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I-Human gut morphogenesis isungula izici ze-crypt-villus ze-3D epithelial microarchitecture kanye nenhlangano yendawo.Lesi sakhiwo esiyingqayizivele siyadingeka ukuze kugcinwe i-homeostasis yamathumbu ngokuvikela i-stem cell niche ku-basal crypt kuma-antigen angaphandle kwama-microbial kanye nama-metabolites awo. Ngakho-ke, ukwakhiwa kabusha kwezakhiwo ze-3D epithelial kubalulekile ekwakhiweni kwamamodeli e-in vitro gut. Ngokuphawulekayo, i-organic mimetic gut-on-a-chip ingabangela okuzenzakalelayo kwe-3D morphogenesis ye-epithelium yamathumbu ngokusebenza okuthuthukisiwe kokusebenza komzimba kanye ne-biomechanics ye-biomechanics ehlinzeka ngokuphindaphinda. i-morphogenesis emathunjini ku-microfluidic chip kanye nakwi-Transwell embedded hybrid chip. ye-microarchitecture yamathumbu esebenzayo ngokulawula ukugeleza kwe-basolateral fluid ye-5 d.Indlela yethu ye-in vitro morphogenesis isebenzisa ukucindezelwa kwe-shear okuhlobene ngokomzimba kanye nokunyakaza komshini futhi ayidingi ubunjiniyela obuyinkimbinkimbi beseli noma ukukhwabanisa, okungase kube ngaphezu kwamanye amasu akhona. ukusetshenziswa kwe-biomedical, umtholampilo, kanye nemithi.
Ukuhlola kubonisa ukuthi amangqamuzana e-Caco-2 e-epithelial emathunjini akhuliswe ku-gut-on-a-chip1,2,3,4,5 noma i-bilayer microfluidic device6,7 angakwazi ukungena ngokuzenzakalelayo kwe-3D morphogenesis in vitro ngaphandle kokuqonda okucacile kwendlela eyisisekelo. i-epithelial morphogenesis in vitro, eye yaboniswa yi-Caco-2 kanye nama-organoids amathumbu atholakala esigulini. Amaseli e-Epithelial aqinisekisiwe.Kulolu cwaningo, sigxile ngokukhethekile ekukhiqizweni kweseli kanye nokusatshalaliswa okugxilile komphikisi we-Wnt onamandla, u-Dickkopf-1 (DKK-1), ku-gut-on-a-chip kanye namadivayisi amancane aguquliwe aqukethe okufakwayo kwe-Transwell, abizwa ngokuthi "I-Hybrid Chip". iphrotheni 1 ehlobene ne-frizzled ehlobene ne-frizzled, noma i-Soggy-1) ku-chip gut ivimbela i-morphogenesis noma iphazamisa ungqimba olwakhiwe ngaphambili lwe-epithelial ye-3D, okusikisela ukuthi ukucindezeleka okuphikisayo phakathi nesiko kunesibopho se-intestinal morphogenesis in vitro.Ngakho-ke, indlela engokoqobo yokufinyelela amazinga aqinile we-mophogenesis e-epithelial ukususa i-epithelial interface eqinile noma ukususwa kwe-epithelial encane ku-epithelial interface. endaweni engaphansi ngokuswayipha okusebenzayo (isb., kumapulatifomu e-gut-on-a-chip noma i-hybrid-on-a-chip platforms) noma ukusabalalisa .Imidiya ye-Basolateral (isb, isuka e-Transwell ifaka kumachibi amakhulu angaphansi kwemithombo emithonjeni).
Kule phrothokholi, sihlinzeka ngendlela enemininingwane yokwenza ama-microdevices amathumbu-on-a-chip kanye nama-Transwell-insertable hybrid chips (izinyathelo 1-5) ukuze kuthuthukiswe amaseli e-epithelial amathumbu ku-polydimethylsiloxane (PDMS)-based based membranes (izinyathelo 6A, 7A, 8, 9) noma ulwelwesi lwe-TranswellB, i-7 ye-polyester B, i-8 I-9) futhi ibangele i-3D morphogenesis in vitro (isinyathelo 10) .Siphinde sahlonza izici zamangqamuzana namangqamuzana ezibonisa i-histogenesis ethize yezicubu kanye nokuhlukaniswa kwamaselula okuncike emgqeni ngokusebenzisa izindlela eziningi ze-imaging (izinyathelo ze-11-24) .Senza i-morphogenesis sisebenzisa i-human intestinal epithelism, i-intestinal organo amaseli we-intestinal organo, ama-intestinal organo-2 amaseli we-intestinal. ngemininingwane yezobuchwepheshe okuhlanganisa ukuguqulwa kwendawo yezingqimba ezimbotshana, ukudalwa kwama-monolayers e-2D, kanye ne-biochemical yamathumbu kanye Nokukhiqizwa kabusha kwe-biomechanical microenvironment.in vitro.Ukuze senze i-3D morphogenesis kusukela ku-2D epithelial monolayers, sisuse abaphikisi be-morphogen kuzo zombili izinhlobo ze-culture ngokugeleza okuphakathi ku-accessories ye-basolateral inikezela indawo yokumelwa kwe-basolateral. i-3D epithelial layer evuselelekayo engasetshenziswa ukwenza imodeli yokukhula kwe-epithelial encike ku-morphogen, i-longitudinal host-microbiome co-cultures, ukutheleleka kwe-pathogen, ukulimala okuvuthayo, ukungasebenzi kahle kwe-epithelial barrier, kanye nemithi yokwelapha esekelwe kuma-probiotic Isibonelo.amathonya.
Iphrothokholi yethu ingase ibe wusizo kuhlu olubanzi lososayensi kusisekelo (isb., i-biology ye-mucosal yamathumbu, i-stem cell biology, ne-developmental biology) kanye nocwaningo olusetshenzisiwe (isb., ukuhlolwa kwezidakamizwa kusengaphambili, ukufanisa izifo, ubunjiniyela bezicubu, kanye ne-gastroenterology) umthelela obanzi.Ngenxa yokuphindaphindeka nokuqina kwephrothokholi yethu ukuze singenise i-3D introphogenesis ye-entrophogenesis yethu isu lobuchwepheshe lingasatshalaliswa ezithamelini ezifunda ukuguquguquka kokusayina kweseli ngesikhathi sokuthuthukiswa kwamathumbu, ukuvuselelwa noma i-homeostasis .Ngaphezu kwalokho, isimiso sethu somthetho siwusizo ekuphenyweni ukutheleleka ngaphansi kwamagciwane ahlukahlukene athathelwanayo afana ne-Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), i-Clostridium diffilera, i-Tymulera Salmonephilla, i-Tymulera 9 noma i-Virgilia. Izethameli ze-pathology yesifo kanye ne-pathogenesis nazo ziwusizo.Ukusetshenziswa kwe-on-chip gut microphysiology system kungase kuvumele i-longitudinal co-culture 10 kanye nokuhlolwa okulandelayo kokuzivikela komninikhaya, izimpendulo zokuzivikela komzimba kanye nokulungiswa kokulimala okuhlobene ne-pathogen ku-gastrointestinal (GI) ipheshana 11 .Ezinye izifo ze-GIceky aguquguqukayo, isifo se-Groaceky, isifo se-Groaceky, isifo se-Groaceky i-ulcerative colitis, i-pouchitis, noma i-irritable bowel syndrome ingenziwa lapho kulungiswa izingqimba ze-3D ze-epithelial zamathumbu kusetshenziswa izingqimba ze-epithelial zamathumbu zesiguli ze-3D, lezi zifo zihlanganisa i-villous atrophy, shortening crypt, umonakalo we-mucosal, noma ukukhubazeka kwe-epithelial barrier. ubunkimbinkimbi obuphezulu bendawo yesifo, abafundi bangase bacabangele ukwengeza izinhlobo zamangqamuzana ahambisana nesifo, njengamaseli egazi esiguli se-peripheral mononuclear (PBMCs), kumamodeli aqukethe i-3D intestinal villus-crypt microarchitectures. amaseli omzimba aqondene nezicubu, 5.
