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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 amancane nama-metabolites awo. Ngakho-ke, ukwakhiwa kabusha kwezakhiwo ze-epithelial ze-3D 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 komzimba kanye ne-biomechanics. I-fluidic chip kanye ne-Transwell embedded chip hybrid. Sichaza izindlela ezinemininingwane zokwenza idivayisi, ukukhuliswa kwe-Caco-2 noma amaseli e-organoid epithelial yamathumbu ezilungiselelweni ezivamile kanye neplathifomu ye-microfluidic, ukufakwa kwe-3D morphogenesis, kanye nokuhlukaniswa kwe-3D epithelia esunguliwe ngokusebenzisa i-multiplear generation control prothocol. ye-5 d.Indlela yethu ye-in vitro morphogenesis isebenzisa ukucindezelwa kwe-shear okuhambisanayo ngokomzimba kanye nokunyakaza komshini futhi akudingi ubunjiniyela beselula obuyinkimbinkimbi noma ukukhwabanisa, okungase kube ngaphezu kwamanye amasu akhona.Sicabanga ukuthi iphrothokholi yethu ehlongozwayo ingaba nomthelela obanzi kumphakathi wocwaningo lwe-biomedical, inikeze indlela yokuvuselela i-3D intestinal epithelical, i-biomedical application ye-biomedical application.
Ukuhlola kubonisa ukuthi amangqamuzana e-Caco-2 e-epithelial emathunjini akhuliswe ku-gut-on-a-chip1,2,3,4,5 noma amadivaysi e-microfluidic ebilayer6,7 angakwazi ukungena ngokuzenzakalelayo kwe-3D morphogenesis in vitro ngaphandle kokuqonda okucacile kwendlela eyisisekelo. sis in vitro, okuye kwaboniswa yi-Caco-2 kanye nama-organoid amathumbu atholakala esigulini.Amaseli e-Epithelial aqinisekisiwe. Kulolu cwaningo, sigxile ngokukhethekile ekukhiqizweni kweseli nasekusabalaliseni 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 ehlobene ne-d 1, noma i-Soggy-1) ku-chip gut ivimbela i-morphogenesis noma iphazamisa ungqimba olwakhiwe ngaphambilini lwe-epithelial ye-3D, okuphakamisa ukuthi ukucindezeleka okuphikiswayo phakathi nesiko kunesibopho se-intestinal morphogenesis in vitro.Ngakho-ke, indlela engokoqobo yokufinyelela i-morphogenesis eqinile ku-epithelial ye-epithelial esebenzayo iwukukhipha isixhumi esibonakalayo esisebenzayo noma ukususa isixhumi esibonakalayo esisebenzayo ukugudluza (isb., kumapulatifomu e-gut-on-a-chip noma e-hybrid-on-a-chip) noma ukusabalalisa .Imidiya ye-Basolateral (isb., isuka e-Transwell ishutheka emanzini amakhulu angaphansi kwemithombo emithonjeni).
Kule phrothokholi, sihlinzeka ngendlela enemininingwane yokwenza ama-microdevices we-gut-on-a-chip kanye nama-Transwell-insertable hybrid chips (izinyathelo 1-5) ukuze enze amaseli e-epithelial amathumbu ku-polydimethylsiloxane (PDMS) -based based membranes (izinyathelo 6A, 7A, 8, 9) noma i-PolyesterB ducts 6 kanye ne-8 I-3D morphogenesis in vitro (isinyathelo se-10) .Siphinde sahlonza izici zamangqamuzana namangqamuzana ezibonisa i-histogenesis eqondene nezicubu kanye nokuhlukaniswa kwamaselula okuncike kulayini ngokusebenzisa izindlela eziningi zokucabanga (izinyathelo ze-11-24) .Senza i-morphogenesis sisebenzisa i-epithelial yamathumbu omuntu, njengemininingwane ye-Cacorous surface modids noma i-membrane yobuchwepheshe be-Caco-2. , 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 ezithuthukisiwe ngokugeleza okuphakathi ku-basolateral compartment yesiko.Ekugcineni, ukumelela okusetshenziselwa kabusha kwe-epithelial D kunganikeza ungqimba olusebenzayo lwe-D olusetshenziswayo. ukukhula kwe-epithelial okuncike ku-phogen, 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, kanye ne-developmental biology) kanye nocwaningo olusetshenzisiwe (isb., ukuhlolwa kwezidakamizwa ngaphambi komtholampilo, ukufanisa izifo, ubunjiniyela bezicubu, kanye ne-gastroenterology) umthelela obanzi.Ngenxa yokuphindaphindeka nokuqina kwephrothokholi yethu ukuze singenise i-3D introphogenesis ye-technical morphogenesis ithunyelelwe izethameli ezifunda ukushintshashintsha kokubonisa amangqamuzana ngesikhathi sokuthuthukiswa kwamathumbu, ukuzalwa kabusha noma i-homeostasis .Ngaphezu kwalokho, isimiso sethu somthetho siwusizo ekuphenyeni ukutheleleka