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O le morphogenesis o le gutu o le tagata e faʻavaeina ai foliga crypt-villus o le 3D epithelial microarchitecture ma le faʻalapotopotoga faʻapitoa. O lenei fausaga tulaga ese e manaʻomia e tausia ai le gut homeostasis e ala i le puipuia o le fatu cell niche i le basal crypt mai exogenous microbial antigens ma o latou metabolites. mucosal surface.O le mea lea, o le toe faia o le 3D epithelial structures e taua tele mo le fausiaina o in vitro gut models. Fa'ailogaina, o le organic mimetic gut-on-a-chip e mafai ona fa'aosofia ai le 3D morphogenesis o le intestinal epithelium ma le fa'aleleia o galuega fa'aletino ma le biomechanics. gut i luga o se microfluidic chip faʻapea foʻi ma se Transwell embedded hybrid chip. Matou te faʻamatalaina auala auʻiliʻili mo le faʻaogaina o masini, faʻatupuina o le Caco-2 poʻo le intestinal organoid epithelial cell i faʻasalalauga masani faʻapea foʻi ma luga o se microfluidic platform, induction of 3D morphogenesis, ma le faʻavasegaina o le faʻavaeina o le 3D epithelia e ala i le faʻaogaina o le tele o faʻataʻitaʻiga faʻataʻitaʻiga. pulea le tafe o le vai basolateral mo 5 d.O matou auala in vitro morphogenesis faʻaaogaina physiologically talafeagai le mamafa o le sele ma le masini faʻainisinia ma e le manaʻomia ai le faʻaogaina o masini faʻapitoa poʻo le faʻaogaina, lea e mafai ona sili atu i isi metotia o loʻo i ai. Matou te vaʻavaʻai atu e mafai ona i ai a matou faʻasalalauga faʻapitoa mo le sosaiete suʻesuʻega biomedical, tuʻuina atu se auala e toe faʻafouina 3D intestinal epithelial, biomedical layers mo le vitroutical layer. talosaga.
O faʻataʻitaʻiga e faʻaalia ai o le intestinal epithelial Caco-2 sela o loʻo faʻatupuina i le gut-on-a-chip1,2,3,4,5 poʻo le bilayer microfluidic devices6,7 e mafai ona oʻo i le 3D morphogenesis in vitro e aunoa ma se malamalamaga manino o le masini faʻavae. epithelial morphogenesis in vitro, lea na faʻaalia e Caco-2 ma maʻi e maua mai i le intestinal organoids. Na faʻamaonia sela Epithelial. I lenei suʻesuʻega, matou te taulaʻi faʻapitoa i le gaosiga o le sela ma le tufatufaina atu o le malosi o le Wnt antagonist, Dickkopf-1 (DKK-1), i totonu o le gut-on-a-chip ma le faʻaogaina o masini microfluidic o loʻo i ai Transwell inserts, ua taʻua o le " Hybrid Chip ". Matou te faʻaalia lena faʻaopoopoga o le exogenous Wnt1 faʻalavelave faʻalilolilo-(Wnt1) palatini e feso'ota'i frizzled 1, po'o Soggy-1) i luga o le atigipusa e taofia ai le morphogenesis po'o fa'alavelaveina le 3D epithelial layer , fa'ailoa mai o le fa'alavelave fa'alavelave i le taimi o le aganu'u e nafa ma le intestinal morphogenesis in vitro. e ala i le fufuluina malosi (faataitaiga, i le gut-on-a-chip poʻo le hybrid-on-a-chip platforms) poʻo le faʻasalalauina o .Basolateral media (faʻataʻitaʻiga, mai Transwell faʻapipiʻi i faʻatanoa basolateral tetele i vaieli).
I totonu o lenei feagaiga, matou te tuʻuina atu se auala auʻiliʻili mo le fausiaina o microdevices gut-on-a-chip ma Transwell-insertable hybrid chips (laasaga 1-5) i le faʻatupuina o sela epithelial intestinal i luga ole polydimethylsiloxane (PDMS) -faʻavae porous membranes (laasaga 6A, 7A, 8, 9) poʻo polyesterBserts o 8, 9) poʻo polyesterBserts (8, 9) poʻo polyesterB6 membranes o polydimethylsiloxane (PDMS). 9) ma faʻaosoina le 3D morphogenesis in vitro (laasaga 10) . Matou te faʻaalia foʻi foliga feaveaʻi ma molecular e faʻaalia ai le histogenesis faʻapitoa faʻapitoa ma le faʻavasegaina o le cellular faʻalagolago i le gafa e ala i le faʻaaogaina o le tele o ata faʻataʻitaʻiga (laasaga 11-24) . Matou te faʻaosofia le morphogenesis e faʻaaoga ai le tino o le intestinal epithelial cell, poʻo i totonu o faʻamatalaga faʻapitoa e lua o le okeni, e pei o Cainaco-okeni e aofia ai faʻamatalaga faʻapitoa. fesuiaiga o membranes porous, faia o 2D monolayers, ma intestinal biochemical ma Toe faia o le biomechanical microenvironment.in vitro.Ina faatosina 3D morphogenesis mai 2D epithelial monolayers, matou aveesea morphogen antagonists i ituaiga uma aganuu e ala i le tafe atu o le ala i totonu o le basolateral vaega o le aganuu. epithelial layer lea e mafai ona faʻaaogaina e faʻataʻitaʻi ai le tuputupu aʻe o le epithelial e faalagolago i le morphogen, faʻatupu faʻatasi o le host-microbiome longitudinal, faʻamaʻi pipisi, faʻamaʻi manuʻa, faʻaleagaina o le epithelial barrier, ma togafitiga faʻavae probiotic Example.influences.
