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Qaab-dhismeedka mindhicirka aadanaha wuxuu dejiyaa astaamaha crypt-villus ee qaab-dhismeedka micro-architecture-ka epithelial-ka 3D iyo qaab-dhismeedka booska. Qaab-dhismeedkan gaarka ah ayaa looga baahan yahay inuu ilaaliyo homeostasis-ka mindhicirka iyadoo la ilaalinayo unugyada asliga ah ee ku jira qarsoodiga aasaasiga ah ee antigens-ka microbial-ka ee dibadda iyo dheef-shiid kiimikaadkooda. Intaa waxaa dheer, villi-ka mindhicirka iyo xabka soo daaya waxay leeyihiin unugyo epithelial ah oo si firfircoon u kala duwan oo leh caqabad ilaalin ah oo ku taal dusha sare ee xuubka mindhicirka. Sidaa darteed, dib u soo nooleynta qaab-dhismeedka epithelial-ka 3D waa mid muhiim u ah dhismaha moodooyinka mindhicirka ee vitro. Waxaa xusid mudan, mimetic gut-on-a-chip-ka dabiiciga ah wuxuu kicin karaa morphogenesis 3D oo iskiis ah oo epithelium-ka mindhicirka ah oo leh hawlo jireed oo la wanaajiyey iyo biomechanics. Halkan, waxaan ku bixinnaa hab-raac la soo saari karo si si adag loogu kiciyo morphogenesis-ka mindhicirka ee ku jira jajab microfluidic ah iyo sidoo kale jajab isku-dhafan oo Transwell ku dhex jira. Waxaan sharraxaynaa hababka faahfaahsan ee wax-soo-saarka qalabka, beerista Caco-2 ama unugyada epithelial-ka organoid-ka xiidmaha ee goobaha caadiga ah iyo sidoo kale madal microfluidic ah, soo saarista morphogenesis-ka 3D, iyo astaamaha 3D ee la aasaasay. epithelia iyadoo la adeegsanayo qaabab sawireed oo badan. Hab-maamuuskani wuxuu ku guuleystaa dib u soo nooleynta microarchitecture-ka mindhicirka oo shaqeynaya isagoo xakameynaya socodka dareeraha salka ah muddo 5 maalmood ah. Habkayaga morphogenesis-ka ee in vitro wuxuu adeegsadaa cadaadis shiidid jireed iyo dhaqdhaqaaq farsamo oo khuseeya mana u baahna injineernimo unug oo adag ama wax-ka-beddel, taasoo ka sarreyn karta farsamooyinka kale ee jira. Waxaan qiyaaseynaa in hab-maamuuskayaga la soo jeediyay uu yeelan karo saameyn ballaaran oo ku saabsan bulshada cilmi-baarista biomedical-ka, isagoo bixinaya hab lagu soo nooleeyo lakabyada epithelial-ka mindhicirka 3D ee in vitro loogu talagalay codsiyada biomedical, kiliinikada, iyo daawooyinka.
Tijaabooyinku waxay muujinayaan in unugyada Caco-2 ee xuubka caloosha ee lagu beero mindhicirka-ku-noole-jiid1,2,3,4,5 ama aaladaha microfluidic-ka laba-lakab leh6,7 ay mari karaan morphogenesis 3D oo iskiis ah in vitro iyada oo aan si cad loo fahmin habka hoose. Daraasaddeenna dhowaan, waxaan ku ogaanay in ka saarista antagonist-yada morphogen-ka ee laga soo saaro aaladaha dhaqanka ay lagama maarmaan tahay oo ku filan in lagu kiciyo morphogenesis-ka 3D ee epithelial in vitro, kaas oo ay muujiyeen Caco-2 iyo organoids-ka mindhicirka ee bukaanka laga helo. Unugyada Epithelial-ka ayaa la xaqiijiyay. Daraasaddan, waxaan si gaar ah diiradda u saarnay wax soo saarka unugyada iyo qaybinta fiirsashada ee antagonist Wnt oo awood leh, Dickkopf-1 (DKK-1), oo ku jira mindhicirka-ku-jir-jiid iyo aaladaha microfluidic ee wax laga beddelay oo ay ku jiraan gelinta Transwell, oo loo yaqaan "Chip Hybrid". Waxaan muujineynaa in ku darista antagonists Wnt oo dibadda ah (sida DKK-1, Wnt repressor 1, borotiinka la xiriira frizzled 1, ama Soggy-1) mindhicirka-ku-jir-jiidku uu joojiyo morphogenesis ama uu carqaladeeyo lakabka epithelial-ka 3D ee hore loo qaabeeyey, taasoo soo jeedinaysa in walbahaarka iska soo horjeeda inta lagu jiro dhaqanka uu mas'uul ka yahay morphogenesis-ka mindhicirka in vitro. Sidaa darteed, hab wax ku ool ah oo lagu gaaro morphogenesis xooggan oo ku yaal is-dhexgalka epithelial waa in laga saaro ama si yar loo ilaaliyo heerarka antagonists Wnt ee qaybta hoose iyadoo la isticmaalayo biyo-shubid firfircoon (tusaale ahaan, goobaha mindhicirka-ku-jir-jiid ama isku-dhafka-ku-jir-jiid) ama faafin. Warbaahinta hoose (tusaale ahaan, laga bilaabo Transwell waxay gelisaa kaydadka waaweyn ee hoose ee ceelasha).
Hab-maamuuskaan, waxaan ku bixinaynaa hab faahfaahsan oo lagu sameeyo qalabka microdevices-ka ee xiidmaha-ku-jira iyo jajabyada isku-dhafan ee Transwell-ku-jira (tallaabooyinka 1-5) si loo beero unugyada epithelial-ka mindhicirka ee xuubka daloolada ku salaysan polydimethylsiloxane (PDMS) (tallaabooyinka 6A, 7A, 8, 9) ama xuubka polyester-ka ee Transwell gelinta (tallaabooyinka 6B, 7B, 8, 9) iyo morphogenesis-ka 3D ee ku-jira in vitro (tallaabada 10). Waxaan sidoo kale aqoonsannay astaamaha unugyada iyo molecular-ka oo tilmaamaya histogenesis-ka gaarka ah ee unugyada iyo kala-soocidda unugyada ku tiirsan lineage-ka annagoo adeegsanayna qaabab sawireed oo badan (tallaabooyinka 11-24). Waxaan kicineynaa morphogenesis annagoo adeegsanayna unugyada epithelial-ka mindhicirka aadanaha, sida Caco-2 ama organoids-ka mindhicirka, laba qaab oo dhaqan ah oo leh faahfaahin farsamo oo ay ku jiraan wax ka beddelka dusha sare ee xuubka daloolada, abuurista monolayers-ka 2D, iyo biochemical-ka mindhicirka iyo Soo saarista microenvironment-ka biomechanical.in vitro. Si aan u kicino morphogenesis-ka 3D ee monolayers-ka epithelial-ka 2D, waxaan ka saarnay Ka-hortagga morphogen-ka ee labada qaab ee la beeray iyagoo ku qulqulaya qaybta hoose ee dhaqanka. Ugu dambeyntii, waxaan bixinnaa matalaad ku saabsan faa'iidada lakabka epithelial-ka 3D ee dib loo soo nooleyn karo kaas oo loo isticmaali karo in lagu daydo koritaanka epithelial-ka ee ku tiirsan morphogen-ka, dhaqamada martida loo yahay ee microbiome-ka ee dheer, caabuqa pathogen-ka, dhaawaca bararka, cilladda caqabadda epithelial, iyo daaweynta ku salaysan probiotic Tusaale ahaan saameyn.
