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Ukwakheka kwamathumbu omntu kuseka iimpawu ze-crypt-villus ze-3D epithelial microarchitecture kunye nokuhlelwa kwendawo. Olu lwakhiwo lukhethekileyo luyimfuneko ukugcina i-homeostasis yamathumbu ngokukhusela i-stem cell niche kwi-basal crypt kwii-antigens ze-microbial zangaphandle kunye ne-metabolites zazo. Ukongeza, i-intestinal villi kunye ne-secreting mucus zivelisa iiseli ze-epithelial ezahlulwe ngokusebenzayo kunye nomqobo okhuselayo kumphezulu we-mucosal yamathumbu. Ke ngoko, ukudala kwakhona izakhiwo ze-epithelial ze-3D kubalulekile ekwakhiweni kweemodeli zamathumbu ze-in vitro. Okuphawulekayo kukuba, i-organic mimetic gut-on-a-chip inokubangela i-3D morphogenesis ezenzekelayo ye-intestinal epithelium ngemisebenzi ephuculweyo yomzimba kunye ne-biomechanics. Apha, sinikezela ngeprotokholi ephindaphindwayo yokukhuthaza ngamandla i-intestinal morphogenesis emathunjini kwi-microfluidic chip kunye nakwi-Transwell embedded hybrid chip. Sichaza iindlela ezineenkcukacha zokwenza isixhobo, ukukhuliswa kweeseli ze-Caco-2 okanye i-intestinal organoid epithelial kwiindawo eziqhelekileyo kunye nakwiqonga le-microfluidic, ukuqaliswa kwe-3D morphogenesis, kunye ukuchazwa kwe-epithelia ye-3D esekwe kusetyenziswa iindlela ezininzi zokuthatha imifanekiso. Le protocol ifezekisa ukuhlaziywa kwe-microarchitecture yamathumbu esebenzayo ngokulawula ukuhamba kolwelo lwe-basolateral kangangeentsuku ezi-5. Indlela yethu ye-in vitro morphogenesis isebenzisa uxinzelelo lwe-shear oluhambelana ne-physiologically kunye nentshukumo yoomatshini kwaye ayifuni ubunjineli beeseli ezintsonkothileyo okanye ukuguqulwa, okunokusebenza ngcono kunezinye iindlela ezikhoyo. Sicinga ukuba iprotocol yethu ecetywayo inokuba nefuthe elibanzi kuluntu lophando lwe-biomedical, ibonelela ngendlela yokuvuselela iileya ze-epithelial zamathumbu ze-3D kwi-vitro kwizicelo ze-biomedical, kliniki, kunye neyeza.
Uvavanyo lubonisa ukuba iiseli ze-epithelial Caco-2 zamathumbu ezikhuliswe kwizixhobo ze-gut-on-a-chip1,2,3,4,5 okanye i-bilayer microfluidic6,7 zinokudlula kwi-3D morphogenesis ezizenzekelayo kwi-vitro ngaphandle kokuqonda ngokucacileyo indlela esisiseko yokusebenza. Kuphononongo lwethu lwakutshanje, sifumanise ukuba ukususwa kwee-basolaterally secreted morphogen antagonists kwizixhobo zenkcubeko kuyimfuneko kwaye kwanele ukukhuthaza i-3D epithelial morphogenesis kwi-vitro, ebonakaliswe yiCaco-2 kunye ne-organoids zamathumbu ezisuka kwisigulane. Iiseli ze-Epithelial ziqinisekisiwe. Kolu phononongo, sigxile ngokukodwa ekuvelisweni kweeseli kunye nokusasazwa koxinzelelo lwe-Wnt antagonist enamandla, iDickkopf-1 (DKK-1), kwi-gut-on-a-chip kunye nezixhobo ze-microfluidic eziguquliweyo ezine-Transwell inserts, ezibizwa ngokuba yi-"Hybrid Chip". Sibonisa ukuba ukongezwa kwee-Wnt antagonists zangaphandle (ezifana ne-DKK-1, i-Wnt repressor 1, i-protein 1 enxulumene ne-frizzled, okanye i-Soggy-1) kwi-on-chip gut kuthintela i-morphogenesis okanye kuphazamisa umaleko we-3D epithelial ocwangcisiweyo, nto leyo ebonisa ukuba uxinzelelo oluchaseneyo ngexesha lenkcubeko lubangela i-intestinal morphogenesis in vitro. Ke ngoko, indlela esebenzayo yokufezekisa i-robust morphogenesis kwi-epithelial interface kukususa okanye ukugcina amanqanaba e-Wnt antagonists kwi-basolateral compartment ngokuyisusa ngokusebenzayo (umz., kwi-gut-on-a-chip okanye kwi-hybrid-on-a-chip platforms) okanye ukusasazeka. I-Basolateral media (umz., evela kwi-Transwell ifaka kwiindawo ezinkulu ze-basolateral reservoirs kwi imithombo).
Kule protocol, sinikezela ngendlela eneenkcukacha yokwenza ii-microdevices ze-gut-on-a-chip kunye nee-Transwell-insertable hybrid chips (amanyathelo 1-5) ukukhulisa iiseli ze-epithelial zamathumbu kwi-polydimethylsiloxane (PDMS)-based porous membranes (amanyathelo 6A, 7A, 8, 9) okanye ii-polyester membranes ze-Transwell inserts (amanyathelo 6B, 7B, 8, 9) kunye ne-induced 3D morphogenesis in vitro (inyathelo 10). Sikwachonge iimpawu zeseli kunye neemolekyuli ezibonisa i-histogenesis ethile yezicubu kunye nokwahlukana kweeseli ezixhomekeke kumgca ngokusebenzisa iindlela ezininzi zokubonisa imifanekiso (amanyathelo 11-24). Sikhuthaza i-morphogenesis sisebenzisa iiseli ze-epithelial zamathumbu omntu, ezifana ne-Caco-2 okanye i-organoids zamathumbu, kwiifomathi ezimbini zenkcubeko ezineenkcukacha zobugcisa kubandakanya ukuguqulwa komphezulu wee-membrane ezine-porous, ukudalwa kwee-monolayers ze-2D, kunye ne-biochemical yamathumbu kunye nokuVeliswa kwe-biomechanical microenvironment.in vitro.Ukukhuthaza i-3D morphogenesis kwi-2D epithelial ii-monolayers, sisuse ii-morphogen antagonists kuzo zombini iindlela ezikhuliswe ngokufaka i-medium kwindawo yesiseko senkcubeko. Okokugqibela, sinikezela ngomfanekiso wokusetyenziswa komaleko we-epithelial we-3D ovuselelekayo onokusetyenziselwa ukubonisa ukukhula kwe-epithelial exhomekeke kwi-morphogen, ii-co-cultures ze-host-microbiome ezinde, usulelo lwe-pathogen, ukwenzakala kokudumba, ukungasebenzi kakuhle kwe-epithelial barrier, kunye ne-probiotic-based therapies Example.influences.