Njengoba i-3D epithelial microstructure ingalungiswa futhi ibonakale ngaphandle kwenqubo yokuhlukanisa, izibukeli ezisebenza nge-spatial transcriptomics kanye ne-high-resolution noma i-super-resolution imaging ingase ibe nentshisekelo ekwenziweni kwethu kwemephu ye-spatiotemporal dynamics yezakhi zofuzo namaprotheni kuma-epithelial niches. Unentshisekelo kwezobuchwepheshe.Ukusabela ku-microbial noma i-immune stimuli.Ngaphezu kwalokho, i-longitudinal host-microbiome crosstalk 10, i-14 eqondisa i-gut homeostasis ingasungulwa kungqimba lwe-3D ye-intestinal mucosal ngokuhlanganisa izinhlobo ezihlukahlukene ze-microbial, imiphakathi ye-microbial noma i-fecal microbiota, ikakhulukazi ku-gut-gut. endaweni yesikhulumi.Le ndlela ikhanga ngokukhethekile ezithamelini ezifunda i-mucosal immunology, gastroenterology, human microbiome, culturomics kanye ne-clinical microbiology efuna ukuhlakulela i-gut microbiota eyayingakaze isetshenziswe endaweni yokucwaninga. gcwalisa ama-basolateral compartments, iphrothokholi ingase futhi isatshalaliswe kulabo abathuthukisa ukuhlolwa kwezemithi, i-biomedical Noma ukuhlolwa okuphezulu noma ukuqinisekiswa kwezinkundla zemboni yezokudla.Njengobufakazi bomgomo, muva nje sibonise ukuthi kungenzeka kwe-multiplex high-throughput addition morphogenesis system scalable to a chip-plate-multiple products have been 24-organ-multiple. Ngakho-ke, ukuqinisekiswa kwendlela yethu ye-in vitro morphogenesis kungasheshiswa futhi okungenzeka kwamukelwe amalabhorethri amaningi ocwaningo, imboni noma uhulumeni kanye nama-ejensi alawulayo ukuze aqonde ukuhlelwa kabusha kwamaselula we-in vitro gut morphogenesis ezingeni le-transcriptomic ukuhlola izidakamizwa noma i-biotherapeutics Ukumuncwa kanye nokuthutha izidakamizwa kusetshenziswa i-gut 3D noma ukuthuthwa kwezidakamizwa kusetshenziswa izidakamizwa. amamodeli we-organ-on-a-chip wokuthengisa ukuze ahlole ukukhiqizwa kabusha kwenqubo ye-gut morphogenesis.
Inani elilinganiselwe lamamodeli okuhlola ahlobene nomuntu liye lasetshenziswa ukutadisha i-epithelial morphogenesis yamathumbu, ikakhulukazi ngenxa yokuntuleka kwezimiso ezilandelwayo zokufaka i-3D morphogenesis in vitro.Eqinisweni, ulwazi oluningi lwamanje mayelana ne-gut morphogenesis lusekelwe ezifundweni zezilwane (isib. okungabazekayo, futhi okubaluleke kakhulu, akunqumi ngokuqondile izinqubo zokuthuthuka komuntu.Lawa mamodeli alinganiselwe kakhulu ekhonweni lawo lokuhlolwa ngezindlela eziningi ezingakaleka.Ngakho-ke, inqubo yethu yokuvuselela ukwakheka kwezicubu ze-3D in vitro ihamba kahle kakhulu kumamodeli ezilwane we-vivo kanye namanye amamodeli amasiko e-2D amile wendabuko.Njengoba kuchaziwe ngaphambili, kwavunyelwa ukusebenzisa isakhiwo sendawo se-3D esihlukile. amangqamuzana e-crypt-villus axis ekuphenduleni i-mucosal ehlukahlukene noma i-immune stimuli.Izingqimba ze-epithelial ze-3D zinganikeza isikhala sokufunda ukuthi amangqamuzana amancane ancintisana kanjani ukuze akhe ama-spatial niches kanye nokuziphendukela kwemvelo ekuphenduleni izici zokubamba (isb, izingqimba ze-mucus zangaphakathi ngokumelene nezangaphandle, ukukhishwa kwe-IgA kanye ne-antimicrobial peptides, i-usermorphology ingakwazi ukuqonda i-epimoretheli ye-3D). i-gut microbiota yakha imiphakathi yayo futhi ikhiqiza ngokuhlanganyela i-microbial metabolites (isb, i-short-chain fatty acids) eyenza inhlangano yamaselula kanye ne-stem cell niches kuma-basal crypts.Lezi zici zingaboniswa kuphela lapho izingqimba ze-epithelial ze-3D zisungulwa ku-vitro.
Ngaphandle kwendlela yethu yokudala izakhiwo ze-epithelial zamathumbu e-3D, kunezindlela eziningana ze-in vitro.Isiko le-organoid yamathumbu iyindlela yesimanje yobunjiniyela bezicubu ezisekelwe ekulinyweni kwamangqamuzana e-intestinal stem ngaphansi kwezimo ezithile ze-morphogen23,24,25.Nokho, ukusetshenziswa kwe-3D organoid models for transport analysis of the constination-microculture intestinal organollenture for transportation of the constination-microculture organoid i-lumen ivalwe ngaphakathi kwe-organoid futhi, ngakho-ke, ukwethulwa kwezingxenye ezikhanyayo ezifana namaseli e-microbial noma ama-antigen angaphandle kunqunyelwe. Ukufinyelela ku-lumens ye-organoid kungathuthukiswa kusetshenziswa i-microinjector, i-26,27 kodwa le ndlela ihlasela futhi idinga abasebenzi futhi idinga ulwazi olukhethekile ukuze yenze.Ngaphezu kwalokho, amasiko endabuko e-organoid agcinwe kuma-scaffold e-hydrogel ngaphansi kwezimo ezimile awabonisi ngokunembile ukusebenza kwe-vivo biomechanics.