Hab-maamuuskayagu wuxuu noqon karaa mid waxtar u leh saynisyahano badan oo ku takhasusay cilmiga aasaasiga ah (tusaale ahaan, bayoolajiga xuubka xiidmaha, bayoolajiga unugyada asliga ah, iyo bayoolajiga horumarka) iyo cilmi-baarista la dabaqay (tusaale ahaan, baaritaanka daawada kahor kiliinikada, qaabaynta cudurka, injineernimada unugyada, iyo gastroenterology) saameyn ballaaran. Sababtoo ah soo-saarista iyo adkeysiga hab-maamuuskayaga si loo kiciyo qaab-dhismeedka 3D ee epithelium-ka mindhicirka in vitro, waxaan qiyaaseynaa in istaraatiijiyadeenna farsamo loo faafin karo dhagaystayaasha baranaya dhaqdhaqaaqa calaamadaynta unugyada inta lagu jiro horumarka mindhicirka, dib-u-cusboonaysiinta ama homeostasis. Intaa waxaa dheer, hab-maamuuskayagu wuxuu waxtar u leeyahay su'aalaha ku saabsan caabuqa iyadoo la adeegsanayo wakiillo kala duwan oo faafa sida Norovirus 8, Cudurka Neefsashada ee Ba'an ee Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 ama Vibrio cholerae. Dadka ka faallooda cudurrada iyo cudur-sidaha ayaa sidoo kale waxtar leh. Isticmaalka nidaamka microphysiology-ga mindhicirka ee ku jira chip-ka ayaa u oggolaan kara dhaqanka wadajirka ah ee dheer 10 iyo qiimeynta xigta ee difaaca martida loo yahay, jawaabaha difaaca jirka iyo hagaajinta dhaawaca la xiriira cudur-sidaha ee marinnada caloosha iyo mindhicirka (GI) 11. Xanuunnada kale ee GI ee la xiriira cilladda mindhicirka ee daadanaysa, cudurka celiac, cudurka Crohn, ulcerative colitis, pouchitis, ama cilladda mindhicirka ee xanaaqa leh ayaa la matali karaa marka lakabyada epithelial-ka mindhicirka ee 3D la diyaariyo iyadoo la isticmaalayo lakabyada epithelial-ka mindhicirka ee 3D ee bukaanka, cudurradan waxaa ka mid ah atrophy villous, gaabinta crypt, dhaawaca xuubka, ama caqabadda epithelial ee daciifka ah. Unugyada mindhicirka ee laga helo unugyada asliga ah ama unugyada stem-ka organoids12,13. Si aad si fiican ugu daydo kakanaanta sare ee deegaanka cudurka, akhristayaashu waxay tixgelin karaan inay ku daraan noocyada unugyada ee khuseeya cudurka, sida unugyada mononuclear-ka dhiigga ee bukaanka (PBMCs), moodooyinka ka kooban microarchitectures-ka villus-crypt ee mindhicirka 3D. unugyada difaaca jirka ee gaarka u ah unugyada, 5.
Maadaama qaab-dhismeedka epithelial-ka 3D la hagaajin karo oo la arki karo iyada oo aan la isticmaalin habka qaybinta, daawadayaasha ka shaqeynaya transcriptionomics-ka booska iyo sawir-qaadista xallinta sare ama xallinta sare waxay xiisayn karaan khariidayntayada dhaqdhaqaaqa spatiotemporal ee hiddo-wadaha iyo borotiinnada ku jira niches-ka epithelial-ka. Xiiseynaya tiknoolajiyada. Jawaabta kicinta microbiome ama difaaca jirka. Intaa waxaa dheer, isku-xidhka 10, 14 ee martigeliyaha-microbiome-ka ee dhererka dheer ee isku-dubaridka homeostasis-ka mindhicirka waxaa lagu dhisi karaa lakabka xuubka mindhicirka ee 3D iyadoo la kobcinayo noocyada kala duwan ee microbial, bulshooyinka microbial ama microbiota saxarada, gaar ahaan mindhicirka-ku-jir-jiid. goobta. Habkani wuxuu si gaar ah u soo jiitaa dhagaystayaasha baranaya difaaca jirka ee xuubka, gastroenterology, microbiome-ka aadanaha, dhaqanka iyo microbiology-ga kiliinikada ee raadinaya inay ku beeraan microbiota mindhicirka ee aan hore loo dhaqmin shaybaarka. Haddii hab-maamuuska morphogenesis-ka ee in vitro-ga lagu habeyn karo qaababka dhaqanka ee la miisaami karo, sida gelinta multiwell ee 24, 96 ama 384 saxan oo ceel ah oo si joogto ah u buuxiya qaybaha hoose, hab-maamuuska waxaa sidoo kale loo faafin karaa kuwa horumarinaya daawooyinka, biomedical ama baaritaan sare ama goobaha xaqiijinta warshadaha cuntada. Sida caddayn-mabda'a, dhawaan waxaan muujinnay suurtagalnimada nidaamka morphogenesis-ka ee multiplex-ka ah oo la miisaami karo qaab saxan 24-ceel ah. Intaa waxaa dheer, alaabooyin badan oo organ-on-a-chip ah ayaa la suuq geeyay16,17,18. Sidaa darteed, ansaxinta habka morphogenesis-ka ee in vitro-ga waxaa la dedejin karaa oo ay isticmaali karaan shaybaarro badan oo cilmi-baaris ah, warshado ama hay'ado dawladeed iyo kuwa sharci dejineed si loo fahmo dib-u-barnaamijka unugyada ee morphogenesis-ka in vitro-ga ee heerka transcriptomik si loo tijaabiyo daawooyinka ama biotherapy-ga nuugista. iyo gaadiidka musharixiinta daroogada ayaa lagu qiimeeyay iyadoo la adeegsanayo daawooyinka 3D ee ku-meel-gaarka ah ama iyadoo la adeegsanayo qaabab gaar ah ama ganacsi oo xubnaha-ku-sameesan si loo qiimeeyo soo-saarista habka morphogenesis-ka mindhicirka.
Tiro kooban oo ah moodooyinka tijaabada ah ee khuseeya aadanaha ayaa loo isticmaalay in lagu barto qaab-dhismeedka epithelial-ka mindhicirka, inta badan sababtoo ah la'aanta hab-raacyada la hirgelin karo si loo kiciyo qaab-dhismeedka 3D ee vitro. Xaqiiqdii, aqoonta badan ee hadda jirta ee ku saabsan qaab-dhismeedka mindhicirka waxay ku salaysan tahay daraasaadka xayawaanka (tusaale ahaan, zebrafish20, jiirarka21 ama digaagga22). Si kastaba ha ahaatee, waa kuwo shaqo badan oo kharash badan, waxaa laga yaabaa in su'aal laga keeno anshaxa, tan ugu muhiimsanna, si sax ah uma go'aamiyaan hababka horumarinta aadanaha. Moodooyinkani sidoo kale aad bay u xaddidan yihiin awooddooda lagu tijaabin karo si dhinacyo badan leh oo la miisaami karo. Sidaa darteed, hab-maamuuskayaga dib-u-soo-nooleynta qaab-dhismeedka unugyada 3D ee vitro wuxuu ka fiican yahay moodooyinka xayawaanka ee vivo iyo sidoo kale moodooyinka dhaqanka unugyada 2D ee caadiga ah ee taagan. Sida hore loo sharraxay, isticmaalka qaab-dhismeedka epithelial-ka 3D wuxuu noo oggolaaday inaan baarno meelaynta booska ee unugyada kala duwan ee ku jira dhidibka crypt-villus iyadoo laga jawaabayo kicinta xuubka ama difaaca jirka ee kala duwan. Lakabyada epithelial-ka 3D waxay bixin karaan meel lagu barto sida unugyada microbial-ku ay ugu tartamaan inay sameeyaan niches-ka booska iyo horumarka deegaanka iyadoo laga jawaabayo arrimaha martida loo yahay (tusaale ahaan, lakabka gudaha iyo kan dibadda ee xabka, dheecaanka IgA iyo peptides-ka lidka ku ah jeermiska). Intaa waxaa dheer, qaab-dhismeedka epithelial-ka 3D wuxuu noo oggolaan karaa inaan fahanno sida microbiota-ga mindhicirka uu u qaabeeyo bulshooyinkeeda isla markaana uu si isku mid ah u soo saaro metabolites-ka microbial (tusaale ahaan, asiidhyada dufanka leh ee silsiladda gaaban) kuwaas oo qaabeeya qaab-dhismeedka unugyada iyo niches-ka unugyada asliga ah ee ku jira sirta aasaasiga ah. Astaamahan waxaa la muujin karaa oo keliya marka lakabyada epithelial-ka 3D la sameeyo in vitro.
