biology developmental) a me ka noiʻi i hoʻopili ʻia (e laʻa, ka hoʻāʻo ʻana i ka lāʻau lapaʻau preclinical, ka hoʻohālike ʻana i nā maʻi, ka ʻenekinia ʻiʻo, a me ka gastroenterology) ka hopena ākea. Ma muli o ka hana hou ʻana a me ka paʻa o kā mākou protocol e hoʻoulu i ka 3D morphogenesis o ka epithelium intestinal in vitro, ke manaʻo nei mākou e hiki ke hoʻolaha ʻia kā mākou hoʻolālā loea i ka poʻe hoʻolohe e aʻo ana i ka dynamics o ka hōʻailona cell i ka wā o ka hoʻomohala ʻana o ka ʻōpū, ka hana hou ʻana a i ʻole ka homeostasis. Eia kekahi, he mea pono kā mākou protocol no ka nīnau ʻana i ka maʻi ma lalo o nā ʻano maʻi like ʻole e like me Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 a i ʻole Vibrio cholerae. He mea pono nō hoʻi ka poʻe hoʻolohe o ka pathology maʻi a me ka pathogenesis. ʻO ka hoʻohana ʻana i kahi ʻōnaehana microphysiology gut on-chip e ʻae i ka longitudinal co-culture 10 a me ka loiloi ma hope o ka pale ʻana o ka host, nā pane pale a me ka hoʻoponopono ʻana i nā hōʻeha e pili ana i ka pathogen i loko o ka gastrointestinal (GI) tract 11. Hiki ke hoʻohālike ʻia nā maʻi GI ʻē aʻe e pili ana i ka leaky gut syndrome, ka maʻi celiac, ka maʻi Crohn, ka ulcerative colitis, pouchitis, a i ʻole ka irritable bowel syndrome i ka wā e hoʻomākaukau ʻia ai nā papa epithelial intestinal 3D me ka hoʻohana ʻana i nā papa epithelial intestinal 3D o ka mea maʻi, ʻo kēia mau maʻi e komo pū ana me ka villous atrophy, crypt shortening, mucosal damage, a i ʻole ka pale epithelial impaired. Biopsy a i ʻole nā ​​​​organoids intestinal i loaʻa mai nā cell stem 12,13. No ka hoʻohālike maikaʻi ʻana i ka paʻakikī kiʻekiʻe o ke kaiapuni maʻi, hiki i ka poʻe heluhelu ke noʻonoʻo e hoʻohui i nā ʻano cell e pili ana i ka maʻi, e like me nā cell mononuclear koko peripheral maʻi (PBMCs), i nā hiʻohiʻona e loaʻa ana nā microarchitectures villus-crypt intestinal 3D. nā cell immune kikoʻī, 5.
Ma muli o ka hiki ke hoʻopaʻa ʻia a ʻike ʻia ka microstructure epithelial 3D me ka ʻole o ke kaʻina hana ʻokiʻoki, makemake paha nā mea nānā e hana ana ma ka transcriptomics spatial a me ke kiʻi kiʻekiʻe-resolution a i ʻole super-resolution i kā mākou palapala ʻāina o ka dynamics spatiotemporal o nā genes a me nā protein ma nā niches epithelial. Makemake i ka ʻenehana. Pane i nā microbial a i ʻole nā ​​​​​​mea hoʻouluulu pale. Eia kekahi, hiki ke hoʻokumu ʻia ka longitudinal host-microbiome crosstalk 10, 14 e hoʻonohonoho ana i ka homeostasis gut i loko o ka papa mucosal intestinal 3D ma o ka hoʻoulu pū ʻana i nā ʻano microbial like ʻole, nā kaiāulu microbial a i ʻole ka fecal microbiota, ʻoi aku hoʻi i ka gut-on-a-chip. ma ke kahua. He mea hoihoi loa kēia ʻano hana i ka poʻe hoʻolohe e aʻo ana i ka mucosal immunology, gastroenterology, human microbiome, culturomics a me ka clinical microbiology e ʻimi nei e mahi i ka microbiota gut i hoʻoulu ʻole ʻia ma ka hale hana. Inā hiki ke hoʻololi ʻia kā mākou protocol morphogenesis in vitro i nā ʻano moʻomeheu scalable, e like me nā hoʻokomo multiwell i loko o nā papa 24, 96 a i ʻole 384 e hoʻopiha mau ana i nā keʻena basolateral, hiki ke hoʻolaha ʻia ka protocol i ka poʻe e hoʻomohala ana i nā kahua lāʻau lapaʻau, biomedical A i ʻole high-throughput screening a i ʻole validation no ka ʻoihana meaʻai. Ma ke ʻano he hōʻoia-o-kumumanaʻo, ua hōʻike koke mākou i ka hiki ke hana i kahi ʻōnaehana morphogenesis multiplex high-throughput scalable i kahi ʻano papa 24-well. Eia kekahi, ua kūʻai ʻia nā huahana organ-on-a-chip he nui16,17,18. No laila, hiki ke hoʻolalelale ʻia ka hōʻoia ʻana o kā mākou ʻano morphogenesis in vitro a hiki ke lawe ʻia e nā keʻena noiʻi he nui, ʻoihana a aupuni a me nā keʻena hoʻoponopono e hoʻomaopopo i ka reprogramming cellular o ka in vitro gut morphogenesis ma ka pae transcriptomic e hoʻāʻo i nā lāʻau lapaʻau a i ʻole biotherapeutics ʻO ka absorption a me ka halihali ua loiloi ʻia nā moho lāʻau lapaʻau me ka hoʻohana ʻana i nā mea pani ʻōpū 3D a i ʻole e hoʻohana ana i nā hiʻohiʻona organ-on-a-chip maʻamau a ʻoihana paha e loiloi i ka hana hou ʻana o ke kaʻina hana morphogenesis ʻōpū.
Ua hoʻohana ʻia kahi helu palena o nā hiʻohiʻona hoʻokolohua pili kanaka e aʻo ai i ka morphogenesis epithelial intestinal, ma muli o ka nele o nā protocols hiki ke hoʻokō ʻia e hoʻoulu i ka morphogenesis 3D i loko o vitro. ʻO ka ʻoiaʻiʻo, ʻo ka hapa nui o ka ʻike o kēia manawa e pili ana i ka morphogenesis gut e pili ana i nā haʻawina holoholona (e laʻa, zebrafish20, mice21 a i ʻole moa22). Eia nō naʻe, he hana nui lākou a he kumukūʻai nui, hiki ke kānalua ʻia ke ʻano pono, a ʻo ka mea nui loa, ʻaʻole e hoʻoholo pololei i nā kaʻina hana hoʻomohala kanaka. He palena loa kēia mau hiʻohiʻona i ko lākou hiki ke hoʻāʻo ʻia ma ke ʻano scalable multi-way. No laila, ʻoi aku ka maikaʻi o kā mākou protocol no ka hoʻoulu hou ʻana i nā ʻano kiko 3D i loko o vitro ma mua o nā hiʻohiʻona holoholona vivo a me nā hiʻohiʻona moʻomeheu cell 2D static kuʻuna. E like me ka mea i wehewehe mua ʻia, ʻo ka hoʻohana ʻana i nā ʻano epithelial 3D i ʻae iā mākou e nānā i ka localization spatial o nā cell i hoʻokaʻawale ʻia i loko o ka axis crypt-villus i ka pane ʻana i nā ʻano mucosal a i ʻole nā ​​​​​​mea hoʻouluulu immune. Hiki i nā papa epithelial 3D ke hāʻawi i kahi e aʻo ai pehea e hoʻokūkū ai nā cell microbial e hana i nā niches spatial a me ka evolution ecological i ka pane ʻana i nā mea hoʻokipa (e laʻa, nā papa mucus i loko a i waho, ka huna ʻana o IgA a me nā antimicrobial peptides). Eia kekahi, hiki i ke ʻano 3D epithelial ke ʻae iā mākou e hoʻomaopopo pehea e hoʻonohonoho ai ka microbiota gut i kona mau kaiāulu a hana synergistically i nā metabolites microbial (e laʻa me nā waikawa momona kaula pōkole) e hoʻopili ana i ka hoʻonohonoho cellular a me nā niches cell stem i loko o nā crypts basal. Hiki ke hōʻike ʻia kēia mau hiʻohiʻona ke hoʻokumu ʻia nā papa epithelial 3D ma vitro.
