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Ukwakheka kwamathumbu omuntu kusungula izici ze-crypt-villus ze-3D epithelial microarchitecture kanye nokuhleleka kwendawo. Lesi sakhiwo esiyingqayizivele siyadingeka ukuze kugcinwe i-homeostasis yamathumbu ngokuvikela i-niche yeseli le-stem ku-basal crypt kuma-antigen angaphandle amagciwane kanye nama-metabolites awo. Ngaphezu kwalokho, i-intestinal villi kanye ne-secreting mucus ziveza amaseli e-epithelial ahlukene ngokusebenza ngesithiyo esivikelayo ebusweni be-mucosal yamathumbu. Ngakho-ke, ukudala kabusha izakhiwo ze-epithelial ze-3D kubalulekile ekwakhiweni kwamamodeli e-in vitro gut. Okuphawulekayo ukuthi i-organic mimetic gut-on-a-chip ingadala i-3D morphogenesis ezenzakalelayo ye-intestinal epithelium ngemisebenzi ethuthukisiwe yomzimba kanye ne-biomechanics. Lapha, sinikeza inqubo ephindaphindwayo yokudala i-intestinal morphogenesis enamandla emathunjini ku-microfluidic chip kanye naku-Transwell embedded hybrid chip. Sichaza izindlela ezinemininingwane zokwenza idivayisi, ukukhulisa amaseli e-Caco-2 noma e-intestinal organoid epithelial ezindaweni ezivamile kanye nasepulatifomu ye-microfluidic, ukuqaliswa kwe-3D morphogenesis, kanye Ukucaciswa kwe-epithelia ye-3D esungulwe kusetshenziswa izindlela eziningi zokuthwebula izithombe. Le phrothokholi ifinyelela ukuvuselelwa kwe-microarchitecture yamathumbu esebenzayo ngokulawula ukugeleza koketshezi lwe-basolateral imizuzu emi-5. Indlela yethu ye-in vitro morphogenesis isebenzisa ukucindezeleka kokugunda okuhlobene nempilo kanye nokunyakaza komshini futhi ayidingi ubunjiniyela bamaseli obuyinkimbinkimbi noma ukuphathwa kabi, okungase kusebenze kangcono kunezinye izindlela ezikhona. Sibona ukuthi iphrothokholi yethu ehlongozwayo ingaba nemiphumela ebanzi emphakathini wocwaningo lwezokwelapha, inikeze indlela yokuvuselela izendlalelo ze-epithelial zamathumbu ze-3D in vitro zezicelo zezokwelapha, zezokwelapha, nezemithi.
Ukuhlolwa kubonisa ukuthi amaseli e-epithelial Caco-2 amathumbu akhuliswe kumadivayisi e-gut-on-a-chip1,2,3,4,5 noma ama-bilayer microfluidic6,7 angadlula kwi-3D morphogenesis ngokuzenzakalelayo ngaphandle kokuqonda okucacile kwendlela eyisisekelo. Ocwaningweni lwethu lwakamuva, sithole ukuthi ukususwa kwe-basolateral morphogen antagonists ekhishwe yi-basolaterally kumadivayisi okukhuliswa kuyadingeka futhi kwanele ukuvusa i-3D epithelial morphogenesis in vitro, okuye kwaboniswa yi-Caco-2 kanye nama-organoids amathumbu atholakala esigulini. Amaseli e-Epithelial aqinisekiswe. Kulolu cwaningo, sigxile kakhulu ekukhiqizweni kwamaseli kanye nokusatshalaliswa kokuhlushwa kwe-Wnt antagonist enamandla, i-Dickkopf-1 (DKK-1), kumadivayisi e-gut-on-a-chip kanye namadivayisi e-microfluidic aguquliwe aqukethe i-Transwell inserts, ebizwa ngokuthi "i-Hybrid Chip". Sibonisa ukuthi ukungezwa kwama-Wnt antagonists angaphandle (njenge-DKK-1, i-Wnt repressor 1, i-protein 1 ehlobene ne-frizzled, noma i-Soggy-1) ku-on-chip gut kuvimbela i-morphogenesis noma kuphazamisa ungqimba lwe-epithelial lwe-3D olwakhiwe ngaphambili, okuphakamisa ukuthi ukucindezeleka okuphikisayo ngesikhathi sokulima kuyisisusa se-intestinal morphogenesis in vitro. Ngakho-ke, indlela esebenzayo yokufeza i-morphogenesis eqinile ku-epithelial interface ukususa noma ukugcina amazinga ama-Wnt antagonists engxenyeni ye-basolateral ngokuwasha okusebenzayo (isb., kumapulatifomu e-gut-on-a-chip noma e-hybrid-on-a-chip) noma ukusabalala. Imidiya ye-Basolateral (isb., evela ku-Transwell ifaka ezindaweni zokugcina ezinkulu ze-basolateral in imithombo).
Kule phrothokholi, sinikeza indlela eningiliziwe yokwakha ama-microdevice e-gut-on-a-chip kanye nama-Transwell-insertable hybrid chips (izinyathelo 1-5) ukuze kukhuliswe amaseli e-epithelial emathunjini kuma-membrane anezimbobo asekelwe ku-polydimethylsiloxane (PDMS) (izinyathelo 6A, 7A, 8, 9) noma ama-membrane e-polyester e-Transwell inserts (izinyathelo 6B, 7B, 8, 9) kanye ne-induced morphogenesis ye-3D in vitro (isinyathelo 10). Siphinde sathola izici zamaseli nama-molecule ezibonisa i-histogenesis ethile yezicubu kanye nokuhlukaniswa kwamaseli okuncike ku-lineage ngokusebenzisa izindlela eziningi zokuthwebula izithombe (izinyathelo 11-24). Sibangela i-morphogenesis sisebenzisa amaseli e-epithelial emathunjini omuntu, njenge-Caco-2 noma ama-organoids emathunjini, ngezindlela ezimbili zokukhulisa ezinemininingwane yobuchwepheshe efaka ukuguqulwa kwendawo yama-membrane anezimbobo, ukudalwa kwama-monolayers e-2D, kanye ne-biochemical yamathumbu kanye nokukhiqizwa kabusha kwe-biomechanical microenvironment.in vitro.Ukukhuthaza i-morphogenesis ye-3D kusuka ku-2D epithelial ama-monolayers, sisuse ama-morphogen antagonists kuzo zombili izinhlobo ezikhuliswe ngokugeleza i-medium iye engxenyeni ye-basolateral yesiko. Okokugcina, sinikeza umfanekiso wokusetshenziswa kwesendlalelo se-epithelial se-3D esivuselelekayo esingasetshenziswa ukukhombisa ukukhula kwe-epithelial okuncike ku-morphogen, ukukhuliswa kwe-co-cultures ye-host-microbiome ende, ukutheleleka kwe-pathogen, ukulimala kokuvuvukala, ukungasebenzi kahle kwesithiyo se-epithelial, kanye nemithi yokwelapha esekelwe kuma-probiotic. Ithonya.
