Ndiyabulela ngokundwendwela i-Nature.com.Uguqulelo lwesikhangeli oyisebenzisayo lunenkxaso elinganiselweyo ye-CSS.Ukufumana amava angcono kakhulu, sincoma ukuba usebenzise i-browser ehlaziyiweyo (okanye ucime imodi yokuhambelana kwi-Internet Explorer). Okwangoku, ukuqinisekisa inkxaso eqhubekayo, siya kubonisa isayithi ngaphandle kwezitayela kunye neJavaScript.
I-Human gut morphogenesis imisela 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 izakhiwo ze-3D ze-epithelial kubalulekile ekwakhiweni kweemodeli ze-in vitro gut. ichip fluidic ngokunjalo nakwiTranswell embedded hybrid chip.Sichaza iindlela ezineenkcukacha zokwenziwa kwesixhobo, ukulima kweCaco-2 okanye iiseli ze-epithelial zamathumbu e-intestinal organoid epithelial kwisethingi eqhelekileyo kunye nakwiqonga le-microfluidic, ukufakwa kwe-3D morphogenesis, kunye nokubonakaliswa kokusekwa kwe-3D epithelia ye-epithelia esebenzayo ngokusebenzisa i-multiple generation yolawulo lwe-microflora. kwi-5 d.Indlela yethu ye-in vitro morphogenesis isebenzisa uxinzelelo lwe-shear olufanelekileyo lwe-physiologically kunye nokunyakaza komatshini kwaye ayifuni ubunjineli beeseli eziyinkimbinkimbi okanye ukuguqulwa, okunokuthi kube ngaphezu kwezinye iindlela ezikhoyo.Sibona ukuba i-protocol yethu ecetywayo ingaba nefuthe elibanzi kuluntu lophando lwe-biomedical, ukubonelela indlela yokuvuselela i-3D intestinal layer, i-biomedical epithelial kunye ne-biomedical application.
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 i-3D morphogenesis in vitro ngaphandle kokuqonda ngokucacileyo indlela ephantsi. sis in vitro, eboniswe yi-Caco-2 kunye ne-organoids yamathumbu ephuma kwisigulane.Kolu phononongo, sigxininise ngokukodwa kwimveliso yeeseli kunye nokusabalalisa koxinzelelo lwe-Wnt antagonist enamandla, i-Dickkopf-1 (DKK-1), kwi-gut-on-a-chip kunye nezixhobo ezitshintshiweyo ze-microfluidic eziqulethe i-Transwell inserts, ebizwa ngokuba yi-"Hybrid Chip" . Iprotheyini enxulumene ne-d 1, okanye i-Soggy-1) kwi-chip gut inhibits morphogenesis okanye iphazamisa i-3D e-epithelial layer, ebonisa ukuba uxinzelelo oluchasayo ngexesha lenkcubeko luxanduva lwe-intestinal morphogenesis in vitro.Ngoko ke, indlela esebenzayo yokufezekisa i-robust morphogenesis kwi-interface esebenzayo kwi-epithelial i-interface esebenzayo kwi-epithelial i-interface esebenzayo kwi-epithelial i-interface esebenzayo kwi-epithelial i-interface encinci ukugungxula (umzekelo, kwiqonga le-gut-on-a-chip okanye kwiplatifti ye-hybrid-on-a-chip) okanye ukusasazwa .Imidiya ye-Basolateral (umzekelo, ukusuka kwi-Transwell ifakela kwimithombo emikhulu ye-basolateral kumaqula).
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-transwellB faka i-polyester , i-7 ye-polyester , i-8, i-9 I-3D morphogenesis in vitro (inyathelo le-10) .Siphinde sachonga iimpawu zeselula kunye ne-molecular ebonisa i-histogenesis ye-tissue-specific kunye ne-lineage-dependent cell differentiation ye-cellular ngokusebenzisa iindlela ezininzi zokucinga (amanyathelo 11-24) .Senza i-morphogenesis ngokusebenzisa i-intestinal epithelial ye-intestinal, njenge-intestinal format okanye i-membrane ye-modnoids ye-podium okanye i-membrane ye-2 ye-intestinal okanye i-membrane ye-24. , ukudalwa kwee-monolayers ze-2D, 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 okuphakathi kwi-compartment basolateral yenkcubeko. Ukukhula kwe-epithelial exhomekeke kwi-phogen, i-longitudinal host-microbiome co-cultures, usulelo lwe-pathogen, ukulimala okuvuthayo, ukungasebenzi kwe-epithelial barrier, kunye ne-probiotic-based therapies Umzekelo.iimpembelelo.