Ma waho aʻe o kā mākou ʻano hana o ka hana ʻana i nā ʻano epithelial intestinal 3D, aia kekahi mau ʻano hana in vitro. ʻO ka moʻomeheu organoid intestinal kahi ʻenehana ʻenekinia ʻiʻo kiʻekiʻe loa e pili ana i ka mahi ʻana o nā cell kumu intestinal ma lalo o nā kūlana morphogen kikoʻī23,24,25. Eia nō naʻe, ʻo ka hoʻohana ʻana i nā hiʻohiʻona organoid 3D no ka nānā ʻana i ka halihali a i ʻole nā ​​​​​​hui host-microbiome co-cultures he mea paʻakikī pinepine no ka mea ua hoʻopuni ʻia ka lumen intestinal i loko o ka organoid a, no laila, ua kaupalena ʻia ka hoʻolauna ʻana o nā ʻāpana luminal e like me nā cell microbial a i ʻole nā ​​​​​​antigens exogenous. Hiki ke hoʻomaikaʻi ʻia ke komo ʻana i nā lumens organoid me ka hoʻohana ʻana i kahi microinjector,26,27 akā he invasive a hana nui kēia ʻano hana a koi i ka ʻike kūikawā e hana ai. Eia kekahi, ʻo nā moʻomeheu organoid kuʻuna i mālama ʻia i loko o nā scaffolds hydrogel ma lalo o nā kūlana static ʻaʻole hōʻike pololei i ka biomechanics in vivo.
ʻO nā ʻano hana ʻē aʻe i hoʻohana ʻia e kekahi mau hui noiʻi e hoʻohana i nā scaffolds hydrogel 3D i hoʻonohonoho mua ʻia e hoʻohālike i ke ʻano epithelial gut ma o ka hoʻoulu ʻana i nā cell intestinal kanaka i hoʻokaʻawale ʻia ma ka ʻili gel. Hana i nā scaffolds hydrogel me ka hoʻohana ʻana i nā ʻōmole 3D-paʻi ʻia, micro-milled, a i ʻole lithographically i hana ʻia. Hōʻike kēia ʻano hana i ka hoʻonohonoho ponoʻī o nā cell epithelial i hoʻokaʻawale ʻia i loko o vitro me nā gradients morphogen pili physiologically, e hoʻokumu ana i kahi ʻano epithelial ratio kiʻekiʻe a me ka crosstalk stroma-epithelial ma ka hoʻokomo ʻana i nā cell stromal i loko o ka scaffold. Eia nō naʻe, ʻo ke ʻano o nā scaffolds i hoʻonohonoho mua ʻia e pale aku i ka hōʻike ʻana o ke kaʻina hana morphogenetic spontaneous ponoʻī. ʻAʻole hāʻawi kēia mau hiʻohiʻona i ka luminal dynamic a i ʻole ke kahe interstitial, nele i ke koʻikoʻi shear fluid e pono ai nā cell intestinal e hana i ka morphogenesis a loaʻa ka hana physiological. Ua hoʻohana kekahi noiʻi hou i nā scaffolds hydrogel i loko o kahi kahua microfluidic a me nā ʻano epithelial intestinal patterned me ka hoʻohana ʻana i nā ʻenehana laser-etching. Hahai nā organoids intestinal mouse i nā ʻano etched e hana i nā ʻano tubular intestinal, a hiki ke hana hou ʻia ke kahe wai intraluminal me ka hoʻohana ʻana i kahi module microfluidics. Eia naʻe, ʻaʻole hōʻike kēia kumu hoʻohālike i ka spontaneous. ʻaʻole hoʻi e komo pū nā kaʻina hana morphogenetic a ʻaʻole hoʻi e komo pū me nā neʻe ʻana o ka mechanobiological gut. Ua hiki i nā ʻenehana paʻi 3D mai ka hui like ke hana i nā ʻōmole ʻōpū liʻiliʻi me nā kaʻina hana morphogenetic spontaneous. ʻOiai ka hana paʻakikī o nā ʻāpana ʻōpū like ʻole i loko o ka ʻōmole, nele pū kēia kumu hoʻohālike i ke kahe wai luminal a me ka deformation mechanical. Eia kekahi, hiki ke kaupalena ʻia ka hana hoʻohālike, ʻoiai ma hope o ka pau ʻana o ke kaʻina hana bioprinting, e hoʻopilikia ana i nā kūlana hoʻokolohua a i ʻole nā ​​​​​​pilina cell-to-cell. Akā, hāʻawi kā mākou protocol i manaʻo ʻia i ka morphogenesis gut spontaneous, ke kaumaha shear pili physiologically, biomechanics e hoʻohālike i ka neʻe ʻana o ka ʻōpū, ke komo ʻana o nā ʻāpana apical a me basolateral kūʻokoʻa, a me ka hana hou ʻana o nā microenvironments biological paʻakikī o ka modularity. No laila, hiki i kā mākou protocol morphogenesis 3D in vitro ke hāʻawi i kahi ala hoʻohui e lanakila ai i nā pilikia o nā ʻano hana e kū nei.
Ke kālele piha nei kā mākou protocol ma ka 3D epithelial morphogenesis, me nā cell epithelial wale nō i ka moʻomeheu a ʻaʻohe ʻano ʻē aʻe o nā cell e hoʻopuni ana e like me nā cell mesenchymal, nā cell endothelial, a me nā cell immune. E like me ka mea i wehewehe mua ʻia, ʻo ke kumu o kā mākou protocol ka hoʻokomo ʻana o ka epithelial morphogenesis ma ka wehe ʻana i nā mea hoʻopaʻa morphogen i hūnā ʻia ma ka ʻaoʻao basolateral o ka medium i hoʻolauna ʻia. ʻOiai ʻo ka modularity ikaika o kā mākou gut-on-a-chip a me ka hybrid-on-a-chip e hiki ai iā mākou ke hana hou i ka papa epithelial 3D undulating, nā paʻakikī olaola hou e like me nā pilina epithelial-mesenchymal33,34, extracellular Matrix (ECM) deposition 35 a, i kā mākou kumu hoʻohālike, nā hiʻohiʻona crypt-villus e lawe ana i nā niches cell stem i nā crypts basal e mau ana e noʻonoʻo hou ʻia. ʻO nā cell Stromal (e laʻa, fibroblasts) i loko o ka mesenchyme e pāʻani i kahi kuleana koʻikoʻi i ka hana ʻana o nā protein ECM a me ka hoʻoponopono ʻana o ka morphogenesis intestinal i vivo35,37,38. ʻO ka hoʻohui ʻana o nā cell mesenchymal i kā mākou kumu hoʻohālike i hoʻonui i ke kaʻina hana morphogenetic a me ka pono o ka hoʻopili ʻana o ke kelepona. ʻO ka papa endothelial (ʻo ia hoʻi, nā capillaries a i ʻole nā ​​lymphatics) he kuleana koʻikoʻi i ka hoʻoponopono ʻana i ka halihali molekala39 a me ka hoʻouluulu ʻana o nā cell immune40 i loko o ka microenvironment gut. Eia kekahi, ʻo nā ʻāpana vasculature i hiki ke hoʻopili ʻia ma waena o nā hiʻohiʻona ʻiʻo he mea pono ia i ka wā e hoʻolālā ʻia ai nā hiʻohiʻona ʻiʻo e hōʻike i nā pilina multi-organ. No laila, pono paha e hoʻokomo ʻia nā cell endothelial e hoʻohālike i nā hiʻohiʻona physiological pololei me ka hoʻonā pae organ. He mea nui nō hoʻi nā cell immune i loaʻa mai ka maʻi no ka hōʻike ʻana i nā pane immune innate, ka hōʻike antigen, ka innate adaptive immune crosstalk, a me ka pale ʻana o ka ʻiʻo i ke ʻano o ka hoʻohālike ʻana i ka maʻi ʻōpū.