ngaphansi kwamagciwane ahlukahlukene athathelwanayo afana ne-Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonellarium cholera noma i-Salmonellae9.Izethameli ze-pathogenesis 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 zamasosha omzimba kanye nokulungiswa kokulimala okuhlobene ne-pathogen ku-gastrointestinal (GI) ipheshana 11 .Ezinye izifo ze-GIAcecolitis, isifo se-Grosaky, isifo se-Grosaky, isifo se-Grosaky i-pouchitis, noma i-irritable bowel syndrome ingalingiswa lapho izingqimba ze-epithelial zamathumbu e-3D zilungiswa kusetshenziswa izingqimba ze-epithelial zesiguli ze-3D, lezi zifo zihlanganisa i-villous atrophy, shortening crypt, umonakalo we-mucosal, noma i-epithelial barrier. cabanga ukwengeza izinhlobo zamaseli 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 yamathumbu emathumbu ngokuhlanganisa izinhlobo ezihlukahlukene ze-microbial, imiphakathi ye-microbial noma i-fecal microbiota-i-gut-on-a-gut-a-gut.endaweni yesikhulumi.Le ndlela iheha kakhulu izethameli ezifunda i-mucosal immunology, i-gastroenterology, i-human microbiome, i-culturomics kanye ne-clinical microbiology efuna ukuhlakulela i-gut microbiota eyayingakaze ibe khona elabhorethri.Uma i-in vitro morphogenesis protocol yethu ingashintshwa ukuze ibe namafomethi we-scalable culture, njenge-multiwell that plates 28 efaka i-basople 49 ku-multiwell, i-basople 49 amakhompathimenti, iphrothokholi ingasatshalaliswa nakulabo abathuthukisa ukuhlolwa kwezemithi, i-biomedical Noma izinga eliphezulu lokuhlola noma ukuqinisekiswa kwezinkundla zemboni yezokudla.Njengobufakazi bomgomo, muva nje sibonise ukuthi kungenzeka i-multiplex high-throughput morphogenesis system scalable to a 24-well-well-throughput screening or validation platforms for the food industry.Njengobufakazi bomthethosisekelo, muva nje sikhombise ukuba nokwenzeka kwe-multiplex high-throughput morphogenesis system scalable to a 24-well-wellplate format.Ngaphezu kwalokho,16-phambili, kunemikhiqizo eminingi yokuhweba,18-phambili, i-multiplex. Ukwenziwa kwendlela yethu ye-in vitro morphogenesis kungasheshiswa futhi kungahle kwamukelwe amalabhorethri amaningi ocwaningo, imboni noma uhulumeni kanye nama-ejensi alawulayo ukuze aqonde ukuhlelwa kabusha kwamaselula kwe-in vitro gut morphogenesis ezingeni le-transcriptomic ukuhlola izidakamizwa noma i-biotherapeutics Ukumuncwa nokuhanjiswa kwezidakamizwa kwahlolwa kusetshenziswa i-3D gut-gut-proondu-process-update yezohwebo inqubo ye-gut morphogenesis.
Inani elilinganiselwe lamamodeli okuhlola ahlobene nomuntu asetshenziselwe ukutadisha i-epithelial morphogenesis yamathumbu, ikakhulukazi ngenxa yokuntuleka kwezivumelwano ezisebenzisekayo zokufaka i-3D morphogenesis in vitro.Eqinisweni, ulwazi oluningi lwamanje mayelana ne-gut morphogenesis lusekelwe ezifundweni zezilwane (isb, i-zebrafish20, i-mice21 noma i-chicken-ingaba-i-labor-ibaluleke kakhulu, i-chicken-esebenza kakhulu2). ly, azinqumi ngokuqondile izinqubo zokuthuthuka komuntu.Lawa mamodeli nawo anomkhawulo kakhulu ekhonweni lawo lokuhlolwa ngezindlela eziningi ezingakala.Ngakho-ke, inqubo yethu yokuvuselela izicubu ze-3D in vitro idlula ukusebenza kahle kumamodeli ezilwane we-vivo kanye namanye amamodeli amasiko weseli e-2D amile.Njengoba kuchaziwe ngaphambili, kusetshenziswa ukwakheka kwe-spatiillus ye-3D kwavumela amaseli e-spatiillus asendaweni ukuhlola i-epithelillus ye-spatiillus yendawo ekuphenduleni i-mucosal ehlukahlukene noma i-immune stimuli.Izingqimba ze-epithelial ze-3D zinganikeza isikhala sokutadisha ukuthi amangqamuzana amancane ancintisana kanjani ukuze akhe izindawo zokuhlala zendawo kanye nokuziphendukela kwemvelo ekuphenduleni izici zokubamba (isb, izingqimba ze-mucus zangaphakathi ngokumelene nezangaphandle, ukukhishwa kwe-IgA kanye nama-peptide e-antimicrobial) . ama-microbial metabolites (isb, ama-fatty chain acids amafushane) akha inhlangano yamaselula kanye ne-stem cell niches kuma-basal crypts.Lezi zici zingaboniswa kuphela uma izingqimba ze-epithelial ze-3D zisungulwa ku-vitro.