Iprothokholi yethu ingaba luncedo kuluhlu olubanzi lwezenzululwazi kwisiseko (umzekelo, i-biology ye-intestinal mucosal, i-biology ye-stem cell, kunye ne-biology yophuhliso) kunye nophando olusetyenzisiweyo (umzekelo, ukuhlolwa kweziyobisi kwangaphambili, ukulinganisa izifo, ubunjineli bezicubu, kunye ne-gastroenterology) impembelelo ebanzi.Ngenxa yokuphindaphinda kunye nokuqina kweprotocol yethu yokuvelisa i-3D kwi-disprovision ye-technical morphogenesis i-entrogenesis ye-technology ukuthunyelwa kubaphulaphuli abafunda i-dynamics ye-cell signaling ngexesha lokuphuhliswa kwamathumbu, ukuvuselelwa okanye i-homeostasis .Ukongezelela, iprotocol yethu iluncedo ekuxilongeni usulelo phantsi kwee-arhente ezosulelayo ezifana ne-Norovirus 8, i-Severe Acute Respiratory Syndrome Coronavirus 2 (i-SARS-CoV-2), i-Clostridium difficile, i-Salmonellae Salmonellaell okanye i-Salmonellae.Abaphulaphuli bezifo zesifo kunye ne-pathogenesis nazo ziluncedo.Ukusetyenziswa kwe-on-chip gut microphysiology system 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-GIAceky, isifo se-ulcer, isifo se-Groceky, isifo se-Groceky, isifo se-ulceace, isifo se-ulcer 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. cinga ukongeza iintlobo zeeseli ezihambelana nesifo, ezifana neeseli zegazi ze-mononuclear zesigulane (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.Ukusabela kwi-microbial okanye i-immune stimuli.Ngaphezu koko, i-longitudinal host-microbiome crosstalk 10, i-14 elungelelanisa i-gut homeostasis inokusekwa kwi-3D intestinal mucosal layer ngokudibanisa iintlobo ezahlukeneyo ze-microbial, i-microbial community okanye i-fecal microbiota-i-gut-gut-i-gut-gut-i-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 inokuhlengahlengiswa kwiifomati zenkcubeko enokuhlanjululwa, njenge-multiwell iplate efakela i-43 kakuhle i-43 eqhubekayo okanye i-4solateral ifaka i-43 eqhubekayo i-compartments, i-protocol ingasasazwa kwabo baphuhlisa amayeza, i-biomedical Okanye i-high-throughput screening okanye iiplatifti zokuqinisekisa kwi-industry yokutya.Njengobungqina bomgaqo-siseko, kutshanje sibonise ukuba nokwenzeka kwe-multiplex high-throughput system morphogenesis scalable to a 24-well-throughput screening or validation platforms for the food industry.Njengoko ubungqina bomgaqo-siseko, kutshanje sibonise ukuba nokwenzeka kwe-multiplex high-throughput system morphogenesis scalable to a 24-well-throughput screening.Phambi kwe-161-imveliso yeplate esemthethweni, kukho i-161-imveliso esemthethweni, i-18-ngaphambili, i-18-imveliso yeplate esemthethweni. Ukwenziwa kwendlela yethu ye-in vitro morphogenesis inokukhawuleziswa kwaye inokuthi yamkelwe ziilabhoratri zophando ezininzi, ishishini okanye urhulumente kunye neearhente ezilawulayo ukuqonda uhlengahlengiso lweselula lwe-in vitro gut morphogenesis kwinqanaba le-transcriptomic ukuvavanya amachiza okanye i-biotherapeutics Ukufunxa kunye nokuthuthwa kwabaviwa beziyobisi kuye kwavavanywa kusetyenziswa i-3D gut-gut-proondus-uvavanyo lwe-3D gut-proondu-assessment okanye i-custom-proondugate yorhwebo. inkqubo 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 (umz., i-zebrafish20, i-mice21 okanye i-chicken-ingaba ibaluleke kakhulu, i-inkukhu ebaluleke kakhulu kwaye i-esebenzayo2). ly, musa ukumisela ngokuchanekileyo iinkqubo zophuhliso lwabantu.Ezi modeli nazo zilinganiselwe kakhulu ekukwazini ukuvavanywa ngeendlela ezininzi ezinobungozi.Ngoko ke, iprothokholi yethu yokuhlaziya izakhiwo ze-3D ze-tissue kwi-vitro zigqithise kwiimodeli zezilwanyana ze-vivo kunye nezinye iimodeli zenkcubeko ye-2D yesiqhelo. 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 izinto ezithintekayo (umzekelo, i-inner against outer mucus layers, secretion of IgA kunye ne-antimicrobial peptides) .Ngaphezu koko, i-3D i-epithelium ye-epithelium ivumela ukuba i-micromorphology ikwazi ukuvelisa i-micromorphology ye-3D ivumela i-epithelium ye-epithelium ye-microbial ivumela ukuba i-micromorphology ikwazi ukuvelisa i-micromorphology. s 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 iileya ze-epithelial ze-3D zisekwe 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-organoid yokuhlalutya i-biomicromen i-hostestinal ivame ukuhlalutya i-coolmen i-cooculclose yokuthutha i-coolmen i-costins d ngaphakathi kwe-organoid kwaye, ngoko ke, ukungeniswa kwamacandelo e-luminal afana neeseli ze-microbial okanye ii-antigens zangaphandle zilinganiselwe.