Iprotokholi yethu inokuba luncedo kuluhlu olubanzi lwezazinzulu kwiziseko (umz., ibhayoloji ye-intestinal mucosal, ibhayoloji ye-stem cell, kunye nebhayoloji yophuhliso) kunye nophando olusetyenzisiweyo (umz., uvavanyo lwamayeza ngaphambi kweklinikhi, imodeli yesifo, ubunjineli bezicubu, kunye ne-gastroenterology) impembelelo ebanzi. Ngenxa yokuzala kwakhona kunye nokuqina kweprotokholi yethu yokukhuthaza i-3D morphogenesis ye-intestinal epithelium in vitro, sicinga ukuba icebo lethu lobuchwephesha linokusasazwa kubaphulaphuli abafunda amandla okubonisa iiseli ngexesha lophuhliso lwamathumbu, ukuvuselelwa okanye i-homeostasis. Ukongeza, iprotokholi yethu iluncedo ekuphenyeni usulelo phantsi kwee-arhente ezahlukeneyo ezisulelayo ezifana neNorovirus 8, iSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), iClostridium difficile, iSalmonella Typhimurium 9 okanye iVibrio cholerae. Abaphulaphuli bezifo kunye ne-pathogenesis nazo ziluncedo. Ukusetyenziswa kwenkqubo ye-microphysiology yesisu esikwi-chip kunokuvumela ukukhula kwe-longitudinal co-culture 10 kunye novavanyo olulandelayo lokuzikhusela komninimzi, iimpendulo zomzimba kunye nokulungiswa kokulimala okunxulumene ne-pathogen kwindlela yesisu (GI) 11. Ezinye iingxaki ze-GI ezinxulumene ne-leaky gut syndrome, isifo se-celiac, isifo sikaCrohn, i-ulcerative colitis, i-pouchitis, okanye i-irritable bowel syndrome zinokulinganiswa xa iileya ze-epithelial zamathumbu ze-3D zilungiswa kusetyenziswa iileya ze-epithelial zamathumbu ze-3D zesigulana, ezi zifo ziquka i-villous atrophy, i-crypt shortening, umonakalo we-mucosal, okanye i-impaired epithelial barrier. I-Biopsy okanye i-stem cell-derived intestinal organoids12,13. Ukuze kuboniswe ngcono ubunzima obuphezulu bendawo yesifo, abafundi banokucinga ngokongeza iintlobo zeeseli ezinxulumene nesifo, ezifana neeseli ze-peripheral blood mononuclear cells (PBMCs), kwiimodeli eziqulethe ii-microarchitectures ze-3D intestinal villus-crypt. iiseli zomzimba ezithile zezicubu, 5.
Ekubeni isakhiwo se-epithelial se-3D sinokulungiswa kwaye sibonwe ngaphandle kwenkqubo yokwahlulahlula, ababukeli abasebenza kwi-spatial transcriptomics kunye ne-high-resolution okanye i-super-resolution imaging banokuba nomdla kwimephu yethu ye-spatiotemporal dynamics ye-genes kunye neeproteni kwi-epithelial niches. Unomdla kwitekhnoloji. Impendulo kwi-microbial okanye i-immune stimuli. Ngaphezu koko, i-longitudinal host-microbiome crosstalk 10, 14 edibanisa i-gut homeostasis inokusekwa kumaleko we-3D intestinal mucosal ngokuhlanganisa iintlobo ezahlukeneyo ze-microbial, uluntu lwe-microbial okanye i-fecal microbiota, ingakumbi kwi-gut-on-a-chip. eqongeni. Le ndlela inomtsalane ngakumbi kubaphulaphuli abafunda nge-immunology ye-mucosal, i-gastroenterology, i-human microbiome, i-culturomics kunye ne-clinical microbiology abafuna ukukhulisa i-gut microbiota engakhuliswanga ngaphambili kwilebhu. Ukuba i-in vitro morphogenesis protocol yethu inokuhlengahlengiswa kwiifomathi zenkcubeko ezikhula ngokukhawuleza, ezifana ne-multiwell inserts kwi-24, 96 okanye 384 well plates ezihlala zigcwalisa ii-basolateral compartments, i-protocol ingasasazwa nakwabo baphuhlisa amayeza, i-biomedical okanye i-high-throughput screening okanye amaqonga okuqinisekisa kwishishini lokutya. Njengobungqina bomgaqo-siseko, kutshanje sibonise ukuba kunokwenzeka inkqubo ye-multiplex high-throughput morphogenesis ekhula ngokukhawuleza kwifomathi ye-24-well plate. Ukongeza, iimveliso ezininzi ze-organ-on-a-chip ziye zathengiswa16,17,18. Ke ngoko, ukuqinisekiswa kwendlela yethu ye-in vitro morphogenesis kunokukhawuleziswa kwaye kusetyenziswe ziilabhoratri ezininzi zophando, ishishini okanye urhulumente kunye neearhente zolawulo ukuqonda ukuhlelwa kwakhona kweseli kwe-in vitro gut morphogenesis kwinqanaba le-transcriptomic ukuvavanya amayeza okanye i-biotherapeutics Ukufunxwa kunye nokuthuthwa kwamayeza afunekayo kuhlolwe kusetyenziswa iindlela zokuxhasa amathumbu ze-3D okanye kusetyenziswa iimodeli zesiko okanye zorhwebo ze-organ-on-a-chip ukuvavanya ukuphinda-phinda kwenkqubo ye-gut morphogenesis.
Inani elilinganiselweyo leemodeli zovavanyo ezihambelana nabantu zisetyenzisiwe ukufunda i-intestinal epithelial morphogenesis, ikakhulu ngenxa yokungabikho kweenkqubo ezinokusetyenziswa zokukhuthaza i-3D morphogenesis in vitro. Enyanisweni, uninzi lolwazi lwangoku malunga ne-gut morphogenesis lusekelwe kwizifundo zezilwanyana (umz., zebrafish20, mice21 okanye iinkukhu22). Nangona kunjalo, zisebenza nzima kwaye zibiza kakhulu, zinokuthandabuzeka ngokwemigaqo yokuziphatha, kwaye okona kubaluleke kakhulu, azichazi ngokuchanekileyo iinkqubo zophuhliso lomntu. Ezi modeli zikwanomda kakhulu kubuchule bazo bokuvavanywa ngendlela enokulinganiswa ngeendlela ezininzi. Ke ngoko, iprotokholi yethu yokuvuselela izakhiwo zezicubu ze-3D in vitro isebenza ngcono kwiimodeli zezilwanyana ze-in vivo kunye nezinye iimodeli zenkcubeko yeeseli ze-2D ezizinzileyo. Njengoko kuchaziwe ngaphambili, ukusebenzisa izakhiwo ze-3D epithelial kusivumele ukuba sihlole indawo yeeseli ezahlukileyo kwi-crypt-villus axis ukuphendula kwiintshukumo ezahlukeneyo ze-mucosal okanye immune. Iileya ze-3D epithelial zinokubonelela ngendawo yokufunda indlela iiseli ze-microbial ezikhuphisana ngayo ukwenza iindawo zesithuba kunye nokuguquka kwendalo ekuphenduleni izinto ezibangela ukuba umntu aphile (umz., inner versus iileya zangaphandle ze-mucus, ukukhutshwa kwe-IgA kunye neepeptides ezilwa ne-antimicrobial).Ngaphezu koko, i-3D epithelial morphology inokusivumela ukuba siqonde indlela i-gut microbiota eyakha ngayo uluntu lwayo kwaye ivelise ngokusebenzisana ii-metabolites ze-microbial (umz., ii-short-chain fatty acids) ezibumba ulungelelwaniso lweeseli kunye nee-niches ze-stem cell kwi-basal crypts. Ezi mpawu zinokubonakaliswa kuphela xa iileya ze-3D epithelial zisekwe kwi-vitro.
Ukongeza kwindlela yethu yokwenza izakhiwo ze-epithelial zamathumbu ze-3D, kukho iindlela ezininzi ze-in vitro. Inkcubeko ye-organoid yamathumbu yindlela yobunjineli bezicubu yanamhlanje esekwe ekukhuliseni iiseli ze-stem zamathumbu phantsi kweemeko ezithile ze-morphogen23,24,25. Nangona kunjalo, ukusetyenziswa kweemodeli ze-organoid ze-3D zohlalutyo lokuthuthwa okanye ii-co-cultures ze-host-microbiome kudla ngokuba nzima kuba i-lumen yamathumbu ivalelwe ngaphakathi kwi-organoid kwaye, ngenxa yoko, ukungeniswa kwezinto ze-luminal ezifana neeseli ze-microbial okanye ii-antigens zangaphandle kunqongophele. Ukufikelela kwi-organoid lumens kunokuphuculwa kusetyenziswa i-microinjector,26,27 kodwa le ndlela ihlasela kwaye ifuna umsebenzi omninzi kwaye ifuna ulwazi olukhethekileyo ukuze isebenze. Ngaphezu koko, iinkcubeko ze-organoid zemveli ezigcinwe kwi-hydrogel scaffolds phantsi kweemeko ezimileyo azibonakalisi ngokuchanekileyo i-biomechanics esebenzayo kwi-vivo.