E mafai ona aoga a matou feagaiga i le tele o saienitisi i le tulaga faavae (faataitaiga, intestinal mucosal biology, stem cell biology, and developmental biology) ma suʻesuʻega faʻaaogaina (faʻataʻitaʻiga, suʻesuʻega faʻamaʻi faʻamaʻi, faʻataʻitaʻiga o faʻamaʻi, faʻainisinia o le tino, ma le gastroenterology) le tele o aʻafiaga. e mafai ona faʻasalalau atu i le au faʻalogo e suʻesuʻe le malosi o le faʻailogaina o le sela i le taimi o le atinaʻeina o le intestinal, toe faʻafouina poʻo le homeostasis .E le gata i lea, o la matou faʻasalalauga e aoga mo le fesiligia o faʻamaʻi pipisi i lalo o vailaʻau faʻamaʻi eseese e pei o le Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile poʻo le Salmonella Typhimu. O tagata faʻalogo o faʻamaʻi faʻamaʻi ma pathogenesis e aoga foi.O le faʻaaogaina o le microphysiology system e mafai ona faʻatagaina le faʻaumiumi faʻatasi-aganuʻu 10 ma suʻesuʻega mulimuli ane o le puipuiga a le au talimalo, tali faʻalavelave ma le toe faʻaleleia o manuʻa e aʻafia ai le pathogen i le gastrointestinal (GI) tract 11 . O isi faʻafitauli GI e fesoʻotaʻi ma le maʻi gut syndrome, maʻi celiac, maʻi faʻamaʻi faʻamaʻi, Crohn's colitis poʻo le maʻi pipisi e mafai ona avea ma maʻi pipisi, ulcerative bowels 3D intestinal epithelial layers are ready using the patient's 3D intestinal epithelial layers , o nei faamaʻi e aofia ai villous atrophy, crypt shortening, mucosal faaleagaina, po o le faaletonu epithelial pa. ma'i peripheral toto mononuclear sela (PBMCs), i faataitaiga o loo i ai 3D intestinal villus-crypt microarchitectures. sela fa'aletino fa'apitoa fa'apitoa, 5.
Talu ai o le 3D epithelial microstructure e mafai ona faʻapipiʻiina ma vaʻaia e aunoa ma le faʻagasologa o le vaega, o tagata matamata o loʻo galulue i luga o faʻasalalauga faʻapitoa ma faʻataʻitaʻiga maualuga poʻo faʻataʻitaʻiga sili e mafai ona fiafia i la tatou faʻafanua o le dynamics spatiotemporal o genes ma proteins i niches epithelial. Fiafia i tekinolosi.Tali i microbial poʻo le immune stimuli.E le gata i lea, longitudinal host-microbiome crosstalk 10, 14 e faʻamaopoopo le gut homeostasis e mafai ona faʻavaeina i le 3D intestinal mucosal layer e ala i le faʻapipiʻiina o ituaiga microbial eseese, microbial community poʻo fecal microbiota-ona-, aemaise i le fecal microbiota-ona. i luga o le tulaga.O lenei auala e sili ona manaia i le au maimoa e suʻesuʻeina mucosal immunology, gastroenterology, microbiome tagata, culturomics ma microbiology falemaʻi saili e faʻaleleia muamua microbiota gut i totonu o le fale suesue. Afai e mafai ona fetuutuunai a tatou in vitro morphogenesis protocol i scalable culture formats, e pei o multiwell inserts i 24, 496 faʻapipiʻi lelei i le 24, 496 ipu faʻapipiʻi lelei, 496 faʻapipiʻi lelei. e mafai foi ona faasalalau atu le feagaiga ia i latou o loo atiina ae pharmaceutical, biomedical Po o le maualuga-throughput screening po o faʻamaoniga tulaga mo le alamanuia meaai.As o se faamaoniga-o-faavae, matou faaalia talu ai nei le mafai o se multiplex maualuga-throughput morphogenesis faiga scalable i se 24-well plate format.In faaopoopo, tele okeni-i-a-ref16 oloa, le faʻatau16 oloa faʻatau, le faʻatau18 oloa. e mafai ona faatelevaveina la matou metotia in vitro morphogenesis ma mafai ona faʻaaogaina e le tele o suʻesuʻega suʻesuʻega, alamanuia poʻo le malo ma faʻalapotopotoga faʻatonutonu e malamalama ai i le cellular reprogramming o in vitro gut morphogenesis i le transcriptomic level e suʻe ai vailaʻau poʻo biotherapeutics O le faʻaaogaina ma le felauaiga o sui o fualaau faasaina na iloiloina e faʻaaoga ai le 3D gut surrogates poʻo le faʻaogaina o le aganuʻu poʻo le faʻataʻitaʻiga faʻatau pisinisi. faagasologa o le morphogenesis.
O se numera faatapulaaina o faʻataʻitaʻiga faʻataʻitaʻiga faʻatatau i tagata na faʻaaogaina e suʻesuʻe ai le intestinal epithelial morphogenesis, e mafua ona o le leai o ni faʻataʻitaʻiga faʻatinoina e faʻaosofia ai le 3D morphogenesis in vitro. fesiligia, ma le mea e sili ona taua, e le fuafuaina tonu faagasologa atinae o tagata. O nei faataitaiga e matua faatapulaaina foi i lo latou gafatia e tofotofoina i se auala scalable manner.O le mea lea, o lo tatou feagaiga mo le toe faafouga 3D fausaga tissue in vitro outperforms i vivo manu faataitaiga faapea foi ma isi tu masani static 2D aganuʻu faʻataʻitaʻiga.As utilizing structure3D faʻatagaina muamua, e utilizing structures3D faʻatagaina le ushelital structure3D faʻatagaina le suʻega 3D faʻatagaina muamua, e utilizing structure3D e faʻatagaina ai le faʻataʻitaʻiga o le suʻega 3D e faʻaaogaina i le faʻataʻitaʻiga muamua. localization o sela eseese i le crypt-villus axis i le tali atu i mucosal eseese po o immune stimuli.3D epithelial layers e mafai ona maua ai se avanoa e suesue ai pe faapefea ona tauva sela microbial e fausia niches spatial ma ecological evolution i le tali atu i mea taua (faʻataʻitaʻiga, totonu versus fafo mucus layers, secretion o IgA ma le antimicrobial epithelial peptides e mafai ai ona malamalama i le peptides antimicrobial, us). pe faʻafefea ona faʻapipiʻiina e le gut microbiota ona nuʻu ma synergistically gaosia microbial metabolites (faʻataʻitaʻiga, pupuʻu filifili gaʻo gaʻo) e faʻapipiʻiina le faʻapipiʻiina o le telefoni feaveaʻi ma niches cell stem i totonu o le basal crypts. O nei foliga e mafai ona faʻaalia pe a faʻavaeina 3D epithelial layers in vitro.