Iphrothokholi yethu ingaba usizo kososayensi abaningi abahlukahlukene (isib., i-intestinal mucosal biology, i-stem cell biology, kanye ne-developmental biology) kanye nocwaningo olusetshenzisiwe (isib., ukuhlolwa kwemithi ngaphambi kokwelashwa, ukumodela izifo, ubunjiniyela bezicubu, kanye ne-gastroenterology) umthelela obanzi. Ngenxa yokuphindaphindeka kanye nokuqina kwephrothokholi yethu yokudala i-3D morphogenesis ye-intestinal epithelium in vitro, sicabanga ukuthi isu lethu lobuchwepheshe lingasakazwa ezilalelini ezifunda ukuguquguquka kokubonakaliswa kwamaseli ngesikhathi sokuthuthukiswa kwamathumbu, ukuvuselelwa noma i-homeostasis. Ngaphezu kwalokho, iphrothokholi yethu iwusizo ekuphenyweni kokutheleleka ngaphansi kwezidakamizwa ezahlukahlukene ezithelelanayo njenge-Norovirus 8, i-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), i-Clostridium difficile, i-Salmonella Typhimurium 9 noma i-Vibrio cholerae. Izilaleli zezifo kanye ne-pathogenesis nazo ziwusizo. Ukusetshenziswa kohlelo lwe-microphysiology yamathumbu olusebenzisa i-on-chip kungavumela ukukhuliswa kwe-longitudinal co-culture 10 kanye nokuhlolwa okulandelayo kokuzivikela komsingathi, izimpendulo zomzimba kanye nokulungiswa kokulimala okuhlobene ne-pathogen endleleni yesisu (GI) 11. Ezinye izifo ze-GI ezihlobene ne-leaky gut syndrome, isifo se-celiac, isifo sikaCrohn, i-ulcerative colitis, i-pouchitis, noma i-irritable bowel syndrome zingalingiswa lapho izingqimba ze-epithelial zamathumbu ze-3D zilungiswa kusetshenziswa izingqimba ze-epithelial zamathumbu zesiguli ze-3D, lezi zifo zifaka phakathi i-villous atrophy, ukufinyeza kwe-crypt, umonakalo we-mucosal, noma i-epithelial barrier engasebenzi kahle. Ama-organoids amathumbu asuselwa ku-Biopsy noma i-stem cell12,13. Ukuze kuboniswe kangcono ubunzima obuphezulu bendawo yesifo, abafundi bangacabanga ngokungeza izinhlobo zamaseli ahlobene nesifo, njengamaseli egazi angaphandle kwesiguli (ama-PBMC), kumamodeli aqukethe ama-microarchitecture e-3D intestinal villus-crypt. amaseli omzimba aqondene nezicubu, 5.
Njengoba isakhiwo se-epithelial se-3D singalungiswa futhi sibonwe ngaphandle kwenqubo yokuhlukanisa, ababukeli abasebenza ku-spatial transcriptomics kanye ne-high-resolution noma i-super-resolution imaging bangaba nentshisekelo ekudwebeni kwethu kwe-spatiotemporal dynamics yezakhi zofuzo namaprotheni kuma-epithelial niches. Banentshisekelo kubuchwepheshe. Impendulo ku-microbial noma i-immune stimuli. Ngaphezu kwalokho, i-longitudinal host-microbiome crosstalk 10, 14 ehlanganisa i-gut homeostasis ingasungulwa ku-3D intestinal mucosal layer ngokukhulisa izinhlobo ezahlukene ze-microbial, imiphakathi ye-microbial noma i-fecal microbiota, ikakhulukazi ku-gut-on-a-chip. epulatifomu. Le ndlela ikhanga kakhulu izilaleli ezifunda nge-immunology ye-mucosal, i-gastroenterology, i-human microbiome, i-culturomics kanye ne-microbiology yezokwelapha ezifuna ukutshala i-gut microbiota engakakhuliswa ngaphambili elabhorethri. Uma inqubo yethu ye-in vitro morphogenesis ingashintshwa ukuze ifane nezakhiwo zesiko ezikhula ngokushesha, njenge-multiwell inserts kuma-plate angu-24, 96 noma angu-384 agcwalisa njalo izingxenye ze-basolateral, le nqubo ingasatshalaliswa nakulabo abathuthukisa amapulatifomu ezokwelapha, ezokwelapha noma e-high-throughput screening noma okuqinisekisa embonini yokudla. Njengobufakazi besimiso, muva nje sibonise ukuthi kungenzeka yini uhlelo lwe-multiplex high-throughput morphogenesis olukhula ngokushesha lube yifomethi ye-plate engu-24. Ngaphezu kwalokho, imikhiqizo eminingi ye-organ-on-a-chip iye yathengiswa16,17,18. Ngakho-ke, ukuqinisekiswa kwendlela yethu ye-in vitro morphogenesis kungasheshiswa futhi kusetshenziswe ama-laboratory amaningi ocwaningo, imboni noma uhulumeni kanye nezinhlangano ezilawulayo ukuqonda ukuhlelwa kabusha kwamaseli kwe-in vitro gut morphogenesis ezingeni le-transcriptomic ukuhlola izidakamizwa noma i-biotherapeutics Ukumuncwa kanye nokuthuthwa kwezidakamizwa ezingasetshenziswa kuhlolwe kusetshenziswa izilinganiso ze-3D gut noma kusetshenziswa amamodeli enziwe ngokwezifiso noma ezentengiselwano eziphathelene nezitho ukuze kuhlolwe ukuphindaphindeka kwenqubo yokwakheka kwamathumbu.
Inani elilinganiselwe lamamodeli okuhlola ahlobene nabantu asetshenziswe ukutadisha i-intestinal epithelial morphogenesis, ikakhulukazi ngenxa yokuntuleka kwezinqubo ezisebenzayo zokubangela i-3D morphogenesis in vitro. Eqinisweni, ulwazi oluningi lwamanje mayelana ne-intestine morphogenesis lusekelwe ezifundweni zezilwane (isb. zebrafish20, mice21 noma izinkukhu22). Kodwa-ke, zisebenza kanzima futhi zibiza kakhulu, zingaba nemibuzo ngokuziphatha, futhi okubaluleke kakhulu, azinqumi ngokunembile izinqubo zokuthuthukiswa komuntu. Lawa mamodeli nawo alinganiselwe kakhulu emandleni awo okuhlolwa ngendlela ebanzi. Ngakho-ke, inqubo yethu yokuvuselela izakhiwo zezicubu ze-3D in vitro isebenza kahle kakhulu kumamodeli ezilwane e-in vivo kanye namanye amamodeli endabuko e-static 2D cell culture. Njengoba kuchaziwe ngaphambili, ukusebenzisa izakhiwo ze-3D epithelial kusivumele ukuthi sihlole indawo yamaseli ahlukene ku-crypt-villus axis ukuphendula ezicini ezahlukahlukene ze-mucosal noma immune. Izingqimba ze-3D epithelial zinganikeza isikhala sokutadisha ukuthi amaseli e-microbial ancintisana kanjani ukwakha izindawo zesikhala kanye nokuvela kwemvelo ekuphenduleni izici zomsingathi (isb., inner versus izingqimba zangaphandle ze-mucus, ukukhiqizwa kwe-IgA kanye nama-peptide alwa namagciwane).Ngaphezu kwalokho, i-3D epithelial morphology ingasenza siqonde ukuthi i-gut microbiota yakha kanjani imiphakathi yayo futhi ikhiqize ngokusebenzisana ama-metabolites amagciwane (isb., ama-fatty acid amafushane) akha ukuhlelwa kwamaseli kanye nama-niches e-stem cell kuma-basal crypts.Lezi zici zingabonakala kuphela uma izingqimba ze-3D epithelial zisungulwa ku-vitro.
Ngaphezu kwendlela yethu yokwakha izakhiwo ze-epithelial zamathumbu ze-3D, kunezindlela eziningana ze-in vitro. Isiko le-organoid lamathumbu liyindlela yobunjiniyela bezicubu yesimanje esekelwe ekutshalweni kwamaseli okuqala amathumbu ngaphansi kwezimo ezithile ze-morphogen23,24,25. Kodwa-ke, ukusetshenziswa kwamamodeli e-organoid e-3D okuhlaziya ezokuthutha noma ama-co-cultures e-host-microbiome kuvame ukuba yinselele ngoba i-lumen yamathumbu ivalwe ngaphakathi kwe-organoid futhi, ngakho-ke, ukwethulwa kwezingxenye ze-luminal ezifana namaseli e-microbial noma ama-antigen angaphandle kunqunyelwe. Ukufinyelela kuma-lumens e-organoid kungathuthukiswa kusetshenziswa i-microinjector,26,27 kodwa le ndlela iyahlasela futhi ifuna umsebenzi omningi futhi idinga ulwazi olukhethekile ukuze isebenze. Ngaphezu kwalokho, amasiko e-organoid endabuko agcinwe kuma-scaffolds e-hydrogel ngaphansi kwezimo ezimile awabonakali kahle ama-biomechanics asebenzayo e-vivo.