Ezinye iindlela ezisetyenziswa ngamaqela ophando amaninzi zisebenzisa ii-scaffolds ze-3D hydrogel ezicwangcisiweyo kwangaphambili ukulinganisa isakhiwo se-epithelial yamathumbu ngokukhulisa iiseli zamathumbu zabantu ezizimeleyo kumphezulu wejeli. Yenza ii-scaffolds ze-hydrogel usebenzisa ii-molds eziprintiweyo ze-3D, ezi-micro-milled, okanye ezenziwe nge-lithographically. Le ndlela ibonisa ulungiselelo oluzilungelelanisileyo lweeseli ze-epithelial ezizimeleyo kwi-vitro ezine-morphogen gradients ezifanelekileyo ngokwasemzimbeni, ziseka isakhiwo se-epithelial esinomlinganiselo ophezulu kunye ne-stroma-epithelial crosstalk ngokufaka iiseli ze-stromal kwi-scaffold. Nangona kunjalo, uhlobo lwee-scaffolds ezicwangcisiweyo kwangaphambili lunokuthintela ukubonakaliswa kwenkqubo ye-morphogenetic ezenzekelayo ngokwayo. Ezi modeli aziboneleli ngokuhamba kwe-luminal okanye kwe-interstitial, zingenalo uxinzelelo lwe-fluid shear olufunekayo kwiiseli zamathumbu ukuze zidlule kwi-morphogenesis kwaye zifumane umsebenzi we-physiological. Olunye uphando lwakutshanje lusebenzise ii-scaffolds ze-hydrogel kwiqonga le-microfluidic kunye nezakhiwo ze-epithelial zamathumbu ezineepateni kusetyenziswa iindlela ze-laser-etching. Ii-organoids zamathumbu empuku zilandela iipatheni ezicwangcisiweyo ukwenza izakhiwo ze-tubular zamathumbu, kwaye ukuhamba kolwelo lwangaphakathi kunokwenza kufuneka iphindwe kusetyenziswa imodyuli ye-microfluidics. Nangona kunjalo, le modeli ayibonisi iinkqubo ze-spontaneous morphogenetic kwaye ayibandakanyi iintshukumo ze-gut mechanobiological. Iindlela zokuprinta ze-3D ezivela kwiqela elinye zikwazile ukwenza iityhubhu zamathumbu ezincinci ezineenkqubo ze-spontaneous morphogenetic. Nangona kwenziwe izinto ezintsonkothileyo zezigaba ezahlukeneyo zamathumbu ngaphakathi kwityhubhu, le modeli ayinayo i-luminal fluid flow kunye ne-mechanical deformation. Ukongeza, ukusebenza kwemodeli kunokulinganiselwa, ngakumbi emva kokuba inkqubo ye-bioprinting igqityiwe, iphazamisa iimeko zovavanyo okanye ukusebenzisana kweseli-kwiseli. Endaweni yoko, iprotokholi yethu ecetywayo ibonelela nge-spontaneous gut morphogenesis, uxinzelelo lwe-shear oluhambelana ne-physiologically, i-biomechanics ezilinganisa ukuhamba kwamathumbu, ukufikeleleka kweendawo ezizimeleyo ze-apical kunye ne-basolateral, kunye nokudalwa kwakhona kwe-complex biological microenvironments of modularity. Ke ngoko, iprotokholi yethu ye-in vitro 3D morphogenesis inokubonelela ngendlela encedisayo yokoyisa imingeni yeendlela ezikhoyo.
Iprotokholi yethu igxile ngokupheleleyo kwi-3D epithelial morphogenesis, kunye neeseli ze-epithelial kuphela kwinkcubeko kwaye akukho ntlobo zeeseli ezijikelezileyo ezifana neeseli ze-mesenchymal, iiseli ze-endothelial, kunye neeseli zomzimba. Njengoko kuchaziwe ngaphambili, isiseko seprotokholi yethu kukungeniswa kwe-epithelial morphogenesis ngokususa ii-morphogen inhibitors ezikhutshwe kwicala le-basolateral le-medium engenisiweyo. Ngelixa i-modularity eqinileyo ye-gut-on-a-chip yethu kunye ne-hybrid-on-a-chip isenza sikwazi ukuphinda senze umaleko we-3D epithelial ongaphantsi, ubunzima obongezelelweyo bebhayoloji obufana nokusebenzisana kwe-epithelial-mesenchymal33,34, i-extracellular Matrix (ECM) deposition35 kwaye, kwimodeli yethu, iimpawu ze-crypt-villus ezihambisa ii-niches ze-stem cell kwi-basal crypts zisaza kuqwalaselwa ngakumbi. Iiseli ze-Stromal (umz., ii-fibroblasts) kwi-mesenchyme zidlala indima ebalulekileyo kwimveliso yeeproteni ze-ECM kunye nokulawulwa kwe-intestinal morphogenesis kwi-vivo35,37,38. Ukongezwa kweeseli ze-mesenchymal kwimodeli yethu kuphucule inkqubo ye-morphogenetic kunye nokusebenza kakuhle kokunamathela kweseli. Umaleko we-endothelial (oko kukuthi, ii-capillaries okanye ii-lymphatics) udlala indima ebalulekileyo ekulawuleni ukuthuthwa kweemolekyuli39 kunye nokufunyanwa kweeseli zomzimba40 kwindawo encinci yamathumbu. Ngaphezu koko, izinto ze-vasculature ezinokudityaniswa phakathi kweemodeli zezicubu ziyimfuneko xa iimodeli zezicubu zenzelwe ukubonisa ukusebenzisana kwamalungu amaninzi. Ke ngoko, iiseli ze-endothelial zinokufuna ukufakwa ukuze zibonise iimpawu zomzimba ezichanekileyo ngesisombululo senqanaba lamalungu. Iiseli zomzimba ezithathwe kwizigulane nazo zibalulekile ekuboniseni iimpendulo zomzimba zangaphakathi, ukubonakaliswa kwe-antigen, i-innate adaptive immune crosstalk, kunye ne-tissue-specific immunity kwimeko yokulinganisa isifo samathumbu.
Ukusetyenziswa kwee-hybrid chips kulula kakhulu kune-gut-on-a-chip kuba ukuseta isixhobo kulula kwaye ukusetyenziswa kwee-Transwell inserts kuvumela inkcubeko ye-epithelium yamathumbu ekhula ngokukhawuleza. Nangona kunjalo, ii-Transwell inserts ezithengiswayo ezine-polyester membranes azinakuba lula kwaye azinakulinganisa iintshukumo ezifana ne-peristaltic. Ngaphezu koko, indawo ye-apical ye-Transwell insert ebekwe kwi-hybrid chip yahlala imile ngaphandle koxinzelelo lwe-shear kwicala le-apical. Ngokucacileyo, iipropati ezizinzileyo kwindawo ye-apical azinakuvumela ukukhuliswa kwebhaktheriya ixesha elide kwii-hybrid chips. Ngelixa sinokubangela ngamandla i-3D morphogenesis kwii-Transwell inserts xa sisebenzisa ii-hybrid chips, ukunqongophala kwe-biomechanics efanelekileyo kwi-physiologically kunye nokuhamba kolwelo lwe-apical kunokunciphisa ukusebenza kweeplatifomu ze-hybrid chip kwizicelo ezinokubakho.