I le faaopoopo atu i la tatou metotia o le fatuina o le 3D intestinal epithelial structures, e tele auala in vitro.Intestinal organoid culture o se tulaga faʻainisinia faʻainisinia faʻainisinia faʻavae e faʻavae i luga o le faʻatupuina o sela o le intestinal i lalo o tulaga faʻapitoa morphogen23,24,25. Ae ui i lea, o le faʻaaogaina o 3D organoid-culture faʻataʻitaʻiga e masani ona faʻataʻitaʻiina mo le felauaiga o faʻataʻitaʻiga o le felauaiga o le faʻataʻitaʻiga masani o le felauaiga o le faʻafeiloaʻiga. Intestinal lumen o loʻo faʻapipiʻiina i totonu o le organoid ma, o le mea lea, o le faʻaofiina o vaega luminal e pei ole microbial cell poʻo antigens exogenous e faʻatapulaʻaina. E mafai ona faʻaleleia le avanoa i lumens organoid e faʻaaoga ai se microinjector,26,27 ae o lenei metotia e faʻalavelave ma galue malosi ma e manaʻomia ai le poto faʻapitoa e faʻatinoina. E le gata i lea, o aganuu masani masani o loʻo tausia i faʻaputuga hydrogel i lalo o tulaga faʻapitoa e le faʻaalia saʻo le gaioiga i vivo biomechanics.
O isi auala o loʻo faʻaaogaina e le tele o vaega suʻesuʻe e faʻaaogaina 3D hydrogel scaffolds e faʻataʻitaʻi i le gut epithelial structure e ala i le faʻatupuina o sela o le intestinal tagata i luga o le gel surface. Fabricate hydrogel scaffolds e faʻaaoga ai le 3D-printed, micro-milled, poʻo le lithographically fabricated molds. morphogen gradients, faatuina o se tulaga maualuga epithelial structure ma stroma-epithelial crosstalk e ala i le aofia ai sela stromal i le fata. ma maua galuega physiological.Ose isi suʻesuʻega talu ai nei na faʻaogaina ai faʻataʻitaʻiga hydrogel i totonu o se microfluidic platform ma mamanu fausaga epithelial intestinal e faʻaaoga ai le laser-etching techniques.Mouse intestinal organoids mulimuli i mamanu etched e fausia ai le intestinal tubular structures, ma intraluminal fluid flows can be recapitulated using a microfluidics procedures neitherowever, spontaneous model.However. mechanobiological movements.3D lomitusi metotia mai le vaega lava lea e tasi na mafai ona faia ni tamai paipa gut with spontaneous morphogenetic process.E ui lava i le lavelave fau o vaega gut eseese i totonu o le paipa, o lenei faataitaiga e leai foi le vai luminal tafe ma deformation masini.E le gata i lea, o le faʻataʻitaʻiga operability e mafai ona faatapulaaina, aemaise lava pe a maeʻa le bioprinting faagasologa ua maeʻa poʻo le faʻataʻitaʻiga faʻataʻitaʻiga tuʻufaʻatasiga tuʻufaʻatasia, faʻataʻitaʻiga. gut morphogenesis, physiologically related shear stress, biomechanics that mimic gut motility, accessibility of independent apical and basolateral compartments, ma le toe faʻafouina o meaola lavelave microenvironments o modularity.O lea, o la tatou in vitro 3D morphogenesis protocol e mafai ona maua ai se auala faʻafesoʻotaʻi e foia ai luitau o metotia o loʻo i ai nei.
O la matou feagaiga e taulaʻi atoa i le 3D epithelial morphogenesis, ma naʻo sela epithelial i le aganuu ma e leai se isi ituaiga o sela o loʻo siomia ai e pei o sela mesenchymal, sela endothelial, ma siama puipuia. gut-on-a-chip ma hybrid-on-a-chip e mafai ai ona tatou toe faia le undulating 3D epithelial layer, faʻalavelave faʻapitoa faʻapitoa e pei o le epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 ma, i la tatou faʻataʻitaʻiga, crypt-villus features that convey stem cell niches stays to be further considering cell crypts. o le mesenchyme o loʻo i ai se sao taua i le gaosiga o le ECM proteins ma le faʻatonutonuina o le intestinal morphogenesis i vivo35,37,38. O le faʻaopoopoga o sela mesenchymal i la tatou faʻataʻitaʻiga na faʻaleleia ai le faagasologa o le morphogenetic ma le faʻapipiʻiina o le tino. microenvironment.E le gata i lea, o vaega vasculature e mafai ona fesoʻotaʻi i le va o faʻataʻitaʻiga o mea e manaʻomia muamua pe a faʻataʻitaʻiina faʻataʻitaʻiga faʻataʻitaʻiga e faʻaalia ai fegalegaleaiga tele-okeni. puipuiga i le tulaga o le fa'ata'ita'iina o fa'ama'i intestinal.
O le faʻaogaina o chips hybrid e sili atu le tuusaʻo nai lo le gut-on-a-chip ona o le seti o masini e faigofie ma o le faʻaogaina o Transwell faʻapipiʻi e mafai ai ona scalable aganuʻu o le gut epithelium. i luga o le itu apical.E manino lava, o meatotino static i le apical compartment seasea mafai ona umi-umi bacteria co-aganuu i hybrid chips.E ui e mafai ona tatou robustly faatosina 3D morphogenesis i Transwell inserts pe a faaaogaina meataalo hybride, o le le lava o le physiologically talafeagai biomechanics ma apical tafe tafe e mafai ona faatapulaaina le mafaia o talosaga hybrid platforms.