ʻOi aku ka maʻalahi o ka hoʻohana ʻana i nā ʻāpana hybrid ma mua o ka gut-on-a-chip no ka mea ʻoi aku ka maʻalahi o ka hoʻonohonoho ʻana o ka hāmeʻa a ʻo ka hoʻohana ʻana i nā hoʻokomo Transwell e ʻae ai i ka moʻomeheu scalable o ka epithelium gut. Eia nō naʻe, ʻaʻole elastic nā hoʻokomo Transwell i loaʻa i ke kālepa me nā membrane polyester a ʻaʻole hiki ke hoʻohālike i nā neʻe peristaltic-like. Eia kekahi, ua noho paʻa ka ʻāpana apical o ka hoʻokomo Transwell i kau ʻia i loko o ka ʻāpana hybrid me ka ʻole o ke kaumaha shear ma ka ʻaoʻao apical. ʻIke loa, ʻaʻole hiki i nā waiwai static i loko o ka ʻāpana apical ke hiki ke hoʻoulu i ka co-culture bacteria lōʻihi i nā ʻāpana hybrid. ʻOiai hiki iā mākou ke hoʻoulu ikaika i ka 3D morphogenesis i nā hoʻokomo Transwell i ka wā e hoʻohana ai i nā ʻāpana hybrid, ʻo ka nele o nā biomechanics pili physiologically a me ke kahe wai apical e kaupalena i ka hiki ke hoʻohana ʻia nā kahua ʻāpana hybrid no nā noi hiki.
ʻAʻole i hoʻokumu piha ʻia nā hana hou piha o ke axis crypt-villus kanaka i loko o nā moʻomeheu gut-on-a-chip a me nā hybrid-on-a-chip. ʻOiai ua hoʻomaka ka morphogenesis mai kahi monolayer epithelial, ʻaʻole pono nā microarchitectures 3D e hāʻawi i ka like morphological i nā crypts in vivo. ʻOiai ua wehewehe mākou i ka heluna cell proliferating kokoke i ka basal crypt domain i loko o ka microengineered 3D epithelium, ʻaʻole i hoʻokaʻawale maopopo ʻia nā ʻāpana crypt a me villous. ʻOiai ʻo nā kahawai kiʻekiʻe ma luna o ka chip e alakaʻi i ka hoʻonui ʻia o ke kiʻekiʻe o ka epithelium microengineered, ua kaupalena ʻia ke kiʻekiʻe kiʻekiʻe loa i ~ 300-400 µm. ʻO ka hohonu maoli o nā crypts intestinal kanaka i loko o nā ʻōpū liʻiliʻi a me nā ʻōpū nui he ~ 135 µm a me ~ 400 µm, kēlā me kēia, a ʻo ke kiʻekiʻe o ka villi intestinal liʻiliʻi he ~ 600 µm41.
Mai kahi kuanaʻike kiʻi, hiki ke kaupalena ʻia ke kiʻi ʻana i ka super-resolution in situ o nā microarchitectures 3D i ka ʻōpū ma kahi chip, ʻoiai ʻo ka mamao hana e pono ai mai ka lens objective a i ka papa epithelial ma ke kauoha o kekahi mau millimeters. No ka lanakila ʻana i kēia pilikia, pono paha kahi pahuhopu mamao. Eia kekahi, he paʻakikī ka hana ʻana i nā ʻāpana lahilahi no ka hoʻomākaukau ʻana i nā specimen kiʻi ma muli o ke kiʻekiʻe o ka elasticity o PDMS. Eia kekahi, ʻoiai ʻo ka microfabrication layer-by-layer o ka ʻōpū ma kahi chip e pili ana i ka hoʻopili mau ʻana ma waena o kēlā me kēia papa, he mea paʻakikī loa ka wehe ʻana a wehe paha i ka papa o luna e nānā i ke ʻano o ka ʻili o ka papa epithelial. No ka laʻana, ma ka hoʻohana ʻana i kahi microscope electron scanning (SEM).
ʻO ka hydrophobicity o PDMS kahi mea palena i nā haʻawina microfluidic e pili ana i nā mole liʻiliʻi hydrophobic, ʻoiai hiki i ka PDMS ke adsorb nonspecifically i kēlā mau mole hydrophobic. Hiki ke noʻonoʻo ʻia nā koho ʻē aʻe i ka PDMS me nā mea polymeric ʻē aʻe. ʻOkoʻa, hiki ke noʻonoʻo ʻia ka hoʻololi ʻana o ka ʻili o PDMS (e laʻa, ka uhi ʻana me nā mea lipophilic 42 a i ʻole poly (ethylene glycol) 43) e hōʻemi i ka adsorption o nā mole hydrophobic.
ʻO ka mea hope loa, ʻaʻole i wehewehe maikaʻi ʻia kā mākou ʻano hana ma ke ʻano o ka hāʻawi ʻana i kahi kānana throughput kiʻekiʻe a i ʻole kahi kahua hoʻokolohua "one-size-fits-all" e aloha ʻia e ka mea hoʻohana. Pono ke kaʻina hana o kēia manawa i kahi pamu syringe no kēlā me kēia microdevice, kahi e lawe ai i kahi i loko o kahi incubator CO2 a pale i nā hoʻokolohua nui. Hiki ke hoʻomaikaʻi nui ʻia kēia palena e ka scalability o nā ʻano moʻomeheu hou (e laʻa, 24-well, 96-well, a i ʻole 384-well porous inserts e ʻae i ka hoʻopiha hou ʻana a me ka wehe ʻana i nā media basolateral).
No ka hoʻoulu ʻana i ka morphogenesis 3D o ka epithelium ʻōpū kanaka ma vitro, ua hoʻohana mākou i kahi mea hana ʻōpū microfluidic chip e loaʻa ana ʻelua microchannels parallel a me kahi membrane porous elastic ma waena e hana i kahi interface lumen-capillary. Hōʻike pū mākou i ka hoʻohana ʻana i kahi mea hana microfluidic hoʻokahi-channel (kahi chip hybrid) e hāʻawi ana i ke kahe basolateral mau ma lalo o nā papa epithelial polarized i ulu ʻia ma nā inserts Transwell. Ma nā kahua ʻelua, hiki ke hōʻike ʻia ka morphogenesis o nā ʻano cell epithelial ʻōpū kanaka ma ka hoʻopili ʻana i ka manipulation directional o ke kahe e wehe i nā antagonists morphogen mai ka ʻāpana basolateral. ʻO ke kaʻina hana hoʻokolohua holoʻokoʻa (Kiʻi 1) he ʻelima mau ʻāpana: (i) microfabrication o ka chip gut a i ʻole ka chip hybrid Transwell insertable (nā ʻanuʻu 1-5; Pahu 1), (ii) ka hoʻomākaukau ʻana o nā cell epithelial ʻōpū (nā cell Caco-2) a i ʻole nā ​​​​organoids ʻōpū kanaka; pahu 2-5), (iii) ka moʻomeheu o nā pūnaewele epithelial intestinal ma nā ʻāpana intestinal a i ʻole nā ​​​​ʻāpana hybrid (nā ʻanuʻu 6-9), (iv) ka hoʻokomo ʻana o ka 3D morphogenesis in vitro (kaʻanuʻu 10) a me (v)) e wehewehe i ka microstructure epithelial 3D (nā ʻanuʻu 11-24). ʻO ka hope loa, ua hoʻolālā ʻia kahi hui hoʻomalu kūpono (i kūkākūkā hou ʻia ma lalo) e hōʻoia i ka pono o ka morphogenesis in vitro ma ka hoʻohālikelike ʻana i ka epithelial morphogenesis i nā kaohi spatial, temporal, conditional, a i ʻole nā ​​​​kaʻina hana.