Marka laga soo tago habkayaga aan u abuurno qaab-dhismeedka epithelial-ka mindhicirka 3D, waxaa jira dhowr habab oo in vitro ah. Dhaqanka organoid-ka mindhicirka waa farsamo injineernimo unug oo casri ah oo ku salaysan beerista unugyada asliga ah ee mindhicirka xaalado gaar ah oo morphogen ah23,24,25. Si kastaba ha ahaatee, isticmaalka moodooyinka organoid-ka 3D ee falanqaynta gaadiidka ama dhaqamada microbiome-ka ee martida loo yahay badanaa waa mid adag sababtoo ah lumen-ka mindhicirka ayaa ku jira organoid-ka, sidaas darteedna, soo bandhigidda qaybaha iftiinka sida unugyada microbial ama antigens-ka dibadda waa xaddidan tahay. Helitaanka lumens-ka organoid-ka waa la hagaajin karaa iyadoo la isticmaalayo microinjector,26,27 laakiin habkani waa mid duullaan ah oo shaqo badan waxayna u baahan tahay aqoon gaar ah si loo qabto. Intaa waxaa dheer, dhaqamada organoid-ka dhaqameed ee lagu hayo saqafyada hydrogel-ka xaaladaha taagan si sax ah uma muujiyaan biomechanics firfircoon in vivo.
Hababka kale ee ay adeegsadaan kooxo cilmi baaris ah oo dhowr ah ayaa adeegsada qaab-dhismeedka hydrogel-ka 3D si loo daydo qaab-dhismeedka epithelial-ka mindhicirka iyadoo la kobcinayo unugyada mindhicirka aadanaha ee go'doonsan dusha sare ee jelka. Samee qaab-dhismeedka hydrogel-ka adoo isticmaalaya qaabab 3D ah oo la daabacay, micro-milled, ama lithographically ah. Habkani wuxuu muujinayaa habaynta is-abaabulan ee unugyada epithelial-ka ee go'doonsan in vitro oo leh jaangooyooyin morphogen oo fiisiyooloji ahaan khuseeya, sameynta qaab-dhismeedka epithelial-ka saamiga sare iyo isku-xidhka stroma-epithelial iyadoo lagu darayo unugyada stromal ee qaab-dhismeedka. Si kastaba ha ahaatee, dabeecadda qaab-dhismeedka qaab-dhismeedka ayaa laga yaabaa inay ka hortagto soo bandhigidda habka morphogenetic-ka ee iskiis u dhaca. Moodooyinkani sidoo kale ma bixiyaan socodka firfircoon ee luminal ama interstitial, iyagoo aan lahayn cadaadiska dareeraha ee unugyada mindhicirka u baahan yihiin inay maraan morphogenetic oo ay helaan shaqo jireed. Daraasad kale oo dhowaan la sameeyay ayaa isticmaashay qaab-dhismeedka hydrogel-ka ee madal microfluidic ah iyo qaab-dhismeedka epithelial-ka mindhicirka ee qaabaysan iyadoo la adeegsanayo farsamooyinka laysarka-qoynta. Jiirarka xiidmaha jiirka ayaa raaca qaababka la jarjaray si ay u sameeyaan qaab-dhismeedka tuubooyinka mindhicirka, socodka dareeraha gudaha luminal-kana waxaa dib loogu celin karaa iyadoo la adeegsanayo module microfluidics ah. Si kastaba ha ahaatee, qaabkani ma muujiyo Hawsha morphogenetic-ga ee iskiis u dhaca kuma jiraan dhaqdhaqaaqyada farsamada mindhicirka. Farsamooyinka daabacaadda 3D ee isla kooxdaas ayaa awooday inay abuuraan tuubooyin mindhicir yar oo leh habab morphogenetic ah oo iskiis u dhaca. In kasta oo sameynta adag ee qaybaha kala duwan ee mindhicirku ku jiro tuubada, qaabkani sidoo kale ma laha socodka dareeraha iftiinka iyo qaab-dhismeedka farsamada. Intaa waxaa dheer, hawlgalka moodeelka ayaa laga yaabaa inuu xaddidan yahay, gaar ahaan ka dib marka habka daabacaadda bioprinting uu dhammaado, taasoo khalkhal gelinaysa xaaladaha tijaabada ama isdhexgalka unugyada-unugyada. Taa beddelkeeda, hab-maamuuskayaga la soo jeediyay wuxuu bixiyaa morphogenesis-ka mindhicirka ee iskiis u dhaca, cadaadiska xiirashada ee jireed ahaan khuseeya, bayoomakiinikada oo ku dayanaya dhaqdhaqaaqa mindhicirka, helitaanka qaybaha apical iyo basolateral ee madaxbannaan, iyo dib-u-abuurista jawiyada bayoolojiga ee adag ee modularity. Sidaa darteed, hab-maamuuska morphogenesis-ka 3D ee vitro wuxuu bixin karaa hab dhammaystiran si looga gudbo caqabadaha hababka jira.
Hab-maamuuskeenu wuxuu si buuxda diiradda u saarayaa qaab-dhismeedka epithelial-ka 3D, iyadoo unugyada epithelial-ka oo keliya ay ku jiraan dhaqanka mana jiraan noocyo kale oo unugyo ku xeeran sida unugyada mesenchymal, unugyada endothelial-ka, iyo unugyada difaaca jirka. Sida hore loogu sharraxay, xudunta hab-maamuuskeenu waa kicinta qaab-dhismeedka epithelial-ka iyadoo laga saarayo inhibitors-ka morphogen-ka ee laga soo saaray dhinaca hoose ee dhexdhexaadka la soo bandhigay. Iyadoo qaab-dhismeedka adag ee xiidmaha-ku-jirta-jilibka iyo isku-dhafka-jilibka-jilibka ay noo oggolaanayso inaan dib u abuurno lakabka epithelial-ka 3D ee aan firfircoonayn, isku-dhafka bayoolojiga ee dheeraadka ah sida isdhexgalka epithelial-mesenchymal33,34, dhigista Matrix-ka ka baxsan unugyada (ECM) 35 iyo, qaabkeenna, astaamaha crypt-villus ee gudbiya niches-ka unugyada asliga ah ee sirta aasaasiga ah ayaa weli la tixgelinayaa. Unugyada Stromal (tusaale ahaan, fibroblasts) ee mesenchyme-ka ayaa door muhiim ah ka ciyaara soo saarista borotiinnada ECM iyo nidaaminta qaab-dhismeedka mindhicirka ee vivo35,37,38. Ku darista unugyada mesenchymal ee qaabkeenna waxay xoojisay habka qaab-dhismeedka iyo ku-xidhnaanta unugyada Waxtarka. Lakabka endothelial-ka (tusaale ahaan, xididdada dhiigga ama lymphatics) wuxuu door muhiim ah ka ciyaaraa nidaaminta gaadiidka molecular-ka39 iyo qorista unugyada difaaca jirka40 ee deegaanka mindhicirka. Intaa waxaa dheer, qaybaha xididdada dhiigga ee lagu xiri karo moodooyinka unugyada waa shuruud marka moodooyinka unugyada loogu talagalay inay muujiyaan isdhexgalka xubnaha badan. Sidaa darteed, unugyada endothelial-ka waxaa laga yaabaa in loo baahdo in lagu daro si loo qaabeeyo astaamaha jireed ee saxda ah oo leh xallinta heerka xubnaha. Unugyada difaaca jirka ee laga helo bukaanka ayaa sidoo kale lagama maarmaan u ah muujinta jawaabaha difaaca jirka ee dabiiciga ah, soo bandhigida antigen-ka, isgoyska difaaca jirka ee la qabsiga, iyo difaaca jirka ee gaarka u ah unugyada marka la eego ku dayashada cudurka mindhicirka.