Toe fa'aleleia atoa o le tagata crypt-villus axis i gut-on-a-chip ma hybrid-on-a-chip aganuu e le'i fa'amautuina atoatoa. Talu ai o le morphogenesis e amata mai i le epithelial monolayer, 3D microarchitectures e le maua ai le tutusa o le morphological i crypts i vivo. 3D epithelium, o le crypt ma villous itulagi e leʻi manino demarcated. E ui o le maualuga o alalaupapa i luga o le vaʻa e taʻitaʻia ai le maualuga maualuga o le microengineered epithelium, o le maualuga maualuga o loʻo faʻatapulaʻaina pea i le ~ 300-400 μm. O le loloto moni o le intestinal crypts o le tagata i totonu o le tootoo laiti ma le lapopoa o le ~m3 ma le tele o le ~m3 ma le tele. maualuga ole intestinal villi e ~600 µm41.
Mai se vaaiga imaging, in situ super-resolution imaging o 3D microarchitectures e mafai ona faatapulaaina i le gut i luga o se pu, talu ai o le galuega manaomia mamao mai le faamoemoega tioata i le epithelial layer o loo i luga o le faasologa o ni nai millimeters. Ina ia foia lenei faafitauli, e mafai ona manaomia se faamoemoega mamao. PDMS.E le gata i lea, talu ai o le microfabrication layer-by-layer o le gutu i luga o se pulou e aofia ai le pipii tumau i le va o vaega taʻitasi, e matua faigata lava ona tatala pe aveese le pito i luga e suʻesuʻe ai le fausaga o le epithelial layer.
O le hydrophobicity o le PDMS ua avea ma mea faʻatapulaʻa i suʻesuʻega faʻavae microfluidic e feagai ma molelaiti laiti hydrophobic, talu ai e mafai e le PDMS ona faʻapipiʻi faʻapitoa ia molelaʻau hydrophobic. Alternatives i le PDMS e mafai ona mafaufauina ma isi mea polymeric. Alternatively, suiga i luga ole PDMS (eg, coating with lipophilic 4) mea e mafai ona manatu i le lipophilic4. faaitiitia adsorption o molelaula hydrophobic.
Ma le mea mulimuli, o la matou metotia e leʻi faʻamaonia lelei i tulaga o le tuʻuina atu o se suʻesuʻega maualuga poʻo le "tasi-size-fits-all" faʻataʻitaʻiga faʻataʻitaʻiga faʻaoga-tagata. 384-well porous inserts e mafai ai ona fa'aauau le toe fa'atumu ma le aveeseina o fa'asalalauga basolateral).
Ina ia faaosofia le 3D morphogenesis o le intestinal epithelium o le tagata in vitro, matou te faʻaaogaina se masini microfluidic chip intestinal o loʻo i ai lua microchannels tutusa ma se membrane porous elastic i le va e fausia ai se lumen-capillary interface. Matou te faʻaalia foi le faʻaogaina o se masini microfluidic tasi-channel (se hybrid chip) lea e maua ai le tafe faifai pea i lalo o le lapisi e lua. platforms, morphogenesis o sela epithelial intestinal eseese tagata e mafai ona faʻaalia e ala i le faʻaaogaina o le faʻaogaina o le faʻaogaina o le tafe e aveese ai le morphogen antagonists mai le vaega basolateral. O le faʻataʻitaʻiga atoa o le faʻataʻitaʻiga (Ata 1) e aofia ai vaega e lima: (i) microfabrication o le gut chip poʻo Transwell insertable hybrid chip (laasaga 1-5) (laasaga 1-5); (Caco-2 sela) poʻo organoids intestinal tagata; pusa 2-5), (iii) aganuu o sela epithelial intestinal i intestinal meataalo po o meataalo hybrid (laasaga 6-9), (iv) induction o le 3D morphogenesis in vitro (laasaga 10) ma (v)) e faʻaalia ai le 3D epithelial microstructure (laasaga 11-24). vitro morphogenesis e ala i le faʻatusatusaina o le epithelial morphogenesis i faʻafanua, faʻaletino, tulaga, poʻo faʻatonuga faʻatonuga.
Na matou faʻaogaina ni faʻavae faʻaleaganuʻu eseese e lua: gut-on-a-chip ma ala saʻo poʻo laina feʻaveaʻi e le laina, poʻo chips hybrid o loʻo iai Transwell (TW) faʻaofi i totonu o se masini microfluidic, faʻapipiʻiina e pei ona faʻamatalaina i le Pusa 1, ma le laasaga 1 -5. organoids intestinal tagata) ma faiga aganuu o loʻo faʻaaogaina i totonu o lenei faʻasalalauga. e mafai ona faʻaogaina le epithelial layers intestinal, mo se faʻataʻitaʻiga, i le faʻavasegaina o le eseesega o le cell, suʻesuʻega o le gut physiology, faʻavaeina o meaola faʻanatura-microbiome, ma faʻataʻitaʻiga faʻamaʻi. Immunofluorescence ata i le "Cell Differentiation" o loʻo faʻaalia ai le nuclei, F-actin ma MUC2 o loʻo faʻaalia i le 3D Caco-2 e faʻaalia i le 3D Caco-2 e faʻaalia ai le faʻailoga o le gut. goblet cell and mucus secreted from mucosal surfaces.Fluorescent images in Gut Physiology show mucus produced by staining for sialic acid and N-acetylglucosamine residues using fluorescent wheat germ agglutinin.The two overlapping images in “Host-Microbe Co-Cultures” show representative host-microbiome co-culture on the co-culture left on the co-culture of the chipculture of the co-culture. coli fa'aalia le polotini fluorescent lanu meamata (GFP) ma microengineered 3D Caco-2 epithelial sela. O le laulau taumatau o loʻo faʻaalia ai le localization o le GFP E. coli faʻatasi ma le 3D Caco-2 epithelial sela, sosoo ai ma le immunofluorescence staining ma F-actin (mumu) ma nuclei (lanu moana). faʻatasi ai ma antigens siama (faʻataʻitaʻiga, lipopolysaccharide, LPS) ma sela puipuia (eg, PBMC; lanumeamata) .Caco-2 sela na faʻatupuina e faʻavae ai se 3D epithelial layer. Scale bar, 50 µm.Ata i le laina pito i lalo: "Differentiation of cell" fetuutuunai ma le faatagaga mai reference.2. Oxford University Press; Toe faia ma le faatagaga mai le Ref.5. NAS; “Host-Microbe Co-Culture” fetuutuunai ma le faatagaga mai ref.3. NAS; "Fa'ata'ita'iga Fa'ama'i" fetu'una'i ma fa'atagaga mai fa'asinomaga.5. NAS.