Ua hoʻohana mākou i ʻelua mau kahua moʻomeheu like ʻole: gut-on-a-chip me nā kahawai pololei a i ʻole nā ​​​​kahawai convoluted nonlinear, a i ʻole nā ​​​​​​chips hybrid e loaʻa ana nā hoʻokomo Transwell (TW) i loko o kahi mea microfluidic, i hana ʻia e like me ka mea i wehewehe ʻia ma ka Pahu 1, a me ka hana 1 -5. "Hōʻike ka Hana Hana" i nā ʻanuʻu nui i ka hana ʻana i kahi chip hoʻokahi a i ʻole kahi chip hybrid. "Culture of Human Intestinal Epithelial Cells" wehewehe i ke kumu cell (Caco-2 a i ʻole nā ​​​​organoids intestinal kanaka) a me ke kaʻina hana moʻomeheu i hoʻohana ʻia ma kēia protocol. "In vitro morphogenesis" hōʻike i nā ʻanuʻu holoʻokoʻa kahi i mahi ʻia ai ʻo Caco-2 a i ʻole nā ​​​​​​cell epithelial i loaʻa i ka organoid ma kahi chip intestinal a i ʻole ma nā hoʻokomo Transwell o kahi chip hybrid, ukali ʻia e ka hoʻokomo ʻana o 3D morphogenesis a me ka hoʻokumu ʻana o kahi ʻano epithelial i hōʻike ʻia. Hōʻike ʻia ka helu ʻanuʻu papahana a i ʻole ka helu pahu ma lalo o kēlā me kēia pua. Hāʻawi ka noi i nā hiʻohiʻona o ke ʻano o ka hoʻohana ʻana i nā papa epithelial intestinal i hoʻokumu ʻia, no ka laʻana, i ke ʻano o ka hoʻokaʻawale ʻana o ke kelepona, nā haʻawina physiology gut, ka hoʻokumu ʻana o nā ʻōnaehana host-microbiome, a me ke kumu hoʻohālike maʻi. Nā kiʻi Immunofluorescence ma "Cell ʻO ka "Differentiation" e hōʻike ana i nā nuclei, F-actin a me MUC2 i hōʻike ʻia ma ka papa epithelial 3D Caco-2 i hana ʻia ma ka ʻāpana ʻōpū. Aia ka hōʻailona MUC2 i loko o nā cell goblet a me ka mucus i hūnā ʻia mai nā ʻili mucosal. Hōʻike nā kiʻi Fluorescent ma Gut Physiology i ka mucus i hana ʻia e ka staining no ka sialic acid a me nā koena N-acetylglucosamine me ka hoʻohana ʻana i ka fluorescent wheat germ agglutinin. Hōʻike nā kiʻi ʻelua e pili ana i ka "Host-Microbe Co-Cultures" i nā co-cultures host-microbiome i loko o ka ʻōpū ma kahi chip. Hōʻike ka panela hema i ka co-culture o E. coli e hōʻike ana i ka protein fluorescent ʻōmaʻomaʻo (GFP) me nā cell epithelial 3D Caco-2 microengineered. Hōʻike ka panela ʻākau i ka localization o GFP E. coli i hoʻoulu ʻia me nā cell epithelial 3D Caco-2, ukali ʻia e ka immunofluorescence staining me F-actin (ʻulaʻula) a me nā nuclei (uliuli). Hōʻike ka hoʻohālike maʻi i ke olakino versus leaky gut i nā chips mumū ʻōpū ma lalo o ka hoʻokūkū physiological me nā antigens bacterial (e.g., lipopolysaccharide, LPS) a me nā cell immune (e.g., PBMC; ʻōmaʻomaʻo). Ua hoʻoulu ʻia nā pūnaewele Caco-2 e hoʻokumu i kahi papa epithelial 3D. Ka pā unahi, 50 µm. Nā kiʻi ma ka lālani lalo: "Differentiation of cells" i hoʻololi ʻia me ka ʻae mai ka kuhikuhi.2. Oxford University Press; Hoʻopuka hou ʻia me ka ʻae mai Ref.5. NAS; "Host-Microbe Co-Culture" i hoʻololi ʻia me ka ʻae mai ref.3. NAS; "Disease Modeling" i hoʻololi ʻia me ka ʻae mai ka kuhikuhi.5. NAS.
Ua hana ʻia nā ʻāpana gut-on-chip a me nā hybrid me ka hoʻohana ʻana i nā replicas PDMS i hoʻohemo ʻia mai nā ʻōmole silicon e ka lithography palupalu1,44 a ua hoʻohālikelike ʻia me SU-8. Hoʻoholo ʻia ka hoʻolālā o nā microchannels i kēlā me kēia chip ma ka noʻonoʻo ʻana i ka hydrodynamics e like me ke kaumaha shear a me ke kaomi hydrodynamic1,4,12. ʻO ka hoʻolālā gut-on-a-chip mua (Extended Data Fig. 1a), nona nā microchannels pololei ʻelua i hoʻohui ʻia, ua ulu aʻe i loko o kahi gut-on-a-chip paʻakikī (Extended Data Fig. 1b) e komo pū ana me kahi pālua o nā microchannels curved e hoʻoulu ai i ka hoʻonui ʻana i ka manawa noho wai, nā ʻano kahe nonlinear, a me ka deformation multiaxial o nā cell i hoʻoulu ʻia (Fig. 2a–f) 12. Ke pono e hana hou ʻia nā biomechanics gut paʻakikī, hiki ke koho ʻia nā gut-on-a-chips paʻakikī. Ua hōʻike mākou e hoʻoulu ikaika ana ka Gut-Chip convoluted i ka 3D morphogenesis i kahi manawa like me ke ʻano like o ka ulu ʻana o ka epithelial i hoʻohālikelike ʻia me ka mea mua. ʻO Gut-Chip, me ka nānā ʻole i ke ʻano o ke kelepona i hoʻoulu ʻia. No laila, no ka hoʻoulu ʻana i ka morphogenesis 3D, hiki ke hoʻololi ʻia nā hoʻolālā ʻōpū linear a paʻakikī ma ka chip. ʻO nā replicas PDMS i hoʻōla ʻia ma nā ʻōmole silicon me nā hiʻohiʻona SU-8 i hāʻawi i nā hiʻohiʻona maikaʻi ʻole ma hope o ka demolding (Fig. 2a). No ka hana ʻana i ka ʻōpū ma kahi chip, ua hoʻopaʻa ʻia ka papa PDMS luna i hoʻomākaukau ʻia i kahi kiʻiʻoniʻoni PDMS porous a laila hoʻohālikelike ʻia me ka papa PDMS haʻahaʻa ma o ka hoʻopaʻa ʻana i hiki ʻole ke hoʻihoʻi ʻia me ka hoʻohana ʻana i kahi corona treater (Fig. 2b–f). No ka hana ʻana i nā chips hybrid, ua hoʻopaʻa ʻia nā replicas PDMS i hoʻōla ʻia i nā paheʻe aniani e hana i nā mea hana microfluidic hoʻokahi-channel e hiki ke hoʻokipa i nā hoʻokomo Transwell (Fig. 2h a me Extended Data Fig. 2). Hana ʻia ke kaʻina hana hoʻopaʻa ma ka mālama ʻana i nā ʻili o ka replica PDMS a me ke aniani me ka plasma oxygen a i ʻole ka mālama ʻana i ka corona. Ma hope o ka sterilization o ka mea hana microfabricated i hoʻopili ʻia i ka paipu silicone, ua mākaukau ka hoʻonohonoho ʻana o ka mea hana e hana i ka morphogenesis 3D o ka epithelium intestinal (Fig. 2g).