Ngaphezu 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 transportation ye-organoid i-hosticulture i-hostestinal imvamisa i-coscullone i-costinal-cosmetology. d 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 iyahlasela futhi idinga umsebenzi omkhulu 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.Yakha ama-scaffolds e-hydrogel usebenzisa i-3D-printed, micro-milled, noma lithographically fabricated mold-self organisation-self organisation. ama-morphogen gradients afanele, 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 ukuvezwa kwenqubo ezenzakalelayo ye-morphogenetic ngokwayo.Lawa mamodeli nawo awanikezi amandla ashukumisayo e-luminal noma e-epithelial adinga ukugeleza kwe-thestinal morphological, i-interstitial genesis edinga ukugeleza kwe-laxaphophysis ye-laxaphogenesis, kanye ne-interstitial genesis ye-langular umsebenzi.Olunye ucwaningo lwakamuva lusebenzise ama-hydrogel scaffolds endaweni yesikhulumi se-microfluidic kanye nezakhiwo ze-epithelial zamathumbu ezinephethini kusetshenziswa amasu okukhipha i-laser.Ama-organoid amathumbu egundane alandela amaphethini amisiwe ukuze enze izakhiwo ze-tubular yamathumbu, futhi ukugeleza koketshezi lwe-intraluminal kungenziwa kabusha kusetshenziswa imojuli ye-microfluidics.Noma kunjalo, i-spophonologies ye-mechanics imodeli ayihlanganisi i-module ye-monophonologic noma i-model ye-module3. amasu e-ing avela eqenjini elifanayo akwazi ukudala amashubhu amancane amathumbu anezinqubo ezizenzakalelayo ze-morphogenetic.Naphezu kokwakhiwa okuyinkimbinkimbi kwezingxenye ezahlukene zamathumbu ngaphakathi kwethubhu, lo modeli futhi awunakho ukugeleza koketshezi olukhanyayo kanye nokuguqulwa komshini.Ukwengeza, ukusebenza kwemodeli kungase kube nomkhawulo, ikakhulukazi ngemva kokuba inqubo ye-bioprinting isiqediwe, iphazamisa izimo zokuhlola noma i-cell-to-cell protocol ihlinzeka nge-protocol ye-cell-to-cell. ukucindezeleka kwe-shear efanele, i-biomechanics elingisa ukuhamba kwamathumbu, ukufinyeleleka kwamakhompathimenti azimele apical kanye ne-basolateral, nokudalwa kabusha kwe-biological microenvironments eyinkimbinkimbi ye-modularity.Ngakho-ke, i-in vitro 3D morphogenesis protocol yethu ingase inikeze indlela ehambisanayo yokunqoba izinselele zezindlela 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 ku-robuast-gudulalatal side yethulwe ku-robuast-medium gudulat yethulwe ku-robusodium yethu. I-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 ama-stem cell niches kumaseli we-basal (i-mebroblast) acatshangelwa njengendima ebalulekile ye-mebroblast, i-mebroblast idlala indima eyinhloko kumaseli we-mebroblast. ekukhiqizeni amaprotheni e-ECM kanye nokulawulwa kwe-morphogenesis yamathumbu ku-vivo35,37,38.Ukwengezwa kwamangqamuzana e-mesenchymal kumodeli yethu kwathuthukisa inqubo ye-morphogenetic kanye nokusebenza kahle kokunamathiselwe kweseli.I-endothelial layer (okungukuthi, ama-capillaries noma i-lymphatics) idlala indima ebalulekile ekulawuleni ukuthuthwa kwamangqamuzana39 kanye ne-immuneviculament cell recruitment can be microrecruitment micro. okuxhunywe phakathi kwamamodeli ezicubu kuyisidingo lapho amamodeli ezicubu eklanyelwe ukubonisa ukusebenzisana kwezitho eziningi.Ngakho-ke, amangqamuzana e-endothelial angase adinge ukufakwa ukuze afanekise izici ezinembile zomzimba ngokulungiswa kwezinga lesitho.Amangqamuzana okuzivikela atholakala esigulini nawo abalulekile ekuboniseni izimpendulo zokuzivikela ezizalwa nazo, isethulo se-antigen, i-innate adaptive immune crosstalk, kanye ne-tissue-immunity immunity intecific intestinfi.
Ukusetshenziswa kwama-chips ayi-hybrid kuqonde kakhulu kune-gut-on-a-chip ngoba ukusethwa kwedivayisi kulula futhi ukusetshenziswa kokufakwa kwe-Transwell kuvumela isiko elingalawuleki 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 efakwe endaweni ene-apical chip compartment ebekwe esiteshini sokucindezeleka se-Apical chip esiteshini se-Criptical. ekuqaleni, izakhiwo ezimile endaweni ye-apical akuvamile ukuthi zinike amandla i-co-culture ye-bacteria yesikhathi eside kuma-chips ayi-hybrid. Nakuba singakwazi ukufaka i-3D morphogenesis ekufakweni kwe-Transwell uma kusetshenziswa ama-chips ahlanganisiwe, ukushoda kwe-physiologically ehambisana ne-biomechanics kanye nokugeleza koketshezi lwe-apical kungase kukhawulele ukubakhona kwezingxenyekazi ze-hybrid chip platforms ezingaba khona ze-chip.
Ukwakhiwa kabusha kwesilinganiso esigcwele se-axis ye-crypt-villus yomuntu kusiko lwe-gut-on-a-chip kanye ne-hybrid-on-a-chip akukasungulwa ngokugcwele. Njengoba i-morphogenesis iqala ku-epithelial monolayer, ama-microarchitectures we-3D awanikezi ngempela ukufana kwe-morphological kuma-crypts in vivo. I-lium, i-crypt nezifunda ezimbi azizange zibekwe imingcele ngokucacile. Nakuba amashaneli aphezulu aphezulu ku-chip eholela ekukhuphukeni kokuphakama kwe-epithelium ene-microengineered, ukuphakama okuphezulu kusakhawulelwe ku-~300–400 µm. I-lli ingu-~600 µm41.
