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Hoʻokumu ka morphogenesis o ke kanaka i nā hiʻohiʻona crypt-villus o 3D epithelial microarchitecture a me ka hui spatial. mucosal surface.No laila, he mea koʻikoʻi ka hana hou ʻana i nā hale epithelial 3D no ke kūkulu ʻana i nā hiʻohiʻona in vitro gut. gut ma ka microfluidic chip e like me ka Transwell embedded hybrid chip.We wehewehe i nā ʻano kikoʻī no ka hana ʻana i nā mea hana, culturing o Caco-2 a i ʻole nā ​​​​pūnaewele epithelial organoid intestinal i nā hoʻonohonoho maʻamau a me kahi microfluidic platform, induction o 3D morphogenesis, a me ke ʻano o ka 3D epithelia i hoʻokumu ʻia e ka hoʻohana ʻana i ka hoʻohana ʻana i nā ʻano hana hoʻohālikelike 3D e hoʻohana ana i ka nui o ka hoʻohana ʻana i nā ʻōnaehana gut. ke hoʻomalu nei i ka kahe wai basolateral no 5 d. ʻO kā mākou ʻano in vitro morphogenesis e hoʻohana i ke koʻikoʻi shear pili i ka physiologically a me ka neʻe mechanical a ʻaʻole koi i ka ʻenekini cell paʻakikī a i ʻole ka manipulation, hiki ke hoʻokō i nā ʻenehana ʻē aʻe. nā noi.
Hōʻike nā hoʻokolohua e hiki i ka intestinal epithelial Caco-2 cell i hoʻoulu ʻia i loko o ka gut-on-a-chip1,2,3,4,5 a i ʻole bilayer microfluidic devices6,7 hiki ke hana i ka 3D morphogenesis in vitro me ka maopopo ʻole o ke kumu kumu. epithelial morphogenesis in vitro, i hōʻike ʻia e Caco-2 a me nā organoids intestinal i loaʻa i ka maʻi. Ua hōʻoia ʻia nā pūnaewele epithelial. Ma kēia haʻawina, ua nānā pono mākou i ka hana ʻana o ka cell a me ka hāʻawi ʻana i ka manaʻo o kahi antagonist Wnt potent, Dickkopf-1 (DKK-1), i loko o ka gut-on-a-chip a me nā mea microfluidic hoʻololi ʻia i loaʻa nā mea hoʻokomo Transwell, i kapa ʻia "Hybrid Chip". ʻO ka protein pili frizzled 1, a i ʻole Soggy-1) i ka ʻōpū on-chip ke kāohi i ka morphogenesis a i ʻole ka hoʻopau ʻana i ka papa epithelial 3D i kūkulu mua ʻia, e hōʻike ana ʻo ke koʻikoʻi antagonistic i ka wā moʻomeheu ke kuleana no ka morphogenesis intestinal in vitro. ma ka hoʻoheheʻe ʻana (e laʻa, i loko o ka ʻōpū-ma-a-chip a i ʻole hybrid-on-a-chip platform) a i ʻole diffusion .Basolateral media (e laʻa, mai Transwell hoʻokomo i loko o nā waihona basolateral nui i loko o nā luawai).
Ma kēia kaʻina hana, hāʻawi mākou i kahi ala kikoʻī no ka hana ʻana i nā microdevices gut-on-a-chip a me Transwell-insertable hybrid chips (step 1-5) i ka moʻomeheu intestinal epithelial cell on polydimethylsiloxane (PDMS) -based porous membranes (step 6A, 7A, 8, 9) a i ʻole polyesterBs, 8, 9) 9) a hoʻokomo i ka morphogenesis 3D in vitro (ʻanuʻu 10). Ua ʻike pū mākou i nā hiʻohiʻona cellular a me nā molecular e hōʻike ana i ka histogenesis kikoʻī kikoʻī kikoʻī a me ka ʻokoʻa cellular e pili ana i ka laina ma o ka hoʻohana ʻana i nā ʻano kiʻi kiʻi he nui (step 11-24). ka hoʻololi ʻana i nā membrane porous, ka hana ʻana i nā monolayers 2D, a me ka biochemical intestinal a me Reproduction o ka microenvironment biomechanical.in vitro.No ka hoʻoulu ʻana i ka morphogenesis 3D mai nā monolayers epithelial 2D, ua wehe mākou i nā antagonists morphogen ma nā ʻano moʻomeheu ʻelua ma ke kahe ʻana i ka waena i loko o ka ʻāpana basolateral o ka moʻomeheu. epithelial layer hiki ke hoʻohana ʻia e hoʻohālike i ka ulu ʻana o ka epithelial e pili ana i ka morphogen, ka longitudinal host-microbiome co-cultures, ka maʻi pathogen, ka ʻeha ʻeha, ka epithelial barrier dysfunction, a me ka probiotic-based therapies Example.influences.