a, Kiʻi hoʻolālā o ka hoʻomākaukau ʻana o nā ʻāpana PDMS mai nā ʻōmole silicon i hoʻohālikelike ʻia me SU-8. Ua ninini ʻia ka hopena PDMS i hoʻōla ʻole ʻia ma luna o kahi ʻōmole silicon (hema), hoʻōla ʻia ma 60 °C (waena) a wāwahi ʻia (ʻākau). Ua ʻoki ʻia ka PDMS i wāwahi ʻia i mau ʻāpana a hoʻomaʻemaʻe ʻia no ka hoʻohana hou ʻana. b, Kiʻi o ka ʻōmole silicon i hoʻohana ʻia e hoʻomākaukau i ka papa luna o PDMS. c, Kiʻi o ka ʻōmole silicon i hoʻohana ʻia e hana i ka membrane porous PDMS. d, He moʻo o nā kiʻi o nā ʻāpana PDMS o luna a me lalo a me ka mea hana ʻōpū ma ka chip i hōʻuluʻulu ʻia. e, Schematic o ke kūlike ʻana o nā ʻāpana PDMS o luna, membrane, a me lalo. Hoʻopaʻa ʻia kēlā me kēia papa e ka mālama ʻana i ka plasma a i ʻole ka corona. f, Schematic o ka mea hana gut-on-a-chip i hana ʻia me nā microchannels convoluted superimposed a me nā keʻena vacuum. g, Hoʻonohonoho o ka gut-on-a-chip no ka moʻomeheu cell microfluidic. Ua kau ʻia ka ʻōpū i hana ʻia ma kahi chip i hōʻuluʻulu ʻia me kahi paipu silicone a me ka syringe ma kahi coverslip. Ua kau ʻia ka mea hana chip ma ke poʻi o kahi Petri 150 mm kīʻaha no ka hana ʻana. Hoʻohana ʻia ka mea hoʻopaʻa e pani i ka paipu silicone.h, Nā kiʻi ʻike maka o ka hana ʻana o ka chip hybrid a me ka morphogenesis 3D me ka hoʻohana ʻana i nā chips hybrid. Ua hoʻokomo ʻia nā hoʻokomo Transwell i hoʻomākaukau kūʻokoʻa ʻia e hoʻoulu i nā monolayers 2D o nā cell epithelial intestinal i loko o ka chip hybrid e hoʻoulu i ka morphogenesis 3D intestinal. Hoʻoheheʻe ʻia ka medium ma o nā microchannels ma lalo o ka papa cell i hoʻokumu ʻia ma ka hoʻokomo Transwell. Ka pā scale, 1 cm.h Paʻi hou ʻia me ka ʻae mai ka kuhikuhi.4. Elsevier.
Ma kēia protocol, ua hoʻohana ʻia ka laina cell Caco-2 a me nā organoids intestinal ma ke ʻano he mau kumu epithelial (Kiʻi 3a). Ua mahi ʻia nā ʻano cell ʻelua me ke kūʻokoʻa (Pahu 2 a me Pahu 5) a hoʻohana ʻia e lūlū i nā microchannels i uhi ʻia e ECM o ka ʻōpū ma luna o ka chip a i ʻole nā ​​​​​​mea hoʻokomo Transwell. Ke hui pū nā cell (>95% uhi i loko o nā flasks), ua hōʻiliʻili ʻia nā cell Caco-2 i mahi mau ʻia (ma waena o nā ala 10 a me 50) i loko o nā T-flasks e hoʻomākaukau i nā hoʻokuʻu cell dissociated e ka wai trypsinization (pahu 2). Ua mahi ʻia nā organoids intestinal kanaka mai nā biopsies intestinal a i ʻole nā ​​​​​​ʻoki ʻoki ʻana i loko o nā domes scaffold Matrigel i loko o nā papa 24-well e kākoʻo i ka microenvironment structural. Ua hoʻohui ʻia nā medium e loaʻa ana nā morphogens koʻikoʻi (e like me Wnt, R-spondin, a me Noggin) a me nā mea ulu i hoʻomākaukau ʻia e like me ka mea i wehewehe ʻia ma ka Pahu 3 i kēlā me kēia lā ʻē aʻe a hiki i ka ulu ʻana o nā organoids i ~ 500 µm ke anawaena. Ua ʻohi ʻia nā organoids i ulu piha ʻia a hoʻokaʻawale ʻia i loko o nā cell hoʻokahi no ka lūlū ʻana ma luna o ka ʻōpū. a i ʻole nā ​​hoʻokomo Transwell ma kahi ʻāpana (Pahu 5). E like me kā mākou i hōʻike mua ai, hiki ke hoʻokaʻawale ʻia e like me ke ʻano maʻi12,13 (e laʻa me ka ulcerative colitis, ka maʻi Crohn, ka maʻi ʻaʻai colorectal, a i ʻole ka mea hāʻawi maʻamau), kahi lesion (e laʻa me ka lesion versus non-lesioned area) a me kahi gastrointestinal i loko o ke ala (e laʻa me ka duodenum, jejunum, ileum, cecum, colon, a i ʻole rectum). Hāʻawi mākou i kahi protocol i hoʻomaikaʻi ʻia ma ka Pahu 5 no ka hoʻoulu ʻana i nā organoids colonic (coloids) e koi pinepine ana i nā ʻano kiʻekiʻe o nā morphogens ma mua o nā organoids ʻōpū liʻiliʻi.
a, Kahe Hana no ka hoʻokomo ʻana i ka morphogenesis ʻōpū i loko o ka ʻāpana ʻōpū. Hoʻohana ʻia ka epithelium ʻōpū kanaka Caco-2 a me nā organoids ʻōpū i loko o kēia protocol e hōʻike i ka morphogenesis 3D. Ua lūlū ʻia nā cell epithelial i hoʻokaʻawale ʻia i loko o ka hāmeʻa gut-on-a-chip i hoʻomākaukau ʻia (hoʻomākaukau ʻana i ka chip). Ke lūlū ʻia nā cell (lūlū ʻia) a hoʻopili ʻia (hoʻopili ʻia) i ka membrane porous PDMS i ka lā 0 (D0), hoʻomaka ʻia ke kahe apical (AP) a mālama ʻia no nā lā 2 mua (kahe, AP, D0-D2). Hoʻomaka pū ʻia ke kahe Basolateral (BL) me nā neʻe hoʻolōʻihi cyclic (hoʻolōʻihi, kahe, AP a me BL) i ka wā i hoʻokumu ʻia ai kahi monolayer 2D piha. Ua hana koke ʻia ka morphogenesis 3D ʻōpū ma hope o 5 mau lā o ka moʻomeheu microfluidic (morphogenesis, D5). Hōʻike nā kiʻi hoʻohālikelike pae i ka morphology ʻelele o nā cell Caco-2 ma kēlā me kēia ʻanuʻu hoʻokolohua a i ʻole ka manawa (kiʻi bar, 100 µm). ʻEhā mau kiʻikuhi schematic e hōʻike ana i nā cascades like o ka morphogenesis ʻōpū (ʻākau luna). ʻO ka dashed ʻO nā pua i loko o ka schematic e hōʻike ana i ke kuhikuhi o ke kahe wai. b, kiʻi SEM e hōʻike ana i ka topology ʻili o ka epithelium 3D Caco-2 i hoʻokumu ʻia (hema). ʻO ka inset e hōʻike ana i ka ʻāpana i hoʻonui ʻia (pahu dashed keʻokeʻo) e hōʻike ana i ka microvilli i hana hou ʻia ma ka papa 3D Caco-2 (ʻākau). c, ʻIke mua Horizontal o Caco-2 3D i hoʻokumu ʻia, claudin (ZO-1, ʻulaʻula) a me nā membrane palena palaki hoʻomau i kapa ʻia ʻo F-actin (ʻōmaʻomaʻo) a me nā nuclei (uliuli) Immunofluorescence confocal visualization o nā cell epithelial ma nā ʻāpana ʻōpū. ʻO nā pua e kuhikuhi ana i ka schematic waena e hōʻike ana i kahi o ka mokulele focal no kēlā me kēia ʻike confocal. d, Ka manawa o nā loli morphological i nā organoids i mahi ʻia ma kahi ʻāpana i loaʻa e ka microscopy ʻokoʻa pae ma nā lā 3, 7, 9, 11, a me 13. ʻO ka inset (ʻākau luna) e hōʻike ana i ka hoʻonui kiʻekiʻe o ke kiʻi i hāʻawi ʻia. e, DIC photomicrograph o ka organoid 3D epithelium i hoʻokumu ʻia i loko o ka ʻōpū ma ka ʻāpana i lawe ʻia ma ka lā 7. f, Nā kiʻi immunofluorescence i uhi ʻia e hōʻike ana i nā māka no nā cell kumu (LGR5; magenta), nā pūnaewele goblet (MUC2; ʻōmaʻomaʻo), F-actin (hina) a me nā nuclei (cyan) i ulu ʻia ma nā ʻāpana ʻōpū no 3 mau lā, kēlā me kēia (Hema) a me 13-lā (waena) organoids ma ka papa epithelial. E nānā pū i ka Extended Data Figure 3, kahi e hōʻike ana i ka hōʻailona LGR5 me ka ʻole o ka hōʻailona MUC2. Nā kiʻi Fluorescence e hōʻike ana i ka microstructure epithelial (ʻākau) o ka epithelium organoid 3D i hoʻokumu ʻia i loko o ka ʻōpū ma kahi ʻāpana ma ka hoʻoluʻu ʻana i ka membrane plasma me ka dye CellMask (ʻākau) ma ka lā 13 o ka moʻomeheu. ʻO ka pā unahi he 50 μm ke ʻole i ʻōlelo ʻia.b Hoʻopuka hou ʻia me ka ʻae mai ka kuhikuhi.2. Oxford University Press; c Hoʻololi ʻia me ka ʻae mai ka Reference.2. Oxford University Press; e a me f i hoʻololi ʻia me ka ʻae ma ka kuhikuhi.12 Ma lalo o ka Creative Commons License CC BY 4.0.