Ngokombono wesithombe, i-in situ super-resolution imaging ye-3D microarchitectures ingase ikhawulelwe emathunjini ku-chip, njengoba ibanga lokusebenza elidingekayo ukusuka ku-lens yenhloso ukuya kungqimba lwe-epithelial lilandelana ngamamilimitha ambalwa. Ukuze unqobe le nkinga, umgomo oqhelile ungase udingeke. Ngaphezu kwalokho, ukwenza izingxenye ezincane zokulinganisa ungqimba lwe-PD ngenxa yokunwebeka okuphezulu ngenxa yokulungiswa kwesithombe kuyaqina. i-microfabrication encane yolwelwesi lwe-gut ku-chip ihilela ukunamathela unomphela phakathi kongqimba ngalunye, kuyinselele kakhulu ukuvula noma ukususa ungqimba olungaphezulu ukuze uhlole isakhiwo esingaphezulu se-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.Okunye, ukuguqulwa kwendawo ye-PDMS, i-polyphilic, i-polyphilic PDMS (isib4poly42, isb. I-3) ingabhekwa njengokunciphisa ukukhangisa kwama-molecule e-hydrophobic.
Okokugcina, indlela yethu ayizange ibonakale kahle mayelana nokuhlinzeka ngokuhlolwa okuphezulu noma "ubukhulu obulingana-bonke" inkundla yokuhlola esebenziseka kalula.Iphrothokholi yamanje idinga iphampu yesirinji nge-microdevice ngayinye, ethatha isikhala ku-CO2 incubator futhi ivimbele ukuhlola okukhulu.Lokhu mkhawulo ungathuthukiswa kakhulu ngokufometha kwe-94well, isiko le-34well, i-34well, i-innovative, i-culture, i-34well, i-94well, i-innovative, i-culture, i-94well, i-94well, i-innovative, ukufakwa kahle kwe-porous okuvumela ukugcwaliswa okuqhubekayo nokususwa kwemidiya ye-basolateral).
Ukuze senze i-3D morphogenesis ye-epithelium yamathumbu omuntu ku-vitro, sisebenzise idivayisi ye-microfluidic chip intestinal equkethe iziteshi ezincane ezimbili ezihambisanayo kanye nolwelwesi olunwebekayo oluyizimbotshana phakathi ukuze sakhe isixhumi esibonakalayo se-lumen-capillary.Siphinde sibonisa ukusetshenziswa kwedivayisi ye-microfluidic yomzila owodwa (i-hybrid chip) ehlinzeka ngeplatifomu egelezayo eqhubekayo yomibili. s, i-morphogenesis yamangqamuzana e-epithelial amathumbu omuntu ahlukahlukene angaboniswa ngokusebenzisa ukuguqulwa kokuqondisa kokugeleza ukuze kukhishwe abaphikisi be-morphogen kusukela ku-basolateral compartment.Yonke inqubo yokuhlola (Umfanekiso 1) iqukethe izingxenye ezinhlanu: (i) i-microfabrication ye-gut chip noma i-Transwell hybrid chip1-5; co-2 amaseli) noma ama-organoid amathumbu omuntu;amabhokisi 2-5), (iii) isiko lamaseli e-epithelial emathunjini kuma-chips amathumbu noma ama-chips ayi-hybrid (izinyathelo 6-9), (iv) ukufakwa kwe-3D morphogenesis in vitro (isinyathelo 10) kanye (v) ) ukuze kubonise ukwakheka kwe-3D epithelial microstructure (izinyathelo 11-24). genesis ngokuqhathanisa i-epithelial morphogenesis nezilawuli zendawo, zesikhashana, ezinemibandela, noma zenqubo.
Sisebenzise izinkundla ezimbili zamasiko ezihlukene: i-gut-on-a-chip enamashaneli aqondile noma amashaneli ahlanganisiwe angaqondile, noma ama-chips ayi-hybrid aqukethe okufakiwe kwe-Transwell (TW) kudivayisi encane ye-microfluidic, eklanywe njengoba kuchazwe ku-Box 1, kanye nesinyathelo 1 -5.”Ukwenziwa Kwedivayisi” kubonisa izinyathelo eziyinhloko ekwenzeni i-chip eyodwa noma i-chip hybrid.”I-Culture of Human cell noma umthombo we-Humancell (umthombo) stinal organoids) kanye nenqubo yesiko esetshenziswa kule protocol.”In vitro morphogenesis” ikhombisa izinyathelo eziphelele lapho amaseli e-Caco-2 noma asuselwa ku-organoid akhiqizwa ku-chip yamathumbu noma ekufakweni kwe-Transwell kwe-chip ye-hybrid, okulandelwa ukwethulwa kwe-3D morphogenesis kanye nokwakheka kwenombolo ye-epithelial ebhalwe ngezansiInombolo ye-epithelial ekhonjiswe ngezansi ihlinzeka ngenombolo ye-epithelial yesakhiwo ngasinye. Izendlalelo ze-epithelial zingasetshenziswa, isibonelo, ekuhlukaniseni ukuhlukaniswa kwamangqamuzana, izifundo ze-gut physiology, ukusungulwa kwe-host-microbiome ecosystems, kanye nemodeli yezifo.Izithombe ze-Immunofluorescence ku-"Cell Differentiation" ezibonisa i-nuclei, i-F-actin ne-MUC2 evezwe ku-3D Caco-2 epithelial epithelial ye-secret epithelial epithelial ephuma ku-mucused chip yamaseli e-secret mucused e-MUcused. ubuso be-mucosal.Izithombe