Hiki i kā mākou protocol ke hoʻohana i kahi ākea o nā ʻepekema ma ke kumu (e laʻa, intestinal mucosal biology, stem cell biology, and developmental biology) a noiʻi noiʻi (eg, preclinical drug testing, maʻi modeling, tissue engineering, and gastroenterology) ākea ākea. hiki ke hoʻolaha ʻia i ka poʻe e aʻo ana i ka dynamics o ka hōʻailona cell i ka wā o ka ulu ʻana o ka ʻōpū, ka hana hou ʻana a i ʻole ka homeostasis. Eia kekahi, pono kā mākou protocol no ka nīnau ʻana i ka maʻi ma lalo o nā ʻano infectious like ʻole e like me Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella choae, Salmonella Tyae. ʻO ka hoʻohana ʻana i kahi ʻōnaehana microphysiology o ka maʻi ma ka chip hiki ke ʻae i ka longitudinal co-culture 10 a me ka loiloi hope o ka pale ʻana o ka mea hoʻokipa, nā pane kūlohelohe a me ka hoʻoponopono ʻeha pili i ka pathogen i ka ʻōpū (GI) tract 11 . ʻO nā maʻi GI ʻē aʻe e pili ana me ka leaky gut syndrome, celiac disease, Crohn's irritation syndrome e hiki ke lilo i ka maʻi maʻi maʻi maʻi maʻi maʻi maʻi maʻi maʻi. Hoʻomākaukau ʻia nā papa epithelial intestinal 3D me ka hoʻohana ʻana i nā papa epithelial intestinal 3D o ka mea maʻi, ʻo kēia mau maʻi e pili ana i ka villous atrophy, crypt shortening, mucosal damage, or impaired epithelial barrier. Biopsy a i ʻole stem cell-derived intestinal organoids12,13. ma'i peripheral blood mononuclear cell (PBMCs), i nā kŘkohu me 3D intestinal villus-crypt microarchitectures. nā ʻiʻo-specific immune cell, 5.
No ka mea hiki ke hoʻopaʻa ʻia a ʻike ʻia ʻo 3D epithelial microstructure me ka ʻole o ke kaʻina ʻāpana, makemake paha ka poʻe nānā e hana ana i nā transcriptomic spatial a me ke kiʻi kiʻi kiʻi kiʻekiʻe a i ʻole super-resolution i kā mākou palapala ʻāina o ka spatiotemporal dynamics o nā genes a me nā protein ma nā epithelial niches. Hoihoi i ka ʻenehana. Ka pane i ka microbial a i ʻole ka hoʻoulu ʻana i ka immune. He mea hoihoi loa keia i ka poe e a'o ana i ka immunology mucosal, gastroenterology, microbiome kanaka, culturomics a me clinical microbiology e imi ana e mahiai i ka microbiota gut mua uncultured in the laboratory. hiki ke hoʻolaha ʻia ka protocol i ka poʻe e hoʻomohala ana i ka lāʻau lapaʻau, biomedical A i ʻole nā ​​​​mea hōʻike kiʻekiʻe kiʻekiʻe a i ʻole nā ​​​​papahana hōʻoia no ka ʻoihana meaʻai. Ma ke ʻano he hōʻoia-o-pono, ua hōʻike koke mākou i ka hiki ʻana o kahi ʻōnaehana morphogenesis multiplex kiʻekiʻe e hiki ke hoʻonui ʻia i kahi format plate 24-well. Hiki ke hoʻonui ʻia kā mākou ʻano in vitro morphogenesis a hiki ke hoʻohana ʻia e nā keʻena noiʻi he nui, ʻoihana a aupuni a me nā keʻena hoʻoponopono e hoʻomaopopo i ka hoʻonohonoho hou ʻana o ka cellular reprogramming o in vitro gut morphogenesis ma ka pae transcriptomic e hoʻāʻo ai i nā lāʻau lapaʻau a i ʻole biotherapeutics Ua loiloi ʻia ka absorption a me ka lawe ʻana i nā moho lāʻau me ka hoʻohana ʻana i ka 3D gut surrogates a i ʻole ka hoʻohana ʻana i ke kumu hoʻohālike o ka reprogutibility maʻamau. kaʻina hana morphogenesis.
Ua hoʻohana ʻia kahi helu liʻiliʻi o nā hiʻohiʻona hoʻokolohua e pili ana i ke kanaka e aʻo ai i ka morphogenesis epithelial intestinal, ma muli o ka nele o nā protocols implementable e hoʻoulu ai i ka morphogenesis 3D in vitro. nīnau, a ʻo ka mea nui loa, ʻaʻole e hoʻoholo pololei i nā kaʻina hoʻomohala kanaka. Ua kaupalena ʻia kēia mau hiʻohiʻona i ko lākou hiki ke hoʻāʻo ʻia ma kahi ʻano scalable manner. localization o ka like 'ole i loko o ka crypt-villus axis ma ka pane ana i ka mucosal a i ole immune stimuli. pehea ka hana ʻana o ka microbiota gut i kona mau kaiaulu a hana pū ʻia nā microbial metabolites (e laʻa, pōkole-chain fatty acids) e hana i ka hui cellular a me nā niches stem cell i loko o nā crypts basal. Hiki ke hōʻike ʻia kēia mau hiʻohiʻona ke hoʻokumu ʻia nā papa epithelial 3D in vitro.