I loko o ka ʻōpū ma kahi ʻāpana, pono e hoʻololi i ka ʻili hydrophobic o ka membrane porous PDMS no ka uhi ʻana o ECM holomua. Ma kēia protocol, hoʻopili mākou i ʻelua mau ʻano like ʻole e hoʻololi i ka hydrophobicity o nā membrane PDMS. No ka mahi ʻana i nā cell Caco-2, ʻo ka hoʻoulu ʻana o ka ʻili e ka mālama ʻana o UV/ozone wale nō ua lawa ia e hōʻemi i ka hydrophobicity o ka ʻili PDMS, e uhi i ka ECM a hoʻopili i nā cell Caco-2 i ka membrane PDMS. Eia nō naʻe, ʻo ka moʻomeheu microfluidic o ka epithelium organoid e pono ai ka hana ʻili e pili ana i ka kemika e hoʻokō i ka waiho ʻana o nā protein ECM ma o ka hoʻopili ʻana i ka polyethyleneimine (PEI) a me ka glutaraldehyde i nā microchannels PDMS. Ma hope o ka hoʻololi ʻana o ka ʻili, ua waiho ʻia nā protein ECM e uhi i ka ʻili PDMS functionalized a laila hoʻokomo ʻia i loko o ka epithelium organoid i hoʻokaʻawale ʻia. Ma hope o ka hoʻopili ʻia ʻana o nā cell, hoʻomaka ka moʻomeheu cell microfluidic ma ka perfusing wale i ka medium i loko o ka microchannel luna a hiki i nā cell ke hana i kahi monolayer piha, ʻoiai ʻo ka microchannel haʻahaʻa e mālama i nā kūlana static. ʻO kēia ʻano hana i hoʻomaikaʻi ʻia no ka hoʻoulu ʻana o ka ʻili a me ka uhi ʻana o ECM e hiki ai i ka hoʻopili ʻana o ka epithelium organoid e hoʻoulu i ka 3D morphogenesis ma ka PDMS. ʻilikai.
Pono pū nā moʻomeheu Transwell i ka uhi ʻana o ECM ma mua o ka lūlū ʻana i nā cell; akā naʻe, ʻaʻole koi nā moʻomeheu Transwell i nā ʻanuʻu hoʻomākaukau paʻakikī e hoʻāla i ka ʻili o nā mea hoʻokomo porous. No ka ulu ʻana o nā cell Caco-2 ma nā mea hoʻokomo Transwell, hoʻolalelale ka uhi ʻana o ECM ma nā mea hoʻokomo porous i ka hoʻopili ʻana o nā cell Caco-2 i hoʻokaʻawale ʻia (<1 hola) a me ka hoʻokumu ʻana o ka pale hui paʻa (<1-2 lā). No ka hoʻoulu ʻana i nā organoids ma nā mea hoʻokomo Transwell, lūlū ʻia nā organoids i hoʻokaʻawale ʻia ma nā mea hoʻokomo i uhi ʻia e ECM, hoʻopili ʻia i ka ʻili membrane (<3 h) a mālama ʻia a hiki i ka hana ʻana o nā organoids i monolayer piha me ka pono o ka pale. Hana ʻia nā moʻomeheu Transwell ma nā papa 24-well me ka ʻole o ka hoʻohana ʻana i nā ʻāpana hybrid.
Hiki ke hoʻomaka ʻia ka morphogenesis 3D in vitro ma ka hoʻopili ʻana i ke kahe wai i ka ʻaoʻao basolateral o kahi papa epithelial i hoʻokumu ʻia. I loko o ka ʻōpū ma kahi chip, ua hoʻomaka ka morphogenesis epithelial i ka wā i hoʻoheheʻe ʻia ai ka medium i loko o nā microchannels o luna a me lalo (Fig. 3a). E like me ka mea i wehewehe mua ʻia, he mea nui e hoʻolauna i ke kahe wai i loko o ka ʻāpana haʻahaʻa (basolateral) no ka wehe mau ʻana o nā mea hoʻopaʻa morphogen i hoʻokuʻu ʻia. No ka hāʻawi ʻana i nā meaʻai lawa a me ka serum i nā cell i hoʻopaʻa ʻia ma nā membrane porous a hoʻoulu i ke kaumaha shear luminal, hoʻopili pinepine mākou i ke kahe pālua i loko o ka ʻōpū ma kahi chip. I loko o nā chips hybrid, ua hoʻokomo ʻia nā hoʻokomo Transwell e loaʻa ana nā monolayers epithelial i loko o nā chips hybrid. A laila, ua hoʻopili ʻia ka medium ma lalo o ka ʻaoʻao basolateral o ka hoʻokomo Transwell porous ma o ka microchannel. Ua hana ʻia ka morphogenesis intestinal 3-5 mau lā ma hope o ka hoʻomaka ʻana o ke kahe basolateral ma nā kahua moʻomeheu ʻelua.