ezikhanyayo ku-Gut Physiology zibonisa amafinyila akhiqizwa ngokungcolisa i-sialic acid nezinsalela ze-N-acetylglucosamine zisebenzisa i-fluorescent wheat germ agglutinin.Izithombe ezimbili ezigqagqene ku-“Host-Microbe Co-Cultures” zibonisa ummeleli omele umsingathi-microbiome gut-culture left on the fluo-microbiome co-culture green ku-co-culture ye-fluo. iphrotheni ye-rescent (GFP) ene-microengineered 3D Caco-2 epithelial cells.Iphaneli elingakwesokudla libonisa ukwenziwa kwasendaweni kwe-GFP E. coli ehlanganiswe ne-3D Caco-2 epithelial cells, elandelwa i-immunofluorescence staining nge-F-actin (red) kanye ne-nuclei (blue) .Isifo imodeling ye-guussiaty illustological chips illustological antibacterial inselela ye-guussiaky antibacterial illustological antibacterial illustological chips. s (isb, lipopolysaccharide, LPS) namaseli omzimba (isb, PBMC;okuluhlaza).Amaseli e-Caco-2 akhuliswe ukuze kusungulwe isendlalelo se-epithelial esingu-3D.Ibha yesikali, 50 µm.Izithombe emgqeni ongezansi: “Umehluko wamaseli” aguqulelwe ngemvume evela kunkomba.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 nama-chips ayi-hybrid akhiwe kusetshenziswa izifaniso ze-PDMS ezadilizwa kusukela ekubunjweni kwe-silicon nge-lithography ethambile1,44 futhi zenziwe iphethini nge-SU-8. Ukuklanywa kwama-microchannel ku-chip ngayinye kunqunywa ngokucabangela i-hydrodynamics efana ne-shear stress kanye ne-hydrodynamic pressure1,4,12. ama-microchannels aqondile ahambisanayo, aguquke abe yi-gut-on-a-chip eyinkimbinkimbi (Idatha Enwetshiwe Fig. 1b) ehlanganisa i-microchannel egobile ukuze ibangele Ukwenyuka kwesikhathi sokuhlala kwamanzi, amaphethini okugeleza okungahambisani, kanye nokuguqulwa kwe-multiaxial yamaseli akhulisiwe (Fig. 2a-f) Sibonise ukuthi i-Gut-Chip ehlanganisiwe iphinda ifake ngokunamandla i-3D morphogenesis ngesikhathi esifanayo ngezinga elifanayo lokukhula kwe-epithelial uma kuqhathaniswa ne-Gut-Chip yasekuqaleni, kungakhathaliseki uhlobo lweseli elikhulisiwe.Ngakho-ke, ukungenisa i-3D morphogenesis, imiklamo eqondile neyinkimbinkimbi ye-chip gut eguquguqukayo enegethivu yamaphethini e-PDMS enikeziwe ngemuva kwe-silicon ye-8 izici ze-silicone.2a).Ukwenza amathumbu ku-chip, ungqimba olulungisiwe lwe-PDMS olulungisiwe luboshwe ngokulandelana kufilimu ye-PDMS enezimbotshana bese luqondaniswa nongqimba oluphansi lwe-PDMS ngokubopha okungabuyiseleki emuva kusetshenziswa i-corona treatment (Fig. 2b-f). Ukuze kwenziwe ama-chips ayingxubevange, ama-replicas e-PDMS aphulukisiwe ahlanganiswe ku-microclimate e-microfluorescent engakwazi ukuhlanganisa ingilazi ye-transfluordal Fig. 2h kanye Nemininingwane Enwetshiwe Fig. 2).Inqubo yokuhlanganisa yenziwa ngokuphatha izindawo ezingaphezulu ze-replica ye-PDMS nengilazi nge-oksijini plasma noma ukwelashwa kwe-corona.Ngemva kokufakwa inzalo kwedivayisi eyenziwe nge-microfabricated exhunywe kushubhu ye-silicone, ukusethwa kwedivayisi kwase kulungele ukwenza i-3D morphogenesis ye-epithelium yamathumbu (Figure 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 ukubumba ungqimba lwe-silicon. dala ulwelwesi olunezimbotshana lwe-PDMS.d, Uchungechunge lwezithombe zezingxenye ze-PDMS ezingenhla neziphansi kanye nedivayisi yamathumbu ehlanganisiwe ku-chip.e, Uhlelo lokuqondanisa kwezingxenye ze-PDMS ezingenhla, ulwelwesi, neziphansi. Ungqimba ngalunye luboshwe ngendlela engenakuhlehliswa yi-plasma noma ukwelashwa kwe-corona. isb, Ukusethwa kwe-gut-on-a-chip ye-microfluidic cell culture.Ithumbu elenziwe ku-chip elihlanganiswe neshubhu le-silicone nesirinji lafakwa ku-coverslip.Idivayisi ye-chip yafakwa esivalweni sesitsha se-Petri esingu-150 mm ukuze sicutshungulwe.I-binder isetshenziselwa ukuvala i-silicone tube.h, i-Visual hybrids hybrids genesis chipsD chipsD chips genesis. okulungiselelwe ngokuzimela kumasiko ama-monolayers angu-2D amaseli e-epithelial amathumbu afakwe ku-chip ye-hybrid ukuze afake i-3D morphogenesis yamathumbu.I-medium ifakwe ngama-microchannels ngaphansi kongqimba lweseli olusungulwe ku-Transwell insert.Ibha yesikali, 1 cm.h Iphrintiwe kabusha ngemvume evela kunkomba.4.Elsevier.