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 ophando aliqela zisebenzisa izikafula ze-3D ze-hydrogel esele zicwangcisiwe ukulinganisa ubume be-epithelial yamathumbu ngokuvelisa iiseli ezizimeleyo zamathumbu omntu kumphezulu wejeli. Yenza izikafula ze-hydrogel usebenzisa i-3D-eprintiweyo, i-micro-milled, okanye i-lithographically fabricated molds-selforganised of molds-self organised. i-morphogen gradients efanelekileyo, 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. umsebenzi.Olunye uphando olutshanje olusetyenzisiweyo i-hydrogel scaffolds kwi-platform ye-microfluidic kunye ne-pattered intestinal epithelial structures usebenzisa i-laser-etching technology ubuchule be-ing ukusuka kwiqela elifanayo bakwazi ukudala iityhubhu ezincinci kunye neenkqubo ze-morphogenetic ezizenzekelayo.Nangona ukuveliswa okuyinkimbinkimbi kwamacandelo ahlukeneyo amathumbu ngaphakathi kwetyhubhu, lo mzekelo nawo awunayo ukuhamba kwamanzi okukhanyayo kunye nokuguqulwa komatshini.Ukongezelela, ukusebenza kwemodeli kunokunciphisa, ngakumbi emva kokuba inkqubo yokushicilela i-bioprinting igqityiwe, iphazamisa iimeko zovavanyo okanye i-cell-to-cell-protocol inikezela nge-cell-to-cell Uxinzelelo lwe-shear olufanelekileyo, i-biomechanics elinganisa ukuhamba kwamathumbu, ukufikeleleka kweendawo ezizimeleyo ze-apical kunye ne-basolateral compartments, kunye nokudalwa kwakhona kwe-biological microenvironments eyinkimbinkimbi ye-modularity.Ngoko ke, i-in vitro yethu ye-3D morphogenesis protocol inokubonelela ngendlela ehambelanayo yokunqoba imingeni yeendlela ezikhoyo.
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 zokuzivikela. i-chip kunye ne-hybrid-on-a-chip ivumela ukuba siphinde siphinde sihlaziye i-3D epithelial layer, ezongezelelweyo ze-biological complexities 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 kwi-basal crypts idlala indima ephambili kwi-cryptsmegstro idlala indima ephambili kwi-basal crypts iseli ye-fibros ithathwa njengeyona nto ibalulekileyo. ekuveliseni iiprotheyini 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 (okt, i-capillaries okanye i-lymphatics) idlala indima ebalulekileyo ekulawuleni ukuthutha i-molecular39 kunye ne-immunevity cell recruit ukuba i-immune cell cell recruit i-microcell recruit. idibaniswe phakathi kweemodeli zezicubu ziyimfuneko xa iimodeli zezicubu zenzelwe ukubonisa ukusebenzisana kwamalungu amaninzi.Ngoko ke, iiseli ze-endothelial zingadinga ukuba zifakwe kwimodeli echanekileyo yeempawu ze-physiological kunye nesisombululo senqanaba le-organ.Iiseli ze-immune eziphuma kwisigulane nazo ziyimfuneko ekuboniseni iimpendulo ze-innate immune, i-antigen presentation, i-innate adaptive immune crosstalk, kunye ne-tissue-immune immune intestinfi.
Ukusetyenziswa kweetshiphusi ezixubeneyo zithe tye ngakumbi kune-gut-on-a-chip kuba ukusekwa kwesixhobo kulula kwaye ukusetyenziswa kofakelo lwe-Transwell kuvumela inkcubeko enobungozi ye-gut epithelium.Nangona kunjalo, ukufakwa ngokurhweba kweTranswell ngeembrane zepolyester azinalastiki kwaye azikwazi ukulinganisa iintshukumo ezifana ne-peristaltic.Ngaphezu koko, i-apical chip plaques ibekwe kwisikhululo se-apical chip kunye ne-apical chip efakwe kwisikhululo soxinzelelo lwe-C. ekuqaleni, iipropati ezimileyo kwikhompatimenti ye-apical kunqabile ukuba zenze inkcubeko yebhaktiriya yexesha elide kwi-chips hybrid chips.Ngelixa sinokuthi singenise ngamandla i-3D morphogenesis kufakelo lwe-Transwell xa kusetyenziswa iitshiphusi ezixubeneyo, ukunqongophala kwe-biomechanics ehambelana ne-physiologically kunye nokuqukuqela kolwelo lwe-apical kunokunciphisa ukwenzeka kweplatform ye-hybrid ye-chip enokubakho.