Ma waho aʻe o kā mākou ʻano hana o ka hana ʻana i nā hale epithelial intestinal 3D, aia kekahi mau ʻano in vitro. Hoʻopili ʻia ka lumen intestinal i loko o ka organoid a, no laila, ua kaupalena ʻia ka hoʻokomo ʻana o nā mea luminal e like me nā microbial cell a i ʻole exogenous antigens. Hiki ke hoʻomaikaʻi ʻia ke komo ʻana i nā lumens organoid me ka microinjector,26,27 akā ʻo kēia ʻano hana invasive a paʻakikī a koi i ka ʻike kūikawā e hana ai.
ʻO nā ala ʻē aʻe i hoʻohana ʻia e kekahi mau pūʻulu noiʻi e hoʻohana i nā scaffolds hydrogel 3D i hoʻonohonoho mua ʻia e hoʻohālike i ke ʻano o ka epithelial o ka ʻōpū ma o ka hoʻoulu ʻana i nā ʻōpū o ke kanaka i kaʻawale ma ka ʻili gel. morphogen gradients, e hoʻokumu ana i kahi kiʻekiʻe hiʻona epithelial structure a me stroma-epithelial crosstalk ma o ka hoʻokomo ʻana i nā cell stromal i loko o ka scaffold. Akā naʻe, ʻo ke ʻano o nā scaffolds i kūkulu mua ʻia hiki ke pale i ka hōʻike ʻana i ke kaʻina morphogenetic spontanetic ponoʻī. a loaʻa ka hana physiological. ʻO kekahi noiʻi hou i hoʻohana i nā scaffolds hydrogel i loko o kahi microfluidic platform a ua hoʻohālikelike ʻia nā hale epithelial intestinal e hoʻohana ana i nā ʻenehana laser-etching. mechanobiological movements.3D printing techniques from the same group was able to create miniature gut tubes with spontaneous morphogenetic procedures.Despite the complex fabrication of different gut segments within the tube, this model is also lack luminal fluid flow and mechanical deformation.Additional, model operability is limited, especially after the bioprinting process is full, persturbing to experimental condition. gut morphogenesis, physiologically shear stress, biomechanics e mimic gut motility, accessibility of independent apical and basolateral compartments, and re-creation of complex biological microenvironments of modularity.
Hoʻopili piha ʻia kā mākou protocol i ka 3D epithelial morphogenesis, me nā cell epithelial wale nō i ka moʻomeheu a ʻaʻohe ʻano ʻē aʻe o nā cell a puni e like me nā mesenchymal cell, endothelial cell, a me nā cell immune. gut-on-a-chip a hybrid-on-a-chip hiki iā mākou ke hana hou i ka undulating 3D epithelial layer, nā mea paʻakikī hou aʻe e like me ka epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 a, i kā mākou kumu hoʻohālike, nā hiʻohiʻona crypt-villus e lawe ana i nā niches stem cell e hoʻomau ʻia i loko o nā cell fibrogStr. ʻO ka mesenchyme ka mea nui i ka hana ʻana o nā protein ECM a me ka hoʻoponopono ʻana i ka morphogenesis intestinal i vivo35,37,38. ʻO ka hoʻohui ʻana o nā cell mesenchymal i kā mākou kumu hoʻohālike i hoʻonui ai i ke kaʻina morphogenetic a me ka hoʻopili ʻana o ka cell. microenvironment. Eia kekahi, pono e hoʻokomo ʻia nā ʻāpana vasculature i hiki ke hoʻopili ʻia ma waena o nā hiʻohiʻona kikoʻī i ka wā i hoʻolālā ʻia ai nā hiʻohiʻona kikoʻī e hōʻike i ka launa pū ʻana o nā mea he nui. immunity i loko o ka pōʻaiapili o ka mimicking intestinal maʻi.
ʻOi aku ka maʻalahi o ka hoʻohana ʻana i nā chips 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 mea hoʻokomo Transwell e hiki ai i ka moʻomeheu scalable o ka epithelium o ka ʻōpū. ma ka ʻaoʻao apical. Maikaʻi, ʻaʻole hiki i nā waiwai paʻa i loko o ka ʻāpana apical ke hiki i ka moʻomeheu lōʻihi o ka bacterial co-culture i nā chips hybrid.
ʻAʻole i hoʻokumu piha ʻia ka hana hou ʻana o ka axis crypt-villus kanaka i loko o nā moʻomeheu gut-on-a-chip a me hybrid-on-a-chip. 3D epithelium, ka crypt a me nā ʻāpana villous ʻaʻole i hoʻokaʻawale ʻia. ʻO ke kiʻekiʻe o ka villi ʻōpū liʻiliʻi he ~600 µm41.