Isticmaalka jajabyada isku-dhafan ayaa ka fudud marka loo eego caloosha-ku-jirta sababtoo ah dejinta qalabku waa mid fudud isticmaalka gelinnada Transwell waxay u oggolaanaysaa dhaqanka la miisaami karo ee epithelium-ka mindhicirka. Si kastaba ha ahaatee, gelinnada Transwell ee ganacsi ahaan laga heli karo oo leh xuubka polyester-ka ma aha kuwo dabacsan mana matali karaan dhaqdhaqaaqyo u eg peristaltic. Intaa waxaa dheer, qaybta dabacsan ee gelinnada Transwell ee lagu dhejiyay jajabka isku-dhafka ah ayaa ahayd mid taagan iyada oo aan lahayn cadaadis go'an dhinaca apical. Sida iska cad, sifooyinka taagan ee qaybta apical-ka ayaa si dhif ah u suurtageliya dhaqanka bakteeriyada muddada dheer ee jajabyada isku-dhafka ah. In kasta oo aan si xooggan u kicin karno qaab-dhismeedka 3D ee gelinnada Transwell marka la isticmaalayo jajabyada isku-dhafka ah, yaraanta biomechanics-ka jireed ee khuseeya iyo socodka dareeraha apical waxay xaddidi kartaa suurtagalnimada goobaha jajabka isku-dhafka ah ee codsiyada suurtagalka ah.
Dib-u-dhiska baaxadda leh ee dhidibka crypt-villus ee aadanaha ee ku jira dhaqamada gut-on-a-chip iyo hybrid-on-a-chip si buuxda looma aasaasin. Maadaama morphogenesis uu ka bilaabmo lakab epithelial ah, microarchitecture-yada 3D daruuri ma aha inay bixiyaan isku mid ahaanshaha morphological ee crypts-ka ee vivo. In kasta oo aan ku sifeynay tirada unugyada ee ku baahsan meel u dhow qaybta crypt-ka aasaasiga ah ee epithelium-ka 3D ee microengineered, gobollada crypt iyo villous si cad looma kala saarin. In kasta oo kanaallada sare ee sare ee chip-ku ay horseedeen dherer kordhay oo epithelium-ka microengineered ah, dhererka ugu badan wali wuxuu ku kooban yahay ~300-400 µm. Qoto dheer ee xiidmaha aadanaha ee xiidmaha yar iyo kuwa waaweyn waa ~135 µm iyo ~400 µm, siday u kala horreeyaan, dhererka villi-ga yar ee xiidmaha yarna waa ~600 µm41.
Marka laga eego dhinaca sawirka, sawir-qaadista heerka sare ah ee qaab-dhismeedka 3D ee gudaha waxaa laga yaabaa inay ku kooban tahay mindhicirka jajabka, maadaama masaafada shaqada ee loo baahan yahay laga bilaabo muraayadda ujeeddada ilaa lakabka epithelial ay tahay dhowr milimitir. Si looga gudbo dhibaatadan, waxaa laga yaabaa in loo baahdo ujeedo fog. Intaa waxaa dheer, sameynta qaybo khafiif ah oo loogu talagalay diyaarinta muunadaha sawir-qaadista waa mid adag sababtoo ah dabacsanaanta sare ee PDMS. Intaa waxaa dheer, maadaama qaab-dhismeedka lakabka-lakabka ee mindhicirka jajabku uu ku lug leeyahay isku-xidhka joogtada ah ee lakab kasta, aad bay u adag tahay in la furo ama laga saaro lakabka sare si loo baaro qaab-dhismeedka dusha sare ee lakabka epithelial. Tusaale ahaan, iyadoo la adeegsanayo mikroskoob elektaroonik ah oo sawiraya (SEM).
Cabsida biyaha ee PDMS waxay ahayd arrin xaddidan oo ku jirta daraasadaha ku salaysan microfluidic ee la xiriira molecules-ka yaryar ee hydrophobic, maadaama PDMS aysan si gaar ah u nuugi karin molecules-ka hydrophobic-ga ah. Beddelka PDMS waxaa laga yaabaa in lagu tixgeliyo agabyada kale ee polymeric-ga ah. Taas beddelkeeda, wax ka beddelka dusha sare ee PDMS (tusaale ahaan, dahaarka walxaha lipophilic 42 ama poly(ethylene glycol) 43) waxaa loo tixgelin karaa inay yareyso nuugista molecules-ka hydrophobic-ga.
Ugu dambeyntii, habkayagu si fiican looma qeexin marka la eego bixinta baaritaan heer sare ah ama goob tijaabo ah oo "hal-cabbir-ku-habboon-dhammaan" oo saaxiibtinimo u leh isticmaalaha. Hab-maamuuska hadda jira wuxuu u baahan yahay bamka sirinjiga ee qalab kasta oo microdevice ah, kaas oo meel ku qaata qalabka CO2 wuxuuna ka hortagaa tijaabooyinka baaxadda weyn. Xaddidaaddan waxaa si weyn loogu hagaajin karaa ballaarinta qaababka dhaqanka ee cusub (tusaale ahaan, 24-ceel, 96-ceel, ama 384-ceel daloolo leh oo u oggolaanaya dib-u-buuxin joogto ah iyo ka saarista warbaahinta hoose).
Si aan u kicino qaab-dhismeedka 3D ee epithelium-ka xiidmaha aadanaha in vitro, waxaan isticmaalnay qalab mindhicir oo microfluidic chip ah oo ka kooban laba microchannels oo isbarbar socda iyo xuub daloolo dabacsan oo u dhexeeya si aan u abuurno is-dhexgal lumen-capillary ah. Waxaan sidoo kale muujineynaa isticmaalka qalab microfluidic ah oo hal-kanaal ah (jilib isku-dhafan) kaas oo bixiya socod joogto ah oo salka hoose ah oo ka hooseeya lakabyada epithelial ee kala qaybsan ee lagu beero gelinta Transwell. Labada gooboodba, qaab-dhismeedka unugyada epithelial ee xiidmaha aadanaha waxaa lagu muujin karaa iyadoo la adeegsanayo hab-dhaqan jiho ah oo socodka ah si looga saaro antagonists-ka morphogen-ka qaybta hoose. Nidaamka tijaabada oo dhan (Jaantuska 1) wuxuu ka kooban yahay shan qaybood: (i) farsamaynta micro-fabrication ee jajabka xiidmaha ama jajabka isku-dhafka ah ee Transwell la gelin karo (tallaabooyinka 1-5; Sanduuqa 1), (ii) diyaarinta unugyada epithelial-ka xiidmaha (unugyada Caco-2) ama organoids-ka xiidmaha aadanaha; sanduuqyada 2-5), (iii) dhaqanka unugyada epithelial-ka mindhicirka ee jajabyada mindhicirka ama jajabyada isku-dhafan (tallaabooyinka 6-9), (iv) kicinta morphogenesis-ka 3D ee in vitro (tallaabada 10) iyo (v)) si loo garto qaab-dhismeedka micro-ka epithelial-ka 3D (tallaabooyinka 11-24). Ugu dambeyntii, koox xakameyn oo ku habboon (oo hoos looga hadlay) ayaa loogu talagalay in lagu xaqiijiyo waxtarka morphogenesis-ka in vitro iyadoo la barbar dhigayo morphogenesis-ka epithelial-ka iyo xakamaynta booska, ku-meel-gaarka ah, shuruudaysan, ama hab-raaca.
