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I-Human gut morphogenesis iseka iimpawu ze-crypt-villus ze-3D epithelial microarchitecture kunye ne-spatial organization.Olu lwakhiwo lukhethekileyo luyafuneka ukuze kugcinwe i-homeostasis yamathumbu ngokukhusela i-stem cell niche kwi-basal crypt kwii-antigens ze-microbial exogenous kunye ne-metabolites yazo. Ngoko ke, ukuphinda kuveliswe i-3D epithelial izakhiwo kubalulekile ekwakhiweni kweemodeli ze-in vitro gut. i-morphogenesis emathunjini kwi-microfluidic chip kunye ne-Transwell embedded hybrid chip.Sichaza iindlela ezicacileyo zokwenza isixhobo, ukulima i-Caco-2 okanye iiseli ze-intestinal organoid epithelial kwiindawo eziqhelekileyo kunye nakwiqonga le-microfluidic, ukufakwa kwe-3D morphogenesis, kunye nokubonakaliswa kwe-imaging ye-multiple protocol esekelwe kwi-3D. ye-microarchitecture esebenzayo yamathumbu ngokulawula ukuhamba kwe-basolateral fluid ye-5 d.Indlela yethu ye-vitro morphogenesis isebenzisa uxinzelelo lwe-shear olufanelekileyo lwe-physiologically kunye nokunyakaza komatshini kwaye ayifuni ubunjineli obuntsonkothileyo beeseli okanye ukuguqulwa, okunokugqithisa ezinye iindlela ezikhoyo. kwi-biomedical, ikliniki, kunye nezicelo zoxubo mayeza.
Iimvavanyo zibonisa ukuba i-intestinal epithelial Caco-2 iiseli ezikhuliswe kwi-gut-on-a-chip1,2,3,4,5 okanye i-bilayer microfluidic devices6,7 inokuhamba ngokukhawuleza kwe-3D morphogenesis in vitro ngaphandle kokuqonda ngokucacileyo indlela engaphantsi. I-epithelial morphogenesis in vitro, ebonakaliswe yiCaco-2 kunye nesigulane esiphuma kwi-intestinal organoids. Kulo cwaningo, sigxininise ngokukodwa kwimveliso yeeseli kunye nokusabalalisa koxinzelelo lwe-Wnt antagonist enamandla, i-Dickkopf-1 (DKK-1), kwi-gut-on-a-chip kunye nezixhobo eziguquliweyo ze-microfluidic eziqulethe i-Transwell inserts, ebizwa ngokuba yi-"Hybrid Chip" . Iprotheyini efihliweyo enxulumene ne-frizzled 1, okanye i-Soggy-1) kwi-chip-gut inhibits morphogenesis okanye iphazamisa i-3D ye-epithelial layer ecwangcisiweyo, ebonisa ukuba uxinzelelo oluchasayo ngexesha lenkcubeko luxanduva lwe-intestinal morphogenesis in vitro.Ngoko ke, indlela esebenzayo yokufezekisa amanqanaba anamandla e-mophogenesis e-epithelial i-epithelial i-interface eyongezelelweyo kwi-epithelial i-epithelial layer. kwi-basolateral compartment ngokugungxulwa okusebenzayo (umzekelo, kwi-gut-on-a-chip okanye i-hybrid-on-a-chip platforms) okanye i-diffusion .Imidiya ye-Basolateral (umzekelo, ukusuka kwi-Transwell ifakela kwi-basolateral reservoirs emaquleni).
Kule protocol, sinika indlela ecacileyo yokwenza i-microdevices ye-gut-on-a-chip kunye ne-Transwell-insertable hybrid chips (amanyathelo 1-5) ukuya kwiiseli ze-epithelial zamathumbu emathunjini kwi-polydimethylsiloxane (PDMS) -based based membranes (amanyathelo 6A, 7A, 8, 9) okanye i-polyester 6 iimbrane ze-polyester, i-7 ye-polyester , i-8 I-9) kunye ne-3D morphogenesis in vitro (inyathelo le-10) .Siye sachonga iimpawu zeselula kunye ne-molecular ebonisa i-histogenesis ye-tissue-specific histogenesis kunye nokwahlukana kweselula exhomekeke kumgca ngokusebenzisa iindlela ezininzi zokucinga (amanyathelo 11-24) . kunye neenkcukacha zobugcisa kubandakanywa ukuguqulwa komphezulu weembrane ezinamaqhekeza, ukudalwa kwe-2D monolayers, kunye ne-intestinal biochemical kunye nokuVeliswa kwe-biomechanical microenvironment.in vitro.Ukwenza i-3D morphogenesis ukusuka kwi-2D epithelial monolayers, sisuse abachasi be-morphogen kuzo zombini iifom zenkcubeko ngokugeleza i-medium kwi-basolateral inikezela i-compartment of the basolateral. uhlaziyo lwe-3D epithelial layer engasetyenziselwa ukulinganisa ukukhula kwe-epithelial exhomekeke kwi-morphogen, i-longitudinal host-microbiome co-cultures, usulelo lwe-pathogen, ukwenzakala okuvuthayo, ukungasebenzi kwe-epithelial barrier dysfunction, kunye ne-probiotic-based therapies Example.influences.
Iprothokholi yethu ingaba luncedo kuluhlu olubanzi lwezenzululwazi kwisiseko (umzekelo, i-biology ye-intestinal mucosal biology, i-stem cell biology, kunye ne-biology yophuhliso) kunye nophando olusetyenzisiweyo (umzekelo, ukuhlolwa kweziyobisi kwangaphambili, imodeli yesifo, ubunjineli bezicubu kunye ne-gastroenterology) impembelelo ebanzi.Ngenxa yokuphindaphinda kunye nokuqina kweprotocol yethu yokuvelisa i-3D kwi-morphogenesis yethu ye-entropino ye-entrophine isicwangciso sobugcisa sinokusasazwa kubaphulaphuli abafunda i-dynamics ye-cell signaling ngexesha lokuphuhliswa kwamathumbu, ukuvuselelwa okanye i-homeostasis .Ukongezelela, i-protocol yethu iluncedo ekuphenyweni ukusuleleka kwintsholongwane phantsi kwee-arhente ezosulelayo ezifana ne-Norovirus 8, i-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), i-Clostridium Salmonelera iTymuphilla, i-Virtool, i-Clostridium difficilla. Abaphulaphuli bezifo zesifo kunye ne-pathogenesis nazo ziluncedo.Ukusetyenziswa kwenkqubo ye-microphysiology ye-chip gut inokuvumela i-longitudinal co-culture 10 kunye novavanyo olulandelayo lokukhusela umkhosi, iimpendulo ze-immune kunye nokulungiswa kokulimala okunxulumene ne-pathogen kwi-gastrointestinal (GI) iphecana 11 .Ezinye izifo ze-GIceky gut, isifo se-Croaceky, isifo se-Groaceky, i-syndrome I-ulcerative colitis, i-pouchitis, okanye i-irritable bowel syndrome inokulinganiswa xa i-3D intestinal epithelial layers ilungiswa ngokusebenzisa i-3D intestinal epithelial layers , ezi zifo ziquka i-villous atrophy, shortening crypt, umonakalo we-mucosal, okanye ukukhubazeka kwe-epithelial barrier. ubunzima obuphezulu bokusingqongileyo kwesifo, abafundi banokuqwalasela ukongeza iintlobo zeeseli ezinxulumene nesifo, ezinjengeeseli zegazi ze-peripheral mononuclear cells (PBMCs), kwiimodeli eziqulethe i-3D intestinal villus-crypt microarchitectures. iiseli zomzimba ezikhethekileyo, 5.