Mai kahi hiʻohiʻona kiʻi, hiki ke kaupalena ʻia ke kiʻi kiʻi super-resolution o 3D microarchitectures i ka ʻōpū ma luna o kahi chip, no ka mea, ʻo ka mamao o ka hana e pono ai mai ka lens pahuhopu a hiki i ka papa epithelial ma ke kauoha o kekahi mau millimeters. PDMS. Eia kekahi, no ka mea, ʻo ka microfabrication layer-by-layer o ka ʻōpū ma luna o ka chip e pili ana i ka hoʻopili mau ʻana ma waena o kēlā me kēia papa, he mea paʻakikī loa ka wehe a wehe ʻana paha i ka papa luna e nānā i ke ʻano o ka ʻili o ka papa epithelial.
ʻO ka hydrophobicity o PDMS he mea kaupalena i ka microfluidic-based haʻawina e pili ana i nā molekole liʻiliʻi hydrophobic, no ka mea, hiki i ka PDMS ke hoʻopili pono ʻole i ia mau mole hydrophobic. Hiki ke noʻonoʻo ʻia nā mea ʻē aʻe i ka PDMS me nā mea polymeric ʻē aʻe. hōʻemi i ka adsorption o nā molekala hydrophobic.
ʻO ka mea hope loa, ʻaʻole i ʻike maikaʻi ʻia kā mākou ala e pili ana i ka hāʻawi ʻana i kahi kānana kiʻekiʻe a i ʻole "hoʻokahi-nui-pono-a pau" hoʻokolohua hoʻokolohua hoʻohana. 384-well porous inserts e ʻae i ka hoʻopiha hou ʻana a me ka wehe ʻana o ka media basolateral).
No ka hoʻoulu ʻana i ka morphogenesis 3D o ka epithelium ʻōpū o ke kanaka in vitro, ua hoʻohana mākou i kahi mea ʻōpū microfluidic chip i loaʻa nā microchannels like ʻelua a me kahi membrane porous elastic ma waena e hana i kahi interface lumen-capillary. Hiki ke hōʻike ʻia ka morphogenesis o nā ʻāpana epithelial ʻokoʻa o ke kanaka ma o ka hoʻohana ʻana i ka manipulation directional o ke kahe e wehe i nā antagonists morphogen mai ka ʻaoʻao basolateral. ʻO ke kaʻina hana hoʻokolohua holoʻokoʻa (Figure 1) he ʻelima mau ʻāpana: (i) microfabrication o ka ʻōpū ʻōpū a i ʻole Transwell insertable hybrid chip (pae 1-5; (Caco-2 cell) a i ʻole nā ​​ʻāpana ʻōpū o ke kanaka; pahu 2-5), (iii) ka moʻomeheu o ka ʻōpū epithelial cell ma nā ʻāpana ʻōpū a i ʻole nā ​​ʻāpana hybrid (ʻanuʻu 6-9), (iv) ka hoʻokomo ʻana o 3D morphogenesis in vitro (step 10) a me (v)) e ʻike i ka microstructure 3D epithelial microstructure (ʻanuʻu 11-24). vitro morphogenesis ma ka hoʻohālikelike ʻana i ka epithelial morphogenesis me nā mana spatial, temporal, conditional, a i ʻole ke kaʻina hana.
Ua hoʻohana mākou i ʻelua mau papa moʻomeheu like ʻole: gut-on-a-chip me nā ala pololei a i ʻole nā ​​​​kānāwai convoluted nonlinear, a i ʻole nā ​​ʻāpana hybrid i loaʻa nā Transwell (TW) hoʻokomo i loko o kahi microfluidic device, i hana ʻia e like me ka wehewehe ʻana ma ka pahu 1, a me ka pae 1 -5. ʻO ka ʻōpū o ke kanaka) a me ke kaʻina hana moʻomeheu i hoʻohana ʻia i loko o kēia protocol. Hiki ke hoʻohana ʻia nā papa epithelial intestinal, no ka laʻana, i ka wehewehe ʻana o ka cell differentiation, gut physiology study, hoʻokumu i ka host-microbiome ecosystems, a me ka hoʻohālikelike ʻana i nā maʻi. Hōʻike nā kiʻi ʻulaʻula i loko o Gut Physiology i ka mucus i hana ʻia ma ka hoʻopaʻa ʻia ʻana no ka ʻakika sialic a me nā koena N-acetylglucosamine me ka hoʻohana ʻana i ka agglutinin germ wheat fluorescent. ʻO nā kiʻi ʻelua i uhi ʻia ma "Host-Microbe Co-Cultures" e hōʻike ana i ka ʻelele host-microbiome co-culture ma ka co-culture o ka co-culture. coli e hōʻike ana i ka protein fluorescent ʻōmaʻomaʻo (GFP) me ka microengineered 3D Caco-2 epithelial cell. Hōʻike ka panel akau i ka localization o GFP E. coli co-cultured me 3D Caco-2 epithelial cell, a ukali ʻia e immunofluorescence staining me F-actin (ʻulaʻula) a me ka nuclei (blue). me nā antigens bacteria (eg, lipopolysaccharide, LPS) a me nā pūnaewele immune (eg, PBMC; ʻōmaʻomaʻo). ʻO 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; "Maʻi Hoʻohālike" i hoʻololi ʻia me ka ʻae ʻia mai ka reference.5. NAS.