Ezinye izindlela ezisetshenziswa amaqembu amaningana ocwaningo zisebenzisa izikafula ezakhiwe kusengaphambili ze-3D hydrogel ukulingisa ukwakheka kwe-epithelial yamathumbu ngokuhlakulela amangqamuzana ahlukene amathumbu omuntu endaweni yejeli.Yenza izikafula ze-hydrogel usebenzisa i-3D-printed, micro-milled, noma lithographically fabricated molds of mold-self-epidthethographical epid methodi-epid ibonisa ukuhlelwa kwesikhunta esizimele. ama-morphogen gradients afanele ngokwempilo, asungula i-aspect ratio ephezulu yesakhiwo se-epithelial kanye ne-stroma-epithelial crosstalk ngokufaka amaseli e-stromal ku-scaffold. Nokho, imvelo yezikafula ezakhiwe ngaphambili ingase ivimbele ukuboniswa kwenqubo ezenzakalelayo ye-morphogenetic ngokwayo. i-morphogenesis futhi izuze umsebenzi wokuphila.Olunye ucwaningo lwakamuva lusebenzise izikafula ze-hydrogel endaweni yesikhulumi se-microfluidic kanye nezakhiwo ze-epithelial zamathumbu ezinephethini zisebenzisa amasu okukhishwa kwe-laser.Ama-organoid amathumbu egundane alandela amaphethini amisiwe ukuze akhe izakhiwo ze-tubular zamathumbu, futhi ukugeleza koketshezi lwe-intraluminal kungenziwa kabusha le modeli ye-modules ye-microfluidics. futhi akubandakanyi ukunyakaza kwe-gut mechanobiological.Izindlela zokunyathelisa ze-3D ezivela eqenjini elifanayo zakwazi ukudala amashubhu amancane amathumbu anezinqubo ezizenzakalelayo ze-morphogenetic.Naphezu kokwakhiwa okuyinkimbinkimbi kwezingxenye ze-gut ezihlukene ngaphakathi kwepayipi, lo modeli futhi awunakho ukugeleza koketshezi olukhanyayo kanye nokuguqulwa kwemishini.Ukwengeza, ukusebenza kwemodeli kungase kukhawulwe, ikakhulukazi ngemva kokuhlolwa kwe-bioprinting-cell, ukuhlolwa kweseli yethu ye-bioprinting-steading kuqedile. Iphrothokholi ehlongozwayo ihlinzeka ngokuzenzakalelayo i-gut morphogenesis, ukucindezelwa kwe-shear okuhlobene ngokomzimba, i-biomechanics elingisa ukuhamba kwamathumbu, ukufinyeleleka kwezingxenye ezizimele ze-apical kanye ne-basolateral, nokudala kabusha kwe-biological microenvironments eyinkimbinkimbi ye-modularity.Ngakho-ke, iphrothokholi yethu ye-in vitro 3D morphogenesis ingase inikeze indlela ehambisanayo yokunqoba izinselele ezikhona.
Iphrothokholi yethu igxile ngokuphelele ku-3D epithelial morphogenesis, enamaseli e-epithelial kuphela esikweni futhi azikho ezinye izinhlobo zamaseli azungezile njengamaseli e-mesenchymal, amangqamuzana e-endothelial, namangqamuzana omzimba omzimba.Njengoba kuchaziwe ngaphambili, umnyombo wephrothokholi yethu ukufakwa kwe-epithelial morphogenesis ngokususa i-morphogen inhibitors ekhishwe kuhlangothi lwethu lwe-robustw olwethulwe ku-robust medium yethulwe. i-gut-on-a-chip kanye ne-hybrid-on-a-chip isivumela ukuthi sidale kabusha ungqimba lwe-epithelial lwe-3D olungaguquki, izinto eziyinkimbinkimbi zebhayoloji ezifana nokusebenzisana kwe-epithelial-mesenchymal33,34, i-extracellular Matrix (ECM) deposition 35 futhi, kumodeli yethu, izici ze-crypt-villus ezidlulisa amaseli e-stem cell acatshangelwa ukuthi amaseli e-crypt fibroblasts) ku-mesenchyme idlala indima ebalulekile ekukhiqizeni amaprotheni e-ECM kanye nokulawulwa kwe-intestinal morphogenesis ku-vivo35,37,38.Ukwengezwa kwamangqamuzana e-mesenchymal kumodeli wethu kuthuthukise inqubo ye-morphogenetic kanye nokusebenza kahle kokunamathiselwe kweseli.I-endothelial layer (okungukuthi, ama-capillaries noma i-lymphatics) idlala indima ebalulekile ye-immune ye-recruit ku-cell recru3 ekuthuthweni kwe-molecule ye-recru3 ye-recrute i-microenvironment.Ngaphezu kwalokho, izingxenye ze-vasculature ezingaxhunywa phakathi kwamamodeli ezicubu ziyisidingo lapho amamodeli ezicubu eklanyelwe ukubonisa ukusebenzisana kwezitho eziningi.Ngakho-ke, amangqamuzana e-endothelial angase adinge ukufakwa ukuze afanekise izici ezinembile zomzimba ngokulungiswa kwezinga lesitho.Amaseli omzimba atholakala esigulini nawo abalulekile ekuboniseni impendulo ye-immune ye-immune, i-adapnate ye-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-antigen, i-anti-immune, i-antigen, i-antigen, i-anti-immune, i-immune kanye ne-anti-immune. ukungatheleleki kumongo wokulingisa isifo samathumbu.
Ukusetshenziswa kwama-chips ayi-hybrid kuqonde kakhulu kune-gut-on-a-chip ngoba ukusethwa kwedivayisi kulula futhi ukusetshenziswa kwe-Transwell inserts kuvumela isiko eliyingozi le-gut epithelium. Nokho, ukufakwa kwe-Transwell okutholakala ngokwentengiso okunolwelwesi lwe-polyester akunwebekanga futhi akukwazi ukulingisa ukunyakaza okufana ne-peristaltic.Ngaphezu kwalokho, i-apical chip chip ibekwe endaweni ene-stress compartment esiteshini se-apical chip efakwe endaweni yokucindezeleka I-apical side.Ngokusobala, izakhiwo ezimile endaweni ye-apical akuvamile ukuthi zivumele ukuhlangana kwebhaktheriya kwesikhathi eside kuma-chips ahlanganisiwe.Yize singakwazi ukufaka i-3D morphogenesis ekufakweni kwe-Transwell lapho kusetshenziswa ama-chips axubile, ukushoda kwe-biomechanics ehambisanayo ngokomzimba kanye nokugeleza koketshezi lwe-apical kungase kukhawulele ukuba nokwenzeka kweplathifomu ye-hybrid engaba khona.
Ukwakhiwa kabusha kwesilinganiso esigcwele se-axis ye-crypt-villus yomuntu kusiko lwe-gut-on-a-chip kanye ne-hybrid-on-a-chip akukakasungulwa ngokugcwele. Njengoba i-morphogenesis iqala ku-epithelial monolayer, ama-microarchitectures we-3D awanikezi ngempela ukufana kwe-morphological nama-crypts in vivo. I-epithelium ye-3D, i-crypt nezifunda ezimbi azinqunywanga ngokucacile. Nakuba amashaneli aphezulu aphezulu ku-chip eholela ekukhuphukeni kobude be-epithelium ene-microengineered, ukuphakama okuphezulu kusakhawulelwe ku-~300–400 µm. Ukujula kwangempela kokufihlwa kwamathumbu omuntu emathunjini amancane namakhulu, ~5mµmµmµ kanye no-05mµ ukujula kwangempela. futhi ukuphakama kwe-villi yamathumbu amancane ngu-~600 µm41.