Ukwakhiwa ngokutsha komyinge ogcweleyo we-crypt-villus axis kwi-gut-on-a-chip kunye neenkcubeko ze-hybrid-on-a-chip azikasekwa ngokupheleleyo. Ekubeni i-morphogenesis iqala kwi-epithelial monolayer, i-3D microarchitectures ayiboneleli ngokufana kwe-morphological kwi-crypts in vivo. i-lium, i-crypt kunye nemimandla ekhohlakeleyo yayingacandwanga ngokucacileyo. Nangona imijelo ephezulu ephezulu kwitshiphu ikhokelela ekuphakameni okwandisiweyo kwe-epithelium eyenziwe nge-microengineered, ubude bobude bomphakamo busalinganiselwe ukuya kuma ~ 300–400 µm. Obona nzulu bobunzulu bamathumbu omntu kumathumbu amancinane namakhulu yi ~µm, encinci kunye nobude obuncinci, kunye nobude obuphakathi kwe-~µm kunye ne-instintium encinci yi-~µm kunye ne-instintium encinci ye-~135 µm kunye nobude obuncinci i-lli 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-microfabrication ye-microfabrication 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 ngokuthe ngqo i-molecules ye-hydrophobic.Ezinye iindlela ze-PDMS zingaqwalaselwa kunye nezinye izinto ze-polymeric. Ngenye indlela, ukuguqulwa kwendawo ye-PDMS okanye i-polyphilic (umzekelo, i-polyphilic PDMS) okanye i-polyphilic (umz. I-3) inokuqwalaselwa ukunciphisa i-adsorption yee-molecule ze-hydrophobic.
Ekugqibeleni, indlela yethu ayizange ibonakaliswe kakuhle ngokubhekiselele ekuboneleleni nge-high-throughput screening okanye "i-one-size-fits-all" iqonga lokulinga lomsebenzisi.Iprotocol yangoku idinga impompo yesirinji kwi-microdevice nganye, ethatha indawo kwi-CO2 incubator kwaye inqande iimvavanyo ezinkulu.Lo mda unokuphuculwa kakhulu nge-scalability ye-scalability ye-94well, i-culture-34well, i-culture, i-34well, i-culture, i-64well, i-culture, i-34well-innovative, i-64well, i-culture format24well, i-34well, i-34well, i-culture, i-34well-innovative, ukufakwa kakuhle kwe-porous evumela ukuzaliswa ngokuqhubekayo kunye nokususwa kwemidiya ye-basolateral).
Ukuphembelela i-3D morphogenesis ye-epithelium yamathumbu omntu kwi-vitro, sasebenzisa isixhobo se-microfluidic chip intestinal equlethe i-microchannels ezimbini ezihambelanayo kunye ne-elastic porous membrane phakathi kokudala i-lumen-capillary interface.Sikwabonisa ukusetyenziswa kwesixhobo se-microfluidic esinye-channel (i-hybrid chip) ebonelela ngokuqhubekayo kwi-platform ye-Translater eqhubekayo. s, i-morphogenesis yeeseli ezahlukeneyo ze-epithelial zamathumbu omntu zingabonakaliswa ngokusebenzisa i-directional manipulation of flow ukususa abachasi be-morphogen ukusuka kwi-basolateral compartment.Yonke inkqubo yovavanyo (Umfanekiso 1) iqulethwe ngamacandelo amahlanu: (i) i-microfabrication ye-chip gut okanye i-Transwell hybrid chip1-5; co-2 iiseli) 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) . genesis ngokuthelekisa i-epithelial morphogenesis kwindawo, ixesha, imiqathango, okanye ulawulo lwenkqubo.
Sisebenzise iiplatifti ezimbini ezahlukeneyo zenkcubeko: i-gut-on-a-chip eneziteshi ezithe tye okanye iitshaneli ezidityanisiweyo ezingezizo, okanye iichips ezixubeneyo ezine-Transwell (TW) ezifakelwayo kwisixhobo se-microfluidic, esenziwe njengoko kuchaziwe kwiBhokisi 1, kunye nenyathelo 1-5. stinal organoids) kunye nenkqubo yenkcubeko esetyenziswa kule protocol "In vitro morphogenesis" ibonisa amanyathelo apheleleyo apho iCaco-2 okanye iiseli ze-epithelial eziphuma kwi-organoid zikhuliswa kwi-chip yamathumbu okanye kwiifakelo ze-Transwell ze-chip hybrid, elandelwa kukufakwa kwe-3D morphogenesis kunye nokwakheka kwenkqubo ebonakalisiweyo ye-epithelial imizekelo ye-epithelial yesicelo esisekiweyo kwibhokisi yesicelo esisezantsi. Iingqimba ze-epithelial zingasetyenziswa, umzekelo, kwi-cell differentiation characterization, izifundo ze-gut physiology, ukusekwa kwe-host-microbiome ecosystems, kunye ne-model yezifo.Imifanekiso ye-Immunofluorescence kwi-"Cell Differentiation" ebonisa i-nuclei, i-F-actin kunye ne-MUC2 echazwe kwi-3D Caco-2 epithelial epithelial epithelial epithelial epithelial epithelial epithelial epithelial epithelial. umphezulu we-mucosal.Imifanekiso ye-Fluorescent kwi-Gut Physiology ibonisa i-mucus eveliswe ngokungcoliswa kwe-asilic acid kunye neentsalela ze-N-acetylglucosamine zisebenzisa i-fluorescent wheat germ agglutinin.Imifanekiso