Ua hana ʻia nā ʻōpū ʻelua a me nā ʻāpana hybrid me ka hoʻohana ʻana i nā replicas PDMS i hoʻokaʻawale ʻia mai nā mold silicon e ka lithography palupalu1,44 a hoʻohālikelike ʻia me SU-8. ʻO ka hoʻolālā ʻana o nā microchannels i kēlā me kēia chip e hoʻoholo ʻia e ka noʻonoʻo ʻana i nā hydrodynamics e like me ka shear stress a me ka hydrodynamic pressure1,4,12. ʻO nā microchannels pololei like ʻole, ua ulu aʻe i loko o kahi puʻupuʻu paʻakikī (Extended Data Fig. 1b) e komo pū ana me 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 moʻomeheu (Fig. 2a-f) gut. hiki ke koho ʻia ka gut-on-a-chips. Ua hōʻike mākou i ka convoluted Gut-Chip e hoʻoikaika ikaika i ka 3D morphogenesis i kahi manawa like me kahi pae like o ka ulu epithelial i hoʻohālikelike ʻia me ka Gut-Chip kumu, me ka nānā ʻole i ke ʻano cell moʻomeheu. Hāʻawi nā ʻano SU-8 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 luna PDMS luna i hoʻopaʻa ʻia i kahi kiʻiʻoniʻoni PDMS porous a laila hoʻopili ʻia me ka papa haʻahaʻa PDMS ma o ka hoʻopaʻa ʻana ʻole me ka hoʻohana ʻana i ka corona treater (Fig. 2b-f). hiki ke hoʻokomo i nā mea hoʻokomo Transwell (Fig. 2h a me Extended Data Fig. 2). Hana ʻia ke kaʻina hana paʻa ma ka mālama ʻana i nā ʻili o ka PDMS replica a me ke aniani me ka oxygen plasma a i ʻole ka mālama corona.
a, Schematic kiʻi o ka hoʻomākaukau ʻana i nā ʻāpana PDMS mai SU-8 patterned silicon molds. The uncured PDMS solution was poured on a silicon mold (hema), hoʻōla ʻia ma 60 °C (waena) a demolded (akau) . hana i ka PDMS porous membrane.d, He moʻo o nā kiʻi o nā ʻāpana PDMS luna a me lalo a me ka mea i hui pū ʻia ma luna o ka ʻōpū. chambers.g, Hoʻonohonoho ʻia o ka puʻupuʻu-ma-a-chip no ka moʻomeheu microfluidic cell. ʻO ka ʻōpū i hana ʻia ma kahi chip i hui ʻia me kahi paipu silicone a me ka syringe i kau ʻia ma luna o kahi uhi uhi. i ka moʻomeheu 2D monolayers o intestinal epithelial cell ua hoʻokomo 'ia i loko o ka hybrid chip e hoʻoulu i ka'ōpū 3D morphogenesis.ʻO ka medium ua perfused ma o microchannels ma lalo o ka cell layer i hoʻokumuʻia ma ka Transwell insert.Scale bar, 1 cm.h Reprinted with the permit from reference.4. Elsevier.
Ma kēia protocol, ua hoʻohana ʻia ka laina kelepona Caco-2 a me nā organoids intestinal e like me nā kumu epithelial (Fig. 3a). 10 a me 50) i loko o ka T-puʻu ua ohi ia e hoomakaukau dissociated cell suspensions ma ka trypsinization wai (pahu 2). Human intestinal organoids mai intestinal biopsies a surgical resections ua moʻomeheu i loko o Matrigel scaffold domes i 24-well papa e kākoʻo i ka structural microenvirgens. ʻO 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 ua hoʻohui ʻia i kēlā me kēia lā a hiki i ka ulu ʻana o nā organoids i ~ 500 µm i ke anawaena. Ua hōʻiliʻili ʻia nā organoids ulu piha a hoʻokaʻawale ʻia i loko o nā ʻāpana hoʻokahi no ka lūlū ʻana ma luna o ka ʻōpū a i ʻole Transwell inserts ma kahi chip (Box 5). colitis, Crohn's disease, colorectal cancer, or normal donor), lesion site (e laʻa, lesion versus non-lesioned area) a me ka ʻōpū o ka ʻōpū ma ka ʻāpana (eg, duodenum, jejunum, ileum, cecum, colon, a rectum). organoids.