Ukwakhiwa ngokutsha okupheleleyo kwe-human crypt-villus axis kwi-gut-on-a-chip kunye ne-hybrid-on-a-chip cultures ayikaqinisekiswa ngokupheleleyo. Ekubeni i-morphogenesis iqala kwi-epithelial monolayer, ii-3D microarchitectures aziboneleli ngokufana kwe-morphological ne-crypts in vivo. Nangona sichaze inani leeseli ezikhulayo kufutshane ne-basal crypt domain kwi-microengineered 3D epithelium, i-crypt kunye ne-villous regions azizange zahlulwe ngokucacileyo. Nangona imijelo ephezulu ephezulu kwi-chip ikhokelela ekuphakameni okuphezulu kwe-microengineered epithelium, ukuphakama okuphezulu kusalinganiselwe kwi-~300–400 µm. Ubunzulu bokwenyani be-crypts yamathumbu omntu kumathumbu amancinci namakhulu yi-~135 µm kunye ne-~400 µm, ngokulandelanayo, kwaye ukuphakama kwe-small intestinal villi yi-~600 µm41.
Ngokwembono yomfanekiso, umfanekiso we-3D microarchitectures kwi-situ unokukhawulelwa emathunjini kwi-chip, kuba umgama ofunekayo wokusebenza ukusuka kwilensi ejolise ngqo ukuya kumaleko we-epithelial ukwi-odolo yeemilimitha ezimbalwa. Ukulungisa le ngxaki, kunokufuneka injongo ekude. Ngaphezu koko, ukwenza amacandelo amancinci okulungiselela isampuli yomfanekiso kunzima ngenxa yokuqina okuphezulu kwe-PDMS. Ngaphezu koko, ekubeni ukwenziwa kwe-microfabrication ye-layer-by-layer ye-gut kwi-chip kubandakanya ukunamathela okungapheliyo phakathi komaleko ngamnye, kunzima kakhulu ukuvula okanye ukususa umaleko ongaphezulu ukuze kuhlolwe isakhiwo somphezulu womaleko we-epithelial. Umzekelo, ngokusebenzisa i-scanning electron microscope (SEM).
Ukungabandakanywa kwamanzi kwe-PDMS kube yinto ethintelayo kwizifundo ezisekelwe kwi-microfluidic ezijongene neemolekyuli ezincinci ze-hydrophobic, kuba i-PDMS inokufunxa iimolekyuli ezinjalo ze-hydrophobic ngokungenangqalelo. Ezinye iindlela eziya kwi-PDMS zinokuqwalaselwa nezinye izinto ze-polymeric. Kungenjalo, ukuguqulwa komphezulu we-PDMS (umz., ukugquma ngezinto ze-lipophilic 42 okanye i-poly(ethylene glycol) 43) kunokuthathwa njengokunciphisa ukufunxwa kweemolekyuli ze-hydrophobic.
Okokugqibela, indlela yethu ayichazwanga kakuhle ngokubonelela ngovavanyo oluphezulu okanye iqonga lovavanyo "elilungele bonke" elisebenziseka lula. Le protocol yangoku ifuna ipompo yesirinji ngesixhobo ngasinye, esithatha indawo kwi-CO2 incubator kwaye sithintele uvavanyo olukhulu. Olu thintelo lunokuphuculwa kakhulu ngokukwazi ukukhulisa iifomathi zenkcubeko ezintsha (umz., i-24-well, i-96-well, okanye i-384-well porous inserts ezivumela ukuphinda kuzaliswe kunye nokususwa kwe-basolateral media).
Ukuze sibangele i-3D morphogenesis ye-epithelium yamathumbu omntu kwi-vitro, sisebenzise isixhobo samathumbu se-microfluidic chip esinee-microchannels ezimbini ezihambelanayo kunye ne-membrane ene-elastic porous phakathi kwayo ukwenza i-lumen-capillary interface. Sikwabonisa ukusetyenziswa kwesixhobo se-single-channel microfluidic (i-hybrid chip) esinika ukuhamba okuqhubekekayo kwe-basolateral phantsi kwee-polarized epithelial layers ezikhuliswe kwi-Transwell inserts. Kuzo zombini iiplatifomu, i-morphogenesis yeeseli ezahlukeneyo ze-epithelial zamathumbu omntu inokubonakaliswa ngokusebenzisa i-directional manipulation ye-flow ukususa ii-morphogen antagonists kwi-basolateral compartment. Inkqubo yonke yovavanyo (Umfanekiso 1) inamacandelo amahlanu: (i) i-microfabrication ye-gut chip okanye i-Transwell insertable hybrid chip (amanyathelo 1-5; Ibhokisi 1), (ii) ukulungiswa kweeseli ze-epithelial zamathumbu (iiseli zeCaco-2) okanye ii-organoids zamathumbu omntu; iibhokisi 2-5), (iii) inkcubeko yeeseli ze-epithelial zamathumbu kwiitships zamathumbu okanye kwiitships ezixutyiweyo (amanyathelo 6-9), (iv) ukuqaliswa kwe-3D morphogenesis in vitro (inyathelo 10) kunye (v) ) ukuchaza isakhiwo se-epithelial se-3D (amanyathelo 11-24). Okokugqibela, iqela lolawulo elifanelekileyo (elixutyushwayo ngezantsi) lenzelwe ukuqinisekisa ukusebenza kwe-in vitro morphogenesis ngokuthelekisa i-epithelial morphogenesis kulawulo lwendawo, lwexesha, olunemiqathango, okanye lwenkqubo.
Sisebenzise amaqonga amabini ahlukeneyo enkcubeko: i-gut-on-a-chip eneendlela ezithe tye okanye iindlela ezidityanisiweyo ezingacwangciswanga, okanye ii-hybrid chips eziqulethe i-Transwell (TW) inserts kwisixhobo se-microfluidic, ezenziwe njengoko kuchaziwe kwiBhokisi 1, kwaye inyathelo 1 -5. "Ukwenziwa kwesixhobo" libonisa amanyathelo aphambili okwenza i-chip enye okanye i-hybrid chip. "Inkcubeko yeeSeli ze-Epithelial zamathumbu omntu" ichaza umthombo weseli (i-Caco-2 okanye ii-organoids zamathumbu omntu) kunye nenkqubo yenkcubeko esetyenzisiweyo kule protocol. "I-in vitro morphogenesis" ibonisa amanyathelo apheleleyo apho iiseli ze-epithelial ezivela kwi-Caco-2 okanye i-organoid ezikhuliswe kwi-intestinal chip okanye kwi-Transwell inserts ye-hybrid chip, elandelwa kukungeniswa kwe-3D morphogenesis kunye nokwakhiwa kwesakhiwo se-epithelial esichaziweyo. Inombolo yenyathelo lenkqubo okanye inombolo yebhokisi iboniswa ngaphantsi kotolo ngalunye. Isicelo sinika imizekelo yendlela ezingasetyenziswa ngayo iileya ze-epithelial zamathumbu ezisekwe, umzekelo, ekuchazeni umahluko weseli, izifundo ze-physiology yamathumbu, ukusekwa kwe-ecosystems ye-host-microbiome, kunye nesifo. ukumodela. Imifanekiso ye-immunofluorescence kwi-"Cell Differentiation" ebonisa ii-nuclei, i-F-actin kunye ne-MUC2 ezivezwe kumaleko we-3D Caco-2 epithelial eveliswe kwi-gut chip. I-MUC2 signaling ikhona kwiiseli ze-goblet kunye ne-mucus ekhutshwe kwiindawo ze-mucosal. Imifanekiso ye-Fluorescent kwi-Gut Physiology ibonisa i-mucus eveliswa ngokudaya i-sialic acid kunye ne-N-acetylglucosamine residues kusetyenziswa i-fluorescent wheat germ agglutinin. Imifanekiso emibini edibeneyo kwi-"Host-Microbe Co-Cultures" ibonisa ii-host-microbiome co-cultures ezimeleyo esiswini kwi-chip. Iphaneli yasekhohlo ibonisa i-co-culture ye-E. coli eveza i-green fluorescent protein (GFP) kunye nee-microengineered 3D Caco-2 epithelial cells. Iphaneli yasekunene ibonisa indawo ye-GFP E. coli ekhuliswe kunye nee-3D Caco-2 epithelial cells, ilandelwa yi-immunofluorescence staining ene-F-actin (ebomvu) kunye nee-nuclei (eluhlaza okwesibhakabhaka). Ukumodela kwesifo kubonisa amathumbu aphilileyo ngokuchasene navuzayo esiswini Iitships zokuvuvukala phantsi komngeni we-physiological kunye nee-antigens zebhaktiriya (umz., i-lipopolysaccharide, i-LPS) kunye neeseli zomzimba (umz., i-PBMC; eluhlaza). Iiseli zeCaco-2 zakhuliswa ukuze kusekwe umaleko we-epithelial we-3D. Ibha yesikali, i-50 µm. Imifanekiso kumqolo ongezantsi: "Ukwahluka kweeseli" kuhlengahlengiswe ngemvume evela kwisalathiso.2. I-Oxford University Press; Iphinde yaveliswa ngemvume evela kwisalathiso.5. I-NAS; "I-Host-Microbe Co-Culture" ihlengahlengiswe ngemvume evela kwisalathiso.3. I-NAS; "Imodeli yeZifo" ihlengahlengiswe ngemvume evela kwisalathiso.5. I-NAS.