Hiki ke kālailai ʻia nā hiʻohiʻona morphological o nā papa epithelial 3D microengineered ma ka hoʻopili ʻana i nā ʻano hana kiʻi like ʻole, me ka microscopy phase contrast, differential interference contrast (DIC) microscopy, SEM, a i ʻole immunofluorescence confocal microscopy (Kiʻi 3 a me 4). Hiki ke hana maʻalahi ʻia ke kiʻi ʻana o ka phase contrast a i ʻole DIC imaging i kēlā me kēia manawa i ka wā o ka moʻomeheu e nānā i ke ʻano a me ka protrusion o nā papa epithelial 3D. Ma muli o ka transparency optical o PDMS a me nā kiʻiʻoniʻoni polyester, hiki i nā kahua gut-on-a-chip a me nā hybrid chip ke hāʻawi i ka imaging in situ manawa maoli me ka ʻole o ka pono no ka ʻoki ʻana a i ʻole ka wehe ʻana o ka hāmeʻa. Ke hana nei i ke kiʻi immunofluorescence (Kiʻi 1, 3c, f a me 4b, c), hoʻopaʻa pinepine ʻia nā cell me 4% (wt/vol) paraformaldehyde (PFA), ukali ʻia e Triton X-100 a me 2% (wt/vol) ) bovine serum albumin (BSA), ma ke kauoha. Ma muli o ke ʻano o ke cell, hiki ke hoʻohana ʻia nā fixatives like ʻole, permeabilizers, a me nā mea pale. ʻO nā antibodies mua e kuhikuhi ana i nā cell lineage-dependent a i ʻole Hoʻohana ʻia nā māka ʻāpana e hōʻike i nā cell i hoʻopaʻa ʻia ma kahi ma ka chip, a ukali ʻia e nā antibodies lua me kahi dye counterstain e kuhikuhi ana i ka nucleus (eg, 4′, 6-diamidino-2-phenylene) indole, DAPI) a i ʻole F-actin (eg, fluorescently labeled phalloidin). Hiki ke hana ʻia ke kiʻi ola e pili ana i ka fluorescence ma kahi e ʻike ai i ka hana mucus (Kiʻi 1, "Cell differentiation" a me "Gut physiology"), ka colonization random o nā cell microbial (Kiʻi 1, "Host-microbe co-culture") , ka hoʻolimalima ʻana o nā cell immune (Kiʻi 1, 'Disease Modeling') a i ʻole nā ​​​​contours o 3D epithelial morphology (Kiʻi 3c, f a me 4b, c). Ke hoʻololi nei i ka ʻōpū ma ka chip e hoʻokaʻawale i ka papa luna mai ka papa microchannel haʻahaʻa, e like me ka mea i wehewehe ʻia ma ref. E like me ka mea i hōʻike ʻia ma ke Kiʻi 2, hiki ke ʻike ʻia ka morphology epithelial 3D a me ka microvilli ma ka palena palaki apical e SEM (Kiʻi 3b). ʻO ka hōʻike ʻana o ka differentiation Hiki ke loiloi ʻia nā māka ma ka hana ʻana i ka PCR5 quantitative a i ʻole ka hoʻonohonoho ʻana o ka RNA cell hoʻokahi. I kēia hihia, ʻohi ʻia nā papa 3D o nā cell epithelial i ulu ʻia i loko o nā ʻāpana ʻōpū a i ʻole nā ​​​​ʻāpana hybrid e ka trypsinization a laila hoʻohana ʻia no ka loiloi molekala a i ʻole genetic.
a, Kahe Hana no ka hoʻokomo ʻana i ka morphogenesis ʻōpū i loko o kahi ʻāpana hybrid. Hoʻohana ʻia ʻo Caco-2 a me nā organoids ʻōpū i loko o kēia protocol e hōʻike i ka morphogenesis 3D i loko o kahi kahua ʻāpana hybrid. Ua lūlū ʻia nā cell epithelial i hoʻokaʻawale ʻia i loko o nā hoʻokomo Transwell i hoʻomākaukau ʻia (TW prep; e ʻike i ke kiʻi ma lalo). I ka wā i lūlū ʻia ai nā cell (seeded) a hoʻopili ʻia i nā membrane polyester i loko o nā hoʻokomo Transwell, ua mahi ʻia nā cell āpau ma lalo o nā kūlana static (TW culture). Ma hope o 7 mau lā, ua hoʻohui ʻia kahi hoʻokomo Transwell hoʻokahi e loaʻa ana kahi monolayer 2D o nā cell epithelial i loko o kahi ʻāpana hybrid e hoʻolauna i kahi kahe basolateral (Flow, BL), ka mea i alakaʻi hope i ka hanauna o kahi papa epithelial 3D (morphogenesis). Nā micrographs hoʻohālikelike Phase e hōʻike ana i nā hiʻohiʻona morphological o nā cell epithelial o ke kino kanaka i loaʻa mai ka mea hāʻawi maʻamau (laina C103) e piʻi ana i ka colon ma kēlā me kēia ʻanuʻu hoʻokolohua a i ʻole ka manawa. Hōʻike nā schematics ma nā papa luna i ka hoʻonohonoho hoʻokolohua no kēlā me kēia ʻanuʻu. b, Hiki i nā ʻāpana Hybrid (schematic hema) ke alakaʻi i ka morphogenesis 3D o nā cell epithelial organoid me nā hiʻohiʻona microscopy confocal top-down i lawe ʻia ma nā kūlana Z like ʻole (luna, waena, a me lalo; e ʻike i ka schematic ʻākau a me nā laina kiko e pili ana). ua hōʻike i nā ʻano morphological maopopo.F-actin (cyan), nucleus (hina).c, Nā micrographs confocal Fluorescence (nānā angled 3D) o nā cell epithelial i loaʻa mai ka organoid i mahi ʻia ma Transwell static (TW; inset i loko o ka pahu dashed keʻokeʻo) versus hybrid chip (pana piha nui loa) e hoʻohālikelike ana i ka morphology 2D versus 3D, kēlā me kēia.ʻO kahi pālua o nā hiʻohiʻona ʻoki kū pololei 2D (inset ma ke kihi ʻākau i luna; "XZ") hōʻike pū i nā hiʻohiʻona 2D a me 3D.Scale bar, 100 µm.c Ua paʻi hou ʻia me ka ʻae mai ka kuhikuhi.4. Elsevier.
Hiki ke hoʻomākaukau ʻia nā kaohi ma ka hoʻoulu ʻana i nā pūnaewele like (Caco-2 a i ʻole nā ​​​​​​pūnaewele epithelial organoid intestinal) i loko o nā monolayers ʻelua-dimensional ma lalo o nā kūlana moʻomeheu static maʻamau. ʻIke ʻia, hiki ke hopena ka emi ʻana o ka meaʻai ma muli o ka palena o ka nui o nā microchannels (ʻo ia hoʻi ~ 4 µL ma ke kahawai luna ma ka hoʻolālā gut-chip mua). No laila, hiki ke hoʻohālikelike ʻia ke ʻano epithelial ma mua a ma hope o ka hoʻopili ʻana o ke kahe basolateral.
Pono e hana ʻia ke kaʻina hana lithography palupalu i loko o kahi lumi maʻemaʻe. No kēlā me kēia papa ma ka chip (nā papa luna a me lalo a me nā membrane) a me nā chips hybrid, ua hoʻohana ʻia nā photomasks like ʻole a hana ʻia ma nā wafers silicon kaʻawale no ka mea he ʻokoʻa ke kiʻekiʻe o nā microchannels. ʻO nā kiʻekiʻe i manaʻo ʻia o nā microchannels luna a me lalo o ka ʻōpū ma ka chip he 500 µm a me 200 µm, kēlā me kēia. ʻO ke kiʻekiʻe i manaʻo ʻia o ke kahawai o ka chip hybrid he 200 µm.
E kau i kahi wafer silicon 3-'īniha i loko o kahi kīʻaha me ka acetone. E wili mālie i ka pā no 30 kekona, a laila e hoʻomaloʻo i ka wafer i ka ea. E hoʻololi i ka wafer i kahi pā me IPA, a laila e wili i ka pā no 30 kekona e hoʻomaʻemaʻe.
Hiki ke hoʻohana ʻia kahi hopena piranha (ka hui ʻana o ka hydrogen peroxide a me ka waikawa sulfuric i hoʻohuihui ʻia, 1:3 (vol/vol)) e hoʻonui i ka wehe ʻana i nā koena organik mai ka ʻili o ka wafer silicon.