emibini ethe gqolo kwi-"Host-Microbe Co-Cultures" ibonisa ummeli we-host-microbiome gut-culture ebonisa i-co-culture eluhlaza kwi-co-culture ye-fluome kwi-co-culture ye-fluo. iprotheyini ye-rescent (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-immunofluorescence staining kunye ne-F-actin (ebomvu) kunye ne-nuclei (i-blue) . s (umzekelo, i-lipopolysaccharide, i-LPS) kunye neeseli ze-immune (umz, i-PBMC;eluhlaza).Iiseli ze-Caco-2 zikhuliswe ukuseka i-3D epithelial layer.I-Scale bar, i-50 µm.Imifanekiso kumqolo ongezantsi: "Ukwahluka kweeseli" zilungelelaniswe ngemvume evela kwireferensi.2.I-Oxford University Press;Iphinde yaveliswa ngemvume evela kwiNgxelo 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 kwaye iphethe i-SU-8.Uyilo lwe-microchannels kwi-chip nganye luchongwa ngokuqwalasela i-hydrodynamics efana noxinzelelo lwe-shear kunye noxinzelelo lwe-hydrodynamic1,4,12.I-design ye-original ye-Data ye-Fixxstededa-design ye-Data yokuqala i-microchannels ehambelanayo eqondileyo, iye yavela kwi-gut-on-a-chip eyinkimbinkimbi (iDatha eyandisiweyo Fig. 1b) equka isibini se-microchannels egobileyo ukuze ibangele Ukwenyuka kwexesha lokuhlala kwamanzi, iipatheni zokuhamba okungahambiyo, kunye ne-multiaxial deformation yeeseli ezikhuliswe (umzobo 2a-f) 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 luhlobo luni lweseli ekhulisiwe.Ngoko ke, ukukhupha i-3D morphogenesis, i-linear kunye ne-complex on-chip gut designs are interchangeable2a) .Ukwenza ithumbu kwitshiphu, umaleko wePDMS ongaphezulu olungisiweyo wadityaniswa ngokulandelelanayo kwifilimu yePDMS eneengxangxasi kwaye emva koko ilungelelaniswe nomgangatho ophantsi wePDMS ngokubopha okungenakujikwa kusetyenziswa i-corona treater (Fig. 2b–f) .Ukwenza iitshiphusi ezixutyiweyo, iikopi zePDMS eziphilisiweyo zadityaniswa kwizixhobo zeglasi ezingakwaziyo ukufaka i-transfluordic edityanisiweyo kwi-microfluorenal i-microfluorenal i-microflora Umzobo we-2h kunye neDatha eyandisiweyo Umfanekiso we-2) . Inkqubo yokubambisana yenziwa ngokuphatha iindawo ze-PDMS replica kunye neglasi nge-oxygen plasma okanye unyango lwe-corona.Emva kokutshatyalaliswa kwesixhobo se-microfabricated esifakwe kwi-tube ye-silicone, ukusekwa kwesixhobo kwakukulungele ukwenza i-3D morphogenesis ye-intestinal epithelium 2g (Figure 2g).
a, Umzobo weSchematic wokulungiswa kweengxenye zePDMS ukusuka kwi-SU-8 enepateni ye-silicon molds.Isisombululo se-PDMS esinganyangekiyo sagalelwa kwi-silicon mold (ekhohlo), yanyangwa kwi-60 °C (phakathi) kwaye yachithwa (ekunene).I-PDMS ediliziweyo yanqunyulwa yaziingceba kwaye yacocwa ukuze isetyenziswe ngakumbi.b, Ifoto esetyenzisiweyo kwi-silicone i-mold mold ye-silicon. yenza i-PDMS i-membrane ene-porous.d, Uthotho lweefoto zamacandelo e-PDMS aphezulu nasezantsi kunye nesixhobo esidityanisiweyo kwi-chip yamathumbu.e, i-Schematic yokulungelelaniswa kwezixhobo eziphezulu, ze-membrane, kunye ne-PDMS engaphantsi. s.g, Ukuseta i-gut-on-a-chip yenkcubeko yeseli ye-microfluidic.Ithumbu elenziwe kwitshiphu elidityaniswe netyhubhu ye-silicone kunye nesirinji yabekwa kwi-coverlip.Isixhobo se-chip sabekwa kwisiciko se-150 mm yesitya sePetri sokucubungula.I-binder isetyenziselwa ukuvala i-silicone tube.h, i-Visual hybrids chipssgenesis ye-chipsd ye-chipsgenesis ye-Visual. ilungiselelwe ngokuzimeleyo kwinkcubeko i-2D monolayers yeeseli ze-epithelial zamathumbu zafakwa kwi-chip hybrid ukuze ifake i-intestinal 3D morphogenesis.I-medium ixutywe nge-microchannels ngaphantsi kwe-cell layer esekelwe kwi-Transwell insert.I-Scale bar, i-1 cm.h Iprintwe kwakhona ngemvume evela kwireferensi.4.