Ekubeni i-microstructure ye-epithelial ye-3D inokulungiswa kwaye ibonwe ngaphandle kwenkqubo yecandelo, ababukeli abasebenza kwi-transcriptomics yendawo kunye ne-high-resolution okanye i-super-resolution imaging inokuba nomdla kwimephu yethu ye-spatiotemporal dynamics ye-genes kunye neeprotheni kwii-epithelial niches. Unomdla kwi-teknoloji.Ukuphendula kwi-microbial okanye i-immune stimuli.Ngaphezu koko, i-longitudinal host-microbiome crosstalk 10, i-14 elungelelanisa i-homeostasis ye-gut inokusekwa kwi-3D intestinal mucosal layer ngokudibanisa iintlobo ezahlukeneyo ze-microbial, uluntu lwe-microbial okanye i-fecal microbiota-i-gut-gut-i-gut-gut-i-gut-gut. eqongeni.Le ndlela inomtsalane ngakumbi kubaphulaphuli abafunda i-mucosal immunology, i-gastroenterology, i-microbiome yabantu, i-culturomics kunye ne-microbiology yeklinikhi efuna ukulima i-gut microbiota eyayingasetyenziswa ngaphambili elabhoratri.Ukuba i-in vitro morphogenesis protocol yethu inokutshintshwa kwiifomathi zenkcubeko ezihlanjululwayo, ezifana ne-multiwell efake i-43 kakuhle, i-2 ifake i-46 ngokuqhubekayo gcwalisa amacandelo e-basolateral, i-protocol ingasasazwa kwabo baphuhlisa amayeza, i-biomedical Okanye i-high-throughput screening okanye iiplatifomu zokuqinisekisa kwi-industry yokutya.Njengobungqina bomgaqo-siseko, kutshanje sibonise ukuba nokwenzeka kwe-multiplex ye-high-throughput yokongeza inkqubo ye-morphogenesis i-scalable kwi-fomati ye-chip-multiple, i-multiple-organ-plate. Ngoko ke, ukuqinisekiswa kwendlela yethu ye-in vitro morphogenesis inokukhawuleziswa kwaye inokuthi yamkelwe ngamaziko amaninzi ophando, imboni okanye urhulumente kunye nee-arhente zokulawula ukuqonda ukuhlelwa kwakhona kweselula ye-in vitro gut morphogenesis kwinqanaba le-transcriptomic ukuvavanya iziyobisi okanye i-biotherapeutics. Iimodeli ze-organ-on-a-chip zokuvavanya ukuveliswa kwakhona kwenkqubo ye-gut morphogenesis.
Inani elilinganiselweyo leemodeli zovavanyo ezichaphazelekayo zomntu ziye zasetyenziselwa ukufunda i-intestinal epithelial morphogenesis, ngokukodwa ngenxa yokungabikho kweeprothokholi ezinokusetyenziswa ukuze kubangele i-3D morphogenesis in vitro. Enyanisweni, ulwazi oluninzi lwangoku malunga ne-gut morphogenesis lusekelwe kwizifundo zezilwanyana (umzekelo, i-zebrafish20, i-mice21 okanye i-chicken-ingaba yi-labor2). ethandabuzekayo, kwaye okona kubaluleke kakhulu, akucacisi ngokuchanekileyo iinkqubo zophuhliso lomntu.Le mizekelo iyancipha kakhulu ekukwazini ukuvavanywa ngeendlela ezininzi ezinobungozi.Ngoko ke, iprotocol yethu yokuhlaziya izakhiwo ze-3D ze-tissue kwi-vitro zigqithise kwiimodeli zezilwanyana ze-vivo kunye neminye imodeli yenkcubeko ye-2D ye-static yendabuko.Njengoko kuchazwe ngaphambili, kuvunyelwe ukusetyenziswa kwe-spatiliation yendawo ye-3D yolwakhiwo lwendawo. iiseli kwi-crypt-villus axis ekuphenduleni kwiintlobo ezahlukeneyo ze-mucosal okanye i-immune stimuli.I-3D epithelial layers inokubonelela ngesithuba sokufunda ukuba iiseli ze-microbial zikhuphisana njani ukuze zenze i-spatial niches kunye ne-ecological evolution ekuphenduleni kwi-host factors (umzekelo, i-inner versus outer mucus layers, secretion of IgA kunye ne-antimicrobial peptides ivumela i-peptides ye-antimicrobial ivumela i-peptides ye-antimicrobial i-user3D ivumela i-peptides ye-antimicrobial). i-gut microbiota iqulunqa uluntu kunye ne-synergistically ivelisa i-microbial metabolites (umzekelo, i-short-chain fatty acids) eyenza umbutho weselula kunye ne-stem cell niches kwi-basal crypts.Ezi mpawu zingabonakaliswa kuphela xa i-3D epithelial layers isekwe kwi-vitro.
Ukongeza kwindlela yethu yokudala i-3D intestinal epithelial structures, kukho iindlela ezininzi ze-in vitro.Inkcubeko ye-organoid ye-intestinal yinkqubo yobunjineli ye-tissue ye-state-of-the-art esekelwe ekulinyweni kweeseli ze-intestinal stem phantsi kweemeko ezithile ze-morphogen23,24,25.Nangona kunjalo, ukusetyenziswa kwe-3D ye-organoid ye-organoid imodeli ye-organoid ye-3D yohlalutyo lwe-biomicroculture i-hostage-i-biomicroculture ivame ukuhlalutya i-coostestinal-models. I-lumen ifakwe ngaphakathi kwe-organoid kwaye, ngoko ke, ukungeniswa kwamacandelo e-luminal afana neeseli ze-microbial okanye i-antigens exogenous. Ukufikelela kwi-lumens ye-organoid kunokuphuculwa ngokusebenzisa i-microinjector, i-26,27 kodwa le ndlela i-invasive kwaye ifuna umsebenzi kwaye idinga ulwazi olukhethekileyo ukwenza.Ngaphezu koko, iinkcubeko ze-organoid zendabuko ezigcinwe kwi-hydrogel scaffolds phantsi kweemeko ze-static azibonakalisi ngokuchanekileyo kwi-vivo biomechanics.