Kule protocol, i-Caco-2 cell line kanye nama-organoid amathumbu asetshenziswe njengemithombo ye-epithelial (Fig. 3a) .Zombili izinhlobo zamaseli zazikhuliswe ngokuzimela (Ibhokisi 2 neBhokisi 5) futhi zisetshenziselwa imbewu ye-ECM-coated microchannels ye-chip gut noma i-Transwell inserts.Lapho amaseli ehlanganisa amaseli e-flasklydbe ehlanganisiwe (> 95% Caverage Caveage), amaseli e-Caverage angu-95%. 0 no-50) kuma-T-flasks avunwa ukuze kulungiswe ukumiswa kwamaseli ahlukanisiwe nge-trypsinization fluid (ibhokisi 2) .Ama-organoid amathumbu omuntu avela ku-biopsies yamathumbu noma ukuhlinzwa okuhlinzayo akhuliswe ku-Matrigel scaffold domes kumapuleti we-24-well ukusekela i-structural structural microenvironment, i-mosphogennt-microenvironment, i-mosphogennt equkethe i-mosphogennt ebalulekile kanye ne-mosphogennt microenvironment ebalulekile equkethe i-mosphogennt equkethe i-mosphogennt ne-mosphogennt ebalulekile. izici zokukhula ezilungiselelwe njengoba kuchazwe eBhokisini lesi-3 zenezelwa zonke izinsuku kuze kube yilapho ama-organoid ekhula aze afike ku-500 µm ububanzi. Ama-organoid akhule ngokugcwele ayavunwa futhi ahlukaniswe abe amangqamuzana awodwa ukuze afakwe emathunjini noma ama-Transwell afakwe ku-chip (Ibhokisi lesi-5).Njengoba sike sabika ngaphambili, singahlukaniswa ngokohlobo lwesifo sohlobo 12,13, isib. indawo (isb., isilonda uma kuqhathaniswa nendawo engeyona izilonda) kanye nendawo yamathumbu epheshaneni (isb, i-duodenum, ijejunum, i-ileum, i-cecum, ikholoni, noma i-rectum). Sihlinzeka ngephrothokholi ethuthukisiwe ku-Box 5 yokukhulisa ama-colonic organoids (ama-coloids) ngokuvamile adinga ukugxila okuphezulu kwama-morphogens noma amancane.
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 ahlanjululwe kudivayisi elungiselelwe i-gut-on-a-chip (ukulungiselela i-chipMS) .Uma amaseli e-seedased0 ngosuku (i-chipMS) efakwe (i-chipd) enamathiselwe ngosuku (i-membrane ehlanganisiwe) I-D0), ukugeleza kwe-apical (AP) kuyaqalwa futhi kugcinwe izinsuku zokuqala ze-2 (ukugeleza, AP, D0-D2). Ukugeleza kwe-Basolateral (BL) nakho kuqalwa kanye nokunyakaza okujikelezayo kwe-cyclic (ukwelula, ukugeleza, i-AP ne-BL) lapho kwakhiwa i-monolayer ye-2D ephelele.Amathumbu e-3D morphogenesis yenzeke izinsuku ze-Micromorphogenesis ezimele ngokuzenzekelayo (i-Micromorphogenesis emele i-5). wamaseli e-Caco-2 esinyathelweni ngasinye sokuhlola noma iphoyinti lesikhathi (igrafu yebha, 100 µm).Imidwebo emine eyisikimu ebonisa i-cascade ehambisanayo ye-gut morphogenesis (phezulu kwesokudla).Imicibisholo edayishiwe kuhlelo imelela isiqondiso sokugeleza koketshezi.b, isithombe se-SEM esibonisa i-topology engaphezulu yendawo emisiwe ye-3D Cacolished (indawo egqanyisiwe ye-Cacoum-2 egqanyisiwe) ibonisa i-microvilli evuselelwe kungqimba lwe-3D Caco-2 (kwesokudla).c, Ukubuka okuvundlile okungaphambili kwe-Caco-2 3D emisiwe, i-claudin (ZO-1, ebomvu) kanye nolwelwesi oluqhubekayo lwebhulashi lomngcele olubhalwe ukuthi F-actin (luhlaza) kanye ne-nuclei (eluhlaza okwesibhakabhaka) ukubonwa kwe-Immunofluorescence confocal kwama-chips epithelial aphakathi nendawo kumcibisholo we-epithelial chips kumcibisholo ngamunye ophakathi nendawo. ukubukwa kwe-confocal.d, Isikhathi sezinguquko ze-morphological ku-organoid ekhuliswe ku-chip etholwe nge-most difference microscopy ezinsukwini ezingu-3, ​​7, 9, 11, kanye no-13.Isethathi (phezulu kwesokudla) sibonisa ukukhuliswa okuphezulu kwesithombe esinikeziwe.e, i-DIC photomicrograph ye-organoid 3D epithelium esungulwe ku-gutrenceid stem L izithombe ezithathwe emgudwini we-overrescence. GR5;i-magenta), amaseli e-goblet (MUC2; oluhlaza okotshani), u-F-actin (ompunga) kanye ne-nuclei (cyan) akhuliswe kuma-gut chips izinsuku ezingu-3, ​​ngokulandelana (Kwesobunxele) kanye nama-organoid ezinsuku eziyi-13 (maphakathi) kungqimba lwe-epithelial.Bheka futhi Umfanekiso 3 Wedatha Enwetshiwe, ogqamisa ukusayinda kwe-LGR5 ngaphandle kophawu lwesakhiwo esincane se-MUC2D. i-organoid epithelium esungulwe emathunjini ku-chip ngokungcolisa ulwelwesi lwe-plasma ngodayi we-CellMask (kwesokudla) ngosuku lwe-13 lwesiko.Ibha yesikali ingu-50 μm ngaphandle kwalapho kuchazwe ngenye indlela.b Iphrintwe kabusha ngemvume evela kunkomba.2.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 enezimbobo ukuze kuphumelele i-ECM coating.Kule protocol, sisebenzisa izindlela ezimbili ezihlukene zokuguqula i-hydrophobicity yama-membrane e-PDMS.Ngokuhlakulela amaseli e-Caco-2, ukusebenza kwe-surface nge-UV/ozone ukwelashwa kuphela kwakwanele ukunciphisa i-hydrophobicity ye-CaCMPD kanye nokunamathisela i-membrane ye-CaCMPD noma kunjalo, isiko le-microfluidic le-organoid epithelium lidinga ukusebenza kwamakhemikhali okusekelwe kumakhemikhali ukuze kuzuzwe ukufakwa kahle kwamaprotheni e-ECM ngokusebenzisa ngokulandelana i-polyethyleneimine (PEI) kanye ne-glutaraldehyde kuma-microchannels e-PDMS. isiko liqala ngokufaka amakha kuphela okuphakathi ku-microchannel engenhla kuze kube yilapho amangqamuzana enza i-monolayer ephelele, kuyilapho i-microchannel ephansi igcina izimo ezimile.Le ndlela elungiselelwe yokwenza kusebenze indawo ephezulu kanye ne-ECM coating ivumela ukunamathiselwa kwe-organoid epithelium ukuze kubangele i-3D morphogenesis endaweni ye-PDMS.