He ʻino loa ka wai Piranha a hoʻopuka i ka wela. Pono nā hana palekana hou aʻe. No ka hoʻolei ʻana i nā ʻōpala, e ʻae i ka wai e hoʻomaʻalili a hoʻoili i kahi pahu ʻōpala maʻemaʻe a maloʻo. E hoʻohana i nā pahu lua a e lepili pono i nā pahu ʻōpala. E ʻoluʻolu e hahai i nā alakaʻi palekana o ka hale no nā kaʻina hana kikoʻī hou aku.
E hoʻomaloʻo i nā wafers ma ke kau ʻana iā lākou ma luna o kahi pā wela 200 °C no 10 min. Ma hope o ka hoʻomaloʻo ʻana, ua lūlū ʻia ka wafer i ʻelima mau manawa i ka lewa e hoʻomaʻalili.
E ninini i ka ~10 g o ka photoresist SU-8 2100 ma ke kikowaena o ka wafer silicon i hoʻomaʻemaʻe ʻia. E hoʻohana i nā tweezers e hohola like i ka photoresist ma luna o ka wafer. I kekahi manawa e kau i ka wafer ma luna o kahi pā wela 65°C e hoʻemi ai i ka pipili o ka photoresist a maʻalahi hoʻi e hohola. Mai kau pololei i ka wafer ma luna o ka pā wela.
Ua hoʻolaha like ʻia ʻo SU-8 ma luna o ka wafer ma o ka holo ʻana i ka uhi wili. E papahana i kahi hoʻohuli e hiki mai ana o ka SU-8 no 5-10 kekona e hoʻolaha ma 500 rpm ma ka wikiwiki o 100 rpm/s. E hoʻonohonoho i ka wili nui no ke ʻano mānoanoa 200 µm ma 1,500 rpm, a i ʻole e hoʻokō i kahi mānoanoa 250 µm (e hana ana i kahi kiʻekiʻe 500 µm no ka papa luna o ka ʻōpū ma ka ʻāpana; e ʻike i nā "ʻanuʻu koʻikoʻi" ma lalo) i hoʻonohonoho ʻia ma ka wikiwiki o 300 rpm/s 30 kekona ma 1,200 rpm.
Hiki ke hoʻololi ʻia ka wikiwiki o ka wili nui e like me ka mānoanoa o ka pahuhopu o ke ʻano SU-8 ma ka wafer silicon.
No ka hana ʻana i nā ʻano SU-8 o 500 µm ke kiʻekiʻe no ka papa o luna o ka ʻōpū ma luna o ka ʻāpana, ua hana hou ʻia nā ʻanuʻu wili a me ka hoʻomoʻa palupalu o kēia Pahu (nā ʻanuʻu 7 a me 8) ma ke ʻano he kauoha (e nānā i ka ʻanuʻu 9) e hana i ʻelua papa o 250 µm He papa mānoanoa o SU-8, hiki ke hoʻohui ʻia a hoʻohui ʻia e ka hōʻike UV ma ka ʻanuʻu 12 o kēia pahu, e hana ana i kahi papa 500 µm ke kiʻekiʻe.
E hoʻomoʻa palupalu i nā wafers i uhi ʻia me ka SU-8 ma ke kau pono ʻana i nā wafers ma luna o kahi pā wela ma 65 °C no 5 min, a laila e hoʻololi i ke hoʻonohonoho i 95 °C a hoʻomoʻa hou no 40 min.
No ka hoʻokō ʻana i ke kiʻekiʻe o 500 μm o ke ʻano SU-8 ma ka microchannel o luna, e hana hou i nā ʻanuʻu 7 a me 8 e hana i ʻelua mau papa SU-8 he 250 μm ka mānoanoa.
Me ka hoʻohana ʻana i ka UV Mask Aligner, e hana i kahi hoʻāʻo kukui e like me nā kuhikuhi a ka mea hana e helu ai i ka manawa hōʻike o ka wafer. (manawa hōʻike, ms) = (ka nui o ka hōʻike, mJ/cm2)/(mana kukui, mW/cm2).
Ma hope o ka hoʻoholo ʻana i ka manawa hōʻike, e kau i ka photomask ma luna o ka mea paʻa mask o ka aligner mask UV a kau i ka photomask ma ka wafer i uhi ʻia ʻo SU-8.
E kau i ka ʻili i paʻi ʻia o ka photomask ma ka ʻaoʻao i uhi ʻia e SU-8 o ka wafer silicon e hōʻemi i ka hoʻopuehu UV.
E hōʻike i ka wafer i uhi ʻia ʻo SU-8 a me ka photomask i luna i ka 260 mJ/cm2 o ke kukui UV no ka manawa hōʻike i hoʻoholo mua ʻia (e nānā i ka hana 10 o kēia pahu).
Ma hope o ka hōʻike ʻana i ka UV, ua hoʻomoʻa ʻia nā wafers silicon i uhi ʻia e SU-8 ma 65°C no 5 min a me 95°C no 15 min ma kēlā me kēia pā wela e hana i nā ʻano me ke kiʻekiʻe o 200 μm. E hoʻolōʻihi i ka manawa ma hope o ka hoʻomoʻa ʻana ma 95°C a i 30 min e hana i nā ʻano me ke kiʻekiʻe o 500 µm.
Ninini ʻia ka mea hoʻomohala i loko o kahi kīʻaha aniani, a waiho ʻia ka wafer i hoʻomoʻa ʻia i loko o ke kīʻaha. Hiki ke ʻokoʻa ka nui o ka mea hoʻomohala SU-8 ma muli o ka nui o ka pā aniani. E hōʻoia e hoʻohana i ka mea hoʻomohala SU-8 lawa e wehe loa i ka SU-8 i hōʻike ʻole ʻia. No ka laʻana, i ka wā e hoʻohana ai i kahi kīʻaha aniani 150 mm ke anawaena me ka mana 1 L, e hoʻohana i ~300 mL o ka mea hoʻomohala SU-8. E hoʻomohala i ka ʻōmole no 25 mau minuke me ka hoʻohuli mālie i kekahi manawa.
E holoi i ka ʻōmole i hoʻomohala ʻia me ~10 mL o ka mea hoʻomohala hou a ukali ʻia e IPA ma ke kāpī ʻana i ka hopena me ka hoʻohana ʻana i kahi pipette.
E kau i ka wafer i loko o kahi mea hoʻomaʻemaʻe plasma a hoʻokuʻu i ka plasma oxygen (kinoea lewa, kaomi i manaʻo ʻia 1 × 10−5 Torr, mana 125 W) no 1.5 min.
E kau i ka wafer i loko o kahi desiccator vacuum me kahi slide aniani i loko. Hiki ke waiho ʻia nā wafers a me nā slide ma ka ʻaoʻao. Inā ua māhele ʻia ka desiccator vacuum i kekahi mau papa e kahi pā, e kau i nā slide ma ke keʻena haʻahaʻa a me nā wafers ma ke keʻena luna. E hoʻokuʻu i 100 μL o ka hopena trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane ma kahi slide aniani a hoʻopili i ka vacuum no ka silanization.
E hoʻoheheʻe i kahi ʻōmole o nā pūnaewele Caco-2 i hoʻopaʻahau ʻia i loko o ka wai ʻauʻau 37°C, a laila e hoʻoili i nā pūnaewele i hoʻoheheʻe ʻia i kahi ʻōmole T75 me 15 mL o ka medium Caco-2 i hoʻomehana mua ʻia he 37°C.
No ka hoʻoili ʻana i nā pūnaewele Caco-2 ma kahi hui ʻana o ~90%, ʻo ka medium Caco-2 mehana mua, PBS, a me 0.25% trypsin/1 mM EDTA i loko o ka ʻauʻau wai 37°C.
E omo i ka medium ma ka vacuum aspiration. E holoi i nā cell i ʻelua manawa me 5 mL o ka PBS mahana ma ka hana hou ʻana i ka vacuum aspiration a me ka hoʻohui ʻana i ka PBS hou.