Ezinye izindlela ezisetshenziswa amaqembu amaningana ocwaningo zisebenzisa ama-scaffolds e-3D hydrogel ahlelwe kusengaphambili ukulingisa isakhiwo se-epithelial yamathumbu ngokukhulisa amangqamuzana amathumbu abantu ahlukaniswe phezu kwejeli. Yakha ama-scaffolds e-hydrogel usebenzisa isikhunta esiphrintiwe nge-3D, esigayiwe nge-micro-milled, noma esenziwe nge-lithographically. Le ndlela ikhombisa ukuhlelwa okuhleliwe kwamaseli e-epithelial ahlukaniswe ngaphakathi kwe-vitro ane-morphogen gradients efanelekile ngokomzimba, okwakha isakhiwo se-epithelial esinobukhulu obuphezulu kanye ne-stroma-epithelial crosstalk ngokufaka amaseli e-stromal ku-scaffold. Kodwa-ke, uhlobo lwama-scaffolds ahlelwe kusengaphambili lungavimbela ukubonakaliswa kwenqubo ye-morphogenetic ezenzakalelayo ngokwayo. Lawa mamodeli awahlinzeki ngokugeleza kwe-luminal noma kwe-interstitial, engenawo ukucindezeleka koketshezi okudingeka amaseli amathumbu adlule ku-morphogenesis futhi athole umsebenzi womzimba. Olunye ucwaningo lwamuva lusebenzise ama-scaffolds e-hydrogel epulatifomu ye-microfluidic kanye nezakhiwo ze-epithelial zamathumbu ezinamaphethini kusetshenziswa amasu okugoba nge-laser. Ama-organoids amathumbu egundane alandela amaphethini aqoshiwe ukwakha izakhiwo ze-tubular zamathumbu, futhi ukugeleza koketshezi lwangaphakathi kungenza kufanele kuphindwe kabili kusetshenziswa imodyuli ye-microfluidics. Kodwa-ke, le modeli ayibonisi izinqubo ze-spontaneous morphogenetic futhi ayifaki ukunyakaza kwe-gut mechanobiological. Amasu okuphrinta e-3D avela eqenjini elifanayo akwazile ukudala amashubhu amancane e-gut anezinqubo ze-spontaneous morphogenetic. Naphezu kokwakhiwa okuyinkimbinkimbi kwezingxenye ezahlukene ze-gut ngaphakathi kwe-tube, le modeli ayinakho ukugeleza koketshezi lwe-luminal kanye nokuguqulwa kwemishini. Ngaphezu kwalokho, ukusebenza kwemodeli kungancishiswa, ikakhulukazi ngemuva kokuba inqubo ye-bioprinting isiphelile, okuphazamisa izimo zokuhlola noma ukusebenzisana kweseli neseli. Esikhundleni salokho, iphrothokholi yethu ehlongozwayo inikeza i-spontaneous gut morphogenesis, ukucindezeleka kwe-shear okuhlobene nempilo, i-biomechanics elingisa ukuhamba kwe-gut, ukufinyeleleka kwezingxenye ezizimele ze-apical kanye ne-basolateral, kanye nokudalwa kabusha kwe-microenvironments eziyinkimbinkimbi ze-biological ze-modularity. Ngakho-ke, iphrothokholi yethu ye-in vitro morphogenesis ye-3D inganikeza indlela ehambisanayo yokunqoba izinselele zezindlela ezikhona.
Iphrothokholi yethu igxile ngokuphelele ku-3D epithelial morphogenesis, ngamaseli e-epithelial kuphela akhona emasikweni futhi azikho ezinye izinhlobo zamaseli azungezile njengamaseli e-mesenchymal, amaseli e-endothelial, namaseli omzimba. Njengoba kuchaziwe ngaphambili, umongo wephrothokholi yethu ukungeniswa kwe-epithelial morphogenesis ngokususa ama-morphogen inhibitors akhishwe ohlangothini lwe-basolateral lwe-medium eyethulwe. Ngenkathi i-modularity eqinile ye-gut-on-a-chip yethu kanye ne-hybrid-on-a-chip isenza sikwazi ukuphinda sakhe ungqimba lwe-3D epithelial oluhambayo, ubunzima obengeziwe bezinto eziphilayo njengokuxhumana kwe-epithelial-mesenchymal33,34, i-extracellular Matrix (ECM) deposition35 kanye, kumodeli yethu, izici ze-crypt-villus ezidlulisa ama-niches eseli le-stem kuma-crypt ayisisekelo zisazocatshangelwa kabanzi. Amaseli e-stromal (isb., ama-fibroblast) ku-mesenchyme adlala indima ebalulekile ekukhiqizweni kwamaprotheni e-ECM kanye nokulawulwa kwe-intestinal morphogenesis ku-vivo35,37,38. Ukwengezwa kwamaseli e-mesenchymal Kumodeli yethu kuthuthukise inqubo ye-morphogenetic kanye nokusebenza kahle kokunamathela kweseli. Ingqimba ye-endothelial (okungukuthi, ama-capillary noma ama-lymphatic) idlala indima ebalulekile ekulawuleni ukuthuthwa kwama-molecule39 kanye nokuqashwa kwamaseli omzimba40 endaweni encane yamathumbu. Ngaphezu kwalokho, izingxenye ze-vasculature ezingaxhunyaniswa phakathi kwamamodeli wezicubu ziyimfuneko lapho amamodeli wezicubu eklanyelwe ukubonisa ukusebenzisana kwezitho eziningi. Ngakho-ke, amaseli e-endothelial kungadingeka afakwe ukuze abonise izici zomzimba ezinembile kakhulu ngesisombululo sezinga lezitho. Amaseli omzimba asuselwa esigulini nawo abalulekile ekuboniseni izimpendulo zamasosha omzimba zangaphakathi, ukwethulwa kwe-antigen, i-innate adaptive immune crosstalk, kanye ne-tissue-specific amasosha omzimba kumongo wokulingisa isifo samathumbu.
Ukusetshenziswa kwama-hybrid chips kulula kakhulu kune-gut-on-a-chip ngoba ukusethwa kwedivayisi kulula futhi ukusetshenziswa kwama-Transwell inserts kuvumela ukukhuliswa kwe-epithelium yamathumbu okungakhuliswa. Kodwa-ke, ama-Transwell inserts atholakala kwezentengiselwano ane-polyester membranes awanwebeka futhi awakwazi ukulingisa ukunyakaza okufana ne-peristaltic. Ngaphezu kwalokho, ingxenye ye-apical ye-Transwell insert ebekwe ku-hybrid chip yahlala imile ngaphandle kokucindezeleka kwe-shear ohlangothini lwe-apical. Ngokusobala, izakhiwo ezimile engxenyeni ye-apical azivami ukuvumela ukukhuliswa kwe-bacterial co-culture yesikhathi eside kuma-hybrid chips. Ngenkathi singadala ngamandla i-3D morphogenesis kuma-Transwell inserts lapho sisebenzisa ama-hybrid chips, ukuntuleka kwama-biomechanics ahlobene nempilo kanye nokugeleza koketshezi lwe-apical kunganciphisa ukusebenziseka kwamapulatifomu e-hybrid chip ezinhlelo zokusebenza ezingaba khona.