Waxaan isticmaalnay laba goobood oo dhaqameed oo kala duwan: mindhicir-ku-jiid-jiid oo leh kanaallo toosan ama kanaallo aan toos ahayn oo isku-dhafan, ama jajabyo isku-dhafan oo ka kooban Transwell (TW) oo lagu geliyo qalab microfluidic ah, oo la sameeyay sida lagu sharaxay Sanduuqa 1, iyo tallaabada 1 -5. "Qaab-dhismeedka Qalabka" wuxuu muujinayaa tallaabooyinka ugu muhiimsan ee sameynta jajab hal ama jajab isku-dhafan." Dhaqanka Unugyada Epithelial-ka Mindhicirka Aadanaha" wuxuu sharxayaa isha unugga (Caco-2 ama organoids-ka mindhicirka aadanaha) iyo habka dhaqanka ee loo isticmaalo hab-maamuuskaan. "In vitro morphogenesis" wuxuu muujinayaa tallaabooyinka guud ee unugyada epithelial-ka Caco-2 ama organoid-ka laga soo saaro organoid lagu beero jajab xiidmaha ama gelinta Transwell ee jajab isku-dhafan, oo ay ku xigto kicinta morphogenesis-ka 3D iyo sameynta qaab-dhismeedka epithelial-ka ee lagu sifeeyay. Lambarka tallaabada barnaamijka ama lambarka sanduuqa waxaa lagu soo bandhigay fallaar kasta hoosteeda. Codsigu wuxuu bixiyaa tusaalooyin sida lakabyada epithelial-ka mindhicirka ee la aasaasay loo isticmaali karo, tusaale ahaan, qeexidda kala-soocidda unugyada, daraasadaha fiisiyoolajiyadda mindhicirka, sameynta nidaamyada deegaanka ee microbiome-ka martida loo yahay, iyo qaabaynta cudurka. Sawirrada difaaca jirka ee "Unugga" Kala duwanaansho” oo muujinaysa nuclei, F-actin iyo MUC2 oo lagu muujiyey lakabka epithelial-ka 3D Caco-2 ee laga soo saaro jajabka mindhicirka. Calaamadaynta MUC2 waxay ku jirtaa unugyada koobka iyo xabka ka soo baxa dusha sare ee xuubka. Sawirrada iftiinka leh ee Fiisiyoolajiga Gut waxay muujinayaan xabka oo laga soo saaro rinjiyeynta sialic acid iyo haraaga N-acetylglucosamine iyadoo la adeegsanayo jeermiska qamadiga fluorescent agglutinin. Labada sawir ee is dulsaaran ee "Host-Microbe Co-Cultures" waxay muujinayaan dhaqanno isku-dhafan oo marti-geliye-microbiome ah oo ku jira mindhicirka jajab. Qaybta bidix waxay muujinaysaa dhaqanka isku-dhafka ah ee E. coli oo muujinaya borotiinka fluorescent cagaaran (GFP) oo leh unugyada epithelial-ka 3D Caco-2 ee microengineered. Qaybta midig waxay muujinaysaa meesha GFP E. coli oo la isku-dhaftay oo leh unugyada epithelial-ka 3D Caco-2, oo ay ku xigto rinjiyeynta immunofluorescence oo leh F-actin (casaan) iyo nuclei (buluug). Qaabka cudurku wuxuu muujinayaa mindhicirka caafimaad qaba iyo kan daadanaya ee jajabyada caabuqa mindhicirka iyadoo la raacayo caqabadda jir ahaaneed ee antigens-ka bakteeriyada (tusaale ahaan, lipopolysaccharide, LPS) iyo unugyada difaaca jirka (tusaale ahaan, PBMC; cagaar). Unugyada Caco-2 ayaa la beeray si ay u sameeyaan lakab epithelial 3D ah. Bar cabbir, 50 µm. Sawirro safka hoose ku jira: "Kala soocidda unugyada" waxaa loo habeeyey ogolaansho laga helay tixraac.2. Oxford University Press; Dib loo soo saaray iyadoo ogolaansho laga helay Ref.5. NAS; "Martigeliyaha-Microbe Co-Culture" waxaa loo habeeyey ogolaansho laga helay tixraac.3. NAS; "Qaabka Cudurrada" waxaa loo habeeyey ogolaansho laga helay tixraac.5. NAS.
Labada jajab ee mindhicirka-ku-jira iyo kuwa isku-dhafan ayaa la sameeyay iyadoo la adeegsanayo nuqullo PDMS ah oo laga soo saaray qaabab silicon ah iyadoo la adeegsanayo lithography jilicsan1,44 oo lagu qaabeeyey SU-8. Naqshadeynta kanaallada yaryar ee jajab kasta waxaa lagu go'aamiyaa iyadoo la tixgelinayo hydrodynamics sida cadaadiska shaararka iyo cadaadiska hydrodynamic1,4,12. Naqshadda asalka ah ee mindhicirka-ku-jira (Sawirka Xogta La-dheereeyay 1a), oo ka koobnayd laba kanaal oo toosan oo isku-dhafan, ayaa isu beddelay mid adag oo caloosha-ku-jira (Sawirka Xogta La-dheereeyay 1b) oo ay ku jiraan labo kanaal oo qaloocan si loo kiciyo waqtiga degenaanshaha dareeraha oo kordha, qaababka socodka aan tooska ahayn, iyo isbeddelka badan ee unugyada la beeray (Sawirka 2a-f) 12. Marka bayoomakiinikada mindhicirka ee adag loo baahan yahay in la abuuro, jajabyada adag ee mindhicirka-ku-jira ayaa la dooran karaa. Waxaan tusnay in Gut-Chip-ka isku-jira uu sidoo kale si xooggan u kiciyo morphogenesis 3D waqti isku mid ah oo leh heer la mid ah koritaanka epithelial marka la barbar dhigo asalka Gut-Chip, iyadoon loo eegin nooca unugyada la beeray. Sidaa darteed, si loo kiciyo qaab-dhismeedka 3D, naqshadaha mindhicirka ee toosan iyo kuwa isku dhafan ayaa la is beddeli karaa. Noocyada PDMS ee lagu daaweeyay qaababka silicon ee leh qaababka SU-8 waxay bixiyeen astaamo taban ka dib markii la burburiyo (Jaantuska 2a). Si loogu sameeyo mindhicirka jajab, lakabka sare ee PDMS ee la diyaariyey waxaa si isdaba joog ah loogu xidhay filim PDMS ah oo daloolsan ka dibna waxaa la waafajiyay lakabka hoose ee PDMS iyadoo la adeegsanayo isku-xidh aan dib loo celin karin iyadoo la adeegsanayo daawayn corona ah (Jaantuska 2b-f). Si loo sameeyo jajabyo isku-dhafan, nuqullada PDMS ee la bogsiiyay waxaa lagu xidhay sawirro galaas ah si loo abuuro aalado microfluidic ah oo hal-kanaal ah oo qaadi kara gelinta Transwell (Jaantuska 2h iyo Xogta La Fidiyay Sawirka 2). Habka isku-xidhka waxaa lagu sameeyaa iyadoo lagu daaweynayo dusha sare ee nuqulka PDMS iyo galaaska balaasmaha oksijiinta ama daaweynta corona. Ka dib markii la nadiifiyo qalabka microfabricated ee ku xiran tuubada silicone, dejinta qalabku waxay diyaar u ahayd inay fuliso qaab-dhismeedka 3D ee epithelium-ka mindhicirka (Jaantuska 2g).