Ngokombono wesithombe, i-in situ super-resolution imaging ye-3D microarchitectures ingase ikhawulelwe emathunjini ku-chip, njengoba ibanga elidingekayo lokusebenza ukusuka ku-lens yenhloso ukuya kungqimba lwe-epithelial lilandelana ngamamilimitha ambalwa. Ukuze unqobe le nkinga, kungase kudingeke umgomo oqhelile. Ngaphezu kwalokho, ukwenza izingxenye ezincane ze-specimen yokuqina kwesithombe ngenxa yokulungiswa okuphezulu kwe-imaging kuya ku-epithelial. PDMS.Ngaphezu kwalokho, njengoba i-microfabrication ye-layer-layer microfabrication ku-chip ihilela ukunamathela unomphela phakathi kongqimba ngalunye, kuyinselele kakhulu ukuvula noma ukususa ungqimba olungaphezulu ukuze uhlole ukwakheka okungaphezulu kwe-epithelial layer.Isibonelo, ngokusebenzisa isibonakhulu se-electron yokuskena (SEM).
I-hydrophobicity ye-PDMS ibe yisici esikhawulelayo ezifundweni ezisekelwe ku-microfluidic ezibhekene nama-molecule amancane e-hydrophobic, njengoba i-PDMS ingakwazi ukukhangisa ngokungaqondile ama-molecule anjalo e-hydrophobic.Izindlela ezihlukile ze-PDMS zingacatshangwa nezinye izinto ze-polymeric. Ngaphandle kwalokho, ukuguqulwa kwendawo ye-coating, i-polyphilic PDMS (isib. glycol) 43 ) ingabhekwa njengokunciphisa ukukhangisa kwama-molecule e-hydrophobic.
Okokugcina, indlela yethu ayizange ibonakale kahle mayelana nokuhlinzeka ngokuhlolwa kokusebenza okuphezulu noma inkundla yokuhlola esebenziseka kalula "ubukhulu obubodwa" 384-ukufakwa kahle kwe-porous okuvumela ukugcwaliswa okuqhubekayo nokususwa kwemidiya ye-basolateral).
Ukuze senze i-3D morphogenesis ye-epithelium yamathumbu omuntu ku-vitro, sisebenzise idivayisi yamathumbu e-microfluidic chip equkethe iziteshi ezincane ezimbili ezihambisanayo kanye nolwelwesi olunwebekayo lwezimbotshana phakathi ukuze sakhe isixhumi esibonakalayo se-lumen-capillary.Siphinde sibonisa ukusetshenziswa kwedivayisi ye-microfluidic yomzila owodwa (i-hybrid chip) ehlinzeka ngokuqhubekayo kwe-Translatelary layer ukufaka.Kuwo womabili amapulatifomu, i-morphogenesis yamangqamuzana e-epithelial amathumbu omuntu ahlukahlukene ingaboniswa ngokusebenzisa ukuguqulwa kokuqondisa kokugeleza ukuze kususwe abaphikisi be-morphogen endaweni ye-basolateral.Yonke inqubo yokuhlola (Umfanekiso 1) iqukethe izingxenye ezinhlanu: (i) i-microfabrication ye-gut chip noma i-Transwellable chip (i-Transwellable chip) i-Transwellable chip; amangqamuzana e-epithelial emathunjini (amaseli e-Caco-2) noma ama-organoid amathumbu omuntu; amabhokisi 2-5), (iii) isiko lamaseli e-epithelial wamathumbu kuma-chips wamathumbu noma ama-chips ayi-hybrid (izinyathelo 6-9), (iv) ukufakwa kwe-3D morphogenesis in vitro (isinyathelo 10) kanye (v) ) ukuze kubonakale ukwakheka kwe-3D epithelial microstructure (izinyathelo 11-24). in vitro morphogenesis ngokuqhathanisa i-epithelial morphogenesis nezilawuli zendawo, zesikhashana, ezinemibandela, noma zenqubo.
Sisebenzise izinkundla ezimbili ezihlukene zamasiko: i-gut-on-a-chip enamashaneli aqondile noma amashaneli ayi-convoluted angaqondile, noma ama-chips ayi-hybrid aqukethe okufakiwe kwe-Transwell (TW) kudivayisi encane ye-microfluidic, eyenziwe njengoba kuchazwe ku-Box 1, kanye nesinyathelo 1-5.”Ukwenziwa Kwedivayisi” kubonisa izinyathelo eziyinhloko ekwenzeni i-chip eyodwa noma i-chip hybrid.” (I-Caco-2 noma i-organoid yamathumbu omuntu) kanye nenqubo yesiko esetshenziswa kule nqubo.”I-In vitro morphogenesis” ikhombisa izinyathelo eziphelele lapho amaseli e-Caco-2 noma asuselwa ku-organoid e-epithelial ekhuliswa ku-chip yamathumbu noma ekufakweni kwe-Transwell kwe-chip ye-hybrid, okulandelwa ukwethulwa kwe-3D morphogenesis kanye nokwakheka kwenombolo ye-epithelial yenombolo ngezansi kwenombolo ye-epithelial ngezansi. Umcibisholo.Uhlelo lokusebenza luhlinzeka ngezibonelo zokuthi izendlalelo ze-epithelial zamathumbu ezimisiwe zingasetshenziswa kanjani, isibonelo, ekuhlukaniseni ukuhlukaniswa kwamaseli, izifundo ze-gut physiology, ukusungulwa kwe-host-microbiome ecosystems, kanye nokumodela kwezifo.Izithombe ze-Immunofluorescence "ku-Cell Differentiation" ezibonisa i-nuclei, i-F-actin kanye ne-3D Capilicoal eveza ungqimba lwe-gut 3D oluvezwe kungqimba lwe-gut-2 Isignali ye-chip.MUC2 ikhona kumaseli e-goblet kanye namafinyila akhiqizwe ebusweni be-mucosal.Izithombe ze-Fluorescent ku-Gut Physiology zibonisa amafinyila akhiqizwa ukungcoliswa kwe-sialic acid nezinsalela ze-N-acetylglucosamine kusetshenziswa igciwane le-fluorescent kakolweni i-agglutinin.Izithombe ezimbili ezigqagqene ku-"Host-Culture-Microbe emele i-Culture-microbe" ithumbu ku-chip.Iphaneli yesokunxele ikhombisa i-co-culture ye-E. coli eveza iphrotheni ye-fluorescent eluhlaza (GFP) enamaseli e-epithelial e-3D Caco-2 microengineered.Iphaneli elingakwesokudla libonisa ukwenziwa kwasendaweni kwe-GFP E. coli ehlanganiswe ne-3D Caco-2 epithelial cells, elandelwa yi-immunofluoresce ne-Fseinclease stain blue (Fseinle-blue). ukumodela kubonisa okunempilo ngokumelene nokuvuza kwamathumbu kuma-chips okuvuvukala amathumbu ngaphansi kwenselele ye-physiological antigens ebhaktheriya (isb, i-lipopolysaccharide, i-LPS) namaseli omzimba (isb., i-PBMC; eluhlaza). Amaseli e-Caco-2 akhuliswe ukuze kusungulwe isendlalelo se-epithelial ye-3D.Ibha yesikali, i-50 µsmage ye-adapta yamaseli aphansi we-Iffered kusuka kumaseli we-"Differ bottom". ireferensi.2. I-Oxford University Press; Ikhiqizwe kabusha ngemvume evela kuRef.5. I-NAS; "Host-Microbe Co-Culture" iguqulelwe ngemvume evela ku-ref.3. I-NAS; “Ukumodela Kwezifo” kuguqulelwe ngemvume evela kunkomba.5. I-NAS.