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 le-5) kwaye zisetyenziselwa imbewu ye-ECM-coated microchannels ye-chip gut okanye i-Transwell inserts.Xa iiseli zihambelanayo (> 955% ye-Call-Callation ye-Call 95%). I-0 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 ukuhlinzwa kokuhlinzwa kwakukhuliswe kwi-Matrigel scaffold domes kwiiplate ze-24-well ukuxhasa i-structural structural organoids, i-microenvironment ye-mospogennt, i-microenvironment ye-mospogennt, i-mospogennt efunekayo kunye ne-mospogennt ebaluleke kakhulu i-microenvironment equkethe i-microenvironment efunekayo. imiba yokukhula elungiselelwe njengoko kuchaziwe kwiBhokisi yesi-3 yongezwa yonke imihla de i-organoids ikhule ukuya kutsho ~ 500 µm ububanzi. I-organoids ekhule ngokupheleleyo iyavunwa kwaye yahlulwe ibe ziiseli enye ukuze ihlwayelwe emathunjini okanye i-Transwell efakwe kwi-chip (Ibhokisi 5) .Njengoko sele sichaze ngaphambili, inokwahlulwa ngokohlobo lwesifo se-12,13, umzekelo, isifo se-ulcernctal, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, isifo somhlaza, umhlaza, isifo somhlaza (umhlaza, isifo somhlaza), kunye nokwahlulwa) indawo (umzekelo, i-lesion ngokubhekiselele kwindawo engabonakaliyo) kunye nendawo yesisu kwi-tract (umzekelo, i-duodenum, i-jejunum, i-ileum, i-cecum, i-colon, okanye i-rectum) .Sibonelela ngeprotocol ephuculweyo kwiBhokisi yesi-5 yokulima i-colonic organoids (i-coloids) efuna ukugxininiswa okuphezulu kwi-morphogens encinci okanye i-morphogens encinci.
a, Ukuhamba komsebenzi wokungeniswa kwe-gut morphogenesis kwi-gut chip.I-Caco-2 i-epithelium yamathumbu omntu kunye ne-intestinal organoids isetyenziswe kule protocol ukubonisa 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 (i-chips) ifakwe kwi-membrane efakwe kwi-membrane efakwe kwi-membrane ngosuku (i-chips). I-D0), ukuhamba kwe-apical (AP) kuqaliswe kwaye kugcinwe kwiintsuku zokuqala ze-2 (ukuhamba, i-AP, i-D0-D2) ukuhamba kwe-Basolateral (BL) kwakhona kuqaliswe kunye ne-cyclic stretching motions (ukwelula, ukugeleza, i-AP kunye ne-BL) xa i-monolayer ye-2D epheleleyo iqulunqwa.I-intestinal 3D morphogenesis yenzeke kwinkcubeko ye-3D ye-morphogenesis yenzeke iintsuku ze-micromorphogenesis ezimele i-micromorphogenesis ngokuzenzekelayo (i-5). yeeseli zeCaco-2 kwinqanaba ngalinye lovavanyo okanye indawo yexesha (igrafu yebha, i-100 µm).Imizobo emine ecwangcisiweyo ebonisa i-cascade ehambelanayo ye-gut morphogenesis (phezulu ekunene) .Iintolo ezidayiweyo kwi-schematic zimele isalathiso se-fluid flow.b, umfanekiso we-SEM obonisa i-topology yomphezulu we-3D esekelwe i-Cacoumset ye-indawo egxininisiweyo (indawo emhlophe ye-cacoumset) ibalaselisa i-3D Cacoumset (indawo emhlophe) ibonisa i-microvilli ehlaziyiweyo kwi-3D Caco-2 umaleko (ekunene) Umbono we-confocal.d, Ixesha lexesha lokutshintsha kwe-morphological kwi-organoids ekhuliswe kwi-chip efunyenwe ngesigaba sokuchasa i-microscopy ngeentsuku ze-3, 7, 9, 11, kunye ne-13. I-inset (phezulu ekunene) ibonisa ukuphakama okuphezulu komfanekiso onikeziweyo. GR5;i-magenta), iiseli ze-goblet (MUC2; eluhlaza), i-F-actin (grey) kunye ne-nuclei (cyan) ekhulile kwii-chips ze-gut iintsuku ze-3, ngokulandelanayo (Ekhohlo) kunye ne-13-day (ephakathi) i-organoids kwi-epithelial layer.Jonga kwakhona i-Data eyandisiweyo Umfanekiso we-3, obonisa ukubonakaliswa kwe-LGR5 ngaphandle kwe-MUluoright ye-microstructure ye-microstructureliD ye-MUC2. i-organoid epithelium esekwe emathunjini kwi-chip ngokungcolisa i-plasma membrane nge-CellMask idayi (ekunene) ngosuku lwe-13 yenkcubeko.I-Scale bar yi-50 μm ngaphandle kokuba kuchazwe ngenye indlela.b Iprintwe kwakhona ngemvume evela kwireferensi.2.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 i-membrane ene-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-membrane ye-CaCMMS kunye ne-CapPD ye-membrane ye-2. ngonaphakade, inkcubeko ye-microfluidic ye-organoid epithelium idinga ukusetyenziswa kweekhemikhali ezisekelwe kwimichiza ukuze kufezekiswe i-deposition esebenzayo yeeprotheni ze-ECM ngokusebenzisa ngokulandelelana i-polyethyleneimine (PEI) kunye ne-glutaraldehyde kwi-PDMS microchannels. inkcubeko iqala ngokuqhola kuphela i-medium kwi-microchannel ephezulu kude kube iiseli zenze i-monolayer epheleleyo, ngelixa i-microchannel esezantsi igcina iimeko ezizinzileyo.Le ndlela ephuculweyo yokusetyenziswa komhlaba kunye ne-ECM yokugqoka yenza ukuba i-epithelium ye-organoid 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 zeCaco-2 kwi-Transwell inserts, i-ECM yokugqoka kwi-porous inserts ikhawuleza i-attachment yeeseli ze-Caco-2 ezidityanisiweyo (<1 iyure) kunye nokwakheka komqobo oqinileyo (<1-2 iintsuku) . <3 h) kwaye igcinwe de i-organoids yenze i-monolayer epheleleyo kunye nengqibelelo yesithintelo .Iinkcubeko zeTranswell zenziwa kwiiplate ze-24-well ngaphandle kokusetyenziswa kweechips ezixutywe.