Zombini ii-gut-on-chip kunye nee-hybrid chips zenziwe kusetyenziswa ii-replicas ze-PDMS ezasuswa kwii-silicon molds nge-soft lithography1,44 kwaye zalungiswa nge-SU-8. Uyilo lwee-microchannels kwi-chip nganye lumiselwa ngokuqwalasela i-hydrodynamics efana noxinzelelo lwe-shear kunye noxinzelelo lwe-hydrodynamic1,4,12. Uyilo lokuqala lwe-gut-on-a-chip (Extended Data Fig. 1a), olwalune-juxtaposed parallel straight microchannels ezimbini, luye lwaguquka lwaba yi-complex gut-on-a-chip (Extended Data Fig. 1b) equka isibini se-curved microchannels ukuze kubangele ukwanda kwexesha lokuhlala kolwelo, iipatheni zokuhamba ezingezizo ezilandelelanayo, kunye nokuguqulwa kwe-multiaxial yeeseli ezikhuliswe (Umzobo 2a–f) 12. Xa kufuneka kuphinde kwenziwe ii-biomechanics ze-gut-on-a-chips ezintsonkothileyo, kunokukhethwa ii-complex gut-on-a-chips. Sibonise ukuba i-convoluted Gut-Chip ikwakhuthaza kakhulu i-3D morphogenesis ngexesha elifanayo kunye nenqanaba elifanayo lokukhula kwe-epithelial xa kuthelekiswa neyokuqala. I-Gut-Chip, nokuba loluphi uhlobo lweseli olukhuliswe ngayo. Ke ngoko, ukuze kubangele i-3D morphogenesis, uyilo lwe-on-chip oluthe ngqo noluntsonkothileyo luyatshintshana. Ii-PDMS replicas ezinyangiweyo kwii-silicon molds ngeepateni ze-SU-8 zibonelele ngeempawu ezimbi emva kokudilizwa (Umzobo 2a). Ukwenza i-gut kwi-chip, umaleko ophezulu we-PDMS olungiselelweyo wadityaniswa ngokulandelelana kwifilimu ye-PDMS enemingxuma waza wahambelana nomaleko osezantsi we-PDMS ngokudityaniswa okungenakuguqulwa kusetyenziswa i-corona treater (Umzobo 2b–f). Ukwenza ii-hybrid chips, ii-PDMS replicas ezinyangiweyo zadityaniswa kwiislayidi zeglasi ukwenza izixhobo ze-microfluidic ze-single-channel ezinokuthatha ii-Transwell inserts (Umzobo 2h kunye nomfanekiso weDatha eyongeziweyo 2). Inkqubo yokudibanisa yenziwa ngokunyanga imiphezulu ye-PDMS replica kunye neglasi nge-oxygen plasma okanye unyango lwe-corona. Emva kokubulala intsholongwane kwesixhobo esenziwe nge-microfabricated esiqhotyoshelwe kwityhubhu ye-silicone, ukuseta isixhobo kwakukulungele ukwenza i-3D morphogenesis ye-intestinal epithelium (Umfanekiso 2g).
a, Umfanekiso ocacileyo wokulungiswa kweendawo zePDMS ezivela kwiimold zesilicon ezineepateni zeSU-8. Isisombululo sePDMS esinganyangwanga sagalelwa kwimold yesilicon (ekhohlo), sanyangwa kwi-60 °C (embindini) saza sasuswa (ekunene). IPDMS esusiweyo yanqunyulwa yaba ziingceba yaza yacocwa ukuze isetyenziswe ngakumbi.b, Umfanekiso wemold yesilicon esetyenziselwa ukulungiselela umaleko ophezulu wePDMS.c, Umfanekiso wemold yesilicon esetyenziselwa ukwenza i-membrane enemingxuma yePDMS.d, Uthotho lweefoto zezinto eziphezulu nezisezantsi zePDMS kunye nesixhobo samathumbu esihlanganisiweyo kwi-chip.e., I-Schematic yokulungelelaniswa kwezinto eziphezulu, i-membrane, kunye nezisezantsi zePDMS. Umaleko ngamnye ubotshelelwe ngokungenakuguquguquka yi-plasma okanye unyango lwe-corona.f, I-Schematic yesixhobo esenziwe nge-gut-on-a-chip esinee-microchannels ezihlanganisiweyo kunye nee-vacuum chambers.g, Ukuseta i-gut-on-a-chip yenkcubeko yeseli ye-microfluidic. I-gut eyenziwe kwi-chip ehlanganiswe ngetyhubhu ye-silicone kunye nesirinji yabekwa kwisiliphu. Isixhobo se-chip sabekwa kwisivalo se Isitya sePetri esiyi-150 mm sokucubungula. I-binder isetyenziselwa ukuvala ityhubhu ye-silicone.h, Iisnapshots ezibonakalayo zokwenza i-hybrid chip kunye ne-3D morphogenesis kusetyenziswa ii-hybrid chips. I-Transwell inserts ezilungiselelwe ngokuzimeleyo ukukhuliswa. Ii-monolayers ze-2D zeeseli ze-epithelial zamathumbu zifakwe kwi-hybrid chip ukuze zibangele i-intestinal 3D morphogenesis. I-medium ixutywe nge-microchannels phantsi komaleko weseli osekwe kwi-Transwell insert. I-Scale bar, 1 cm.h Ishicilelwe kwakhona ngemvume evela kwireferensi.4. Elsevier.