a, Workflow for the induction of gut morphogenesis in the gut chip.Caco-2 human intestinal epithelium and intestinal organoids hoʻohana ʻia i kēia protocol e hōʻike i ka 3D morphogenesis.The isolated epithelial cell were seeded in the prepared gut-on-a-chip device (chip preparation). (D0), hoʻomaka a mālama ʻia ke kahe apical (AP) no nā lā 2 mua (ke kahe, AP, D0-D2). Hoʻomaka pū ʻia ke kahe Basolateral (BL) me nā neʻe cyclic stretching (stretch, flow, AP a me BL) ke hoʻokumu ʻia kahi monolayer 2D piha. morphology hōʻike o Caco-2 cell ma kēlā me kēia hoʻokolohua ʻanuʻu a i ʻole ka manawa manawa (ka pakuhi pahu, 100 µm). ʻEhā kiʻi schematic e hōʻike ana i nā cascades pili o ka morphogenesis o ka ʻōpū (luna ʻākau). ʻO nā pua i hoʻopaʻa ʻia i ka schematic e hōʻike ana i ke kuhikuhi o ka kahe wai. (pahu keʻokeʻo keʻokeʻo) hōʻike i ka microvilli regenerated ma ka 3D Caco-2 papa (akau). E hōʻike i kahi o ka mokulele kikoo no kēlā me kēia confocal view.d, Time course of morphological changes in organoids moʻomeheu ʻia ma ka chip i loaʻa ma ka phase contrast microscopy ma nā lā 3, 7, 9, 11, a me 13. ʻO ka inset (luna ʻākau) hōʻike i ka hoʻonui kiʻekiʻe o ke kiʻi i hāʻawi ʻia. ʻO nā kiʻi immunofluorescence e hōʻike ana i nā hōʻailona no nā pūnaewele kumu (LGR5; magenta), nā kīʻaha kīʻaha (MUC2; ʻōmaʻomaʻo), F-actin (hina) a me ka nuclei (cyan) i ulu ʻia ma nā ʻōpū ʻōpū no nā lā 3, kēlā me kēia (Left) a me 13-lā (waena) organoids ma ka papa epithelial. hōʻailona.Fluorescence kiʻi e hōʻike ana i ka epithelial microstructure ('ākau) o 3D organoid epithelium i hoʻokumuʻia i loko o ka'ōpū ma luna o ka chip ma ka hoʻopaʻaʻana i ka membrane plasma me ka CellMask dye ('ākau) i ka lā 13 o ka moʻomeheu. ʻO Oxford University Press; c Hoʻololi ʻia me ka ʻae mai ka Reference.2. ʻO Oxford University Press; ua hoʻololi ʻia ka e a me ka f me ka ʻae ʻia e ka manaʻo.12 Ma lalo o Creative Commons License CC BY 4.0.
I loko o ka ʻōpū ma ka chip, pono ia e hoʻololi i ka ʻili hydrophobic o ka membrane porous PDMS no ka uhi ʻana i ka ECM kūleʻa. Ma kēia protocol, hoʻohana mākou i ʻelua mau ala like ʻole e hoʻololi ai i ka hydrophobicity o nā membranes PDMS. Membrane. Akā naʻe, pono ka microfluidic moʻomeheu o ka organoid epithelium i ka hoʻohana ʻana i ka ʻili ma muli o ke kemika e hoʻokō pono ai 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. i hoʻopili ʻia, hoʻomaka ka moʻomeheu microfluidic cell ma ka hoʻopili wale ʻana i ka waena i loko o ka microchannel luna a hiki i ka hana ʻana o nā cell i kahi monolayer piha, ʻoiai ka microchannel haʻahaʻa e mālama i nā kūlana static. ʻO kēia ala i hoʻopaʻa ʻia no ka hoʻoulu ʻana i ka ʻili a me ka uhi ECM hiki ke hoʻopili i ka epithelium organoid e hoʻoulu i ka morphogenesis 3D ma ka ʻili PDMS.
Pono nō hoʻi nā moʻomeheu Transwell i ka uhi ʻana o ECM ma mua o ke kanu ʻana i ke kelepona; akā naʻe, ʻaʻole koi nā moʻomeheu Transwell i nā ʻanuʻu pretreatment paʻakikī e hoʻāla i ka ʻili o nā inserts porous. ECM-coated inserts, pili i ka membrane surface (<3 h) a mālamaʻia a hiki i ka hanaʻana o nā organoids i kahi monolayer piha me ka paʻa paʻa.
Hiki ke hoʻomaka i ka morphogenesis in vitro 3D ma o ka hoʻopili ʻana i ka kahe wai i ka ʻaoʻao basolateral o kahi papa epithelial i hoʻokumu ʻia. No ka hāʻawi ʻana i nā meaʻai kūpono a me ka serum i nā cell i hoʻopaʻa ʻia ma nā membrane porous a hoʻoulu i ke koʻikoʻi luminal shear, hoʻohana maʻamau mākou i nā kahe pālua ʻelua i loko o ka ʻōpū ma kahi chip. kahe basolateral ma nā paepae moʻomeheu ʻelua.