Ezinye iindlela ezisetyenziswa ngamaqela amaninzi ophando zisebenzisa i3D hydrogel scaffolds ukulinganisa ubume be-epithelial yamathumbu ngokuvelisa iiseli ezizimeleyo zamathumbu omntu kumphezulu wejeli. Fabricate hydrogel scaffolds usebenzisa i-3D-printed, micro-milled, okanye lithographically fabricated molds-self organised of molds self-organized. I-physiologically relevant morphogen gradients, ukuseka umlinganiselo ophezulu we-epithelial structure kunye ne-stroma-epithelial crosstalk ngokubandakanya iiseli ze-stromal kwi-scaffold.Nangona kunjalo, ubume be-scaffolds obucwangcisiweyo bunokuthintela ukubonakaliswa kwenkqubo ye-morphogenetic ezenzekelayo ngokwayo. I-morphogenesis kunye nokufumana umsebenzi we-physiological.Olunye uphando olutshanje lusetyenzise i-hydrogel scaffolds kwiqonga le-microfluidic kunye ne-pattered intestinal epithelial structures usebenzisa iindlela ze-laser-etching.I-Mouse intestinal organoids ilandela iipatheni ezihleliweyo ukwenza izakhiwo ze-tubular zamathumbu, kunye ne-intraluminal fluid flow can be recapitulated le modyuli ye-modules, imodyuli ye-modules ye-module. kwaye ayibandakanyi ukunyakaza kwe-gut mechanobiological.Iendlela zokushicilela ze-3D ezivela kwiqela elifanayo zakwazi ukwenza iibhubhu ezincinci zamathumbu kunye neenkqubo ezizenzekelayo ze-morphogenetic.Nangona ukuveliswa okuyinkimbinkimbi kwamacandelo ahlukeneyo amathumbu ngaphakathi kombhobho, lo mzekelo awunako ukuhamba kwe-luminal fluid kunye ne-mechanical deformation.Ukongezelela, imodeli yokusebenza inokukhawulelana, ngakumbi emva kokuba inkqubo ye-bioprinting ye-cell-steading igqityiwe, inkqubo ye-bioprinting yeseli igqityiwe. Iprothokholi ecetywayo ibonelela ngokuzenzekela kwe-morphogenesis yamathumbu, uxinzelelo lwe-shear olufanelekileyo, i-biomechanics elinganisa ukuhamba kwamathumbu, ukufikeleleka kwe-apical apical kunye ne-basolateral compartments, kunye nokudalwa kwakhona kwe-biological microenvironments enzima ye-modularity.Ngoko ke, i-in vitro 3D morphogenesis protocol inokubonelela ngendlela ehambelanayo yendlela ekhoyo yokunqoba.
Iprotocol yethu igxininise ngokupheleleyo kwi-3D epithelial morphogenesis, kunye neeseli ze-epithelial kuphela kwinkcubeko kwaye azikho ezinye iintlobo zeeseli ezijikelezileyo ezifana neeseli ze-mesenchymal, iiseli ze-endothelial, kunye neeseli ze-immune.Njengoko kuchazwe ngaphambili, ingundoqo yeprotocol yethu kukungeniswa kwe-epithelial morphogenesis ngokususa i-morphogen inhibitors efihliweyo kwi-modularity yethu ye-robust. i-gut-on-a-chip kunye ne-hybrid-on-a-chip ivumela ukuba siphinde senze i-3D epithelial layer engaguqukiyo, izinto eziyinkimbinkimbi zebhayoloji ezifana ne-epithelial-mesenchymal interactions33,34, i-extracellular Matrix (ECM) idipozithi ye-35 kwaye, kwimodeli yethu, iimpawu ze-crypt-villus ezihambisa i-stem cell niches, i-cryptstroll cell niches. i-fibroblasts) kwi-mesenchyme idlala indima ebalulekileyo ekuveliseni iiprotheni ze-ECM kunye nokulawulwa kwe-intestinal morphogenesis kwi-vivo35,37,38. Ukongezwa kweeseli ze-mesenchymal kumzekelo wethu kuphuculwe inkqubo ye-morphogenetic kunye nokusebenza kakuhle kwe-cell attachment.I-endothelial layer (oko kukuthi, i-capillaries okanye i-lymphatics) idlala indima ebalulekileyo ye-immune ye-cell regulat kwi-cell recruit9 kwi-cell regulate93 indima ebalulekileyo kwi-cell recruit. I-microenvironment.Ngaphezu koko, iinqununu ze-vasculature ezinokudibaniswa phakathi kweemodeli zezicubu ziyimfuneko xa iimodeli zezicubu zenzelwe ukubonisa ukusebenzisana kwamalungu amaninzi.Ngoko ke, iiseli ze-endothelial zingadinga ukufakwa ukuze zenze imodeli echanekileyo yeempawu ze-physiological kunye nesisombululo somgangatho we-organ. ukungakhuseleki kwimeko yokulinganisa isifo samathumbu.
Ukusetyenziswa kweetshiphusi ezixubeneyo zichaneke ngakumbi kune-gut-on-a-chip ngenxa yokuba ukuseta isixhobo kulula kwaye ukusetyenziswa kokufakwa kwe-Transwell kuvumela inkcubeko enobungozi ye-gut epithelium.Nangona kunjalo, ukufakwa ngokurhweba kweTranswell ngeembrane zepolyester azinalastiki kwaye azikwazi ukulinganisa iintshukumo ezinjenge-peristaltic.Ngaphezu koko, i-apical chip efakwe kwisikhululo soxinzelelo lwe-apical yahlala kwisikhululo soxinzelelo lwe-apical nokufakwa kwisikhululo Ngokucacileyo, iipropati ezimileyo kwikhompatimenti ye-apical kunqabile ukuba zenze inkcubeko yebhaktiriya yexesha elide kwi-chips hybrid chips.Ngelixa sinokuthi singenise ngamandla i-3D morphogenesis kufakelo lweTranswell xa kusetyenziswa iitshiphusi ezixutyiweyo, ukunqongophala kwe-biomechanics ehambelana ne-physiologically kunye nokuhamba kolwelo lwe-apical kunokunciphisa ukwenzeka kweplatform ye-hybrid enokubakho.
Ukwakhiwa kwakhona ngokupheleleyo kwe-axis ye-crypt-villus yomntu kwi-gut-on-a-chip kunye neenkcubeko ze-hybrid-on-a-chip azikasekwa ngokupheleleyo. Ekubeni i-morphogenesis iqala kwi-epithelial monolayer, i-microarchitectures ye-3D ayinikezeli ngokufana kwe-morphological kwi-crypts in vivo. I-epithelium ye-3D, i-crypt kunye nemimandla ekhohlakeleyo yayingacalulwanga ngokucacileyo.Nangona imijelo ephezulu ephezulu kwitshiphu ikhokelela ekuphakameni okwandisiweyo kwe-epithelium ene-microengineered, owona mphakamo uphezulu usakhawulelwe ukuya ku- ~300–400 µm. Obona nzulu bobunzulu bamathumbu omntu kumathumbu amancinane namakhulu, ~1mµmµmµinkulu, intlonipho yi ~1mµmµm, i-0mµm, i-0mµm, i-5mµm, i-0mµm kunye ne-5mµm, i-0mµm, i-50m, i-50mµm, i-0m kwaye umphakamo we-intestinal villi encinci yi ~600 µm41.