Kokubili ama-gut-on-chip kanye nama-hybrid chips akhiwe kusetshenziswa izifaniso ze-PDMS ezadilizwa kusukela ekubunjweni kwe-silicon nge-lithography ethambile1,44 futhi iphethini nge-SU-8. Umklamo wama-microchannel ku-chip ngayinye kunqunywa ngokucabangela i-hydrodynamics efana ne-shear stress kanye ne-hydrodynamic pressure1,4,12. ama-microchannels amabili aqondile afanayo ahlangene, aguqukele ku-gut-on-a-chip eyinkimbinkimbi (Idatha Enwetshiwe Fig. 1b) ehlanganisa i-microchannel egobile ukuze ibangele Ukwenyuka kwesikhathi sokuhlala kwamanzi, amaphethini okugeleza angenawo umugqa, kanye nokuguqulwa kwe-multiaxial kwamangqamuzana akhulisiwe (Fig. 2a-f) kudinga i-biocreated more complex, i-biochannel more complex, i-12. ama-gut-on-a-chips angakhethwa.Sibonise ukuthi i-convoluted Gut-Chip iphinde idonse ngokuqinile i-3D morphogenesis ngesikhathi esifanayo nesilinganiso esifanayo sokukhula kwe-epithelial uma kuqhathaniswa ne-Gut-Chip yasekuqaleni, kungakhathaliseki uhlobo lwamaseli akhulisiwe.Ngakho-ke, ukuze kufakwe i-3D morphogenesis, i-linear kanye ne-complex ye-silicon eguquguqukayo ku-chipPD idizayini ye-silicone eguquguqukayo. ngamaphethini e-SU-8 anikeze izici ezingezinhle ngemva kokudilizwa (Fig. 2a).Ukwenza ithumbu ku-chip, isendlalelo esiphezulu se-PDMS esilungisiwe sasiboshwe ngokulandelana kufilimu ye-PDMS enezimbotshana bese siqondaniswe nesendlalelo esiphansi se-PDMS ngokubopha okungenakuhlehliswa kusetshenziswa i-corona treater (Fig. 2b–f) Ukuze kwenziwe ama-chips ahlanganisiwe engilazi ukuze kuhlanganiswe ama-chips e-cuplired enziwe ngengilazi ye-PDMS ehlanganisiwe. amadivaysi e-microfluidic esiteshi esisodwa angamukela ukufakwa kwe-Transwell (Umfanekiso we-2h kanye Nomfanekiso Wedatha Enwetshiwe. 2). Inqubo yokuhlanganisa yenziwa ngokuphatha indawo engaphezulu ye-PDMS replica nengilazi nge-oxygen plasma noma ukwelashwa kwe-corona.Ngemva kokuvalwa komshini we-microfabricated exhunywe kushubhu ye-silicone, ukusetha idivayisi ye-3D e-epillite mortico ilungele ukwenza ukusethwa kwe-silicone (Umfanekiso 2g).
a, Umdwebo weSchematic wokulungiswa kwezingxenye ze-PDMS ezivela esikhunjeni se-silicon enephethini ye-SU-8.Isixazululo se-PDMS esinganqatshiwe sathelwa esikhunjeni se-silicon (kwesokunxele), selashwe ngo-60 °C (maphakathi) futhi sadilizwa (kwesokudla).I-PDMS ediliziwe yasikwa yaba yizicucu futhi yahlanzwa ukuze isetshenziswe ngokuqhubekayo.b, Isithombe se-silicon esetshenziselwa ungqimba lwe-silicon. isikhunta se-silicon esisetshenziselwa ukwakha ulwelwesi olunezimbotshana lwe-PDMS.d, Uchungechunge lwezithombe zezingxenye ze-PDMS ezingaphezulu neziphansi kanye nedivayisi yamathumbu ehlanganiswe ku-chip.e, Uhlelo lokuqondanisa kwezingxenye ze-PDMS ezingenhla, ulwelwesi, nezingezansi. ama-microchannels ahlanganisiwe kanye nama-vacuum chambers.g, Ukusethwa kwe-gut-on-a-chip ye-microfluidic cell culture.Amathumbu enziwe ku-chip ahlanganiswe neshubhu le-silicone nesipetu abekwe phezu kwe-coverlip.Idivayisi ye-chip yafakwa esivalweni sesitsha sePetri esingu-150 mm ukuze sicutshungulwe.I-chips hybrid silicone isetshenziselwa ukuvala i-silicone ye-silicone esetshenziselwa ukuvala. ukwakhiwa kanye ne-3D morphogenesis kusetshenziswa ama-chips ayi-hybrid.Okufakwayo kwe-Transwell okulungiselelwe ngokuzimele kumasiko ama-monolayers angu-2D amaseli e-epithelial amathumbu afakwe ku-chip ye-hybrid ukuze kufakwe i-3D morphogenesis yamathumbu.Okuphakathi kufakwe perfused ngokusebenzisa ama-microchannels ngaphansi kongqimba lweseli olusungulwe kusithenjwa se-Transwell.Ibha yesikali, iphrintwe kabusha ngo-4 cm. Elsevier.
Kule protocol, i-Caco-2 cell line kanye nama-organoids amathumbu asetshenziswa njengemithombo ye-epithelial (Fig. 3a) .Zombili izinhlobo zamaseli zakhiwe ngokuzimela (Ibhokisi 2 neBhokisi 5) futhi zisetshenziselwa imbewu ye-ECM-coated microchannels ye-on-chip gut noma i-Transwell inserts.Lapho amaseli ehlangene (> 95%) amaseli e-Cacoco (phakathi kwamavesi 10 no-50) kuma-T-flasks avunwa ukuze kulungiswe ukumiswa kwamaseli ahlukanisiwe nge-trypsinization fluid (ibhokisi 2). Ama-organoid amathumbu omuntu avela ku-intestinal biopsies noma ukukhishwa okuhlinzayo akhuliswe ezindlini ze-Matrigel scaffold kumapuleti angu-24-well ukuze asekele i-mossstrucment esemqoka (i-microenvironment). I-R-spondin, ne-Noggin) kanye nezici zokukhula ezilungiselelwe njengoba kuchazwe eBhokisini lesi-3 zenezelwa zonke izinsuku kuze kube yilapho ama-organoid ekhula aze afike ku-~500 µm ububanzi. Isifo sikaCrohn, umdlavuza wesikhumba, noma umnikeli ojwayelekile), indawo yesilonda (isb., isilonda uma kuqhathaniswa nendawo engenazilonda) kanye nendawo yokugaya emathunjini epheshaneni (isb., i-duodenum, ijejunum, ileum, i-cecum, ikholoni, noma irectum). ama-organoids emathunjini.