a, Sawir sawireed oo ku saabsan diyaarinta qaybaha PDMS ee ka imanaya qaababka silicon ee SU-8. Xalka PDMS ee aan la daaweyn ayaa lagu shubay qaab silicon ah (bidix), oo lagu daaweeyay 60 °C (dhexe) oo la dumiyay (midig). PDMS-kii la dumiyay ayaa la jarjaray oo la nadiifiyay si loo sii isticmaalo.b, Sawirka qaabka silicon ee loo isticmaalay in lagu diyaariyo lakabka sare ee PDMS.c, Sawirka qaabka silicon ee loo isticmaalay in lagu sameeyo xuubka daloolsan ee PDMS.d, Taxane sawirro ah oo ka kooban qaybaha PDMS ee sare iyo hoose iyo qalabka mindhicirka ee la isku keenay ee ku yaal jajabka.e, Sawirka isku-xidhka qaybaha PDMS ee sare, xuubka, iyo hoose. Lakab kasta waxaa si aan dib loo celin karin ugu xidhma plasma ama daaweynta corona.f, Sawirka qalabka mindhicirka ee la sameeyay oo leh kanaal microcomputed ah oo la dul saaray iyo qolalka faakiyuumka.g, Dejinta mindhicirka-ku-jira-jiibka dhaqanka unugyada microfluidic. Mindhicirka la sameeyay oo ku yaal jajab lagu soo ururiyay tuubo silicon ah iyo sirinji ayaa la saaray dabool. Qalabka jajabka waxaa la dhigay daboolka Saxan Petri ah oo 150 mm ah oo loogu talagalay farsamaynta. Xidhmada waxaa loo isticmaalaa in lagu xiro tuubada silicone.h, Sawiro muuqaal ah oo ku saabsan sameynta jajabka isku-dhafan iyo qaab-dhismeedka 3D iyadoo la adeegsanayo jajabyada isku-dhafan. Gelida Transwell si madax-bannaan loogu diyaariyey dhaqanka 2D monolayers ee unugyada epithelial-ka mindhicirka ayaa la geliyay jajabka isku-dhafka ah si loo kiciyo qaab-dhismeedka 3D ee mindhicirka. Dhexdhexaadinta waxaa lagu shubaa iyada oo loo marayo kanaallo yar yar oo ka hooseeya lakabka unugyada ee lagu sameeyay gelinta Transwell. Barka cabbirka, 1 cm.h Dib ayaa loo daabacay iyadoo la oggolaanayo tixraaca.4. Elsevier.
Hab-maamuuskaan, khadka unugyada Caco-2 iyo organoids-ka xiidmaha ayaa loo isticmaalay ilo epithelial ah (Jaantuska 3a). Labada nooc ee unugyada waxaa si madax-bannaan loo beeray (Sanduuqa 2 iyo Sanduuqa 5) waxaana loo isticmaalay in lagu beero kanaallada yaryar ee ECM-da ku dahaaran ee mindhicirka ku jira ama gelinta Transwell. Marka unugyadu ay isku milmaan (>95% daboolida dhalooyinka), unugyada Caco-2 ee si joogto ah loo beero (inta u dhaxaysa marinnada 10 iyo 50) ee ku jira dhalooyinka T-ka ayaa la goostaa si loogu diyaariyo joojinta unugyada kala go'ay iyadoo la adeegsanayo dareeraha trypsinization (sanduuqa 2). Unugyada xiidmaha aadanaha ee laga soo qaaday biopsy-ga xiidmaha ama qalliinnada ayaa lagu beeray godadka qaab-dhismeedka Matrigel ee saxanno 24-bool ah si loo taageero deegaanka yar ee qaab-dhismeedka. morphogens-ka dhexdhexaadka ah ee muhiimka ah (sida Wnt, R-spondin, iyo Noggin) iyo arrimaha koritaanka ee loo diyaariyey sida lagu sharraxay Sanduuqa 3 ayaa la kabay maalin kasta oo kale ilaa organoids-ku ay koraan ~500 µm dhexroor ahaan. Unugyada si buuxda u koray ayaa la goostaa oo la kala saaraa Unugyo keli ah oo lagu beero mindhicirka ama Transwell oo ku jira jajab (Sanduuqa 5). Sidaan hore u soo sheegnay, waxaa loo kala saari karaa iyadoo loo eegayo nooca cudurka 12,13 (tusaale ahaan boogta caloosha, cudurka Crohn, kansarka mindhicirka, ama deeq-bixiyaha caadiga ah), goobta dhaawaca (tusaale ahaan, nabarka iyo aagga aan dhaawaca lahayn) iyo goobta caloosha iyo mindhicirka ee marinnada (tusaale, duodenum, jejunum, ileum, cecum, mindhicirka, ama malawadka). Waxaan ku bixinnaa hab-maamuus la hagaajiyay Sanduuqa 5 si loo beero organoids-ka mindhicirka (coloids) kuwaas oo caadiyan u baahan fiirsasho sare oo morphogens ah marka loo eego organoids-ka mindhicirka yar.
a, Socodka shaqada ee kicinta morphogenesis-ka mindhicirka ee jajabka mindhicirka. Caco-2 epithelium-ka mindhicirka aadanaha iyo organoids-ka mindhicirka ayaa loo isticmaalaa hab-raacan si loo muujiyo morphogenesis-ka 3D. Unugyada epithelial-ka ee go'doonsan ayaa lagu beeray qalabka la diyaariyey ee mindhicirka-ku-samee-jilibka (diyaarinta jajabka). Marka unugyada la beero (la beero) oo lagu lifaaqo (lagu lifaaqo) xuubka daloolsan ee PDMS maalinta 0 (D0), socodka apical (AP) ayaa la bilaabaa oo la ilaaliyaa 2da maalmood ee ugu horreeya (socodka, AP, D0-D2). Socodka salka hoose (BL) ayaa sidoo kale la bilaabaa iyadoo lala socdo dhaqdhaqaaqyada fidinta wareegga ah (fidinta, socodka, AP iyo BL) marka la sameeyo lakab dhammaystiran oo 2D ah. Morogenesis-ka 3D ee mindhicirka ayaa si iskiis ah u dhacay ka dib 5 maalmood oo dhaqanka microfluidic ah (morphogenesis, D5). Sawirrada isbarbardhigga wejiga waxay muujinayaan qaab-dhismeedka matalaya unugyada Caco-2 tallaabo kasta oo tijaabo ah ama waqti kasta (garaafka baarka, 100 µm). Afar jaantus oo jaantus ah oo muujinaya cascades u dhigma ee mindhicirka morphogenesis (midigta sare). Fallaadhaha jajaban ee ku jira jaantuska waxay matalaan jihada socodka dareeraha.b, Sawirka SEM wuxuu muujinayaa dusha sare ee epithelium-ka Caco-2 ee la aasaasay (bidix). Qaybta hoose ee iftiiminaysa aagga la weyneeyay (sanduuqa cad ee jajaban) waxay muujinaysaa microvilli-ga dib loo soo nooleeyay ee lakabka 3D Caco-2 (midig).c, Aragtida hore ee toosan ee Caco-2 3D ee la aasaasay, claudin (ZO-1, casaan) iyo xuubka xuduudaha ee joogtada ah ee burushka lagu calaamadeeyay F-actin (cagaar) iyo nuclei (buluug) Immunofluorescence muuqaal confocal ah oo unugyada epithelial ah oo ku yaal jajabyada mindhicirka. Fallaadhaha tilmaamaya jaantuska dhexe waxay tilmaamayaan goobta diyaaradda diiradda saaran ee aragti kasta oo confocal ah.d, Waqtiga isbeddelka qaab-dhismeedka ee organoids-ka lagu beeray jajab laga helay mikroskoobka isbarbardhigga marxaladda maalmaha 3, 7, 9, 11, iyo 13. Qaybta hoose (midigta sare) waxay muujinaysaa weynaynta sare ee sawirka la bixiyay.e, DIC photomicrograph ee epithelium-ka organoid 3D ee lagu aasaasay mindhicirka Qayb laga qaaday maalinta 7.f, Sawirrada difaaca jirka ee dul saaran oo muujinaya calaamado unugyada asliga ah (LGR5; magenta), unugyada goblet (MUC2; cagaar), F-actin (cawlan) iyo nuclei (cyan) oo lagu beero jajabyada mindhicirka muddo 3 maalmood ah, siday u kala horreeyaan (Bidix) iyo 13-maalmood (dhexe) organoids-ka lakabka epithelial-ka. Sidoo kale eeg Xogta La Fidiyay Jaantuska 3, kaas oo iftiiminaya calaamadaynta LGR5 iyada oo aan lahayn calaamadaynta MUC2. Sawirrada fluorescence-ka oo muujinaya qaab-dhismeedka epithelial-ka (midig) ee epithelium-ka organoid-ka 3D ee laga sameeyay mindhicirka jajab iyadoo lagu rinjiyeynayo xuubka balaasmaha iyadoo la isticmaalayo rinjiga CellMask (midig) maalinta 13aad ee dhaqanka. Barka cabbirka waa 50 μm haddii aan si kale loo sheegin.b Dib loo daabacay ogolaansho laga helay tixraac.2. Oxford University Press; c La qabsaday ogolaansho laga helay Tixraaca.2. Oxford University Press; e iyo f ayaa la waafajiyay ogolaansho laga helay tixraac.12 Hoosta Shatiga Creative Commons CC BY 4.0.