O le gut-on-chip ma hybrid chips na faʻaaogaina e faʻaaoga ai le PDMS replicas na faʻaumatia mai le silicon molds e lithography malu1,44 ma faʻataʻitaʻiina i le SU-8. O le mamanu o microchannels i totonu o vaʻa taʻitasi e faʻamoemoeina e ala i le iloiloina o hydrodynamics e pei o le mamafa o le shear ma le hydrodynamic pressure1,4,12. The original gut-on-a-chip design 1,4,12. fa'atasi fa'atasi microchannels sa'o sa'o, ua fa'atupuina i se fa'alavelave fa'alavelave (Extended Data Fig. 1b) lea e aofia ai se pea o microchannels curved e fa'aosofia ai le Fa'ateleina o le taimi e nofo ai le vai, fa'asologa o le tafe e le laina, ma multiaxial deformation o sela fa'aleaganu'u (Fig. 2a-f) toe lavelave gut. gut-on-a-chips e mafai ona filifilia.Ua matou faʻaalia o le Gut-Chip faʻafefeteina e faʻamalosia malosi foi le 3D morphogenesis i se taimi tutusa ma se tikeri tutusa o le tuputupu aʻe o le epithelial pe a faʻatusatusa i le Gut-Chip muamua, e tusa lava po o le a le ituaiga cell culture. SU-8 mamanu na maua ai foliga le lelei pe a uma ona faʻamalo (Fig. 2a). Ina ia faʻapipiʻi le gutu i luga o se pu, o le pito i luga o le PDMS layer sa faʻapipiʻiina faʻasolosolo i se ata porous PDMS ma faʻapipiʻi ma le pito i lalo o le PDMS e ala i le faʻaogaina o le corona treater (Fig. 2b-f). e mafai ona faʻaogaina faʻapipiʻi Transwell (Fig. 2h ma Extended Data Fig. 2). O le faʻaogaina o le faʻagasologa o loʻo faia e ala i le togafitia o luga o le PDMS replica ma tioata faʻatasi ma le okesene plasma poʻo le togafitiga o le corona. Ina ua maeʻa le faʻamaʻiina o le masini microfabricated faʻapipiʻi i le silicone tube, ua saunia le seti masini e faʻatino 3D morphFigurlogenesis epithelium2 intestinagogenesis.
a, Faʻataʻitaʻiga Schematic o le saunia o vaega PDMS mai SU-8 patterned silicon molds.O le PDMS solution uncured sa sasaa i luga o se silicon mold (agavale), faʻamalolo i le 60 °C (ogatotonu) ma demolded (taumatau). O le demolded PDMS na tipiina ma faʻamama mo le faʻaogaina atili.b, Ata o le silicon mold faʻaaoga e saunia ai. fauina le PDMS porous membrane.d, O se faasologa o ata o vaega pito i luga ma lalo o le PDMS ma le faʻapipiʻiina i luga o le masini intestinal device.e, Schematic of the alignment of the upper, membrane, and lower PDMS parts.Each layer is irreversibly bonded by plasma or corona treatment.f, Schematic of the fabricated gut-on-a-imchannel device with vase microcuum-on-a-imchaposed device. chambers.g, Setup of gut-on-a-chip for microfluidic cell culture.The fabricated gut on a chip assembled with a silicone tube and syringe sa tuu i luga o se coverslip.O le masini chip na tuu i luga o le tapuni o le 150 mm Petri dish mo le faagasologa.O le fusifusia e faaaoga e tapuni ai le silicone tube.h, Vaaiga ata ma ata o le 3D hybrid chipmorph saunia. i aganuu 2D monolayers o intestinal epithelial sela na faaofiina i totonu o le atigi pusa e faaosofia ai le intestinal 3D morphogenesis.O le ala o loʻo faʻafefeteina e ala i microchannels i lalo ifo o le cell layer na faʻavaeina i luga o le Transwell insert.Scale bar, 1 cm.h Toe lolomi ma faʻatagaga mai reference.4. Elsevier.
I lenei feagaiga, o le Caco-2 cell line ma organoids intestinal na faʻaaogaina e avea ma puna epithelial (Fig. 3a). O ituaiga uma e lua na faʻatupuina tutoʻatasi (Pusa 2 ma Pusa 5) ma faʻaaogaina e fatu ai le ECM-coated microchannels of on-chip gut poʻo Transwell inserts. Pe a faʻapipiʻi sela (> 95% le faʻaogaina o le aganuʻu (> 95% o le faʻaogaina o le aganuʻu i totonu o le aganuʻu masani (> 95% o le faʻaogaina o le aganuʻu). 10 ma le 50) i T-funu'u e seleseleina e saunia suspensions cell dissociated e trypsinization vai (pusa 2) .Tagata intestinal organoids mai intestinal biopsies po o taotoga tipitipi sa aganuu i Matrigel scaffold domes i 24-well ipu e lagolago ai le structural microenviring. R-spondin, ma Noggin) ma mea tuputupu aʻe saunia e pei ona faamatalaina i le Pusa 3 sa faaopoopo i isi aso uma seia oo ina tuputupu aʻe le okeni i le ~ 500 μm i le lautele. O organoids matua tupu atoa e seleseleina ma vavaeina i sela e tasi mo le totoina i luga o le gut po o Transwell inserts i luga o se pulou (Pusa 5). colitis, Crohn's disease, colorectal cancer, poʻo le foaʻi masani), nofoaga o manuʻa (faʻataʻitaʻiga, lesion versus lesioned area) ma le gastrointestinal location i totonu o le ala (faʻataʻitaʻiga, duodenum, jejunum, ileum, cecum, colon, poʻo le rectum). okeni.