Ukusuka kwimbono yokucinga, kwi-situ super-resolution imaging ye-3D microarchitectures inokulinganiselwa kwi-gut kwi-chip, ekubeni umgama ofunekayo wokusebenza ukusuka kwi-lens yenjongo ukuya kumaleko we-epithelial ungomyalelo weemilimitha ezimbalwa. I-PDMS.Ngaphezu koko, ekubeni i-microfabrication ye-malay-by-layer ye-gut kwi-chip ibandakanya ukunamathela ngokusisigxina phakathi komgangatho ngamnye, kunzima kakhulu ukuvula okanye ukususa umaleko ophezulu ukuze uhlolisise isakhiwo somgangatho we-epithelial layer.Ngokomzekelo, ngokusebenzisa i-electron microscope (SEM) yokuskena.
I-hydrophobicity ye-PDMS ibe yinto ekhawulelayo kwizifundo ezisekelwe kwi-microfluidic ezijongene ne-hydrophobic iimolekyuli ezincinci, ekubeni i-PDMS ingakwazi ukudibanisa ngokukodwa i-molecules ye-hydrophobic. glycol) 43 ) inokuthathwa njengokunciphisa i-adsorption yee-molecule ze-hydrophobic.
Ekugqibeleni, indlela yethu ayizange ibonakaliswe kakuhle ngokubhekiselele ekuboneleleni nge-high-throughput screening okanye "ubukhulu obunye-bonke" iqonga lokulinga lomsebenzisi.Iprotocol yangoku idinga impompo yesirinji kwi-microdevice nganye, ethatha indawo kwi-CO2 incubator kwaye ithintele iimvavanyo ezinkulu.Lo mda ungaphuculwa kakhulu nge-scalability ye-scalability ye-9well, i-culture, i-64well, i-culture, i-64well, i-culture, i-64well, i-culture-9 Ukufakwa kwe-384-well porous evumela ukuzaliswa ngokuqhubekayo kunye nokususwa kweendaba ezisisiseko).
Ukwenza i-3D morphogenesis ye-epithelium yamathumbu omntu kwi-vitro, sisebenzise isixhobo se-microfluidic chip intestinal equlethe i-microchannels ezimbini ezihambelanayo kunye ne-elastic porous membrane phakathi ukudala i-lumen-capillary interface. ukufakwa.Kuzo zombini iiplatifomu, i-morphogenesis yeeseli ezahlukeneyo ze-epithelial zamathumbu omntu zingabonakaliswa ngokusetyenziswa kwe-directional manipulation of flow ukususa abachasi be-morphogen ukusuka kwi-compartment ye-basolateral.Yonke inkqubo yovavanyo (Umfanekiso 1) iqulethwe ngamacandelo amahlanu: (i) i-microfabrication ye-gut chip okanye i-Transwellable chip (i-chip) i-Transwellable chip (5) i-chipwellable hybrid (i-chip) i-5. iiseli ze-epithelial zamathumbu (iiseli zeCaco-2) okanye i-organoids yamathumbu omntu; iibhokisi ze-2-5), (iii) inkcubeko yeeseli ze-intestinal epithelial kwi-intestinal chips okanye i-chips hybrid (amanyathelo 6-9), (iv) ukufakwa kwe-3D morphogenesis in vitro (inyathelo le-10) kunye (v)) ukubonisa i-3D epithelial microstructure (amanyathelo 11-24) . in vitro morphogenesis ngokuthelekisa i-epithelial morphogenesis kwindawo, ixesha, imiqathango, okanye ulawulo lwenkqubo.
Sisebenzise amaqonga enkcubeko amabini ahlukeneyo: i-gut-on-a-chip enetshaneli ezithe tye okanye amajelo adityanisiweyo angenamgca, okanye iitshiphusi ezixubeneyo ezinee-Transwell (TW) ezifakelwayo kwisixhobo se-microfluidic, esenziwe njengoko kuchaziwe kwiBhokisi 1, kunye nenyathelo 1-5. (I-Caco-2 okanye i-organoids yamathumbu omntu) kunye nenkqubo yenkcubeko esetyenziswe kule protocol."In vitro morphogenesis" ibonisa amanyathelo apheleleyo apho iCaco-2 okanye iiseli ze-epithelial eziphuma kwi-organoid zikhuliswa kwi-chip yamathumbu okanye kwii-Transwell ezifakwe kwi-chip hybrid chip, elandelwa kukufakelwa kwe-3D morphogenesis kunye nokwakheka kwenombolo ye-epithe yebhokisi ye-epithelial ebonisa inombolo engezantsi. utolo.Isicelo sibonelela ngemizekelo yendlela enokusetyenziswa ngayo iileya ze-epithelial zamathumbu, umzekelo, kwi-cell differentiation characterization, i-gut physiology studies, ukusekwa kwe-host-microbiome ecosystems, kunye nemodeli yezifo.Imifanekiso ye-Immunofluorescence "kwi-Cell Differentiation" ebonisa i-nuclei, i-F-actin kunye ne-3D Capicoal evezwe kwi-ecosystem ye-3D Capicoal evezwe kwi-ecosystem2. I-chip.MUC2 ibonakaliso ikhona kwiiseli ze-goblet kunye ne-mucus efihliweyo kwi-mucosal surfaces.Imifanekiso ye-Fluorescent kwi-Gut Physiology ibonisa i-mucus eveliswa ngokungcoliswa kwe-asilic acid kunye ne-N-acetylglucosamine intsalela usebenzisa i-fluorescent yengqolowa intsholongwane i-agglutinin. ithumbu kwi-chip.Iphaneli ekhohlo ibonisa i-co-culture ye-E. coli ebonisa iprotheni ye-fluorescent eluhlaza (GFP) kunye ne-microengineered 3D Caco-2 iiseli ze-epithelial.Iphaneli elungileyo ibonisa indawo ye-GFP E. coli edibeneyo kunye ne-3D Caco-2 iiseli ze-epithelial, ilandelwa yi-immunofluored kunye ne-FDiincleaseicle ye-FDiincleasein blue imodeli ibonisa impilo echasene ne-gut evuzayo kwi-gut ukudumba chips phantsi komngeni we-physiological kunye ne-antigens ye-bacterial (umzekelo, i-lipopolysaccharide, i-LPS) kunye neeseli ze-immune (umzekelo, i-PBMC; eluhlaza). isalathiso.2. I-Oxford University Press; Iphinde yaveliswa ngemvume evela kwiSiza.5. I-NAS; "I-Host-Microbe Co-Culture" iguqulelwe ngemvume evela kwi-ref.3. I-NAS; "Umzekelo weSifo" ulungelelaniswe ngemvume evela kwireferensi.5. I-NAS.