Amasiko eTranswell nawo adinga ukumbozwa kwe-ECM ngaphambi kokutshala kweseli;Nokho, amasiko akwaTranswell awadingi izinyathelo zokwelashwa eziyinkimbinkimbi ukuze kusebenze ubuso bokufakwa kwezimbotshana.Ngokukhula kwamaseli e-Caco-2 ekufakweni kwe-Transwell, ukumbozwa kwe-ECM kuma-porous inserts kusheshisa ukunamathiselwa kwamaseli e-Caco-2 ahlukanisiwe (<1 ihora) kanye nokwakheka kwesithiyo esiqinile (<1-2 izinsuku) <3 h) futhi inakekelwe kuze kube yilapho ama-organoid enza i-monolayer ephelele ene-barrier integrity .Amasiko e-Transwell enziwa ngamapuleti 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 kuchazwe ngaphambilini, kubalulekile ukwethula ukugeleza kwe-secret or morphogenment ye-secret morphogenesis (ukususwa kwe-secret morphogenesis) ukukhishwa kwe-secret morphogenesis (ukususwa kwe-continuous fluid). Ukuhlinzeka ngemisoco eyanele kanye ne-serum emangqamuzaneni aboshwe kulwelwesi olunezimbotshana futhi akhiqize ukucindezelwa kwe-shear ukukhanya, ngokuvamile sisebenzisa ukugeleza okumbaxambili emathunjini ku-chip.Kuma-chips ayingxubevange, ukufakwa kwe-Transwell okuqukethe ama-monolayers e-epithelial afakwa kuma-chips ayi-hybrid. Ngemva kwalokho, okuphakathi kwasetshenziswa ngaphansi kohlangothi lwe-basolateral lwe-porous Transwell-insection ye-porous Transwell-insestination ye-microphorous 3 insestination ye-microphorous 3 insestination ye-microphorous. zombili izinkundla zamasiko.
Izici ze-morphological zezendlalelo ze-epithelial ze-3D ezincane ezine-microengineered zingahlaziywa ngokusebenzisa izindlela zokucabanga ezihlukahlukene, okuhlanganisa imakroskopi yokugqama kwesigaba, i-differial interference difference (DIC) microscopy, SEM, noma i-immunofluorescence confocal microscopy (Figure 3 kanye 4).Ukungafani kwesigaba noma imaging ye-DIC phakathi nenkathi ye-Duli ye-protrupision3D ingenziwa kalula. e ekukhanyeni kokubonakalayo 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, 3c, f no-4b, i-fixedt/hypovolde% ye-PF), amaseli alandelwa yi-Triftal/hyde ton X-100 kanye no-2% (wt/vol) ) i-bovine serum albumin (BSA), ngokulandelana.Ngokuya ohlotsheni lweseli, izinto ezilungiswayo ezihlukene, ama-permeabilizer, nama-blocking agents angasetshenziswa.Amasosha omzimba ayinhloko aqondise kumaseli ancike emugqeni noma omaka besifunda asetshenziselwa ukugqamisa amaseli anganyakaziswanga ku-situ ku-chip, i-counter-grade 6, i-counter-day i-diamidino-2-phenylene) indole, i-DAPI) noma i-F-actin (isb, ebhalwe nge-fluorescently phalloidin). Ukuthwebula bukhoma okusekelwe ku-Fluorescence nakho kungenziwa ku-situ ukuze kutholwe ukukhiqizwa kwamafinyila (Fig.I-1, "Ukuhlukaniswa kwamaseli" kanye "ne-Gut physiology"), ukwenziwa kwekoloni okungahleliwe kwamaseli e-microbial (Fig. 1, "Host-microbe co-culture"), ukubuthwa kwamangqamuzana omzimba omzimba (Fig. 1, 'Imodeli Yezifo') noma i-contours ye-3D epithelial morphology (Fig. 4bt ku-modifyc, uhlukanisa ungqimba lwe-epithelial kusuka ku-ungqimba we-3D kuya ku-gupper). ungqimba lwe-microchannel ephansi, njengoba kuchazwe ku-ref.Njengoba kuboniswe ku-Fig. 2, i-3D epithelial morphology kanye ne-microvilli emngceleni webhulashi le-apical ingabonwa nge-SEM (Fig. 3b) .Inkulumo yomaka bokuhlukanisa ingahlolwa ngokwenza i-quantitative PCR5 noma ama-chips e-hybrid yeseli eyodwa ye-RNA ama-chips e-hybrid ekhulayo amaseli e-RNA akhule. zivunwa nge-trypsinization bese zisetshenziselwa ukuhlaziywa kwamangqamuzana noma ufuzo.