Ukwakhiwa kabusha okugcwele kwe-axis ye-crypt-villus yomuntu emathunjini amancane kanye nakwa-hybrid-on-a-chip akukaqinisekiswa ngokugcwele. Njengoba i-morphogenesis iqala kusuka ku-epithelial monolayer, ukwakheka kwama-microarchitecture e-3D akunikezi ngempela ukufana kwe-morphological nama-crypts in vivo. Nakuba sichaze inani lamaseli akhulayo eduze kwesizinda se-basal crypt ku-epithelium ye-3D engineered microengineered, izindawo ze-crypt kanye nezindawo ze-villous azizange zihlukaniswe ngokucacile. Nakuba iziteshi eziphezulu eziphezulu ku-chip ziholela ekuphakameni okuphezulu kwe-epithelium engineered microengineered, ukuphakama okuphezulu kusalinganiselwe ku-~300–400 µm. Ukujula kwangempela kwama-crypts emathunjini amancane namakhulu ku-~135 µm kanye no-~400 µm, ngokulandelana, kanti ukuphakama kwe-villi yamathumbu amancane ku-~600 µm41.
Ngokombono wezithombe, izithombe ze-in situ super-resolution ze-3D microarchitectures zingase zikhawulelwe emathunjini ku-chip, njengoba ibanga lokusebenza elidingekayo ukusuka kulensi eqondiwe kuya ku-epithelial layer lingamamilimitha ambalwa. Ukuze kunqotshwe le nkinga, kungadingeka i-objective ekude. Ngaphezu kwalokho, ukwenza izingxenye ezincane zokulungiselela isampula yezithombe kuyinselele ngenxa yokuqina okuphezulu kwe-PDMS. Ngaphezu kwalokho, njengoba i-microfabrication ye-layer-by-layer ye-gut ku-chip ihilela ukunamathela okuhlala njalo phakathi kwe-layer ngayinye, kuyinselele kakhulu ukuvula noma ukususa i-layer ephezulu ukuze kuhlolwe isakhiwo sobuso be-epithelial layer. Isibonelo, ngokusebenzisa i-scanning electron microscope (SEM).
Ukungathandi amanzi kwe-PDMS kube yisici esivimbelayo ezifundweni ezisekelwe ku-microfluidic ezibhekene nama-molecule amancane angathandi amanzi, njengoba i-PDMS ingamunca ama-molecule anjalo angathandi amanzi. Ezinye izindlela ze-PDMS zingacatshangelwa nezinye izinto ze-polymeric. Ngaphandle kwalokho, ukuguqulwa kwendawo ye-PDMS (isb., ukumbozwa ngezinto ze-lipophilic 42 noma i-poly(ethylene glycol) 43) kungacatshangelwa ukunciphisa ukumuncwa kwama-molecule angathandi amanzi.
Okokugcina, indlela yethu ayizange ichazwe kahle ngokuhlinzeka ngokuhlolwa okuphezulu noma ipulatifomu yokuhlola "efanela bonke ngobukhulu obufanayo". Iphrothokholi yamanje idinga ipompo yesirinji ngedivayisi encane, ethatha isikhala ku-incubator ye-CO2 futhi ivimbele ukuhlolwa okukhulu. Lo mkhawulo ungathuthukiswa kakhulu ngokukhula kwefomethi yesiko elisha (isb., i-inserts enezimbobo ezingu-24, i-96, noma i-384-well evumela ukugcwaliswa okuqhubekayo nokususwa kwemidiya ye-basolateral).
Ukuze sibangele ukwakheka kwe-3D kwe-epithelium yamathumbu omuntu nge-vitro, sisebenzise idivayisi yamathumbu ye-chip ye-microfluidic equkethe iziteshi ezimbili ezihambisanayo kanye ne-membrane enezimbobo ezinwebekayo phakathi ukuze sakhe i-lumen-capillary interface. Siphinde sibonise ukusetshenziswa kwedivayisi ye-microfluidic yesiteshi esisodwa (i-chip ye-hybrid) enikeza ukugeleza okuqhubekayo kwe-basolateral ngaphansi kwezingqimba ze-epithelial ezihlanganisiwe ezikhuliswe kuma-Transwell inserts. Kuzo zombili izinkundla, ukwakheka kwamaseli e-epithelial amathumbu omuntu ahlukahlukene kungabonakaliswa ngokusebenzisa ukuphathwa kokuqondisa kokugeleza ukuze kususwe abaphikisi be-morphogen engxenyeni ye-basolateral. Inqubo yonke yokuhlola (Isithombe 1) iqukethe izingxenye ezinhlanu: (i) ukwakheka kwe-chip yamathumbu noma i-chip ye-hybrid efakwe yi-Transwell (izinyathelo 1-5; Ibhokisi 1), (ii) ukulungiswa kwamaseli e-epithelial yamathumbu (amaseli e-Caco-2) noma ama-organoids amathumbu omuntu; amabhokisi 2-5), (iii) ukukhuliswa kwamaseli e-epithelial emathunjini kuma-chip emathunjini noma kuma-chip ahlanganisiwe (izinyathelo 6-9), (iv) ukuqaliswa kwe-morphogenesis ye-3D in vitro (isinyathelo 10) kanye (v) ) ukuze kuchazwe isakhiwo esincane se-epithelial se-3D (izinyathelo 11-24). Okokugcina, iqembu lokulawula elifanele (okuxoxwe ngalo ngezansi) laklanywa ukuqinisekisa ukusebenza kahle kwe-morphogenesis in vitro ngokuqhathanisa i-morphogenesis ye-epithelial nezilawuli zendawo, zesikhathi, ezinemibandela, noma zenqubo.
Sisebenzise amapulatifomu amabili ahlukene okukhulisa: i-gut-on-a-chip eneziteshi eziqondile noma iziteshi ezihlanganisiwe ezingezona eziqondile, noma ama-hybrid chips aqukethe i-Transwell (TW) inserts kudivayisi ye-microfluidic, eyenziwe njengoba kuchaziwe ku-Box 1, kanye nesinyathelo 1 -5. "Ukwakhiwa Kwedivayisi" kukhombisa izinyathelo eziyinhloko zokwenza i-chip eyodwa noma i-hybrid chip. "Isiko Lamaseli Epithelial Amathumbu Abantu" kuchaza umthombo weseli (i-Caco-2 noma ama-organoids amathumbu abantu) kanye nenqubo yokukhulisa esetshenziswa kule protocol. "I-in vitro morphogenesis" kukhombisa izinyathelo eziphelele lapho amaseli e-epithelial asuselwa ku-Caco-2 noma ama-organoids asuselwa ku-organoid akhuliswa khona ku-chip yamathumbu noma ku-Transwell inserts ye-chip ehlanganisiwe, kulandelwa ukuqaliswa kwe-morphogenesis ye-3D kanye nokwakheka kwesakhiwo se-epithelial esibonakalayo. Inombolo yesinyathelo sohlelo noma inombolo yebhokisi iboniswa ngaphansi komcibisholo ngamunye. Uhlelo lokusebenza lunikeza izibonelo zendlela izingqimba ze-epithelial zamathumbu ezisungulwe zingasetshenziswa ngayo, isibonelo, ekuchazeni umehluko wamaseli, izifundo ze-physiology yamathumbu, ukusungulwa kwezindawo zemvelo ze-host-microbiome, kanye nezifo. ukumodela. Izithombe ze-immunofluorescence ku-"Cell Differentiation" ezibonisa i-nuclei, i-F-actin kanye ne-MUC2 ezivezwe kungqimba lwe-epithelial lwe-3D Caco-2 olukhiqizwe ku-gut chip. I-MUC2 signaling ikhona kuma-goblet cells kanye ne-mucus ekhishwe ezindaweni ze-mucosal. Izithombe ze-fluorescent ku-Gut Physiology zibonisa i-mucus ekhiqizwa ngokudaya i-sialic acid kanye ne-N-acetylglucosamine residues kusetshenziswa i-fluorescent wheat germ agglutinin. Izithombe ezimbili ezihambisanayo ku-"Host-Microbe Co-Cultures" zibonisa ama-co-cultures amele i-host-microbiome emathunjini ku-chip. Iphaneli yesobunxele ibonisa i-co-culture ye-E. coli eveza iphrotheni eluhlaza okwesibhakabhaka (i-GFP) enamaseli e-epithelial e-3D Caco-2 akhiwe nge-microengineered. Iphaneli yesokudla ibonisa indawo ye-GFP E. coli ekhuliswe ngamaseli e-epithelial e-3D Caco-2, kulandelwe i-immunofluorescence staining ene-F-actin (ebomvu) kanye ne-nuclei (eluhlaza okwesibhakabhaka). Ukumodela kwesifo kubonisa i-gut enempilo uma kuqhathaniswa nokuvuza emathunjini. Ama-chip okuvuvukala ngaphansi kwenselele yomzimba ngama-antigen ebhaktheriya (isb., i-lipopolysaccharide, i-LPS) kanye namaseli omzimba (isb., i-PBMC; eluhlaza). Amaseli e-Caco-2 akhuliswa ukuze kusungulwe ungqimba lwe-epithelial lwe-3D. Ibha yesikali, 50 µm. Izithombe emgqeni ongezansi: “Ukwehlukaniswa kwamaseli” kulungiswe ngemvume evela kunkomba.2. I-Oxford University Press; Kukhiqizwe kabusha ngemvume evela kunkomba.5. I-NAS; “I-Host-Microbe Co-Culture” kulungiswe ngemvume evela kunkomba.3. I-NAS; “Imodeli Yezifo” kulungiswe ngemvume evela kunkomba.5. I-NAS.