a, Ukugeleza komsebenzi wokufakwa kwe-gut morphogenesis ku-gut chip.I-Caco-2 i-epithelium yamathumbu omuntu kanye nama-organoid amathumbu asetshenziswa kule protocol ukuze abonise i-3D morphogenesis.Amangqamuzana e-epithelial ahlukanisiwe afakwe kumshini olungiselelwe we-gut-on-a-chip (ukulungiswa kwe-chipMS) .Uma amaseli e-seedased) anamathiselwe kwi-podtache (i-chipMS) enamathiselwe (i-membrane) ngosuku lwe-0 (D0), ukugeleza kwe-apical (AP) kuyaqalwa futhi kugcinwe izinsuku zokuqala ze-2 (ukugeleza, AP, D0-D2).Ukugeleza kwe-Basolateral (BL) nakho kuqalwa kanye nokunyakaza kwe-cyclic stretching (ukwelula, ukugeleza, i-AP ne-BL) lapho kwakhiwa i-monolayer ye-2D ephelele.I-Intestinal 3D ye-micromorphogenesis yezinsuku ze-5 yenzeka i-micromorphogenesis yezinsuku ze-5 D5).Izithombe ezihlukile zesigaba zibonisa i-morphology emele amaseli e-Caco-2 esinyathelweni ngasinye sokuhlola noma iphoyinti lesikhathi (igrafu yebha, 100 µm).Imidwebo emine eyisikimu ebonisa ukuqhuma okuhambisanayo kwe-gut morphogenesis (phezulu kwesokudla).Imicibisholo edayishiwe kuhlelo imelela isiqondiso sokugeleza koketshezi.b, SEM of the scematic surface ebonisa i-eft3D ephezulu. isethi egqamisa indawo ekhulisiwe (ibhokisi elinedeshi elimhlophe) sibonisa i-microvilli evuselelwe kusendlalelo se-3D Caco-2 (kwesokudla).c, Ukubuka okuvundlile okungaphambili kwe-Caco-2 3D emisiwe, i-claudin (ZO-1, ebomvu) kanye nolwelwesi lwebhulashi oluqhubekayo olubhalwe ukuthi F-actin (oluhlaza) kanye ne-nuclei (blue) yamaseli e-conductible e-conductible e-conceptous cell. ama-chips.Imicibisholo ekhomba uhlaka oluphakathi nendawo ibonisa indawo yendiza egxile ekubukeni ngakunye okuhlangene.d, Isikhathi sesikhathi sezinguquko ze-morphological ku-organoid ekhuliswe ku-chip etholwe nge-makroskopu yokuqhathanisa yesigaba ezinsukwini 3, 7, 9, 11, kanye no-13. ocezwini oluthathwe ngosuku lwe-7.f, izithombe ze-immunofluorescence Ezimboziwe ezibonisa izimpawu zamaseli e-stem (LGR5; i-magenta), amaseli e-goblet (MUC2; oluhlaza okotshani), u-F-actin (ompunga) kanye nama-nuclei (cyan) atshalwe kuma-gut chips izinsuku ezingu-3, ​​ngokulandelana (Kwesobunxele) kanye nezinsuku eziyi-13 (maphakathi) nazo ezigqamisa ungqimba lwe-Epigure. Isignali ye-LGR5 ngaphandle kophawu lwe-MUC2.Izithombe ze-Fluorescence ezibonisa i-epithelial microstructure (kwesokudla) ye-3D organoid epithelium esungulwe emathunjini ku-chip ngokungcolisa ulwelwesi lwe-plasma ngodayi we-CellMask (kwesokudla) ngosuku lwe-13 lwesiko.Ibha yesikali ingu-50 μm ngaphandle uma kushiwo ngenye indlela kusukela kunkomba.b I-Oxford University Press; c Ithathelwe ngemvume evela kuNkomba.2. I-Oxford University Press; e kanye no f kwashintshwa ngemvume ngereferensi.12 Ngaphansi Kwelayisense Ye-Creative Commons CC BY 4.0.
Emathunjini ku-chip, kuyadingeka ukuguqula indawo ye-hydrophobic ye-PDMS ye-membrane ye-porous yokugqoka i-ECM ephumelelayo.Kule protocol, sisebenzisa izindlela ezimbili ezihlukene zokuguqula i-hydrophobicity ye-membrane ye-PDMS.Ngokuhlakulela amangqamuzana e-Caco-2, ukusebenza kwe-surface nge-UV / ozone ukwelashwa kuphela kwakwanele ukunciphisa i-hydrophobicity ye-hydrophobicity kanye nokunamathisela amaseli e-Caat hydrophobicity, i-coat-caat cell ye-Caco-CM2 I-membrane ye-PDMS.Nokho, isiko le-microfluidic ye-organoid epithelium idinga ukusebenza kwamakhemikhali okusekelwe kumakhemikhali ukuze kuzuzwe ukufakwa kahle kwamaprotheni e-ECM ngokusebenzisa ngokulandelanayo i-polyethyleneimine (PEI) kanye ne-glutaraldehyde kuma-microchannels we-PDMS.Ngemva kokuguqulwa kwendawo, amaprotheni e-ECM afakwa ukuze amboze i-facelid esebenzayo noma i-PDMSA efakwe i-isoothesis esebenzayo noma i-PDMSA efakwe ku-isosodium esebenzayo. amaseli anamathiselwe, i-microfluidic cell culture iqala ngokufaka amakha okuphakathi kuphela ku-microchannel engenhla kuze kube yilapho amangqamuzana enza i-monolayer ephelele, kuyilapho i-microchannel ephansi igcina izimo ezimile.Le ndlela elungiselelwe yokwenza kusebenze ubuso obuphezulu kanye ne-ECM coating yenza ukunamathiselwa kwe-organoid epithelium kubangele i-3D morphogenesis ebusweni be-PDMS.
Amasiko eTranswell nawo adinga ukumbozwa kwe-ECM ngaphambi kokutshala kweseli; kodwa-ke, amasiko akwaTranswell awadingi izinyathelo zokwelashwa eziyinkimbinkimbi ukuze kusebenze indawo yokufaka okunezimbotshana.Ngokukhula kwamaseli e-Caco-2 ekufakweni kwe-Transwell, ukumbozwa kwe-ECM ekufakweni okunezimbotshana kusheshisa ukunamathiselwa kwamaseli e-Caco-2 ahlukanisiwe (<1 ihora) kanye nokwakheka komgoqo oqinile we-junction (<1-2 izinsuku). ebusweni be-membrane (<3 h) futhi igcinwe kuze kube yilapho ama-organoid enza i-monolayer ephelele ene-barrier integrity .Amasiko e-Transwell enziwa kumapuleti anemithombo engu-24 ngaphandle kokusebenzisa ama-chips ahlanganisiwe.
I-in vitro 3D morphogenesis ingaqalwa ngokusebenzisa ukugeleza koketshezi kusici se-basolateral sengqimba ye-epithelial emisiwe.Ethunjini ku-chip, i-epithelial morphogenesis yaqala lapho i-medium ifakwe ngama-microchannels angaphezulu naphansi (Fig. 3a) .Njengoba kuchaziwe ngaphambili, kubalulekile ukwethula ukugeleza kwe-completial eqhubekayo yokugeleza kwe-colateral (ukugeleza kwe-comferial) kwe-continuous fluid (ukugeleza kwe-comferical) secreted morphogen inhibitors.Ukuhlinzeka ngemisoco eyanele kanye ne-serum kumaseli aboshwe kulwelwesi olunezimbotshana futhi kukhiqize ukucindezelwa kwe-luminal shear, sivamise ukusebenzisa ukugeleza okumbaxambili emathunjini ku-chip.Kuma-chips ahlanganisiwe, ukufakwa kwe-Transwell okuqukethe ama-epithelial monolayers kwafakwa kuma-chips ahlanganisiwe.Kwabe sekufakwa okuphakathi kwasetshenziswa ngaphansi kohlangothi lwe-basolateral lwe-microgenesis ye-3 Izinsuku ze-Transphowell ze-Porousgene I-Transphowell. ngemuva kokuqaliswa kokugeleza kwe-basolateral kuzo zombili izinkundla zesiko.