Xiidmaha oo ku yaal jajab, waxaa lagama maarmaan ah in wax laga beddelo dusha sare ee xuubka daloolsan ee PDMS si loo helo dahaarka ECM ee guuleysta. Hab-maamuuskaan, waxaan ku dabaqnaa laba hab oo kala duwan si aan wax uga beddelno hydrophobicity ee xuubka PDMS. Si loo kobciyo unugyada Caco-2, kicinta dusha sare ee daaweynta UV/ozone oo keliya ayaa ku filnayd in la yareeyo hydrophobicity ee dusha sare ee PDMS, lagu dahaadho ECM oo lagu dhejiyo unugyada Caco-2 xuubka PDMS. Si kastaba ha ahaatee, dhaqanka microfluidic ee epithelium organoid wuxuu u baahan yahay shaqo-ku-saleysan dusha sare ee kiimikada si loo gaaro dhigista hufan ee borotiinada ECM iyadoo si isdaba joog ah loogu marinayo polyethyleneimine (PEI) iyo glutaraldehyde kanaalka PDMS. Ka dib wax ka beddelka dusha sare, borotiinada ECM waxaa la dhigay si loo daboolo dusha sare ee PDMS ee shaqeynaya ka dibna lagu soo bandhigay epithelium organoid go'doonsan. Ka dib marka unugyada la xiro, dhaqanka unugyada microfluidic wuxuu ku bilaabmaa isagoo kaliya ku shubaya dhexdhexaadka microchannel-ka sare ilaa unugyadu ay sameeyaan hal lakab oo dhammaystiran, halka kanaalka hoose uu ilaaliyo xaaladaha taagan. Habkan loo habeeyay ee kicinta dusha sare iyo dahaarka ECM wuxuu awood u siinayaa ku xidhnaanta organoid. epithelium si uu u kiciyo qaab-dhismeedka 3D ee dusha sare ee PDMS.
Dhaqanka Transwell sidoo kale wuxuu u baahan yahay dahaarka ECM ka hor inta aan la beerin unugyada; si kastaba ha ahaatee, dhaqamada Transwell uma baahna tallaabooyin daaweyn ka hor ah si loo kiciyo dusha sare ee gelinta daloolada. Si loo koriyo unugyada Caco-2 ee gelinta Transwell, dahaarka ECM ee gelinta daloolada waxay dardar gelisaa ku xidhnaanta unugyada Caco-2 ee kala go'ay (<1 saac) iyo sameynta caqabado isku xiran (<1-2 maalmood). Si loo beero organoids-ka gelinta Transwell, organoids-ka go'doonsan waxaa lagu beeraa gelinta dahaarka leh ee ECM, oo lagu dhejiyo dusha sare ee xuubka (<3 saacadood) oo la ilaaliyo ilaa organoids-ku ay sameeyaan hal lakab oo dhammaystiran oo leh hufnaan caqabad. Dhaqanka Transwell waxaa lagu sameeyaa saxanno 24-bool ah iyada oo aan la isticmaalin jajabyo isku dhafan.
In vitro 3D morphogenesis waxaa lagu bilaabi karaa iyadoo la marinayo socodka dareeraha dhinaca hoose ee lakabka epithelial ee la aasaasay. Mindhicirka ku yaal jajab, morphogenesis epithelial wuxuu bilaabmay markii dhexdhexaadka lagu shubay kanaalka sare iyo kan hoose ee microchannels (Sawirka 3a). Sida hore loo sharraxay, waa muhiim in la soo bandhigo socodka dareeraha ee qaybta hoose (basolateral) si looga saaro si joogto ah daawooyinka morphogen ee jihooyinku soo daayaan. Si loo siiyo nafaqooyin iyo seeram ku filan unugyada ku xiran xuubka daloolada oo loo abuuro cadaadis xiirto iftiin leh, waxaan caadi ahaan ku shubnaa socodka laba-laabka ah ee mindhicirka jajab. Chips-yada isku-dhafka ah, gelinta Transwell oo ka kooban monolayers-ka epithelial ayaa la geliyay jajabyada isku-dhafka ah. Kadib, dhexdhexaadka waxaa lagu dabaqay dhinaca hoose ee gelinta daloolka Transwell iyada oo loo marayo kanaalka microchannel. morphogenesis-ka mindhicirka wuxuu dhacay 3-5 maalmood ka dib markii la bilaabay socodka salka ee labada goobood ee dhaqanka.
Astaamaha qaab-dhismeedka ee lakabyada epithelial-ka 3D ee microengineered waxaa lagu falanqeyn karaa iyadoo la adeegsanayo habab sawireed oo kala duwan, oo ay ku jiraan microscopy-ga isbarbardhigga wejiga, microscopy-ga kala duwanaanshaha faragelinta kala duwan (DIC), SEM, ama immunofluorescence confocal microscopy (Jaantusyada 3 iyo 4). Kala duwanaanshaha wejiga ama sawir-qaadista DIC si fudud ayaa loo samayn karaa wakhti kasta inta lagu jiro dhaqanka si loola socdo qaabka iyo soo bixitaanka lakabyada epithelial-ka 3D. Sababtoo ah hufnaanta indhaha ee PDMS iyo filimada polyester, labada goobood ee jajabka caloosha-ku-jira iyo kuwa isku-dhafan waxay bixin karaan sawir-qaadista waqtiga-dhabta ah iyada oo aan loo baahnayn in la kala saaro ama la kala furo qalabka. Marka la samaynayo sawir-qaadista immunofluorescence (Jaantusyada 1, 3c, f iyo 4b, c), unugyada waxaa caadi ahaan lagu hagaajiyaa paraformaldehyde 4% (wt/vol) (PFA), oo ay ku xigto Triton X-100 iyo 2% (wt/vol) ) serum serum bovine (BSA), si nidaamsan. Iyada oo ku xidhan nooca unugyada, hagaajin kala duwan, permeabilizers, iyo xannibaadda. Iyada oo ku xidhan nooca unugyada, hagaajin kala duwan, permeabilizers, iyo xannibaadda. Iyada oo ku xidhan nooca unugyada, hagaajin kala duwan, permeabilizers, iyo xannibaadda. Iyada oo ku xidhan nooca unugyada, hagaajin kala duwan, permeabilizers, iyo xannibaadda. Iyada oo ku xidhan nooca unugyada, hagaajin kala duwan, permeabilizers, iyo xannibaadda. wakiilada waa la isticmaali karaa. Unugyada difaaca jirka ee aasaasiga ah ee bartilmaameedsanaya calaamadaha unugyada ama gobolka ee ku xiran hiddaha ayaa loo isticmaalaa in lagu muujiyo unugyada aan dhaqdhaqaaq lahayn ee ku jira hiddaha, oo ay ku xigto unugyada difaaca jirka ee labaad oo ay weheliso midab ka-hortagga wasakhda oo bartilmaameedsanaya nucleus (tusaale ahaan, 4′, 6-diamidino-2-phenylene) indole, DAPI) ama F-actin (tusaale ahaan, phalloidin oo lagu calaamadeeyay fluorescent). Sawir-qaadista tooska ah ee ku salaysan fluorescent-ka ayaa sidoo kale lagu samayn karaa in situ si loo ogaado wax soo saarka xabka (Sawirka 1, "Kala-soocidda Unugyada" iyo "Fiisiyoolajiyadda Gut"), gumeysi aan kala sooc lahayn oo unugyada microbial ah (Sawirka 1, "Isku-dhafka-microbe-ka ee Marti-gelinta"), qorista unugyada difaaca jirka (Sawirka 1, 'Moodaynta Cudurrada') ama qaababka qaab-dhismeedka epithelial 3D (Sawirka 3c, f iyo 4b, c). Marka la beddelayo mindhicirka ku yaal jajabka si loo kala saaro lakabka sare iyo lakabka hoose ee microchannel, sida lagu sharaxay tixraaca. Sida lagu muujiyay Sawirka 2, qaab-dhismeedka epithelial 3D iyo sidoo kale microvilli-ga ku yaal Xuduudda burushka apical waxaa lagu arki karaa SEM (Jaantuska 3b). Muujinta calaamadaha kala soocidda waxaa lagu qiimeyn karaa iyadoo la samaynayo isku xigxiga PCR5 ee tirada ama hal unug ee RNA. Xaaladdan oo kale, lakabyada 3D ee unugyada epithelial ee ku koray jajabyada mindhicirka ama jajabyada isku dhafan ayaa la goostaa iyadoo la adeegsanayo trypsinization ka dibna loo isticmaalo falanqaynta molecular ama hidde-side.