Hiki ke kālailai ʻia nā hiʻohiʻona morphological o nā papa epithelial 3D microengineered ma o ka hoʻohana ʻana i nā ʻano ʻano kiʻi like ʻole, me ka microscopy phase contrast, microscopy interference contrast (DIC), SEM, a i ʻole immunofluorescence confocal microscopy (Figure 3 a me 4). layers.Ma muli o ka ʻike maka o ka PDMS a me nā kiʻiʻoniʻoni polyester, hiki i ka gut-on-a-chip a me ka hybrid chip platform ke hāʻawi i ka manawa maoli i ka imaging situ me ka ʻole o ka pono o ka ʻāpana a i ʻole ka wehe ʻana o ka mea. (PFA), ukali ʻia e Triton X-100 a me 2% (wt/vol) ) bovine serum albumin (BSA), ma ke ʻano. Ma muli o ke ʻano o ke kelepona, hiki ke hoʻohana ʻia nā mea hoʻoponopono like ʻole, permeabilizers, a me nā mea pale. (e laʻa, 4′,6-diamidino-2-phenylene) indole, DAPI) a i ʻole F-actin (eg, fluorescently labeled phalloidin) .Fluorescence-based live imaging hiki ke hana ʻia ma kahi e ʻike ai i ka hana mucus (Fig. co-culture") , ka recruitment o immune cell (Fig. 1, 'Disease Modeling') aiʻole nā ​​contours o 3D epithelial morphology (Fig. 3c, f a me 4b, c). I ka hoʻololiʻana i ka'ōpū ma ka chip e hoʻokaʻawale i ka papa luna mai ka papa microchannel lalo, e like me ka mea i hōʻikeʻia ma ka ref. hiki ke ʻike ʻia ka palena ʻāpana apical e SEM (Fig. 3b). Hiki ke loiloi ʻia ka hōʻike o nā māka ʻokoʻa ma o ka hana ʻana i ka quantitative PCR5 a i ʻole hoʻokahi-cell RNA sequencing. I kēia hihia, ʻohi ʻia nā papa 3D o nā epithelial cell i ulu i loko o nā ʻāpana ʻōpū a i ʻole nā ​​ʻāpana hybrid e ka trypsinization a laila hoʻohana ʻia no ka molecular a genetic analysis.
a, Workflow no ka induction o ka morphogenesis intestinal i loko o ka hybrid chip.Caco-2 a me ka intestinal organoids ua hoʻohana 'ia i loko o kēia protocol e hōʻike 3D morphogenesis i loko o ka hybrid chip platform. Dissociated epithelial cell were seeded in prepared Transwell inserts (TW prep; see kiʻi ma lalo). kūlana (TW culture).Ma hope o 7 lā, hoʻokahi Transwell hoʻokomo i loko o ka 2D monolayer o epithelial cell i hoʻohuiʻia i loko o ka hybrid chip e hoʻolauna i ka basolateral kahe (Flow, BL), i ka hopena i alakai i ka hanauna o ka 3D epithelial layer (morphogenesis). ʻanuʻu hoʻokolohua a i ʻole ka manawa manawa. ʻO nā schematics ma nā papa luna e hōʻike ana i ka hoʻonohonoho hoʻokolohua no kēlā me kēia step.b, Hiki i nā ʻāpana Hybrid (ka ʻaoʻao 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 a me nā laina kiko kūpono). Ua hōʻike ʻia nā hiʻohiʻona morphological maopopo. kihi ʻākau; “XZ”) hōʻike pū i nā hiʻohiʻona 2D a me 3D. Paʻa Scale, 100 µm.c Ua paʻi hou ʻia me ka ʻae ʻia mai ka kuhikuhi.4. Elsevier.
Hiki ke hoʻomākaukau ʻia nā mana ma ka hoʻoulu ʻana i nā cell like (Caco-2 a i ʻole nā ​​​​pūnaewele organoid epithelial intestinal) i loko o nā monolayers ʻelua-dimensional ma lalo o nā kūlana moʻomeheu static maʻamau. e hoohalikeia.
Pono e hana ʻia ke kaʻina hana lithography palupalu i loko o kahi lumi maʻemaʻe. No kēlā me kēia papa ma luna o ka chip (nā papa luna a me lalo a me nā membrane) a me nā chip hybrid, ua hoʻohana ʻia nā photomasks like ʻole a hana ʻia ma nā wafer silika ʻokoʻa no ka mea ʻokoʻa nā kiʻekiʻe o nā microchannels. 200 µm.
E kau i ka wafer silicon 3-inihi i loko o kahi pā me ka acetone. E hoʻowili mālie i ka pā no 30 kekona, a laila e hoʻomaloʻo i ka wai.
Hiki ke ho'ohana 'ia ka hopena piranha (ka hui 'ana o ka hydrogen peroxide a me ka sulfuric acid concentrated, 1:3 (vol/vol)) no ka ho'onui 'ana i ka wehe 'ana i nā koena mea ola mai ka ili wafer silika.
ʻO ka hopena Piranha he meaʻino loa a hoʻoulu i ka wela. Pono nā mea palekana hou.
E hoʻomaloʻo i nā wafers ma ke kauʻana iā lākou ma kahi pā wela 200 ° C no 10 min. Ma hope o ka maloʻoʻana, ua hoʻoluliluliʻia ka wafer iʻelima manawa i ka ea e hoʻomaha.