Izici ze-morphological ze-microengineered 3D epithelial layers zingahlaziywa ngokusebenzisa izindlela zokucabanga ezihlukahlukene, okuhlanganisa imicroscope yesigaba sokungafani, i-differential interference difference (DIC) microscopy, SEM, noma i-immunofluorescence confocal microscopy (Figure 3 kanye 4) . Izendlalelo ze-epithelial.Ngenxa yokucaca kokubona kwe-PDMS namafilimu e-polyester, zombili izinkundla ze-gut-on-a-chip kanye ne-hybrid chip zingahlinzeka ngesithombe sesikhathi sangempela ku-situ ngaphandle kwesidingo sokuhlukaniswa noma ukuqaqa idivayisi.Lapho kwenziwa imaging ye-immunofluorescence (Izibalo 1, 3bly, 4b, 4b, c, c kanye namaseli ahlanganisiwe) (wt/vol) paraformaldehyde (PFA), elandelwa i-Triton X-100 kanye no-2% (wt/vol) ) i-bovine serum albumin (BSA), ngokulandelana.Ngokuya ngohlobo lweseli, ukulungisa okuhlukene, ama-permeabilizers, nama-agent avimbelayo angasetshenziswa.Amasosha omzimba ayinhloko aqondise kumaseli ancike emugqeni ukuze kugqanyiswe amangqamuzana e-chip asetshenziswa kumaseli we-chip asetshenziswa kumaseli wesifunda. kanye nodayi we-counterstain oqondise noma iyiphi i-nucleus (isb, 4′,6-diamidino-2-phenylene) indole, DAPI) noma i-F-actin (isb., okulebulwe nge-fluorescently phalloidin). Ukuthwebula bukhoma okusekelwe ku-Fluorescence nakho kungenziwa endaweni ukuze kutholwe ukukhiqizwa kwamafinyila (Umfanekiso ohlukile we-colon "Fig. 1", "Fig. amaseli e-microbial (Fig. 1, “Host-microbe co-culture”) , ukubuthwa kwamaseli omzimba (Fig. 1, 'Disease Modeling') noma ama-contours we-3D epithelial morphology (Fig. 3c,f and 4b,c) .Lapho ulungisa amathumbu ku-chip ukuze uhlukanise ungqimba lwe-microf oluphezulu oluchazwe njengesendlalelo esingaphansi. 2, i-3D epithelial morphology kanye ne-microvilli emngceleni webhulashi le-apical ingabonwa nge-SEM (Fig. 3b).Inkulumo yomaka bokuhlukanisa ingahlolwa ngokwenza i-PCR5 yobuningi noma ukulandelana kwe-single-cell RNA.Kulokhu, izingqimba ze-3D ze-epithelicular chips noma ama-chips e-hybrid asetshenziselwa ama-chips e-chips noma ama-chips okuvunwa bese etshalwa kuma-chips e-guest ukuhlaziywa kofuzo.
a, Ukugeleza komsebenzi wokufakwa kwe-intestinal morphogenesis ku-chip hybrid.I-Caco-2 kanye nama-organoid amathumbu asetshenziswa kule protocol ukuze kuboniswe i-3D morphogenesis endaweni yesikhulumi se-hybrid chip.Amaseli e-epithelial ahlukanisiwe afakwe imbewu ekufakweni kwe-Transwell okulungiselelwe (i-TW prep; bheka umfanekiso ngezansi) .Uma amaseli embewu efakwe emangqamuzaneni e-polyseed (amaseli e-polyseed anamathiselwe) afakwa kumaseli e-polyseed. ikhule ngaphansi kwezimo ezimile (isiko le-TW) .Ngemva kwezinsuku ezingu-7, okufakiwe okukodwa kwe-Transwell okuqukethe i-2D monolayer yamaseli e-epithelial kwahlanganiswa ku-chip ye-hybrid ukwethula ukugeleza kwe-basolateral (Flow, BL), ekugcineni okuholele ekukhiqizeni ungqimba lwe-3D epithelial (morphogenesis). (Umugqa we-C103) ikholoni ekhuphukayo esinyathelweni ngasinye sokuhlola noma iphoyinti lesikhathi.I-schematics ezisezendlalelo ezingaphezulu zibonisa ukucushwa kokuhlola kwesinyathelo ngasinye.b, Ama-chips e-Hybrid (uhlelo lwesokunxele) lungaholela ku-3D morphogenesis yamaseli e-organoid epithelial ane-top-down confocal microscopy ukubukwa okuthathwe ezindaweni ezihlukene ze-Z, i-scheme ephansi kanye nemigqa ehambisanayo ye-Z ibonise izici ezisobala ze-morphological.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled view) yamaseli e-epithelial asuselwa ku-organoid akhuliswe ku-static Transwell (TW; inset ngaphakathi kwebhokisi elidashi elimhlophe) ngokumelene ne-hybrid chip (ishothi enkulu kunazo zonke egcwele) uma kuqhathaniswa ne-3D morphology2D. ukubuka okuqondile okuphambene (okufakwe ekhoneni eliphezulu kwesokudla; ​​“XZ”) futhi kubonisa izici ze-2D ne-3D.Ibha yesikali, 100 µm.c Iphrintwe kabusha ngemvume evela kunkomba.4. Elsevier.
Izilawuli zingalungiselelwa ngokuhlakulela amangqamuzana afanayo (i-Caco-2 noma amaseli e-organoid epithelial yamathumbu) abe ama-monolayers anezinhlangothi ezimbili ngaphansi kwezimo ezivamile zesiko elimile.Ngokuphawulekayo, ukuncipha komsoco kungase kubangele ngenxa yomthamo olinganiselwe wevolumu yamashaneli amancane (okungukuthi ~4 µL esiteshini esiphezulu esiteshini sokuqala sokugeleza kwe-gut-chippisoli ngaphambi komklamo we-gut-chippisoli ongasemuva kanyeLapho ngaphambi kokwakheka kwe-cancer ye-cancer). nazo ziqhathaniswe.
Inqubo ye-lithography ethambile kufanele yenziwe ekamelweni elihlanzekile.Kusendlalelo ngasinye ku-chip (izendlalelo ezingaphezulu nezingaphansi kanye nolwelwesi) kanye nama-chips ayingxube, ama-photomasks ahlukene asetshenziswa futhi enziwa kumawafa e-silicon ahlukene ngenxa yokuthi ubude bama-microchannel babuhlukile.Ubude obuqondisiwe bemigudu emincane engaphezulu nephansi ye-gut ku-chip ingu-500 µmµubude obuqondiwe ngo-500mµ I-hybrid chip ingu-200 µm.
Faka i-wafer ye-silicon engama-intshi angu-3 esitsheni esine-acetone.Phendukisa kahle ipuleti imizuzwana engu-30, bese womisa iwafa emoyeni.Dlulisa iwafa epuletini eline-IPA, bese usonta ipuleti imizuzwana engu-30 ukuze uyihlanze.
Isixazululo se-piranha (ingxube ye-hydrogen peroxide ne-concentrated sulfuric acid, 1:3 (vol/vol)) ngokuzithandela singasetshenziselwa ukukhulisa ukukhishwa kwezinsalela zezinto eziphilayo endaweni eyi-silicon wafer.