a, Galulue mo le faʻaosoina o le morphogenesis i totonu o le pu.Caco-2 tagata intestinal epithelium ma intestinal organoids o loʻo faʻaaogaina i lenei faʻataʻitaʻiga e faʻaalia ai le 3D morphogenesis. O sela epithelial vavae ese na faʻatoʻaina i totonu o le masini gut-on-a-chip saunia (sauniuniga chip) . (D0), o le apical (AP) tafe e amata ma tausia mo le 2 aso muamua (tafe, AP, D0-D2). Basolateral (BL) tafe mai e amata foi faatasi ma cyclic faalautele gaioiga (faʻaloaloa, tafe, AP ma BL) pe a faia se monolayer atoatoa 2D ua fausia. Intestinal 3D morphogenesis faʻaalia ata o le micromorphic5D morphogenesis faʻaalia faʻafuaseʻi pe a maeʻa 5 aso contraidic5). sui morphology o Caco-2 sela i laasaga faataitai taitasi po o le taimi (kalafa pa, 100 µm). Fa ata schematic e faʻaalia ai cascades tutusa o le gut morphogenesis (taumatau pito i luga). O aū faʻamavae i le schematic o loʻo faʻatusalia le itu o le tafe o le vai.b, SEM ata o loʻo faʻaalia ai le topology luga o le epithelium e3D Casetle-2 faʻamautu. (pusa pa'epa'e pa'epa'e) o lo'o fa'aalia ai le microvilli toe fa'afouina i luga o le 3D Caco-2 layer (taumatau).c, Fa'asagaga fa'asaga i luma o le fa'avaeina o Caco-2 3D, claudin (ZO-1, mumu) ma fa'aauau pea le pulumu tuaoi membranes fa'aigoaina F-actin (lanumeamata) ma le nuclei (lanumoana) Immunofluorescence confocal va'aiga va'aiga i le va'aiga va'aiga o le epitestAll schematic va'aiga i le va'aiga o le epithell schematic. faailoa mai le nofoaga o le vaalele taulai mo vaaiga confocal taitasi.d, Taimi taimi o suiga morphological i organoids aganuu i luga o se vaʻa maua e vaega faʻatusatusa microscopy i aso 3, 7, 9, 11, ma 13. O le inset (taumatau pito i luga) o loʻo faʻaalia ai le faʻalauteleina maualuga o le ata ua saunia.e, DIC photomicrograph of organoid slice 3D epithelium na faʻatuina i le aso gut7. ata immunofluorescence o loʻo faʻaalia ai faʻailoga mo sela (LGR5; magenta), sela ipu (MUC2; lanumeamata), F-actin (efuefu) ma nuclei (cyan) tupu i luga o gut chips mo 3 aso, faasologa (Agavale) ma 13-aso (ogatotonu) organoids i luga o le epithelial layer.Vaʻai foi Faʻamatalaga Faʻalautele LG2 Faʻamatalaga Faʻamatalaga Faʻamatalaga faailoilo.Fluorescence ata o loʻo faʻaalia ai le epithelial microstructure (taumatau) o le 3D organoid epithelium o loʻo faʻavaeina i totonu o le gut i luga o se vaʻa e ala i le pisia o le plasma membrane i le CellMask dye (taumatau) i le aso 13 o le aganuu. O le paʻu fua e 50 μm seʻi vagana ua taʻua.b Toe lolomiina ma le faʻatagaga mai le faʻamatalaga.2. Oxford University Press; i Fetuuna'i ma le fa'atagaga mai Fa'asinomaga.2. Oxford University Press; u ma f fetuutuunai ma le faatagaga e ala ile faasinomaga.12 I lalo ole Creative Commons License CC BY 4.0.
I totonu o le gutu i luga o se pu, e manaʻomia le suia o le hydrophobic surface o le PDMS porous membrane mo le manuia o le ECM coating. I lenei faʻasalalauga, matou te faʻaogaina auala eseese e lua e faʻaleleia ai le hydrophobicity o membranes PDMS. membrane.Ae ui i lea, microfluidic culture o organoid epithelium e manaʻomia ai le faʻaogaina o luga ole kemikolo e ausia ai le lelei o le teuina o palatini ECM e ala ile faʻaogaina faʻasolosolo o le polyethyleneimine (PEI) ma le glutaraldehyde i le PDMS microchannels. Ina ua maeʻa suiga i luga, na teu ai polotini ECM e ufiufi ai le faʻaogaina o le PDMS o le epilium o loʻo tuʻuina atu i luga o le epilium o le PDMS. faʻapipiʻi, amata le aganuu cell microfluidic i perfusing na o le ala i luga o le microchannel pito i luga seia oo ina fausia sela se monolayer atoatoa, ae o le microchannel pito i lalo o loo tausia tulaga static.O lenei auala optimized mo le faatoaagaina i luga ma le ECM coating e mafai ai le faapipiiina o le epithelium organoid e faatosina 3D morphogenesis i luga o le PDMS luga.
O aganu'u Transwell e mana'omia fo'i le ufiufi ECM a'o le'i totoina sela; ae ui i lea, o aganuu a Transwell e le manaʻomia ni laʻasaga lavelave e faʻagaoioia ai le faʻaogaina o mea faʻapipiʻi porous. Mo le tuputupu aʻe o Caco-2 sela i luga o faʻapipiʻi Transwell, faʻapipiʻi ECM i luga o mea faʻapipiʻi porous faʻavavevaveina le faʻapipiʻiina o sela Caco-2 dissociated (<1 itula) ma le faʻapipiʻiina o pa puipui faʻapipiʻi (<1-2 aso). Faʻapipiʻi faʻapipiʻi ECM, faʻapipiʻi i luga o le membrane (<3 h) ma tausia seia oʻo ina fausia e le organoids se monolayer atoatoa ma le faʻamaoni pa puipui .O aganuu Transwell e faia i 24-well plates e aunoa ma le faʻaogaina o meataalo hybrid.
In vitro 3D morphogenesis e mafai ona amataina e ala i le faʻaogaina o le tafe o le vai i le vaega basolateral o se epithelial layer ua faʻamautuina. I totonu o le gutu i luga o se pu, na amata le epithelial morphogenesis pe a faʻafefeina le ala ile pito i luga ma lalo ole microchannels (Fig. 3a). Iniseti. Ina ia maua le lava meaʻai ma serum i sela fusifusia i luga o porous membranes ma maua ai le mamafa o le vili luminal, e masani ona tatou faaaogaina le tafe lua i le gut i luga o se chip.In hybrid chips, Transwell inserts o loo i ai epithelial monolayers sa faaofiina i totonu o meataalo hybride. Ona, o le ala na faʻaaogaina i lalo o le itu basolateral o le porous Transwell faʻaofiina i aso o le microinaltimorph. tafeaga i totonu o tulaga uma e lua.