Zombini i-gut-on-chip kunye ne-hybrid chips zenziwe kusetyenziswa ii-replicas ze-PDMS eziye zachithwa kwi-silicon molds nge-lithography ethambileyo1,44 kunye nepateni kunye ne-SU-8.Uyilo lwe-microchannels kwi-chip nganye luchongwa ngokuqwalasela i-hydrodynamics efana noxinzelelo lwe-shear kunye noxinzelelo lwe-hydrodynamic1,4,12. ezimbini juxtaposed parallel ngqo microchannels, iye yavela kwi-gut-on-a-chip entsonkothileyo (Idatha Eyongeziweyo Fig. 1b) equka iperi ye-microchannels egobileyo ukuze ibangele Ukwenyuka kwexesha lokuhlala kwamanzi, iipatheni zokuhamba okungahambiyo, kunye ne-multiaxial deformation yeeseli zenkcubeko (Umfanekiso we-2a-f) idinga i-biocreated ngakumbi, i-biocreated idinga i-biocreated ngakumbi i-12. I-gut-on-a-chips ingakhethwa.Siye sabonisa ukuba i-Gut-Chip edibeneyo iphinda ibangele i-3D morphogenesis ngexesha elifanayo kunye neqondo elifanayo lokukhula kwe-epithelial xa kuthelekiswa ne-original Gut-Chip, kungakhathaliseki ukuba yiyiphi i-celled cell type.Ngoko ke, ukubangela i-3D morphogenesis, i-linear kunye ne-complex ye-silicone eguquguqukayo kwi-silicone eguquguqukayo. kunye neepatheni ze-SU-8 zibonelele ngeempawu ezingalunganga emva kokudilizwa (Umfanekiso 2a) .Ukwenza umgudu kwi-chip, i-PDMS ephezulu elungiselelweyo idityaniswe ngokulandelelanayo kwifilimu ye-PDMS ene-porous kwaye emva koko ilungelelaniswe ne-PDMS ephantsi yomaleko ngokudibanisa okungaguqukiyo usebenzisa i-corona treater (Fig. 2b-f) Ukudala i-chips hybriddes ye-glass PDMS ehlanganisiweyo kwi-chips ye-glass PDMS ehlanganisiweyo. Izixhobo ze-microfluidic ze-single-channel ezinokuthi zifake ukufakwa kwe-Transwell (umzobo we-2 kunye nedatha eyandisiweyo ye-Fig. 2) . Inkqubo yokudibanisa iqhutywe ngokuphatha iindawo ze-PDMS replica kunye neglasi nge-oxygen plasma okanye unyango lwe-corona.Emva kokutshatyalaliswa kwesixhobo se-microfabricated esifakwe kwi-tube ye-silicone, ukuseta isixhobo se-silicone i-epigene sikulungele ukwenza isixhobo se-3D e-morphligene silungele (Umfanekiso 2g).
a, Umzobo weSchematic wokulungiswa kweengxenye zePDMS ukusuka kwi-SU-8 iipatheni ze-silicon molds.Isisombululo esinganyangekiyo se-PDMS sagalelwa kwi-silicon mold (ekhohlo), yanyangwa kwi-60 °C (ephakathi) kwaye yachithwa (ekunene).I-PDMS ediliziweyo yanqunyulwa yaziingceba kwaye yacocwa ukuze isetyenziswe ngakumbi.b, Ifoto esetyenziswe kwi-silicon ye-mold. ukungunda kwesilicon esetyenziselwa ukwenza iPDMS inwebu evuzayo.d, Uthotho lweefoto zamalungu aphezulu nasezantsi ePDMS kunye nesixhobo esidityanisiweyo se-chip.e, iSchematic yokulungelelaniswa kwezixhobo zePDMS eziphezulu, inwebu, kunye nezisezantsi. microchannels convoluted kunye vacuum chambers.g, Setup of gut-on-a-chip for microfluidic cell culture.Ithumbu eyenziweyo kwi-chip edityaniswe netyhubhu ye-silicone kunye nesirinji yafakwa kwi-coverlip.Isixhobo se-chip safakwa kwisiciko se-150 mm isitya sePetri sokucubungula.I-binder hybrids ye-silicone isetyenziselwa ukuvala i-silicone ye-chips isetyenziselwa ukuvala. Ukwenziwa kunye ne-3D morphogenesis usebenzisa i-chips hybrid.Ukufakwa kweTranswell kulungiselelwe ngokuzimeleyo kwinkcubeko ye-2D i-monolayers yeeseli ze-epithelial zamathumbu zafakwa kwi-chip hybrid ukuze ifake amathumbu e-3D morphogenesis.I-medium ixutywe nge-microchannels ngaphantsi kwe-cell layer esekwe kwi-Transwell i-reference bar. Elsevier.
Kule protocol, i-cell line ye-Caco-2 kunye ne-organoids yamathumbu yayisetyenziswe njengemithombo ye-epithelial (umzobo 3a) .Zombini iintlobo zeeseli zazikhuliswe ngokuzimeleyo (Ibhokisi 2 kunye neBhokisi 5) kwaye zisetyenziselwa ukuhlwayela i-ECM-coated microchannels ye-chip gut okanye i-Transwell inserts.Xa iiseli zihambelanayo (> 995% yeCacocell cell coverage) (phakathi kweendinyana ze-10 kunye ne-50) kwii-T-flasks zivunwa ukulungiselela ukumiswa kweeseli ezidityanisiweyo nge-trypsinization fluid (ibhokisi 2) .I-organoids yamathumbu omntu avela kwi-intestinal biopsies okanye ukukhutshwa koqhaqho kwakhuliswa kwi-Matrigel scaffold domes kwi-24-well plates ukuxhasa i-mossgenment microenvironment ukuxhasa i-microenvironment microenvironment. I-R-spondin, kunye ne-Noggin) kunye nezinto zokukhula ezilungiselelwe njengoko zichazwe kwiBhokisi 3 zongezwa yonke imihla de i-organoids ikhule ukuya kwi ~ 500 µm ububanzi. Isifo sikaCrohn, umhlaza wesisu, okanye umnikeli oqhelekileyo), indawo yesilonda (umzekelo, i-lesion ngokubhekiselele kwindawo engabonakaliyo) kunye nendawo yesisu kwiphecana (umzekelo, i-duodenum, i-jejunum, i-ileum, i-cecum, i-colon, okanye i-rectum) i-organoids yamathumbu.