I-in vitro i-3D morphogenesis ingaqaliswa ngokusebenzisa ukuhamba kwamanzi kwi-basolateral ye-epithelial layer esekelwe. Kwi-gut kwi-chip, i-epithelial morphogenesis yaqala xa i-medium yaxutywa kwi-microchannels ephezulu nangaphantsi (umzobo 3a) .Njengoko kuchazwe ngaphambili, kubalulekile ukuzisa ukuhanjiswa kwe-secret morphogenesis kwi-secret morphogenesis (ukukhutshwa kwe-secret morphogenesis) ukukhutshwa kwe-secret morphogenesis kwi-secret morphogenesis. Ukubonelela ngezondlo ezaneleyo kunye ne-serum kwiiseli ezibophelelwe kwi-membrane e-porous kwaye zenze uxinzelelo lwe-shear luminal, sisebenzisa ukuhamba kabini emathunjini kwi-chip.Kwi-chips hybrid, i-Transwell ifakela i-epithelial monolayers ifakwe kwi-chips hybrid.Emva koko, i-medium isetyenziswe phantsi kwecala le-basolateral le-porous Transwell-i-insestination yeentsuku ze-porous Transwell. omabini amaqonga enkcubeko.
Iimpawu ze-morphological ze-microengineered 3D epithelial layers zingahlalutywa ngokusebenzisa iindlela ezahlukeneyo zokucinga, kubandakanywa ukwahlukana kwesigaba microscopy, differential interference difference (DIC) microscopy, SEM, okanye immunofluorescence confocal microscopy (Figure 3 kunye 4) .Phase umahluko okanye i-DIC imaging ingenziwa ngexesha le-protrusion ye-protrusion ye-protrusion ye-DIC ngexesha le-protrusion ye-protrusion ye-protrusion ye-DIC. e kwi-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 (Figure 1, 3c, f kunye ne-4b, i-fixedt / hyde) iiseli ezilandelwa yi-Trixt (Amanani e-1, i-3c, i-f kunye ne-4b, i-fixedt / hyde%) ilandelwa yi-fixedt / hyde ton X-100 kunye ne-2% (wt / vol) ) i-albumin ye-bovine serum (BSA), ngokulandelelana.Ngokuxhomekeka kuhlobo lweeseli, izilungiso ezahlukeneyo, i-permeabilizers, kunye ne-blocking agents zinokusetyenziswa.Ii-antibodies eziphambili ezijolise kumgca we-cell okanye iimpawu zommandla zisetyenziselwa ukugqamisa iiseli ezingenakunyakaziswa kwi-situ kwi-situ kwi-chip, i-counter-decoction ye-nucleus, i-counter-anti-6, i-counter-decoction, i-counter-anti-6, i-countering , i-counter-6, i-anti-antigen i-diamidino-2-phenylene) indole, i-DAPI) okanye i-F-actin (umzekelo, i-fluorescently labeled phalloidin) .I-Fluorescence-based based imaging live ingenziwa kwakhona kwi-situ ukufumana ukuveliswa kwe-mucus (Fig.I-1, "Ukwahlula kweeseli" kunye ne "Gut physiology"), i-colonization engahleliweyo yeeseli ze-microbial (umzobo 1, "i-Host-microbe co-culture"), ukuqeshwa kweeseli ze-immune (umzobo 1, 'iModeli yeSifo') okanye i-contours ye-3D epithelial morphology (umzobo 4bc kwi-modifying, i-fig. umaleko we-microchannel osezantsi, njengoko kuchaziwe kwi-ref.Njengoko kuboniswe kwi-Fig.2, i-3D epithelial morphology kunye ne-microvilli kumda we-apical brush inokubonakala nge-SEM (Fig. 3b) zivunwa nge-trypsinization kwaye zisetyenziselwa uhlalutyo lwemolekyuli okanye lwemfuzo.