Kokubili ama-gut-on-chip kanye nama-hybrid chips enziwe kusetshenziswa ama-replica e-PDMS akhishwe kuma-silicon molds nge-soft lithography1,44 futhi adwetshwa nge-SU-8. Umklamo wama-microchannels ku-chip ngayinye unqunywa ngokubheka ama-hydrodynamics afana nokucindezeleka kwe-shear kanye ne-hydrodynamic pressure1,4,12. Umklamo wokuqala we-gut-on-a-chip (Extended Data Fig. 1a), owawuneziteshi ezimbili eziqondile ezihambisanayo, uguquke waba yi-gut-on-a-chip eyinkimbinkimbi (Extended Data Fig. 1b) ehlanganisa iziteshi ezimbili ezigobile ukuze kubangele isikhathi sokuhlala koketshezi, amaphethini okugeleza okungeyona i-linear, kanye nokuguqulwa kwe-multiaxial kwamaseli akhuliswe (Fig. 2a–f) 12. Lapho i-biomechanics yamathumbu eyinkimbinkimbi idinga ukwenziwa kabusha, ama-gut-on-a-chips ayinkimbinkimbi angakhethwa. Sikhombisile ukuthi i-convoluted Gut-Chip ikhuthaza kakhulu i-3D morphogenesis ngesikhathi esifanayo ngezinga elifanayo lokukhula kwe-epithelial uma kuqhathaniswa neyokuqala. I-Gut-Chip, kungakhathaliseki uhlobo lweseli elikhuliswe. Ngakho-ke, ukuze kubangele ukwakheka kwe-3D, ukwakheka kwesisu okuqondile nokuyinkimbinkimbi kwe-on-chip kuyashintshana. Ama-replica e-PDMS alashwe ezikhumbeni ze-silicon ngamaphethini e-SU-8 anikeze izici ezingezinhle ngemva kokudilizwa (Isithombe 2a). Ukuze kwakhiwe i-gut ku-chip, ungqimba oluphezulu lwe-PDMS olulungisiwe lwahlanganiswa ngokulandelana kwifilimu ye-PDMS enezimbobo bese luhambisana nongqimba oluphansi lwe-PDMS ngokubopha okungenakuguqulwa kusetshenziswa i-corona treater (Isithombe 2b–f). Ukuze kwakhiwe ama-hybrid chips, ama-replica e-PDMS alashiwe ahlanganiswa kuma-slide engilazi ukuze kudalwe amadivayisi e-microfluidic esiteshi esisodwa angamukela ama-Transwell inserts (Isithombe 2h kanye ne-Extended Data Fig. 2). Inqubo yokubopha yenziwa ngokwelapha ubuso be-replica ye-PDMS nengilazi nge-oxygen plasma noma ukwelashwa kwe-corona. Ngemva kokuhlanza idivayisi eyenziwe nge-microfabricated enamathiselwe ku-silicone tube, ukusethwa kwedivayisi kwase kulungele ukwenza i-3D morphogenesis ye-epithelium yamathumbu (Isithombe 2g).
a, Umfanekiso weskimu wokulungiswa kwezingxenye ze-PDMS kusuka ezikhunjini ze-silicon ezinephethini ze-SU-8. Isixazululo se-PDMS esingakaphekwa sathelwa esikhunjeni se-silicon (kwesobunxele), saphiliswa ku-60 °C (phakathi) sabe sesisuswa (kwesokudla). I-PDMS esusiwe yanqunywa yaba yizicucu futhi yahlanzwa ukuze isetshenziswe kabanzi.b, Isithombe sesikhunjeni se-silicon esisetshenziselwa ukulungiselela ungqimba oluphezulu lwe-PDMS.c, Isithombe sesikhunjeni se-silicon esisetshenziselwa ukwakha ulwelwesi olunezimbobo ze-PDMS.d, Uchungechunge lwezithombe zezingxenye ze-PDMS eziphezulu nezingezansi kanye nedivayisi yamathumbu ehlanganisiwe ye-on-chip.e., Iskimu sokuqondaniswa kwezingxenye ze-PDMS eziphezulu, ulwelwesi, kanye nezingezansi. Ungqimba ngalunye luboshwe ngokungenakulungiseka nge-plasma noma ukwelashwa kwe-corona.f, Iskimu sedivayisi ye-gut-on-a-chip eyenziwe eneziteshi ezincane ezihlanganisiwe kanye namakamelo okuhlanza.g, Ukusethwa kwe-gut-on-a-chip yokukhula kwamaseli e-microfluidic. Isisu esenziwe ku-chip esihlanganiswe ne-silicone tube kanye nesirinji sabekwa esivikelweni. Idivayisi ye-chip yabekwa esivalweni se Isitsha se-Petri esingu-150 mm sokucubungula. I-binder isetshenziselwa ukuvala ishubhu le-silicone.h, Izithombe ezibonakalayo zokwenziwa kwe-chip exubile kanye ne-morphogenesis ye-3D kusetshenziswa ama-chip e-hybrid. Ama-Transwell inserts alungiselelwe ngokuzimela ukukhuliswa. Ama-monolayer e-2D amaseli e-epithelial amathumbu afakwe ku-chip exubile ukuze abangele i-morphogenesis yamathumbu ye-3D. Le medium ifakwa ngeziteshi ezincane ngaphansi kwesendlalelo seseli esisungulwe ku-Transwell insert. Ibha yesikali, 1 cm.h Iphrintwe kabusha ngemvume evela ku-reference.4. Elsevier.