Kule protocol, umgca weseli yeCaco-2 kunye nee-organoids zamathumbu zisetyenziswe njengemithombo ye-epithelial (Umzobo 3a). Zombini ezi ntlobo zeeseli zakhuliswa ngokwazo (Ibhokisi 2 kunye neBhokisi 5) kwaye zasetyenziselwa ukuhlwayela ii-microchannels ezigqunywe yi-ECM ze-on-chip gut okanye ii-Transwell inserts. Xa iiseli zidibene (>95% ukugubungela kwiiflasks), iiseli zeCaco-2 ezikhuliswe rhoqo (phakathi kweendlela ze-10 kunye ne-50) kwiiflasks ze-T ziyavunwa ukulungiselela ukumiswa kweeseli ezihlukanisiweyo ngolwelo lwe-trypsinization (ibhokisi 2). Ii-organoids zamathumbu abantu ezivela kwi-intestinal biopsies okanye ukususwa kotyando zakhuliswa kwi-Matrigel scaffold domes kwiiplates ezingama-24-well ukuxhasa i-microenvironment yesakhiwo. Ii-morphogens ezibalulekileyo eziphakathi (ezifana ne-Wnt, i-R-spondin, kunye ne-Noggin) kwaye izinto zokukhula ezilungiselelwe njengoko kuchaziwe kwiBhokisi 3 zongezwa yonke imihla de ii-organoids zikhule zibe yi-~500 µm ububanzi. Ii-organoids ezikhule ngokupheleleyo ziyavunwa kwaye zahlulwe zibe ziiseli ezizimeleyo ukuze zityalwe emathunjini okanye kwi-Transwell inserts kwi-chip (Ibhokisi 5). Njengoko besitshilo ngaphambili, zinokwahlulwa ngokwesifo sohlobo 12,13 (umz. i-ulcerative colitis, isifo sikaCrohn, umhlaza we-colorectal, okanye umntu oqhelekileyo onikelayo), indawo yesilonda (umz., isilonda xa kuthelekiswa nendawo engenasilonda) kunye nendawo yesisu kwindlela yokugaya ukutya (umz., i-duodenum, i-jejunum, i-ileum, i-cecum, i-colon, okanye i-rectum). Sinikezela ngenkqubo ephuculweyo kwiBhokisi 5 yokukhulisa ii-organoids ze-colonic (ii-coloids) ezidla ngokufuna ubuninzi be-morphogens kune-organoids zamathumbu amancinci.
a, Ukuhamba komsebenzi wokungenisa i-morphogenesis yamathumbu kwi-chip yamathumbu. I-Caco-2 intestinal epithelium kunye ne-organoids yamathumbu zisetyenziswa kule protocol ukubonisa i-morphogenesis ye-3D. Iiseli ze-epithelial ezizimeleyo zityalwe kwisixhobo esilungisiweyo se-gut-on-a-chip (ukulungiswa kwe-chip). Nje ukuba iiseli zityalwe (zityalwe) kwaye zincamathiselwe (zincamathiselwe) kwi-membrane enemingxuma ye-PDMS ngomhla we-0 (D0), ukuhamba kwe-apical (AP) kuyaqaliswa kwaye kugcinwe kwiintsuku ezi-2 zokuqala (ukuhamba, i-AP, i-D0-D2). Ukuhamba kwe-Basolateral (BL) kukwaqaliswa kunye neentshukumo zokolula nge-cyclic (ukolula, ukuhamba, i-AP kunye ne-BL) xa kwakheka i-monolayer epheleleyo ye-2D. I-morphogenesis yamathumbu ye-3D yenzeka ngokuzenzekelayo emva kweentsuku ezi-5 zenkcubeko ye-microfluidic (i-morphogenesis, i-D5). Imifanekiso yomahluko wesigaba ibonisa i-morphology emele iiseli ze-Caco-2 kwinqanaba ngalinye lovavanyo okanye ixesha (igrafu yebha, i-100 µm). Iidayagram ezine zeskimu ezibonisa ii-cascades ezifanelekileyo ye-morphogenesis yamathumbu (phezulu ngasekunene). Iintolo eziqhekekileyo kwi-schematic zimele indlela yokuhamba kolwelo.b, umfanekiso we-SEM obonisa i-topology yomphezulu we-3D Caco-2 epithelium esekwe (ekhohlo). I-inset egxininisa indawo ekhulisiweyo (ibhokisi emhlophe eqhekekileyo) ibonisa i-microvilli evuselelweyo kumaleko we-3D Caco-2 (ekunene).c, Umbono othe tye we-Caco-2 3D esekwe, i-claudin (ZO-1, ebomvu) kunye ne-continuous brush border membranes ebhalwe i-F-actin (eluhlaza) kunye ne-nuclei (eluhlaza okwesibhakabhaka) Umbono we-immunofluorescence confocal weeseli ze-epithelial kwii-intestinal chips. Iintolo ezikhomba kwi-middle schematic zibonisa indawo ye-focal plane nganye ye-confocal view.d, Ixesha lotshintsho lwe-morphological kwi-organoids ezikhuliswe kwi-chip efunyenwe nge-phase contrast microscopy kwiintsuku ze-3, 7, 9, 11, kunye ne-13. I-inset (ekunene phezulu) ibonisa ukukhulisa okuphezulu komfanekiso onikiweyo.e, i-DIC photomicrograph ye-organoid 3D epithelium esekwe emathunjini kwisilayi esithathwe ngomhla wesi-7.f, Imifanekiso ye-immunofluorescence egqunywe ngaphezulu ebonisa iimpawu zeeseli zesiqu (LGR5; magenta), iiseli zegoblet (MUC2; luhlaza), i-F-actin (grey) kunye ne-nuclei (cyan) ezikhuliswe kwiitships zamathumbu iintsuku ezi-3, ngokulandelelana (ekhohlo) kunye nee-organoids zeentsuku ezili-13 (eziphakathi) kumaleko we-epithelial.Jonga kwakhona uMfanekiso weDatha eyongeziweyo 3, obonisa i-LGR5 signaling ngaphandle kwe-MUC2 signaling. Imifanekiso ye-Fluorescence ebonisa i-epithelial microstructure (ekunene) ye-3D organoid epithelium esekwe emathunjini kwi-chip ngokufaka i-plasma membrane nge-CellMask dye (ekunene) ngomhla we-13 wenkcubeko. I-Scale bar yi-50 μm ngaphandle kokuba kuchazwe ngenye indlela.b Ishicilelwe kwakhona ngemvume evela kwisalathiso.2. I-Oxford University Press; c Ilungiswe ngemvume evela kwisalathiso.2. I-Oxford University Press; e kunye ne-f ilungiswe ngemvume evela kwisalathiso.12 Phantsi kweLayisensi yeCreative Commons CC BY 4.0.
Emathunjini kwi-chip, kuyimfuneko ukutshintsha umphezulu we-hydrophobic we-membrane enemingxuma ye-PDMS ukuze kubekho ugqubuthelo lwe-ECM oluphumelelayo. Kule protocol, sisebenzisa iindlela ezimbini ezahlukeneyo ukutshintsha ugqubuthelo lwe-hydrophobic lwee-membrane ze-PDMS. Ukukhulisa iiseli ze-Caco-2, ukusebenza komphezulu ngonyango lwe-UV/ozone kuphela kwanele ukunciphisa ugqubuthelo lwe-hydrophobic kumphezulu we-PDMS, ukugqubuthelo lwe-ECM kunye nokuncamathisela iiseli ze-Caco-2 kwi-membrane ye-PDMS. Nangona kunjalo, inkcubeko ye-microfluidic ye-epithelium ye-organoid ifuna ukusebenza komphezulu okusekwe kwiikhemikhali ukuze kufezekiswe ukufakwa ngokufanelekileyo kweeproteni ze-ECM ngokusebenzisa i-polyethyleneimine (PEI) kunye ne-glutaraldehyde ngokulandelelana kwiitshaneli ezincinci ze-PDMS. Emva kokuguqulwa komphezulu, iiproteni ze-ECM zafakwa ukuze zigqume umphezulu we-PDMS osebenzayo zaza zangeniswa kwi-epithelium ye-organoid ehlukanisiweyo. Emva kokuba iiseli ziqhotyoshelwe, inkcubeko yeseli ye-microfluidic iqala ngokufaka kuphela i-medium kwi-microchannel ephezulu de iiseli zenze i-monolayer epheleleyo, ngelixa i-microchannel esezantsi igcina iimeko ezizinzileyo. Le ndlela ilungiselelwe ukusebenza komphezulu kunye nogqubuthelo lwe-ECM ivumela ukunamatheliswa kwe-organoid. i-epithelium ukuze ibangele i-3D morphogenesis kumphezulu we-PDMS.