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 ahlukene afakwa imbewu ekufakweni kwe-Transwell okulungiselelwe (i-TW prep; bheka umfanekiso ongezansi) .Uma amaseli e-seed afakwe ngaphansi kwezimo ze-polystatic afakwe ngaphansi kwezimo ze-polyseed (amaseli ahlanganisiwe) (isiko le-TW) .Ngemuva kwezinsuku ezingu-7, okufakiwe okukodwa kwe-Transwell okuqukethe i-2D monolayer yamaseli e-epithelial kwahlanganiswa ku-chip ye-hybrid ukuze kwethulwe ukugeleza kwe-basolateral (Flow, BL), okwaholela ekugcineni ekukhiqizeni ungqimba lwe-epithelial lwe-3D (morphogenesis) .Isigaba sokuqhathanisa ama-micrographs abonisa izici ze-morphological ze-morphological zamangqamuzana e-colon3 e-depice yesitho somuntu ngasinye) isinyathelo noma iphoyinti lesikhathi.I-schematics ezingqimbeni ezingaphezulu ibonisa ukucushwa kokuhlola kwesinyathelo ngasinye.b, Ama-chips e-Hybrid (uhlelo lwesokunxele) lungaholela ku-3D morphogenesis yamaseli e-organoid epithelial anokubukwa kwe-microscopy econfocal ephezulu ukuya phansi ethathwe ezindaweni ezahlukene Z (phezulu, maphakathi, nangaphansi;bheka isikimu esingakwesokudla nemigqa enamachashazi ehambisanayo).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 engaphakathi kwebhokisi elidashi elimhlophe) ngokumelene ne-hybrid chip (ishothi enkulu kunazo zonke egcwele) ngokuqhathanisa i-3D pairivevertical morphus2 ukubukwa (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 zingalungiswa ngokuhlakulela amangqamuzana afanayo (i-Caco-2 noma amaseli e-organoid epithelial yamathumbu) abe ama-monolayers amabili-dimensional ngaphansi kwezimo ezivamile zesiko le-static. Ngokuphawulekayo, ukuncipha komsoco kungase kubangele ngenxa yomthamo wevolumu elinganiselwe yama-microchannels (okungukuthi ~4 µL kusiteshi esiphezulu esiteshini sokuqala se-gut-chippithe kanye ne-bemorphology yohlelo lokusebenza ngaphambi kokuqhathaniswa nomklamo we-bemorphology we-belateral.
Inqubo ye-lithography ethambile kufanele yenziwe ekamelweni elihlanzekile.Kusendlalelo ngasinye ku-chip (izendlalelo ezingaphezulu nezingaphansi nolwelwesi) nama-chips ayingxube, ama-photomasks ahlukene asetshenziswa futhi enziwa kumawafa e-silicon ahlukene ngenxa yokuthi ubude bama-microchannel babuhlukile.Ukuphakama okuhlosiwe kwama-microchannels angaphezulu naphansi e-gut ku-chip angu-500 chip µubude obuyingxube ye-chipµ ngokulinganayo yi-chipµmµ ngokulinganayo. 200µm.
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 sidala ukushisa.Izinyathelo zokuphepha ezengeziwe ziyadingeka.Ekulahleni kukadoti, vumela isixazululo ukuthi siphole futhi sidluliselwe esitsheni semfucumfucu esihlanzekile, esomile.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 kuka-5–10 s ukuze isakazeke ku-500 rpm ngokusheshisa okungu-100 rpm/s. Setha i-main spin ka-200 µm ukujiya kwephethini ngo-1,500m³ 2pm m ubude bongqimba olungaphezulu lwamathumbu ku-chip; bona "Izinyathelo ezibucayi" ngezansi) zibekwe ngokusheshisa okungu-300 rpm/s imizuzwana engu-30 ku-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 kushiphu, ukujiya kwe-spin nezinyathelo zokubhaka ezithambile zaleli Bhokisi (izinyathelo 7 no-8) ziphindwe ngokulandelana (bona isinyathelo 9) ukuze kukhiqizwe izendlalelo ezimbili zika-250 µm Ungqimba oluwugqinsi lwe-SU-8, olungakwazi ukuvezwa ungqimba ongu-5 we-UV2 ngebhokisi elingu-0 futhi lihlanganiswe ngu-0 .
Bhaka ama-wafers 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, bese iwafa ebhakiwe ibekwe esitsheni.Ivolumu yonjiniyela we-SU-8 ingahluka kuye ngosayizi wepuleti lengilazi.Qiniseka ukuthi usebenzisa unjiniyela we-SU-8 owanele ukususa ngokuphelele i-SU-8.Isibonelo, uma usebenzisa isitsha sengilazi esingu-150 mm esinomthamo ongu-1 L, sebenzisa u-~300 wonjiniyela we-rold amaminithi angu-300 we-development emnene. .
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, ukucindezela okuhlosiwe kwe-1 × 10-5 Torr, amandla angu-125 W) ku-1.5 min.
Faka i-wafer ku-vacuum desiccator ene-slide yengilazi 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 i-100 μL ye-trichlor1oct,Hylfluox,H2H, ingilazi ye-trichloroct,H2H shelela 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.