a, Ukuhamba komsebenzi wokufakelwa kwe-gut morphogenesis kwi-gut chip.I-Caco-2 i-epithelium yamathumbu omntu kunye ne-intestinal organoids isetyenziswe kule protocol yokubonisa i-3D morphogenesis.Iiseli ze-epithelial ezizimeleyo zahlwaywa kwisixhobo esilungisiweyo se-gut-on-a-chip (ukulungiswa kwe-chip) . Kanye emva kokuba iiseli zifakwe kwi-membrane efakwe kwi-membrane efakwe kwi-membrane ye-podta (i-chips). ngosuku lwe-0 (D0), ukuhamba kwe-apical (AP) kuqaliswe kwaye kugcinwe kwiintsuku zokuqala ze-2 (ukuhamba, i-AP, i-D0-D2) ukuhamba kwe-Basolateral (BL) nayo iqaliswe kunye ne-cyclic stretching motions (ukwelula, ukuhamba, i-AP kunye ne-BL) xa i-monolayer ye-2D epheleleyo yenziwa.I-Intestinal 3D yenkcubeko ye-micromorphogenesis ye-spopogenesis yenzeke emva kweentsuku ze-3D ze-micromorphogenesis zenzeke emva kwe-micromorphogenesis. I-D5) .Imifanekiso echasene nesigaba ibonisa i-morphology emele iiseli ze-Caco-2 kwinqanaba ngalinye lokulinga okanye ixesha (igrafu yebha, i-100 µm) Imifanekiso emine ye-schematic ebonisa i-cascade ehambelanayo ye-gut morphogenesis (phezulu ekunene) .Iintolo ezidayiweyo kwi-schematic zimela isalathiso sokuhamba kwamanzi.b, SEMcoli yomfanekiso obonisa umphezulu we-eft 3D. i-inset egxininisa indawo eyandisiweyo (ibhokisi edayiweyo emhlophe) ibonisa i-microvilli ehlaziyiweyo kwi-3D Caco-2 umaleko (ekunene) .c, Umbono othe tyaba wangaphambili we-Caco-2 3D esekiweyo, i-claudin (ZO-1, ebomvu) kunye ne-brush eqhubekayo yeembrane zemida ebhalwe F-actin (eluhlaza) kunye ne-nuclei (i-bluence) ye-impiostinalization ye-conductors ebonakalayo Iitshiphusi.Iintolo ezilathe kwisicwangciso esisembindini zibonisa indawo yenqwelomoya egxininisekileyo kumbono ngamnye weconfocal.d, Ixesha lexesha lotshintsho lwemophological kwi-organoids ekhuliswe kwitshiphu efunyenwe ngesigaba sokuchasana kwemakroskopu ngeentsuku 3, 7, 9, 11, kunye 13.Iseti (phezulu ekunene) ibonisa ukwandiswa okuphezulu kwefotoid enikeziweyo ye-3D ekwi-epicopy ekwi-DIC okanye i-Dganoid yefoto ecikizekileyo kwi-epig. kwisiqwenga esithathwe ngosuku lwe-7.f, Imifanekiso ye-Immunofluorescence eNgqunyiweyo ebonisa iimpawu zeeseli ze-stem (LGR5; i-magenta), iiseli ze-goblet (MUC2; eluhlaza), i-F-actin (grey) kunye ne-nuclei (cyan) ekhuliswe kwi-gut chips iintsuku ze-3, ngokulandelanayo (Ekhohlo) kunye ne-13-day (ephakathi) kunye ne-organolitendes i-organorendis i-Expresse layer3 kwi-Datase. Ukubonisa i-LGR5 ngaphandle komqondiso we-MUC2.Imifanekiso ye-Fluorescence ebonisa i-epithelial microstructure (ekunene) ye-3D organoid epithelium esekwe emathunjini kwi-chip ngokungcolisa i-plasma membrane nge-CellMask idayi (ekunene) ngosuku lwe-13 yenkcubeko.Ibha yokulinganisa yi-50 μm ngaphandle kokuba kuchazwe ngenye indlela. I-Oxford University Press; c Ilungelelaniswe ngemvume esuka kwiNgcaciso.2. I-Oxford University Press; e kunye no f ihlengahlengiswe ngemvume ngereferensi.12 Ngaphantsi kweLayisensi yeCreative Commons CC BY 4.0.
Kwi-gut kwi-chip, kuyimfuneko ukuguqula i-hydrophobic surface ye-PDMS ye-membrane e-porous ye-coating ye-ECM eyimpumelelo.Kule protocol, sisebenzisa iindlela ezimbini ezahlukeneyo zokuguqula i-hydrophobicity ye-membrane ze-PDMS.Ukulima iiseli ze-Caco-2, ukusetyenziswa kwendawo yonyango ye-UV / ozone yodwa kwakwanele ukunciphisa i-hydrophobicity ye-hydrophobicity kunye ne-coatco-cell ye-caatco ye-Caatco-cell kunye ne-coatco-cell ye-Caatco ye-caatco I-membrane ye-PDMS.Nangona kunjalo, inkcubeko ye-microfluidic ye-organoid epithelium idinga i-chemical-based surface functionalization ukuze ifezekise i-deposition esebenzayo yeeprotheni ze-ECM ngokusebenzisa ngokulandelelana i-polyethyleneimine (PEI) kunye ne-glutaraldehyde kwi-PDMS microchannels.Emva kokuguqulwa kwendawo, iiprotheni ze-ECM zifakwe ukugubungela i-functionalized surface okanye i-isopidi efakwe kwi-isopidi ye-PDMSA efakwe kwi-isoothe. iiseli ziqhotyoshelweyo, inkcubeko yeseli ye-microfluidic iqala ngokufaka i-perfusing kuphela i-medium kwi-microchannel ephezulu kuze kube yilapho iiseli zenza i-monolayer epheleleyo, ngelixa i-microchannel ephantsi igcina i-static conditions.Le ndlela ephuculweyo yokusetyenziswa komphezulu kunye ne-ECM yokugqoka yenza ukuba i-attachment ye-organoid epithelium ifake i-3D morphogenesis kwindawo ye-PDMS.
Iinkcubeko zeTranswell nazo zifuna ukugquma kwe-ECM ngaphambi kokuhlwayelwa kweseli; nangona kunjalo, iinkcubeko zeTranswell azifuni amanyathelo angaphambili anzima ukuze kusebenze umphezulu wokufakwa kwe-porous.Ukukhula kweeseli ze-Caco-2 kwi-Transwell inserts, i-ECM yokugqoka kwi-porous inserts ikhawuleza i-attachment yeeseli ze-Caco-2 ezidityanisiweyo (<1 iyure) kunye nokwakheka komqobo ongqongqo (<1-2 iintsuku) . ukuya kwi-membrane surface (<3 h) kwaye igcinwe de i-organoids yenze i-monolayer epheleleyo kunye nengqibelelo yomqobo .Iinkcubeko zeTranswell zenziwa kwiiplate ze-24-well ngaphandle kokusetyenziswa kweechips hybrid.