Ii-Transwell cultures nazo zifuna i-ECM coating ngaphambi kokuba kuhlwayelwe iiseli; nangona kunjalo, ii-Transwell cultures azidingi manyathelo anzima onyango ukuze kusebenze umphezulu wee-inserts ezineembobo. Ukuze kukhuliswe iiseli zeCaco-2 kwii-inserts zeTranswell, i-ECM coating kwii-inserts ezineembobo ikhawulezisa ukunamathela kweeseli zeCaco-2 ezidityanisiweyo (
Iimpawu ze-morphological ze-microengineered 3D epithelial layers zinokuhlalutywa ngokusebenzisa iindlela ezahlukeneyo zokujonga imifanekiso, kubandakanya i-phase contrast microscopy, i-differential interference contrast (DIC) microscopy, i-SEM, okanye i-immunofluorescence confocal microscopy (Imifanekiso 3 kunye ne-4). I-Phase contrast okanye i-DIC imaging inokwenziwa ngokulula nangaliphi na ixesha ngexesha lokulima ukujonga imo kunye nokuvela kwe-3D epithelial layers. Ngenxa yokukhanya okubonakalayo kwe-PDMS kunye neefilimu ze-polyester, zombini iiplatifomu ze-gut-on-a-chip kunye ne-hybrid chip zinokubonelela nge-real-time in situ imaging ngaphandle kwesidingo sokuhlukanisa okanye ukukhupha isixhobo. Xa senza i-immunofluorescence imaging (Imifanekiso 1, 3c, f kunye ne-4b, c), iiseli zihlala zilungisiwe nge-4% (wt/vol) paraformaldehyde (PFA), ilandelwa yiTriton X-100 kunye ne-2% (wt/vol) ) bovine serum albumin (BSA), ngokulandelelana. Kuxhomekeke kuhlobo lweseli, izinto ezahlukeneyo zokulungisa, Ii-permeabilizers, kunye nee-blocking agents zingasetyenziswa. Ii-antibodies eziphambili ezijolise kumgca-siseko weseli okanye iimpawu zengingqi zisetyenziselwa ukuqaqambisa iiseli ezinganyakaziyo kwindawo ethile kwi-chip, zilandelwa zii-antibodies zesibini kunye nedayi ye-counterstain ejolise kwi-nucleus (umz., 4′, 6-diamidino-2-phenylene) indole, DAPI) okanye i-F-actin (umz., i-phalloidin ebhalwe nge-fluorescently). I-Fluorescence-based live imaging inokwenziwa kwindawo ethile ukuze kufunyanwe imveliso ye-mucus (Umzobo 1, “Ukwahluka kweeseli” kunye “ne-Gut physiology”), i-random colonization yeeseli ze-microbial (Umzobo 1, “I-Host-microbe co-culture”), ukufunyanwa kweeseli zomzimba (Umzobo 1, ‘Imodeli yeZifo’) okanye i-contours ye-3D epithelial morphology (Umzobo 3c, f kunye no-4b, c). Xa uguqula isisu kwi-chip ukuze wahlukanise umaleko ongaphezulu kumaleko osezantsi we-microchannel, njengoko kuchaziwe kwi-ref. Njengoko kubonisiwe kwi-Fig. 2, i-3D epithelial imofoloji kunye ne-microvilli kumda webhrashi ye-apical inokubonwa yi-SEM (Umzobo 3b). Ukubonakaliswa kweempawu zokwahluka kunokuvavanywa ngokwenza i-quantitative PCR5 okanye i-single-cell RNA sequencing. Kule meko, iileya ze-3D zeeseli ze-epithelial ezikhuliswe kwiitships zamathumbu okanye kwiitships ze-hybrid zivunwa nge-trypsinization kwaye emva koko zisetyenziselwa uhlalutyo lweemolekyuli okanye lwemfuza.
a, Ukuhamba komsebenzi wokungenisa i-intestinal morphogenesis kwi-hybrid chip. I-Caco-2 kunye ne-intestinal organoids zisetyenziswa kule protocol ukubonisa i-3D morphogenesis kwiqonga le-hybrid chip. Iiseli ze-epithelial ezihlukanisiweyo zatyalwa kwii-Transwell inserts ezilungisiweyo (i-TW prep; jonga umfanekiso ongezantsi). Nje ukuba iiseli zityalwe (zityalwe) kwaye zinamathele kwiimembrane ze-polyester kwi-Transwell inserts, zonke iiseli zatyalwa phantsi kweemeko ezimileyo (inkcubeko ye-TW). Emva kweentsuku ezi-7, i-Transwell insert enye equlethe i-2D monolayer yeeseli ze-epithelial yadityaniswa kwi-hybrid chip ukuze ingenise i-basolateral flow (Flow, BL), ekugqibeleni ekhokelele ekudalweni kwe-3D epithelial layer (morphogenesis). Ii-phase contrast micrographs ezibonisa iimpawu ze-morphological zeeseli ze-epithelial zomzimba womntu ezithathwe kwi-normal donor (C103 line) ascending colon kwinyathelo ngalinye lovavanyo okanye ixesha. Iischematics kwiileya eziphezulu zibonisa uqwalaselo lovavanyo lwenyathelo ngalinye.b, ii-Hybrid chips (left schematic) zinokukhokelela kwi-3D morphogenesis ye-organoid epithelial iiseli ezineembono ze-confocal microscopy ezithathwe kwiindawo ezahlukeneyo ze-Z (eziphezulu, eziphakathi, nezisezantsi; jonga imigca ye-schematic esekunene kunye nemigca ehambelanayo enamachaphaza). zibonise iimpawu ezibonakalayo ze-morphological. I-F-actin (i-cyan), i-nucleus (grey).c, ii-Fluorescence confocal micrographs (umbono we-3D angled) zeeseli ze-epithelial ezivela kwi-organoid ezikhuliswe kwi-static Transwell (TW; i-inset ngaphakathi kwebhokisi emhlophe enemigca) ngokuchasene ne-hybrid chip (i-full shot enkulu) ethelekisa i-2D ngokuchasene ne-3D morphology, ngokulandelelana. Isibini seembono ze-2D ezithe nkqo ezinqamlezileyo (i-inset kwikona ephezulu ngasekunene; "XZ") nazo zibonisa iimpawu ze-2D kunye ne-3D. Ibha yesikali, i-100 µm.c Ishicilelwe kwakhona ngemvume evela kwisalathiso.4. Elsevier.
Ulawulo lunokulungiswa ngokuhluza iiseli ezifanayo (iiseli ze-Caco-2 okanye iiseli ze-epithelial ze-organoid zamathumbu) zibe zii-monolayers ezimbini phantsi kweemeko zenkcubeko eqhelekileyo ye-static. Okuphawulekayo kukuba, ukuphelelwa zizondlo kunokubangelwa kumthamo omncinci we-microchannels (oko kukuthi ~4 µL kumjelo ophezulu kuyilo lokuqala lwe-gut-chip). Ke ngoko, i-epithelial morphology ngaphambi nasemva kokusetyenziswa kwe-basolateral flow nayo inokuthelekiswa.
Inkqubo ye-lithography ethambileyo kufuneka yenziwe kwigumbi elicocekileyo. Kwimaleko nganye kwi-chip (iileya eziphezulu nezisezantsi kunye nee-membranes) kunye nee-hybrid chips, kusetyenziswe ii-photomasks ezahlukeneyo kwaye zenziwe kwii-silicon wafers ezahlukeneyo kuba ukuphakama kwee-microchannels kwahlukile. Ukuphakama okujoliswe kuko kwee-microchannels eziphezulu nezisezantsi ze-gut kwi-chip yi-500 µm kunye ne-200 µm, ngokwahlukeneyo. Ukuphakama okujoliswe kuko kwe-channel chip ye-hybrid yi-200 µm.