Kule phrothokholi, umugqa weseli i-Caco-2 kanye nama-organoid amathumbu asetshenziswe njengemithombo ye-epithelial (Isithombe 3a). Zombili izinhlobo zamaseli zakhuliswa ngokuzimela (Ibhokisi 2 kanye neBhokisi 5) futhi zasetshenziswa ukutshala iziteshi ezincane ezimbozwe yi-ECM ze-on-chip gut noma i-Transwell inserts. Lapho amaseli ehlangana (>95% ukumbozwa kuma-flask), amaseli e-Caco-2 akhuliswe njalo (phakathi kwezindawo 10 no-50) kuma-T-flasks avunwa ukuze kulungiselelwe ukumiswa kwamaseli ahlukanisiwe ngoketshezi lwe-trypsinization (ibhokisi 2). Ama-organoid amathumbu abantu avela kuma-biopsies amathumbu noma ukususwa kokuhlinzwa akhuliswe kuma-domes e-Matrigel scaffold emapuletini angu-24-well ukusekela imvelo encane yesakhiwo. Ama-morphogens aphakathi nendawo aqukethe okubalulekile (njenge-Wnt, i-R-spondin, ne-Noggin) kanye nezinto zokukhula ezilungiselelwe njengoba kuchaziwe kuBhokisi 3 zanezelwa njalo ngemva kwezinsuku ezimbili kuze kube yilapho ama-organoid ekhula abe ngu-~500 µm ububanzi. Ama-organoid akhule ngokugcwele ayavunwa futhi zihlukaniswe zibe amaseli angawodwa ukuze zitshalwe emathunjini noma kuma-Transwell inserts ku-chip (Ibhokisi 5). Njengoba sibike ngaphambilini, zingahlukaniswa ngokuya ngohlobo lwesifo12,13 (isb. i-ulcerative colitis, isifo sikaCrohn, umdlavuza we-colorectal, noma umnikeli ojwayelekile), indawo yezilonda (isb., izilonda uma kuqhathaniswa nendawo engenazo izilonda) kanye nendawo yesisu emgudwini (isb., i-duodenum, i-jejunum, i-ileum, i-cecum, i-colon, noma i-rectum). Sinikeza inqubo elungiselelwe kahle kuBhokisi 5 yokukhulisa ama-organoids e-colonic (ama-coloids) avame ukudinga amazinga aphezulu e-morphogens kune-organoids yamathumbu amancane.
a, Ukuhamba komsebenzi wokungeniswa kokwakheka kwesisu ku-gut chip. I-Caco-2 intestinal epithelium kanye nama-organoids amathumbu asetshenziswa kule phrothokholi ukukhombisa ukwakheka kwe-3D. Amaseli e-epithelial ahlukanisiwe ahlwanyelwe kudivayisi elungiselelwe i-gut-on-a-chip (ukulungiswa kwe-chip). Uma amaseli esehlwanyelwe (ahlwanyelwe) futhi enamathiselwe (anamathiselwe) ku-membrane enezimbobo ze-PDMS ngosuku 0 (D0), ukugeleza kwe-apical (AP) kuyaqalwa futhi kugcinwe izinsuku ezimbili zokuqala (ukugeleza, i-AP, i-D0-D2). Ukugeleza kwe-Basolateral (BL) nakho kuqaliswa kanye nokunyakaza kokwelula okujikelezayo (ukwelula, ukugeleza, i-AP kanye ne-BL) lapho kwakheka i-monolayer ephelele ye-2D. Ukwakheka kwe-intestinal 3D kwenzeka ngokuzenzakalelayo ngemva kwezinsuku ezi-5 zesiko le-microfluidic (i-morphogenesis, i-D5). Izithombe zokungafani kwesigaba zibonisa ukwakheka okumele kwamaseli e-Caco-2 esinyathelweni ngasinye sokuhlola noma iphuzu lesikhathi (igrafu yebha, i-100 µm). Imidwebo emine yeskimu ekhombisa ama-cascade ahambisanayo kwe-morphogenesis yamathumbu (phezulu kwesokudla). Imicibisholo edwetshwe ku-schematic imelela indlela yokugeleza koketshezi.b, isithombe se-SEM esibonisa i-topology engaphezulu kwe-epithelium ye-3D Caco-2 esungulwe (kwesobunxele). I-inset egqamisa indawo ekhulisiwe (ibhokisi elimhlophe elidwetshwe) ikhombisa i-microvilli evuselelwe ku-3D Caco-2 layer (kwesokudla).c, Umbono oqondile wangaphambili we-Caco-2 3D esungulwe, i-claudin (ZO-1, ebomvu) kanye ne-continuous brush border membranes ebhalwe ukuthi i-F-actin (eluhlaza) kanye ne-nuclei (eluhlaza okwesibhakabhaka) Ukuboniswa kwe-immunofluorescence confocal kwamaseli e-epithelial kuma-chips amathumbu. Imicibisholo ekhomba ku-schematic ephakathi ikhombisa indawo yendiza egxile kuyo yonke i-confocal view.d, Isikhathi sesikhathi sokushintsha kwe-morphological kuma-organoids akhuliswe ku-chip atholwe nge-phase contrast microscopy ezinsukwini 3, 7, 9, 11, kanye no-13. I-inset (phezulu kwesokudla) ikhombisa ukukhuliswa okuphezulu kwesithombe esinikeziwe.e, i-DIC photomicrograph ye-organoid 3D epithelium esungulwe emathunjini esiqeshaneni esithathwe ngosuku lwe-7.f, Izithombe ezimboziwe ze-immunofluorescence ezibonisa izimpawu zamaseli okuqala (LGR5; magenta), amaseli e-goblet (MUC2; luhlaza), i-F-actin (grey) kanye ne-nuclei (cyan) ezikhuliswe kuma-gut chips izinsuku ezi-3, ngokulandelana (Kwesobunxele) kanye nama-organoids ezinsuku eziyi-13 (phakathi) kungqimba lwe-epithelial.Bheka futhi i-Extended Data Figure 3, eqokomisa ukusayinwa kwe-LGR5 ngaphandle kokusayinwa kwe-MUC2. Izithombe ze-Fluorescence ezibonisa isakhiwo se-epithelial microstructure (kwesokudla) se-epithelium ye-organoid ye-3D esakhiwe emathunjini ku-chip ngokufaka i-plasma membrane ngedayi ye-CellMask (kwesokudla) ngosuku lwe-13 lwesiko. Ibha yesikali ingu-50 μm ngaphandle kokuthi kuchazwe ngenye indlela.b Iphrintwe kabusha ngemvume evela ku-reference.2. I-Oxford University Press; c Iguqulwe ngemvume evela ku-Reference.2. I-Oxford University Press; e kanye ne-f ilungiswe ngemvume ngokubhekisela.12 Ngaphansi kwe-Creative Commons License CC BY 4.0.
Emathunjini e-chip, kuyadingeka ukuguqula ubuso obungenamanzi be-membrane ye-PDMS enezimbobo ukuze kuhlanganiswe i-ECM ngempumelelo. Kule phrothokholi, sisebenzisa izindlela ezimbili ezahlukene ukuguqula ukungabi namanzi kwe-membrane ye-PDMS. Ukuze kukhuliswe amaseli e-Caco-2, ukusebenza kwendawo ngokwelashwa kwe-UV/ozone kuphela kwakwanele ukunciphisa ukungabanda kwamanzi kwendawo ye-PDMS, ukumboza i-ECM nokunamathisela amaseli e-Caco-2 ku-membrane ye-PDMS. Kodwa-ke, isiko le-microfluidic le-epithelium ye-organoid lidinga ukusebenza kwendawo okusekelwe kumakhemikhali ukuze kufezwe ukufakwa okuphumelelayo kwamaprotheni e-ECM ngokusebenzisa i-polyethyleneimine (PEI) kanye ne-glutaraldehyde ngokulandelana kuma-microchannel e-PDMS. Ngemva kokuguqulwa kwendawo, amaprotheni e-ECM afakwa ukuze amboze indawo ye-PDMS esebenzayo abese efakwa kwi-epithelium ye-organoid ehlukanisiwe. Ngemva kokuba amaseli enamathiselwe, isiko le-microfluidic cell liqala ngokufaka kuphela i-medium ku-microchannel ephezulu kuze kube yilapho amaseli akha i-monolayer ephelele, kuyilapho i-microchannel engezansi igcina izimo ezimile. Le ndlela elungiselelwe ukusebenza kwendawo kanye nokumboza kwe-ECM kwenza kube lula ukunamathisela i-organoid. i-epithelium ukuze ibangele ukwakheka kwe-3D ebusweni be-PDMS.