Isixazululo se-Piranha sigqwala kakhulu futhi sikhiqiza ukushisa.Izinyathelo zokuphepha ezengeziwe ziyadingeka.Ekulahleni kukadoti, vumela isixazululo ukuthi siphole futhi sidluliselwe esitsheni esihlanzekile, esomile semfucumfucu.Sebenzisa iziqukathi zesibili bese ulebula kahle iziqukathi zemfucuza.Sicela ulandele imihlahlandlela yezokuphepha yale ndawo ukuze uthole izinqubo ezinemininingwane eyengeziwe.
Dlulisa amanzi ama-wafer ngokuwabeka epuleti elishisayo elingu-200 ° C imizuzu engu-10. Ngemva kokuphelelwa amanzi emzimbeni, i-wafer yanyakaziswa izikhathi ezinhlanu emoyeni ukuze iphole.
Thela u-~10 g we-photoresist SU-8 2100 phakathi nendawo ye-silicon wafer ehlanziwe.Sebenzisa ama-tweezers ukuze usakaze i-photoresist ngokulinganayo ku-wafer.Ngezinye izikhathi beka iwafa kupuleti elishisayo elingu-65°C ukuze wenze i-photoresist inganamatheli futhi ibe lula ukusisakaza.Ungafaki iwafa ngqo epuleti elishisayo.
I-SU-8 yasatshalaliswa ngokulinganayo ku-wafer ngokugijima i-spin coating.Hlela ukuzungezisa okungenayo kwe-SU-8 ngamasekhondi angu-5–10 ukuze isakazeke ku-500 rpm ngokusheshisa okungu-100 rpm/s. Setha i-spin eyinhloko ka-200 µm ukujiya kwephethini ngo-1,500m2pm 500 µm ukuphakama kongqimba olungaphezulu lwamathumbu ku-chip; bona “Izinyathelo ezibucayi” ngezansi) isethwe ngokusheshisa okungu-300 rpm/s imizuzwana engu-30 ngo-1,200 rpm.
Isivinini sokujikeleza esiyinhloko singalungiswa ngokuya ngokujiya okuhlosiwe kwephethini ye-SU-8 ku-wafer ye-silicon.
Ukwakha amaphethini e-SU-8 obude obungu-500 µm kungqimba olungaphezulu lwamathumbu ku-chip, ukujiya nge-spin nezinyathelo zokubhaka ezithambile zaleli Bhokisi (izinyathelo 7 no-8) ziphindaphindwe ngokulandelana (bona isinyathelo 9) ukuze kukhiqizwe izendlalelo ezimbili zika-250 µm Ungqimba oluwugqinsi lwe-SU-8, olunganwetshwa nge-UV1 lesendlalelo esingu-0 futhi sihlanganiswe ngu-0. µm ukuphakama.
Bhaka amawafa ahlanganiswe nge-SU-8 ngokucophelela ngokubeka amawafa epuleti elishisayo elingu-65 °C imizuzu emi-5, bese ushintsha ukulungiselelwa kube ku-95 °C bese ufukamela eminye imizuzu engama-40 eyengeziwe.
Ukuze uthole ukuphakama okungu-500 μm kwephethini ye-SU-8 kusiteshi esincane esingaphezulu, phinda izinyathelo 7 no-8 ukuze ukhiqize izendlalelo ezimbili ezingu-250 μm eziwugqinsi ze-SU-8.
Usebenzisa i-UV Mask Aligner, yenza ukuhlola kwesibani ngokuya ngemiyalelo yomkhiqizi ukuze ubale isikhathi sokuchayeka sewafa.(isikhathi sokuchayeka, ms) = (umthamo wokuchayeka, mJ/cm2)/(amandla welambu, mW/cm2).
Ngemuva kokunquma isikhathi sokuchayeka, beka imaski yesibambi semaski yokuqondanisa imaski ye-UV bese ubeka imaski yesithombe kusinkwa esiyisicwecwana se-SU-8.
Beka indawo ephrintiwe ye-photomask ngqo ohlangothini oluboshwe lwe-SU-8 lwe-silicon wafer ukuze unciphise ukuhlakazeka kwe-UV.
Veza i-wafer egqinsiwe ye-SU-8 kanye nemaski esithombe ngokuqondile ku-260 mJ/cm2 wokukhanya kwe-UV ngesikhathi esinqunyelwe kusengaphambili (bona isinyathelo 10 saleli bhokisi).
Ngemva kokuchayeka kwe-UV, amawafa e-silicon ajikijelwe nge-SU-8 abhakwa ku-65°C imizuzu engu-5 no-95°C imizuzu engu-15 kupuleti ngalinye elishisayo ukuze enze amaphethini anobude obungu-200 μm. Nweba isikhathi sangemva kokubhaka ku-95 °C kuya kumaminithi angu-30 ukuze wenze amaphethini anobude obungu-500 µm.
Umthuthukisi uthelwa endishini yengilazi, futhi iwafa ebhakiwe ifakwa esitsheni.Ivolumu yonjiniyela we-SU-8 ingahluka kuye ngosayizi wepuleti lengilazi.Qiniseka ukuthi usebenzisa unjiniyela we-SU-8 owanele ukususa ngokuphelele i-SU-8 engavezwanga.Ngokwesibonelo, uma usebenzisa isitsha sengilazi esingu-150 mm esinomthamo ongu-1 L, sebenzisa u-~300 mthuthuthu kanjiniyela wemizuzu engu-300 mL ukujikeleza okuthambile ngezikhathi ezithile.
Hlanza isikhunta esithuthukisiwe nge ~10 mL kanjiniyela omusha kulandele i-IPA ngokufafaza isisombululo usebenzisa i-pipette.
Faka i-wafer endaweni yokuhlanza i-plasma bese uveza i-oxygen plasma (igesi yasemkhathini, ingcindezi ehlosiwe 1 × 10−5 Torr, amandla 125 W) ye-1.5 min.
Faka i-wafer ku-vacuum desiccator ene-slide sengilazi ngaphakathi.Ama-wafers nama-slides angabekwa eceleni.Uma i-vacuum desiccator ihlukaniswa ngezingqimba eziningana ngepuleti, beka ama-slides ekamelweni elingezansi kanye namawafa ekamelweni eliphezulu.Yehlisa u-100 μL we-trichloro,1H, 2H, 2H-perfluorooctyl)isixazululo se-silane kusilayidi sengilazi bese usebenzisa i-vacuum yokwenza i-silanization.
Ncibilikisa ibhodlela lamaseli e-Caco-2 aqandisiwe endaweni yokugeza yamanzi engu-37°C, bese udlulisela amaseli ancibilikisiwe kuflask ye-T75 equkethe u-15 mL we-37°C medium of Caco-2 efudunyeziwe.
Ukuze udlule amaseli e-Caco-2 ku-~90% confluency, i-Caco-2 medium efudumele yokuqala, i-PBS, ne-trypsin engu-0.25%/1 mM EDTA kubhavu wamanzi ongu-37°C.
Fudumeza i-medium by vacuum aspiration.Geza amaseli kabili ngo-5 mL we-PBS efudumele ngokuphinda ukuphefumula kwe-vacuum nokungeza i-PBS entsha.