Beka i-wafer ye-silicon eyi-intshi ezi-3 kwisitya esine-acetone. Jikelezisa ipleyiti kancinci imizuzwana engama-30, uze uyomise ngomoya i-wafer. Dlulisa i-wafer kwipleyiti ene-IPA, uze ujike ipleyiti imizuzwana engama-30 ukuze icoceke.
Isisombululo sepiranha (umxube wehydrogen peroxide kunye ne-concentrated sulfuric acid, 1:3 (vol/vol)) singasetyenziswa ngokuzithandela ukususa iintsalela zezinto eziphilayo kumphezulu we-silicon wafer.
Isisombululo sePiranha siyatsha kakhulu kwaye sivelisa ubushushu. Kufuneka amanyathelo okhuseleko ongezelelweyo. Ukuze kulahlwe inkunkuma, vumela isisombululo siphole kwaye usidlulisele kwisitya senkunkuma esicocekileyo nesomileyo. Sebenzisa izikhongozeli zesibini kwaye ubhale iilebhile ezifanelekileyo kwizikhongozeli zenkunkuma. Nceda ulandele izikhokelo zokhuseleko zesikhungo ukuze ufumane iinkqubo ezineenkcukacha ezithe vetshe.
Nciphisa amanzi kwiiwafers ngokuzibeka kwipleyiti eshushu engama-200 °C imizuzu eli-10. Emva kokuphelelwa ngamanzi emzimbeni, iwafer yashukunyiswa kahlanu emoyeni ukuze iphole.
Galela i-10 g ye-photoresist SU-8 2100 embindini we-silicon wafer ecociweyo. Sebenzisa ii-tweezers ukusasaza i-photoresist ngokulinganayo kwi-wafer. Ngamanye amaxesha beka i-wafer kwi-hot plate engama-65°C ukuze i-photoresist inganamatheli kakhulu kwaye kube lula ukuyisasaza. Musa ukuyibeka ngqo kwi-hot plate.
I-SU-8 yasasazwa ngokulinganayo kwi-wafer ngokusebenzisa i-spin coating. Cwangcisa ujikelezo olungenayo lwe-SU-8 imizuzwana emi-5-10 ukuze isasazeke kwi-500 rpm ngesantya se-100 rpm/s. Misela i-main spin ukuze i-200 µm ubukhulu bube yi-1,500 rpm, okanye ufikelele kubukhulu be-250 µm (ukwenza ukuphakama kwe-500 µm kumaleko ongaphezulu wesisu kwi-chip; jonga "Amanyathelo abalulekileyo" ngezantsi) Misela isantya se-300 rpm/s imizuzwana engama-30 kwi-1,200 rpm.
Isantya esiphambili sokujikeleza singalungiswa ngokwesilinganiselo sobukhulu bephethini ye-SU-8 kwi-silicon wafer.
Ukuze kwenziwe iipateni ze-SU-8 zokuphakama kwe-500 µm kumaleko ongaphezulu wesisu kwi-chip, i-spin coating kunye namanyathelo okubhaka athambileyo ale Bhokisi (amanyathelo 7 kunye no-8) aphindaphindwe ngokulandelelana (jonga inyathelo 9) ukuvelisa amaleko amabini e-250 µm Umaleko otyebileyo we-SU-8, onokubekwa kwaye udityaniswe kukuvezwa kwe-UV kwinyathelo 12 lale bhokisi, okwenza umaleko ube yi-500 µm ukuphakama.
Bhaka ii-wafers ezigqunywe yi-SU-8 ngononophelo ngokuzibeka ngononophelo kwipleyiti eshushu kwi-65 °C imizuzu emi-5, uze utshintshele kwi-95 °C uze uzifumise imizuzu engama-40 eyongezelelweyo.
Ukuze ufumane ubude obuyi-500 μm bephethini ye-SU-8 kwi-microchannel ephezulu, phinda amanyathelo 7 kunye no-8 ukuze uvelise iileya ezimbini ze-SU-8 ezinobukhulu obuyi-250 μm.
Usebenzisa i-UV Mask Aligner, yenza uvavanyo lwesibane ngokwemiyalelo yomenzi ukuze ubale ixesha lokuvezwa kwe-wafer. (ixesha lokuvezwa, ms) = (idosi yokuvezwa, mJ/cm2)/(amandla esibane, mW/cm2).
Emva kokugqiba ixesha lokuvezwa, beka i-photomask kwisibambi semaski se-UV mask aligner uze ubeke i-photomask kwi-wafer egqunywe yi-SU-8.
Beka umphezulu oprintiweyo we-photomask ngqo kwicala eligqunywe yi-SU-8 le-silicon wafer ukunciphisa ukusasazeka kwe-UV.
Veza i-wafer egqunywe yi-SU-8 kunye ne-photomask ngokuthe nkqo ukuya kwi-260 mJ/cm2 yokukhanya kwe-UV ngexesha elimiselweyo lokuvezwa (jonga inyathelo le-10 lale bhokisi).
Emva kokuvezwa yi-UV, ii-wafers ze-silicon ezigqunywe yi-SU-8 zibhakwe kwi-65°C imizuzu emi-5 kunye ne-95°C imizuzu eli-15 kwisitya ngasinye esishushu ukuze kwenziwe iipateni ezinokuphakama kwe-200 μm. Yandisa ixesha emva kokubhaka kwi-95°C ukuya kwi-30 imizuzu ukuze kwenziwe iipateni ezinokuphakama kwe-500 µm.
Umphuhlisi ugalelwa kwisitya seglasi, kwaye i-wafer ebhakiweyo ifakwa kwisitya. Umthamo womphuhlisi we-SU-8 unokwahluka ngokuxhomekeke kubukhulu bepleyiti yeglasi. Qinisekisa ukuba usebenzisa umphuhlisi we-SU-8 owaneleyo ukususa ngokupheleleyo i-SU-8 engatyhilekanga. Umzekelo, xa usebenzisa isitya seglasi esinobubanzi be-150 mm esinomthamo we-1 L, sebenzisa ~300 mL yomphuhlisi we-SU-8. Phuhlisa isikhunta imizuzu engama-25 ngokujikeleza kancinci ngamanye amaxesha.
Hlamba isikhunta esiphuhlisiweyo nge-~10 mL yomphuhlisi omtsha uze ulandelise nge-IPA ngokufafaza isisombululo usebenzisa i-pipette.
Beka i-wafer kwisicoci se-plasma uze uyibeke kwi-oxygen plasma (igesi yomoya-bume, uxinzelelo olujoliswe kuyo 1 × 10−5 Torr, amandla 125 W) imizuzu eli-1.5.
Beka i-wafer kwi-vacuum desiccator ene-glass slide ngaphakathi. Ii-wafer kunye nee-slides zingabekwa ecaleni kwelinye. Ukuba i-vacuum desiccator yahlulwe yaba ziileya ezininzi ngepleyiti, beka ii-slides kwigumbi elisezantsi kunye nee-wafers kwigumbi eliphezulu. Lahla i-100 μL yesisombululo se-trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane kwi-glass slide uze ufake i-vacuum ukuze i-silanization ihlale.
Nyibilikisa ibhotile yeeseli zeCaco-2 ezikhenkcezisiweyo kwindawo yokuhlambela ngamanzi engama-37°C, uze udlulisele iiseli ezinyibilikisiweyo kwiflaski yeT75 equlethe i-15 mL ye-37°C eshushu ngaphambili yeCaco-2.
Ukuze kudlule iiseli zeCaco-2 xa zidibana nge-90%, kuqala kufuneka kufakwe i-Caco-2 medium efudumeleyo, i-PBS, kunye ne-0.25% trypsin/1 mM EDTA kwindawo yokuhlambela ngamanzi engama-37°C.
Hlamba iiseli kabini nge-5 mL ye-PBS efudumeleyo ngokuphinda uphefumle nge-vacuum uze wongeze i-PBS entsha.
Ixesha lokuthumela: Julayi-16-2022