E ninini i ka ~10 g o ka photoresist SU-8 2100 ma ke kikowaena o ka wafer silicon i hoʻomaʻemaʻe ʻia.
Ua māhele like ʻia ka SU-8 ma ka wafer ma ka hoʻopili ʻana i ka milo. E hoʻopololei i ka hoʻololi ʻana o ka SU-8 no 5-10 s e hoʻolaha ma 500 rpm ma ka wikiwiki o 100 rpm/s. µm kiʻekiʻe no ka papa luna o ka ʻōpū ma ka chip;
Hiki ke hoʻololi ʻia ka wikiwiki milo nui e like me ka mānoanoa o ke ʻano SU-8 ma ka wafer silika.
No ka hana ʻana i nā hiʻohiʻona SU-8 o 500 µm kiʻekiʻe no ka papa luna o ka ʻōpū ma ka chip, ua hana hou ʻia ka uhi milo a me nā ʻanuʻu bake palupalu o kēia pahu (nā ʻanuʻu 7 a me 8) (e ʻike i ka ʻanuʻu 9) e hana i ʻelua papa o 250 µm He papa mānoanoa o SU-8, hiki ke hoʻopaʻa ʻia i ka pahu 10 µm. kiʻekiʻe.
E hoʻomoʻa mālie i nā wafers SU-8 i uhi ʻia ma ke kau ʻana i nā wafers ma kahi pā wela ma 65 °C no 5 min, a laila e hoʻololi i ka hoʻonohonoho i 95 °C a e hoʻomoʻa no 40 mau minuke hou.
No ka loaʻa ʻana o kahi kiʻekiʻe 500 μm o ke ʻano SU-8 i ka microchannel luna, e hana hou i nā ʻanuʻu 7 a me 8 e hana i ʻelua mau papa SU-8 mānoanoa 250 μm.
Ke hoʻohana nei i ka UV Mask Aligner, e hana i kahi hoʻāʻo kukui e like me nā ʻōlelo a ka mea hana e helu ai i ka manawa hoʻolaha o ka wafer.
Ma hope o ka hoʻoholo ʻana i ka manawa hoʻolaha, kau i ka photomask ma luna o ka mea paʻa maka o ka UV mask aligner a kau i ka photomask ma ka wafer uhi ʻia SU-8.
E kau pololei i ka ʻili paʻi o ka photomask ma ka ʻaoʻao i uhi ʻia ʻo SU-8 o ka wafer silicon e hōʻemi i ka hoʻopuehu ʻana o UV.
E hōʻike i ka wafer i uhi ʻia i ka SU-8 a me ka pale paʻi kiʻi i ka 260 mJ/cm2 o ke kukui UV no ka manawa hoʻolaha i koho mua ʻia (e nānā i ka ʻanuʻu 10 o kēia pahu).
Ma hope o ka hoʻolaha ʻana o UV, ua kālua ʻia nā wafers silicon SU-8-coated ma 65 ° C no 5 min a me 95 ° C no 15 min ma kēlā me kēia pā wela e hana i nā hiʻohiʻona me ke kiʻekiʻe o 200 μm. E hoʻolōʻihi i ka manawa ma hope o ka bake ʻana ma 95 ° C i 30 min e hana i nā kumu me ke kiʻekiʻe o 500 µm.
Ua 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 hoʻololi ʻia ka nui o ka mea hoʻomohala SU-8 ma muli o ka nui o ka pā aniani.
Holoi i ka ʻōpala i hoʻomohala ʻia me ka ~ 10 mL o ka mea hoʻomohala hou a ukali ʻia e IPA ma ke kāpī ʻana i ka hopena me ka pipette.
E kau i ka wafer i loko o kahi mea hoʻomaʻemaʻe plasma a hōʻike i ka plasma oxygen (ke kinoea lewa, ke kaomi pahuhopu 1 × 10−5 Torr, mana 125 W) no 1.5 min.
E kau i ka wafer i loko o ka vacuum desiccator me ka pahe aniani i loko. Hiki ke waiho i na wafers a me na pahee ma ka aoao. vacuum no ka silani.
E hoʻoheheʻe i kahi hue o nā kelepa Caco-2 paʻa i loko o kahi ʻauʻau wai 37°C, a laila e hoʻoneʻe i nā pūnaewele i hoʻoheheʻe ʻia i loko o kahi hue T75 i loaʻa he 15 mL o 37°C ʻo Caco-2 i hoʻomaʻamaʻa mua ʻia.
No ka hele ʻana i nā pūnaewele Caco-2 ma ~90% confluency, mehana Caco-2 mua, PBS, a me 0.25% trypsin/1 mM EDTA i loko o kahi ʻauʻau wai 37°C.
E hoʻomoʻi i ka mea waena ma ka aspiration vacuum. E holoi i nā keena i ʻelua manawa me 5 mL o ka PBS mahana ma ka hana hou ʻana i ka hoʻoulu ʻana a me ka hoʻohui ʻana i ka PBS hou.