I-in vitro i-3D morphogenesis inokuqaliswa ngokusebenzisa ukuhamba kwamanzi kwinqanaba le-basolateral ye-epithelial layer esekelwe. Kwi-gut kwi-chip, i-epithelial morphogenesis yaqala xa i-medium yaxutywa kwi-microchannels ephezulu kunye nesezantsi (Umfanekiso 3a) .Njengoko kuchazwe ngaphambili, kubalulekile ukungenisa i-introlateral fluid kwi-continual discation ye-completial flowing (i-continual fluid) ukususwa kwe-completial flowing (i-continual fluid) ukukhupha i-completeor eqhubekayo. I-secreted morphogen inhibitors.Ukubonelela ngezondlo ezaneleyo kunye ne-serum kwiiseli ezibophelelwe kwi-membrane ene-porous kwaye zivelise uxinzelelo lwe-luminal shear, sisebenzisa ngokuqhelekileyo ukuhamba kabini emathunjini kwi-chip.Kwi-chips hybrid, ukufakwa kwe-Transwell okuqulethe i-epithelial monolayers kwafakwa kwi-chips ye-hybrid. emva kokuqaliswa kokuhamba kwesiseko kuwo omabini amaqonga enkcubeko.
Iimpawu ze-morphological ze-microengineered 3D epithelial layers zingahlalutywa ngokusebenzisa iindlela ezahlukeneyo zokucinga, kubandakanywa ukuchasana kwe-microscopy yesigaba, i-different interference difference (DIC) microscopy, i-SEM, okanye i-immunofluorescence confocal microscopy (Amanani 3 kunye ne-4) . I-epithelial layers.Ngenxa ye-optical transparency ye-PDMS kunye neefilimu ze-polyester, zombini i-gut-on-a-chip kunye ne-hybrid chip platforms inokubonelela ngexesha langempela kwi-imaging ye-situ ngaphandle kwesidingo sokwahlula okanye ukuchithwa kwesixhobo.Xa usenza umfanekiso we-immunofluorescence (Amanani 1, 3b ngokuqhelekileyo, i-4b, i-c, i-c, i-c, i-f (wt / vol) paraformaldehyde (PFA), elandelwa yi-Triton X-100 kunye ne-2% (wt / vol) ) i-albumin ye-bovine serum (BSA), ngokulandelelana.Ngokuxhomekeka kuhlobo lweseli, ukulungiswa okuhlukeneyo, i-permeabilizers, kunye ne-blocking agents zinokusetyenziswa.Ii-antibodies eziphambili ezijolise kumgca-oxhomekeke kumgca we-cell okanye i-chip markers ezisetyenziswayo kwiiseli ze-second immobiling zisetyenziselwa ukulinganisa iiseli ze-sixia kunye nedayi ye-counterstain ejolise nokuba yi-nucleus (umzekelo, i-4′, i-6-diamidino-2-phenylene) indole, i-DAPI) okanye i-F-actin (umzekelo, i-fluorescently labeled phalloidin) .I-Fluorescence-based imaging live ingenziwa kwakhona kwi-situ ukufumanisa ukuveliswa kwe-mucus (umzobo ohlukeneyo we-colonology ye-mucus), i-Fig. iiseli microbial (Fig. 1, "Host-microbe co-culture") , ukugaywa kwamaseli omzimba (umzobo 1, 'Umzekelo wesifo') okanye i-contours ye-3D epithelial morphology (Fig. 3c, f kunye ne-4b, c) .Xa uguqulela i-gut kwi-chip ukuze uhlukanise i-microf layer echazwe njenge-microf. I-2, i-3D epithelial morphology kunye ne-microvilli kumda we-apical brush inokubonwa yi-SEM (Umfanekiso 3b) uhlalutyo lwemfuza.
a, Workflow for induction of intestinal morphogenesis in a hybrid chip.Caco-2 kunye ne-intestinal organoids zisetyenziswa kule protocol ukubonisa i-3D morphogenesis kwiplatifti ye-hybrid chip. Iiseli ze-epithelial ezidityanisiweyo zahlwayelwa kwii-Transwell inserts ezilungiselelwe (i-TW prep; jonga umfanekiso ongezantsi) . Kanye emva kokuba iiseli zembewu zifakwe kwi-polyseed celleds (iiseli ezifakwe kwi-polyseed). ikhuliswe phantsi kweemeko ze-static (inkcubeko ye-TW) .Emva kweentsuku ze-7, i-Transwell ifake enye equkethe i-2D monolayer yeeseli ze-epithelial yadityaniswa kwi-chip hybrid ukwazisa ukuhamba kwe-basolateral (Flow, BL), ekugqibeleni kwakhokelela ekuveliseni i-3D epithelial layer (morphogenesis) . (umgca we-C103) ukunyuka kwekholoni kwinqanaba ngalinye lokulinga okanye ixesha.I-schematics kwiingqimba eziphezulu zibonisa ukucwangciswa kovavanyo kwinqanaba ngalinye.b, Ii-chips ze-Hybrid (i-schematic ekhohlo) inokukhokelela kwi-3D morphogenesis yeeseli ze-organoid epithelial kunye ne-top-down confocal microscopy imibono ethathwe kwiindawo ezahlukeneyo ze-microscopy ezihambelanayo, i-doupper ehambelanayo kunye nemigqaliselo ye-doupper ephantsi, i-Z. ibonise iimpawu ezicacileyo zemophological.F-actin (cyan), nucleus (grey) .c, Fluorescence confocal micrographs (3D angled view) yeeseli ze-epithelial ezivela kwi-organoid ezikhuliswe kwi-static Transwell (TW; i-inset ngaphakathi kwebhokisi edayiweyo emhlophe) ngokuchasene ne-hybrid chip (ishot enkulu egcweleyo) xa kuthelekiswa ne-3D morphology2D morphology. iimboniselo ezithe nkqo ezinqamlezileyo (ukufakwa kwikona ephezulu ngasekunene; “XZ”) ikwabonisa i-2D kunye neempawu ze-3D.Ibar yesikali, 100 µm.c Ishicilelwe kwakhona ngemvume evela kwireferensi.4. Elsevier.
Ulawulo lunokulungiswa ngokuhlakulela iiseli ezifanayo (i-Caco-2 okanye i-intestinal organoid epithelial cells) kwii-monolayers ezimbini-dimensional phantsi kweemeko zenkcubeko ye-static yesiqhelo. nayo ithelekiswe.
Inkqubo ye-lithography ethambileyo kufuneka yenziwe kwigumbi elicocekileyo.Kumaleko ngamnye kwi-chip (imigangatho ephezulu kunye nesezantsi kunye ne-membrane) kunye ne-chips hybrid chips, ii-photomasks ezahlukeneyo zisetyenzisiwe kwaye zenziwe kwii-silicone wafers ezihlukeneyo ngenxa yokuba ukuphakama kwe-microchannels kwahlukile.Ukuphakama okujoliswe kuyo kwe-microchannels ephezulu kunye nesezantsi kwi-gut kwi-chip yi-500 µm ubude kunye nobude obujoliswe kwi-500 µm kunye nobude obujoliswe kwi-500 µm. itshiphu exutyiweyo ngama-200 µm.
Beka i-wafer ye-silicon ye-intshi ezi-3 kwisitya esine-acetone. Jika ipleyiti ngobunono imizuzwana engama-30, uze womise iwafer emoyeni. Dlulisa i-wafer kwipleyiti ene-IPA, uze ujikelezise ipleyiti i-30 s ukuyicoca.