E mafai ona suʻesuʻeina uiga faʻapitoa o microengineered 3D epithelial layers e ala i le faʻaaogaina o ata faʻataʻitaʻiga eseese, e aofia ai vaega faʻatusatusa microscopy, faʻalavelave faʻalavelave faʻalavelave (DIC) microscopy, SEM, poʻo immunofluorescence confocal microscopy (Ata 3 ma 4) .Phase contrast poʻo DIC imaging e mafai ona faigofie ona faʻatinoina i le taimi faʻataʻitaʻiga o le DIC ma faʻataʻitaʻiga i le taimi o le faʻataʻitaʻiga o le DIC e mafai ona faigofie ona faʻatinoina i le taimi faʻataʻitaʻiga o le DIC. layers.Due to the optical transparency of PDMS and polyester films, both the gut-on-a-chip and hybrid chip platforms can provide real-time in situ imaging e aunoa ma le manaomia o se vaega po o le disassembly o le masini.When faia immunofluorescence imaging (Ata 1, 3c, f ma 4b, c), sela e faʻapipiʻiina (paradehytly% typically) (PFA), sosoo ai ma le Triton X-100 ma le 2% (wt/vol) ) bovine serum albumin (BSA), i le faasologa. (faʻataʻitaʻiga, 4′,6-diamidino-2-phenylene) indole, DAPI) poʻo le F-actin (eg, fluorescently labeled phalloidin).Fluorescence-based imaging ola e mafai foi ona faia i totonu e iloa ai le gaosiga o le mucus (Fig. 1, "Cell differentiation" ma le "Gut physiology"), faʻaogaina faʻafuaseʻi (Fig-biology cell), faʻaogaina faʻafuaseʻi (Fig-biology cell), faʻaogaina faʻafuaseʻi (Fig. co-culture"), le faʻafaigaluegaina o sela puipuia (Fig. 1, 'Disease Modeling') poʻo le faʻasologa o le 3D epithelial morphology (Fig. 3c, f ma 4b, c) . Pe a suia le gutu i luga o le chip e vavae ese ai le pito i luga mai le pito i lalo microchannel layer, e pei ona faʻamatalaina i le ref.E pei ona faʻaalia i le Fig3D evillimorphology. e mafai ona vaʻaia le tuaoi o le pulumu apical e SEM (Fig. 3b) .O le faʻaaliga o faʻailoga eseese e mafai ona suʻesuʻeina e ala i le faia o le PCR5 quantitative poʻo le tasi-cell RNA sequencing. I lenei tulaga, o le 3D layers o sela epithelial e tupu i totonu o le gut chips poʻo le hybrid chips e seleseleina e le trypsinization ona faʻaaogaina lea mo suʻesuʻega mole mole poʻo genetic.
a, Galulue mo le induction o le intestinal morphogenesis i totonu o se pulou hybrid.Caco-2 ma organoids intestinal o loʻo faʻaaogaina i lenei protocol e faʻaalia ai le 3D morphogenesis i totonu o se tuʻufaʻatasiga vaʻaia. tulaga (aganuu TW) . Ina ua mavae aso 7, o se tasi Transwell insert o loo i ai se 2D monolayer o sela epithelial ua tuufaatasia i totonu o se pulou hybrid e faailoa atu ai se tafega basolateral (Flow, BL), lea na iu lava ina taitai atu ai i le fausiaina o se 3D epithelial layer (morphogenesis) .Phase contrast micrographs showing morphological features of human organ epithelial epithelial 3 line or cell normal. laʻasaga faʻataʻitaʻi poʻo le taimi taimi.O le schematics i luga o le laulau faʻataʻitaʻiga le faʻataʻitaʻiga faʻataʻitaʻiga mo laasaga taʻitasi.b, meataalo Hybrid (agavale schematic) e mafai ona taʻitaʻia 3D morphogenesis o sela epithelial organoid ma pito i lalo confocal microscopy vaaiga ave i tulaga Z eseese (luga, ogatotonu, ma le pito i lalo; vaai schematic taumatau ma laina togitogi tutusa). fa'aalia uiga manino morphological.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled view) of organoid- maua mai sela epithelial cultured in static Transwell (TW; inset within white dashed box) versus hybrid chip (tele tele ata atoa) faatusatusa 2D versus 3D vaaiga pito i luga o le morphology. tulimanu taumatau; “XZ”) o lo'o fa'aalia fo'i foliga 2D ma le 3D. Pa'u fua, 100 µm.c Toe lolomi fa'atasi ma le fa'atagaga mai fa'amatalaga.4. Elsevier.
E mafai ona saunia le pulea e ala i le faʻatupuina o sela tutusa (Caco-2 poʻo le intestinal organoid epithelial cell) i le lua-dimensional monolayers i lalo o tulaga masani faʻaleaganuʻu masani. fa'atusaina.
E tatau ona faia le faagasologa o le lithography vaivai i totonu o se potu mama. Mo vaega taʻitasi i luga o le pu (pito i luga ma pito i lalo ma membranes) ma chips hybrid, na faʻaaogaina photomasks eseese ma faʻapipiʻiina i luga o vaʻa silikoni eseese ona o le maualuga o le microchannels e eseese. 200 µm.
Tu'u se 3-inisi mafolafola silikoni i totonu o se ipu o lo'o iai le acetone. Fa'alili lemu le ipu mo le 30 sekone, ona fa'amamago lea i luga o le ea.
O se vaifofo piranha (fa'afefiloi o le hydrogen peroxyide ma le sulfuric acid concentrated, 1:3 (vol/vol)) e mafai ona fa'aogaina e fa'ateleina ai le aveesea o toega oona mai luga o le silicon wafer surface.