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 ezilungiselelwe (i-TW prep; jonga umfanekiso ongezantsi) . Emva kokuba iiseli zembewu zifakwe kwiiseli ze-polyster kunye neeseli ezifakwe kwi-polystered. (inkcubeko ye-TW) .Emva kweentsuku ze-7, i-Transwell ifake enye equlethe i-2D monolayer yeeseli ze-epithelial yadityaniswa kwi-chip hybrid ukwazisa ukuhamba kwe-basolateral (Flow, BL), ekugqibeleni kwakhokelela ekuvelisweni kwe-3D ye-epithelial layer (morphogenesis) .Isigaba sokuchasana kwe-micrographs ebonisa iimpawu ze-morphological ze-colonline organor C epithelial ye-3 ye-colonline ye-depithelial ebonisa iimpawu ze-3 ze-coloni ye-depithelial ephuma kwi-epithelial ye-human epithelial. inyathelo okanye indawo yexesha.I-schematics kwiileyile eziphezulu zibonisa ukucwangciswa kovavanyo kwinqanaba ngalinye.b, Iichips zeHybrid (i-schematic ekhohlo) inokukhokelela kwi-3D morphogenesis yeeseli ze-organoid epithelial ezineembono eziphezulu ze-confocal ze-microscopy ezithathwe kwiindawo ezahlukeneyo ze-Z (phezulu, phakathi, nangaphantsi;jonga isicwangciso sasekunene kunye nemigca echokoziweyo engqinelanayo).ibonise iimpawu ezicacileyo ze-morphological.F-actin (cyan), nucleus (grey) .c, Fluorescence confocal micrographs (3D angled view) yeeseli ze-epithelial ze-organoid-derived ezikhuliswe kwi-static Transwell (TW; inset ngaphakathi kwebhokisi edayiweyo emhlophe) ngokuchasene ne-hybrid chip (ishot enkulu epheleleyo) kuthelekisa i-3D vertical morphus2. iimboniselo (ukufakwa kwikona ephezulu ngasekunene; “XZ”) ikwabonisa iimpawu ze-2D kunye ne-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 eziqhelekileyo zenkcubeko ye-static.Ngokucacileyo, ukuchithwa kwezondlo kungabangela ngenxa yomthamo olinganiselweyo we-microchannels (okt ~ 4 µL kumjelo ophezulu kwi-channel ephezulu kwi-gut-chippiso ye-original ye-original kunye ne-design ye-chippiso ye-original ngaphambi kokuba kuqhathaniswe noyilo lwe-bamorphology ye-before.
Inkqubo ye-lithography ethambileyo kufuneka yenziwe kwigumbi elicocekileyo.Kumaleko ngamnye kwi-chip (amaleko aphezulu nasezantsi kunye ne-membrane) kunye ne-chips ezixubileyo, ii-photomasks ezahlukeneyo zisetyenzisiwe kwaye zenziwe kwii-silicone wafers ezahlukeneyo ngenxa yokuba ukuphakama kwe-microchannels kwahlukile.Ubude obujoliswe kuyo be-microchannels ephezulu kunye nesezantsi kwi-chip yi-500 chip µm ukuphakama kwe-chip yi-500 µm µm ubude be-chip. 200µm.
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.
Dlulisa ama-wafers ngokuwabeka kwi-200 ° C eshushu iplate 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 ngokulungeleleneyo kwiwafer ngokubaleka i-spin coating. Inkqubo yokujikeleza okungenayo kwe-SU-8 ye-5–10 s ukusasaza nge-500 rpm ngesantya se-100 rpm/s. Seta eyona spin ye-200 µm ubukhulu bepateni nge-1,500m µm2 okanye ukuphumeza i-500 rpm m ubude bomgangatho ongaphezulu we-gut kwi-chip, jonga "Amanyathelo abalulekileyo" ngezantsi) ebekwe kwi-acceleration ye-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 obungama-500 µm kumphezulu we-gut kwitshiphu, i-spin coating kunye namanyathelo okubhaka athambileyo ale Bhokisi (amanyathelo esi-7 nesesi-8) aye aphinda-phindwa ngokulandelelana (jonga inyathelo lesi-9) ukuvelisa iileya ezimbini ze-250 µm Umaleko oshinyeneyo we-SU-8, onokuthi ufakwe kwi-2 ye-UV1 kwi-2 ye-leleko ye-0 µm kwaye idityaniswe ngu-0 µm. .
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 elimiselweyo (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 bepleyiti 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 yemizuzu ye-rovelop2-mld. .
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 kunye ne-slide yeglasi ngaphakathi.I-Wafers kunye ne-slides zinokubekwa ecaleni.Ukuba i-vacuum desiccator ihlulwe ibe ngamacandelo amaninzi ngeplate, faka iislayidi kwigumbi elisezantsi kunye ne-wafers kwigumbi eliphezulu.Ukulahla i-100 μL ye-trichloro, i-2H, i-glass, i-Hsiper, i-Hyper, isisombululo tyibilikisa 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.