Ama-Transwell cultures nawo adinga ukuhlanganiswa kwe-ECM ngaphambi kokutshala amaseli; noma kunjalo, ama-Transwell cultures awadingi izinyathelo eziyinkimbinkimbi zokwelashwa ngaphambi kokusebenzisa ubuso bezinto ezifakwe imigodi. Ukuze kukhule amaseli e-Caco-2 kuma-Transwell inserts, ukuhlanganiswa kwe-ECM kuma-insert anemigodi kusheshisa ukunamathela kwamaseli e-Caco-2 ahlukanisiwe (
Izici zesimo sezendlalelo ze-3D epithelial ezisebenzisa i-microengineered zingahlaziywa ngokusebenzisa izindlela ezahlukene zokuthwebula izithombe, okuhlanganisa i-phase contrast microscopy, i-differential interference contrast (DIC) microscopy, i-SEM, noma i-immunofluorescence confocal microscopy (Izithombe 3 no-4). Ukuthwebula izithombe ze-Phase contrast noma i-DIC kungenziwa kalula nganoma yisiphi isikhathi ngesikhathi sokukhuliswa ukuze kuqashwe ukuma nokuvela kwezingqimba ze-3D epithelial. Ngenxa yokucaca okubonakalayo kwamafilimu e-PDMS namafilimu e-polyester, amapulatifomu e-gut-on-a-chip kanye ne-hybrid chip anganikeza isithombe sesikhathi sangempela ngaphandle kwesidingo sokuhlukaniswa noma ukuhlukaniswa kwedivayisi. Lapho kwenziwa isithombe se-immunofluorescence (Izithombe 1, 3c, f kanye no-4b, c), amaseli ngokuvamile aqiniswa nge-4% (wt/vol) paraformaldehyde (PFA), kulandelwe yi-Triton X-100 kanye ne-2% (wt/vol) ) bovine serum albumin (BSA), ngokulandelana. Kuye ngohlobo lweseli, izinto ezihlukile zokulungisa, Ama-permeabilizer, kanye nama-blocking agent angasetshenziswa. Ama-antibodies ayinhloko aqondisa izimpawu zeseli noma zesifunda ezincike ku-lineage asetshenziswa ukugqamisa amaseli anganyakazi endaweni ethile ku-chip, kulandelwe ama-antibodies esibili kanye nodayi we-counterstain oqondisa i-nucleus (isb., 4′, 6-diamidino-2-phenylene) indole, DAPI) noma i-F-actin (isb., i-phalloidin ebhalwe nge-fluorescent). Izithombe ezibukhoma ezisekelwe ku-fluorescence zingenziwa endaweni ethile ukuthola ukukhiqizwa kwe-mucus (Isithombe 1, “Ukwehlukaniswa kwamaseli” kanye “ne-Gut physiology”), ukwakheka kwamaseli amagciwane ngokungahleliwe (Isithombe 1, “I-Host-microbe co-culture”), ukuqashwa kwamaseli omzimba (Isithombe 1, ‘Imodeli Yezifo’) noma imidwebo ye-3D epithelial morphology (Isithombe 3c, f kanye no-4b, c). Lapho uguqula isisu ku-chip ukuze uhlukanise ungqimba olungaphezulu nongqimba oluphansi lwe-microchannel, njengoba kuchazwe ku-ref. Njengoba kuboniswe ku-Fig. 2, i-epithelial ye-3D i-morphology kanye ne-microvilli emngceleni webhulashi le-apical kungabonakala nge-SEM (Isithombe 3b). Ukuvezwa kwezimpawu zokuhlukanisa kungahlolwa ngokwenza i-PCR5 eningi noma i-single-cell RNA sequencing. Kulesi simo, izendlalelo ze-3D zamaseli e-epithelial akhuliswe kuma-gut chips noma kuma-hybrid chips zivunwa nge-trypsinization bese zisetshenziselwa ukuhlaziywa kwama-molecular noma kwezakhi zofuzo.
a, Ukuhamba komsebenzi wokungeniswa kokwakheka kwamathumbu ku-chip ehlanganisiwe. I-Caco-2 kanye nama-organoids amathumbu asetshenziswa kule phrothokholi ukukhombisa ukwakheka kwe-3D epulatifomu ye-chip ehlanganisiwe. Amaseli e-epithelial ahlukanisiwe ahlwanyelwa ezitsheni ze-Transwell ezilungisiwe (ukulungiselela kwe-TW; bheka isithombe esingezansi). Lapho amaseli esehlwanyelwe (ahlwanyelwe) futhi enamathiselwe kuma-membrane e-polyester kuma-inserts e-Transwell, wonke amaseli akhuliswa ngaphansi kwezimo ezimile (isiko le-TW). Ngemuva kwezinsuku eziyi-7, i-Transwell eyodwa equkethe ungqimba olulodwa lwe-2D lwamaseli e-epithelial yahlanganiswa kwi-chip ehlanganisiwe ukuze kwethulwe ukugeleza kwe-basolateral (Flow, BL), okwagcina kuholele ekwakhiweni kwengqimba ye-epithelial ye-3D (i-morphogenesis). Ama-micrographs okungafani kwesigaba abonisa izici zesimo samaseli e-epithelial ezitho zomuntu atholakala ku-donor ojwayelekile (umugqa we-C103) okhuphukayo esinyathelweni ngasinye sokuhlola noma iphuzu lesikhathi. Ama-schematics ezingqimbeni eziphezulu abonisa ukucushwa kokuhlola kwesinyathelo ngasinye.b, ama-Hybrid chips (i-schematic yesobunxele) angaholela ekuguqulweni kwe-3D kwe-epithelial ye-organoid amaseli anemibono ye-confocal microscopy ephezulu-phansi ethathwe ezindaweni ezahlukene ze-Z (ephezulu, ephakathi, nangaphansi; bheka imigqa ye-schematic yesokudla kanye nemigqa enamachashazi ahambisanayo). abonise izici ezicacile ze-morphological. I-F-actin (i-cyan), i-nucleus (grey).c, ama-micrograph e-confocal e-Fluorescence (umbono we-angled we-3D) wamaseli e-epithelial atholakala ku-organoid akhuliswe ku-static Transwell (TW; i-inset ngaphakathi kwebhokisi elimhlophe eline-dashed) ngokumelene ne-hybrid chip (isithombe esigcwele esikhulu kunazo zonke) eqhathanisa i-2D ngokumelene ne-3D morphology, ngokulandelana. I-pair yemibono ye-2D eqondile enqamulayo (i-inset ekhoneni eliphezulu kwesokudla; "XZ") nayo ikhombisa izici ze-2D kanye ne-3D. Ibha yesikali, 100 µm.c Iphrintwe kabusha ngemvume evela ku-reference.4. Elsevier.
Izilawuli zingalungiswa ngokuhlela amaseli afanayo (amaseli e-Caco-2 noma amaseli e-epithelial e-intestinal organoid) abe yizingqimba ezimbili ngaphansi kwezimo ezivamile zokukhuliswa kwe-static. Okuphawulekayo ukuthi, ukuncipha kwezakhamzimba kungabangelwa umthamo olinganiselwe weziteshi ezincane (okungukuthi ~4 µL esiteshini esiphezulu kumklamo wokuqala we-gut-chip). Ngakho-ke, isimo se-epithelial ngaphambi nangemva kokusetshenziswa kokugeleza kwe-basolateral nakho kungaqhathaniswa.
Inqubo ye-lithography ethambile kufanele yenziwe ekamelweni elihlanzekile. Ngesendlalelo ngasinye ku-chip (izendlalelo eziphezulu nezingezansi kanye nama-membrane) kanye nama-chip e-hybrid, kusetshenziswe ama-photomasks ahlukene futhi enziwa kuma-wafer ahlukene e-silicon ngoba ukuphakama kwama-microchannels kwakuhlukile. Ukuphakama okuqondiwe kwama-microchannels aphezulu naphansi esiswini ku-chip kungu-500 µm no-200 µm, ngokulandelana. Ukuphakama okuqondiwe kwesiteshi se-chip e-hybrid kungu-200 µm.