Isisombululo sepiranha (umxube wehydrogen peroxide kunye ne-sulfuric acid egxininisiweyo, 1:3 (vol/vol)) ingasetyenziswa ngokuzithandela ukunyusa ukususwa kweentsalela zezinto eziphilayo kumphezulu we-silicon wafer.
Isisombululo sePiranha sinoburharha ngokugqithisileyo kwaye sivelisa ubushushu.Ukhuseleko olongezelelweyo luyimfuneko.Ukulahlwa kwenkunkuma, vumela isisombululo siphole kwaye sidluliselwe kwisingxobo senkunkuma esicocekileyo, esomileyo.Sebenzisa izikhongozeli zesibini kwaye uleyibhelishe ngokufanelekileyo izikhongozeli zenkunkuma.Nceda ulandele izikhokelo zokhuseleko lweziko ukuze ufumane iinkqubo ezineenkcukacha.
Dehydrate ama-wafers ngokuwabeka kwi-200 ° C eshushu i-plate ye-10 min. Emva kokuphelelwa ngamanzi emzimbeni, i-wafer yatshitshiswa kahlanu emoyeni ukuze ipholile.
Galela ~ 10 g ye photoresist SU-8 2100 embindini we wafer yesilicon ecociweyo.Sebenzisa i-tweezers ukusasaza iphotoresist ngokulinganayo kwi-wafer.Ngamathuba athile ubeke iwafa kwi-65°C ipleyiti eshushu ukwenza i-photoresist ingancangathi kwaye ibe lula ukuyisasaza.Musa ukubeka iwafer ngqo kwipleyiti eshushu.
I-SU-8 yasasazwa ngokulinganayo kwi-wafer ngokubaleka i-spin coating. Inkqubo yokujikeleza okungenayo kwe-SU-8 ye-5-10 s ukusasaza kwi-500 rpm ngesantya se-100 rpm/s. Seta eyona spin i-200 µm ubukhulu bepateni nge-1,500m2 500 µm ubude bomgangatho ongaphezulu wamathumbu kwitshiphu; bona “Amanyathelo abalulekileyo” ngezantsi) iseti ngesantya esingama-300 rpm/s imizuzwana engama-30 kwi-1,200 rpm.
Esona santya sijikelezayo sinokulungelelaniswa ngokobukhulu obujoliswe kuko bepateni ye-SU-8 kwi-silicon wafer.
Ukwenza iipateni ze-SU-8 zobude be-500 µm zobude obungaphezulu be-gut kwitshiphu, i-spin coating kunye namanyathelo okubhaka athambileyo ale Bhokisi (amanyathelo esi-7 nesesi-8) aye aphindwa ngokulandelelana (jonga inyathelo lesi-9) ukuvelisa iileya ezimbini ze-250 µm. µm phezulu.
Ukubhaka okuthambileyo ii-wafers ezigqunywe nge-SU-8 ngokubeka ngononophelo ii-wafers kwipleyiti eshushu kwi-65 °C kangangemizuzu emi-5, emva koko utshintshe ukuseta ukuya kwi-95 °C kwaye ufukamele i-40 min eyongezelelweyo.
Ukufezekisa i-500 μm ubude bepateni ye-SU-8 kwi-microchannel ephezulu, phinda amanyathelo 7 kunye ne-8 ukuvelisa ama-250 μm angqingqwa e-SU-8.
Ukusebenzisa i-UV Mask Aligner, yenza uvavanyo lwesibane ngokwemiyalelo yomenzi ukubala ixesha lokuvezwa kwe-wafer.
Emva kokumisela ixesha lokuvezwa, beka i-photomask kwisibambi semaski ye-UV imask aligner kwaye ubeke i-photomask kwi-wafer ecatyiswe yi-SU-8.
Beka indawo eprintiweyo ye-photomask ngqo kwicala le-SU-8 eligqunywe kwisiqwenga se-silicon ukunciphisa ukusasazwa kwe-UV.
Veza i-SU-8 egqunywe nge-wafer kunye ne-photomask ngokuthe nkqo ukuya kwi-260 mJ/cm2 yesibane se-UV ngexesha elibekiweyo lokuvezwa (jonga inyathelo le-10 lale bhokisi).
Emva kokuvezwa kwe-UV, ii-wafers ze-silicon ezigqunywe nge-SU-8 zabhakwa kwi-65 ° C ngemizuzu emi-5 kunye ne-95 ° C ngemizuzu eyi-15 kwipleyiti nganye eshushu ukwenza iipateni ezinobude obungama-200 μm.
Umphuhlisi ugalelwa kwisitya seglasi, kwaye i-wafer ebhakiweyo ifakwe kwisitya.Umthamo womphuhlisi we-SU-8 unokwahluka ngokuxhomekeke kubukhulu beplate yeglasi.Qinisekisa ukuba usebenzise ngokwaneleyo umphuhlisi we-SU-8 ukususa ngokupheleleyo i-SU-8.Ngokomzekelo, xa usebenzisa i-150 mm isitya seglasi ububanzi kunye nomthamo we-1 L, sebenzisa i- ~ 300 mold2 i-mold . ukujikeleza okuthambileyo ngamaxesha athile.
Hlanza i-mold ephuhlisiwe nge ~ 10 mL yomphuhlisi omtsha olandelwa yi-IPA ngokutshiza isisombululo usebenzisa i-pipette.
Beka i-wafer kwi-plasma yokucoca kwaye uveze i-oxygen plasma (igesi ye-atmospheric, uxinzelelo olujoliswe kuyo 1 × 10-5 Torr, amandla 125 W) kwi-1.5 min.
Beka i-wafer kwi-vacuum desiccator ene-slide yeglasi ngaphakathi.I-Wafers kunye ne-slides zinokubekwa ngapha nangapha.Ukuba i-vacuum desiccator ihlulwe ngokwemigangatho emininzi ngepleyiti, faka iislayidi kwigumbi elisezantsi kunye neefafasi kwigumbi eliphezulu.Ukulahla i-100 μL ye-trichloro, H, 2H, i-1H 2H-perfluorooctyl)isisombululo se-silane kwisilayidi seglasi kwaye ufake ivacuum ukwenza i-silanization.
Nyibilikisa ibhoyile yeeseli zeCaco-2 ezikhenkcezisiweyo kwindawo yokuhlambela yamanzi engama-37°C, emva koko udlulisele iiseli ezinyibilikisiweyo kwiflaski ye-T75 equlethe i-15 mL ye-37°C eshushu ngaphambili ephakathi kweCaco-2.
Ukudlula iiseli ze-Caco-2 kwi-~ 90% confluency, i-Caco-2 yokuqala efudumeleyo ephakathi, i-PBS, kunye ne-0.25% ye-trypsin / 1 mM EDTA kwi-37 ° C yokuhlamba amanzi.
Aspirate medium by vacuum aspiration.Geza iiseli kabini nge 5mL ye PBS eshushu ngokuphinda vacuum aspiration kunye nokongeza iPBS entsha.