O le vaifofo o le Piranha e matua'i pala ma fa'atupu ai le vevela.E mana'omia ni puipuiga fa'aopoopo mo le lafoa'iina o otaota, fa'ataga le vaifofo e mālūlū ma tu'u atu i se atigipusa mama mamago. Fa'aoga koneteina lona lua ma fa'aigoa lelei koneteina otaota. Fa'amolemole mulimuli i ta'iala mo le saogalemu o le fale mo fa'amatalaga auiliili.
Fa'amama le vai e ala i le tu'u i luga o se ipu vevela 200 °C mo le 10 min. A mae'a le fa'amama, sa luluina le wafer i le lima taimi i le ea e malulu.
Liligi ~ 10 g o le photoresist SU-8 2100 i luga o le ogatotonu o le mama silicon wafer. Faʻaaoga tweezers e faʻasalalau le photoresist tutusa i luga o le wafer.Tuu i nisi taimi le wafer i luga o le 65 ° C vevela ipu ina ia faaitiitia pipii le photoresist ma faigofie ona salalau.
SU-8 na tufa tutusa i luga o le wafer e ala i le faʻaogaina o le ufiufi vili. Polokalama se suiga o le SU-8 mo le 5-10 s e faʻasalalau i le 500 rpm i le saoasaoa o le 100 rpm / s. Seti le vili autu mo le 200 µm mafiafia mamanu i le 1,500 rpm 50 (ma maua le mafiafia o le 1,500 rpm) µm maualuga mo le vaega pito i luga o le pu i luga o le pu; taga'i i "Laasaga taua" i lalo) seti i le saoasaoa o le 300 rpm/s 30 sekone i le 1,200 rpm.
E mafai ona fetu'una'i le saoasaoa tele o le milo e tusa ai ma le mafiafia o le SU-8 mamanu i luga o le fafie silikoni.
Ina ia faia ni mamanu SU-8 o le 500 µm maualuga mo le vaega pito i luga o le gutu i luga o le pu, o le vili vili ma laasaga tao tao vaivai o lenei Pusa (laasaga 7 ma le 8) sa toe fai faasolosolo (silasila i le Laasaga 9) e maua ai ni laulau se lua o le 250 μm O se vaega mafiafia o le SU-8, lea e mafai ona faʻapipiʻi i totonu o le laasaga 12 ma faʻapipiʻi le UV 5 layer. maualuga.
Fa'amama fa'asusu le su-8 fa'apipi'i fa'apipi'i e ala i le tu'u ma le fa'aeteete o ma'i mafolafola i luga o se ipu vevela i le 65 °C mo le 5 min, ona sui lea o le fa'atulagaina i le 95 °C ma fa'asusu mo se isi 40 min.
Ina ia ausia le maualuga o le 500 μm o le SU-8 mamanu i le microchannel pito i luga, toe fai laasaga 7 ma le 8 e faʻatupu ai le 250 μm mafiafia SU-8 layers.
I le fa'aaogaina o le UV Mask Aligner, fai se su'ega molī e tusa ai ma fa'atonuga a le kamupani gaosi mea e fa'atatau ai le taimi e fa'aalia ai le fa'ama'i.
A maeʻa ona fuafuaina le taimi e faʻaalia ai, tuʻu le photomask i luga o le ufimata o loʻo i ai le UV mask aligner ma tuʻu le photomask i luga o le SU-8 coated wafer.
Tuu sa'o le pito lomitusi o le ufimata i luga o le itu SU-8 o lo'o ufiufiina o le fafie silikoni e fa'aitiitia ai le fa'asalalauina o le UV.
Fa'aali sa'o le su-8 masi fa'apipi'i fa'apea ma le ufimata ata i luga i le 260 mJ/cm2 o le malamalama UV mo le taimi e fa'ailoa ai muamua (silasila i le Laasaga 10 o lenei pusa).
Ina ua maeʻa le faʻaalia o le UV, na taoina le su-8-coated silicon wafers i le 65 ° C mo le 5 min ma le 95 ° C mo le 15 min i luga o ipu vevela taʻitasi e faʻapipiʻi mamanu ma le maualuga o le 200 μm. Faʻalautele le taimi o le taoina i le 95 ° C i le 30 min e fai ai mamanu ma le maualuga o le 500 μm.
E sasaa le tagata atiae i totonu o se ipu tioata, ma tuu le fasi falaoa tao tao i totonu o le ipu.O le voluma o le SU-8 developer e mafai ona fesuisuiai e faalagolago i le tele o le ipu tioata. Ia mautinoa ia faʻaaoga le lava SU-8 developer e aveese atoa le SU-8.Mo se faʻataʻitaʻiga, pe a faʻaaogaina se ipu tioata 150 mm lautele ma le 1 L le gafatia, faʻaaoga ~ 300 mL o le suʻesuʻega faʻataʻamilosaga 5.
Fufulu le limu ua atia'e i le ~10 mL o le mea fou fa'atupu fa'ato'a soso'o ai ma le IPA e ala i le sasaina o le vaifofo e fa'aoga ai le paipa.
Tuu le wafer i totonu o le plasma cleaner ma faʻaalia i le plasma okesene (kasa o le ea, faʻatonuga 1 × 10−5 Torr, mana 125 W) mo le 1.5 min.
Tuu le wafer i totonu o se masini faʻamama ma se tioata faʻataʻitaʻi i totonu. E mafai ona tuʻu faʻatasi ma faʻataʻitaʻiga faʻamaʻi. gaogao mo silani.
Liu se fagu o sela Caco-2 aisa i totonu o le vai taele e 37°C, ona ave lea o sela ua liusuavai i se fagu T75 o loo iai le 15 mL o le 37°C o le medium Caco-2 ua uma ona faamafanafanaina.
Ina ia pasi Caco-2 sela i le ~ 90% faʻatasi, muamua mafanafana Caco-2 medium, PBS, ma 0.25% trypsin / 1 mM EDTA i totonu o le vai taele 37 ° C.
Fufulu le ala e ala i le fa'amama gaogao. Fufulu fa'alua sela i le 5 mL o le PBS mafanafana e ala i le toe faia o le manava fa'amama ma fa'aopoopo le PBS fou.