Beka i-wafer ye-silicon engamasentimitha angu-3 esitsheni esine-acetone. Phendula ipuleti kancane imizuzwana engama-30, bese uyomisa ngomoya i-wafer. Dlulisa i-wafer epuletini ene-IPA, bese uphenduphendula ipuleti imizuzwana engama-30 ukuze ihlanzeke.
Isixazululo se-piranha (ingxube ye-hydrogen peroxide kanye ne-sulfuric acid egxilile, 1:3 (vol/vol)) singasetshenziswa ngokuzithandela ukuze kususwe kakhulu izinsalela zezinto eziphilayo ebusweni be-silicon wafer.
Isixazululo sePiranha siyagqwala kakhulu futhi sikhiqiza ukushisa. Kudingeka izinyathelo zokuphepha ezengeziwe. Ukuze kulahlwe imfucuza, vumela isixazululo siphole bese usidlulisela esitsheni semfucuza esihlanzekile, esomile. Sebenzisa izitsha zesibili bese ubhala amalebula kahle ezitsheni zemfucuza. Sicela ulandele imihlahlandlela yokuphepha yesikhungo ukuze uthole izinqubo ezinemininingwane eminingi.
Hlikihla amanzi kuma-wafer ngokuwabeka epuletini elishisayo elingu-200 °C imizuzu eyi-10. Ngemva kokuphelelwa amanzi, i-wafer yanyakaziswa kahlanu emoyeni ukuze iphole.
Thela cishe amagremu ayi-10 e-photoresist SU-8 2100 phakathi nendawo ye-silicon wafer ehlanziwe. Sebenzisa ama-tweezers ukusabalalisa i-photoresist ngokulinganayo ku-wafer. Ngezikhathi ezithile beka i-wafer epuletini elishisayo elingu-65°C ukuze wenze i-photoresist inganamatheli kakhulu futhi kube lula ukuyisakaza. Ungabeki i-wafer ngqo epuletini elishisayo.
I-SU-8 yasatshalaliswa ngokulinganayo ku-wafer ngokusebenzisa i-spin coating. Hlela ukujikeleza okungenayo kwe-SU-8 imizuzwana emi-5-10 ukuze kusabalale ku-500 rpm ngokusheshisa okungu-100 rpm/s. Setha i-main spin ukuze kube namaphethini obukhulu obungu-200 µm ku-1,500 rpm, noma ufinyelele ubukhulu obungu-250 µm (okwenza ukuphakama okungu-500 µm kwengqimba engenhla yesisu ku-chip; bheka “Izinyathelo ezibalulekile” ngezansi) ezibekwe ekusheshiseni okungu-300 rpm/s imizuzwana engama-30 ku-1,200 rpm.
Isivinini esikhulu sokujikeleza singalungiswa ngokuya ngobukhulu obuqondiwe bephethini ye-SU-8 ku-silicon wafer.
Ukuze kwakhiwe amaphethini e-SU-8 anokuphakama okungu-500 µm kwengqimba engenhla yesisu ku-chip, ukumbozwa kwe-spin kanye nezinyathelo zokubhaka ezithambile zaleli Bhokisi (izinyathelo 7 no-8) kwaphindwa ngokulandelana (bheka isinyathelo 9) ukuze kukhiqizwe izendlalelo ezimbili ezingu-250 µm. Ingqimba ejiyile ye-SU-8, engahlanganiswa futhi ihlanganiswe nokuchayeka kwe-UV esinyathelweni 12 saleli bhokisi, okwenza ungqimba lube ngu-500 µm ukuphakama.
Bhaka ama-wafer ambozwe yi-SU-8 ngobumnene ngokubeka ama-wafer ngokucophelela epuletini elishisayo ku-65 °C imizuzu emi-5, bese ushintsha isethingi ibe ngu-95 °C bese uwafukamela eminye imizuzu engama-40.
Ukuze ufinyelele ukuphakama okungu-500 μm kwephethini ye-SU-8 ku-microchannel ephezulu, phinda izinyathelo 7 no-8 ukuze ukhiqize izendlalelo ezimbili ze-SU-8 ezinobukhulu obungu-250 μm.
Usebenzisa i-UV Mask Aligner, yenza isivivinyo sesibani ngokwemiyalelo yomenzi ukuze ubale isikhathi sokuchayeka kwe-wafer. (isikhathi sokuchayeka, ms) = (isilinganiso sokuchayeka, mJ/cm2)/(amandla esibani, mW/cm2).
Ngemva kokunquma isikhathi sokuvezwa, beka i-photomask esibambi semaski se-aligner yemaski ye-UV bese ubeka i-photomask ku-wafer eboshwe nge-SU-8.
Beka indawo ephrintiwe ye-photomask ngqo ohlangothini olumbozwe yi-SU-8 lwe-silicon wafer ukuze unciphise ukusabalala kwe-UV.
Veza i-wafer eboshwe nge-SU-8 kanye ne-photomask ngokuqondile ku-260 mJ/cm2 yokukhanya kwe-UV ngesikhathi sokuchayeka esinqunyiwe (bheka isinyathelo 10 saleli bhokisi).
Ngemva kokuchayeka ku-UV, ama-wafer e-silicon ambozwe yi-SU-8 abhakwa ku-65°C imizuzu emi-5 kanye no-95°C imizuzu eyi-15 epuletini ngalinye elishisayo ukuze kwenziwe amaphethini anokuphakama okungu-200 μm. Nweba isikhathi sangemuva kokubhaka ku-95°C kuya ku-30 imizuzu ukuze kwenziwe amaphethini anokuphakama okungu-500 µm.
Umthuthukisi uthululwa esitsheni sengilazi, bese i-wafer ebhakiwe ifakwa esitsheni. Umthamo womthuthukisi we-SU-8 ungahluka kuye ngobukhulu bepuleti lengilazi. Qiniseka ukuthi usebenzisa umthuthukisi we-SU-8 owanele ukususa ngokuphelele i-SU-8 engakabonakali. Isibonelo, uma usebenzisa isitsha sengilazi esingu-150 mm ububanzi esinomthamo we-1 L, sebenzisa ~300 mL womthuthukisi we-SU-8. Yakha isikhunta imizuzu engama-25 ngokujikeleza okuthambile ngezikhathi ezithile.
Hlanza isikhunta esithuthukisiwe nge-~10 mL yomshini omusha osetshenziswayo bese ulandela i-IPA ngokufafaza isisombululo usebenzisa i-pipette.
Beka i-wafer kumshini wokuhlanza i-plasma bese uyibeka ku-oxygen plasma (igesi yomoya-mpilo, ingcindezi eqondiwe engu-1 × 10−5 Torr, amandla angu-125 W) imizuzu eyi-1.5.
Faka i-wafer ku-vacuum cleaner ene-glass slide ngaphakathi. Ama-wafer nama-slide angabekwa eceleni. Uma i-vacuum cleaner ihlukaniswe ngezingqimba eziningana ngepuleti, beka ama-slide ekamelweni elingezansi kanye nama-wafer ekamelweni eliphezulu. Lahla isixazululo se-trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane esingu-100 μL ku-glass slide bese ufaka i-vacuum ukuze i-silanization ihlale injalo.
Ncibilikisa ibhodlela lamaseli e-Caco-2 aqandisiwe ebhavini lamanzi elingu-37°C, bese udlulisela amaseli ancibilikisiwe ebhodleleni le-T75 eliqukethe i-15 mL ye-37°C efudumele ngaphambili.
Ukuze kudlule amaseli e-Caco-2 lapho kuhlangana khona amaseli angama-90%, okokuqala faka i-Caco-2 medium efudumele, i-PBS, kanye ne-0.25% trypsin/1 mM EDTA emanzini okugeza angama-37°C.
Geza i-medium ngokufutha nge-vacuum. Geza amaseli kabili nge-5 mL ye-PBS efudumele ngokuphinda ukufutha nge-vacuum bese ufaka i-PBS entsha.
Isikhathi sokuthunyelwe: Julayi-16-2022