a, Socodka shaqada ee kicinta morphogenesis-ka xiidmaha ee jajab isku-dhafan. Caco-2 iyo organoids-ka xiidmaha ayaa loo isticmaalaa hab-raacan si loo muujiyo morphogenesis-ka 3D ee goob jajab isku-dhafan ah. Unugyada epithelial-ka ee kala go'ay ayaa lagu beeray gelinta Transwell ee la diyaariyey (diyaarinta TW; eeg sawirka hoose). Markii unugyada la beeray (la beeray) oo lagu dhejiyay xuubka polyester-ka ee gelinta Transwell, dhammaan unugyada waxaa lagu beeray xaalado taagan (dhaqanka TW). 7 maalmood ka dib, hal gelin Transwell ah oo ka kooban hal lakab oo 2D ah oo unugyada epithelial ah ayaa lagu daray jajab isku-dhafan si loo soo bandhigo socodka salka (Flow, BL), taasoo ugu dambeyntii keentay soo saarista lakabka epithelial-ka 3D (morphogenesis). Micrographs-ka isbarbardhigga wejiga oo muujinaya sifooyinka morphological ee unugyada epithelial-ka xubnaha aadanaha ee laga soo qaatay deeq-bixiyaha caadiga ah (khadka C103) oo kor u kacaya xiidmaha tallaabo kasta oo tijaabo ah ama waqti kasta. Jaantusyada ku jira lakabyada sare waxay muujinayaan qaabaynta tijaabada ee tallaabo kasta.b, Chips-ka isku-dhafan (qaabka bidix) waxay horseedi karaan morphogenesis-ka 3D ee unugyada epithelial-ka ee organoid oo leh confocal sare-hoos ah Aragtiyada mikroskoobka ee laga qaaday boosaska kala duwan ee Z (sare, dhexe, iyo hoose; fiiri xariiqyada dhibcaha ee midig iyo kuwa u dhigma). waxay muujiyeen astaamo morphological ah oo muuqda. F-actin (cyan), nucleus (cawlan).c, Micrographs confocal fluorescence (muuqaal 3D ah) ee unugyada epithelial-ka ee laga soo qaatay organoid oo lagu beeray Transwell static (TW; gudaha sanduuqa cad ee jajaban) iyo chip isku-dhafan (sawirka buuxa ee ugu weyn) oo isbarbardhigaya qaab-dhismeedka 2D iyo 3D, siday u kala horreeyaan. Labo aragti oo toosan oo 2D ah oo isgoys ah (ku yaal geeska sare ee midig; "XZ") ayaa sidoo kale muujinaya astaamo 2D iyo 3D ah. Bar cabbirka, 100 µm.c Dib ayaa loo daabacay iyadoo ogolaansho laga helayo tixraaca.4. Elsevier.
Xakamaynta waxaa lagu diyaarin karaa iyadoo la beerayo unugyo isku mid ah (Caco-2 ama unugyada epithelial-ka mindhicirka) oo loo qaybiyo lakabyo laba-geesood ah oo hoos yimaada xaaladaha dhaqanka caadiga ah ee aan joogtada ahayn. Waxaa xusid mudan in yaraanta nafaqooyinka ay ka dhalan karto awoodda xaddidan ee kanaallada yaryar (tusaale ahaan ~4 µL ee kanaalka sare ee naqshadda mindhicirka-jarista asalka ah). Sidaa darteed, qaab-dhismeedka epithelial ka hor iyo ka dib codsiga socodka hoose ayaa sidoo kale la barbar dhigi karaa.
Habka lithography-ga jilicsan waa in lagu sameeyaa qol nadiif ah. Lakab kasta oo ku yaal jajabka (lakabyada sare iyo hoose iyo xuubka) iyo jajabyada isku-dhafka ah, maaskaro sawireed oo kala duwan ayaa la isticmaalay oo lagu sameeyay wafers silicon ah oo kala duwan sababtoo ah dhererka kanaallada yaryar way kala duwanaayeen. Dhererka bartilmaameedka ee kanaallada sare iyo hoose ee mindhicirka ee jajabka waa 500 µm iyo 200 µm, siday u kala horreeyaan. Dhererka bartilmaameedka kanaalka ee jajabka isku-dhafka ah waa 200 µm.
Saxan silikoon ah oo 3-inji ah ku rid saxan leh acetone. Si tartiib ah u rog saxanka muddo 30 ilbiriqsi ah, ka dibna hawo ku qalaji waferka. Waferka ku wareeji saxan leh IPA, ka dibna saxanka ku wareeji 30 ilbiriqsi si aad u nadiifiso.
Xalka piranha (isku darka hydrogen peroxide iyo aashitada sulfuric ee xoogga leh, 1:3 (vol/vol)) ayaa ikhtiyaar ahaan loo isticmaali karaa si loo kordhiyo ka saarista haraaga dabiiciga ah ee dusha sare ee wafer-ka silicon.
Xalka Piranha aad buu u daxal badan yahay wuxuuna dhaliyaa kuleyl. Taxaddar dheeraad ah oo badbaado ayaa lagama maarmaan ah. Si aad u tuurto qashinka, u ogolow xalka inuu qaboojiyo oo uu u wareejiyo weel qashin oo nadiif ah oo qalalan. Isticmaal weelasha labaad oo si habboon u calaamadee weelasha qashinka. Fadlan raac tilmaamaha badbaadada ee xarunta si aad u hesho habraacyo faahfaahsan.
Faleebada nuuga ku rid saxan kulul oo 200 °C ah muddo 10 daqiiqo ah. Ka dib fuuqbaxa, keega waxaa la ruxay shan jeer hawada si uu u qaboojiyo.
Ku shub ~10 g oo ah suufka iska caabiya sawirka SU-8 2100 bartamaha waferka silikoon ee la nadiifiyey. Isticmaal birqayayaal si aad si siman ugu fidiso suufka iska caabiya sawirka. Mararka qaarkood waferka saar saxan kulul oo 65°C ah si aad u yareyso dhegdhegga sawirka oo aad u fududaato in la faafiyo. Ha dhigin waferka si toos ah saxanka kulul.
SU-8 si siman ayaa loogu qaybiyay wafer-ka iyadoo la adeegsanayo dahaarka wareegga. Barnaamiji wareeg soo socda oo SU-8 ah 5-10 ilbiriqsi si loogu faafiyo 500 rpm xawaare dhan 100 rpm/s. Deji wareegga ugu weyn qaabaynta dhumucda 200 µm 1,500 rpm, ama gaar dhumucda 250 µm (samaynta dherer 500 µm ah lakabka sare ee mindhicirka ee ku yaal jajabka; eeg "Tallaabooyinka Muhiimka ah" ee hoose) oo lagu dejiyay xawaare dhan 300 rpm/s 30 ilbiriqsi 1,200 rpm.
Xawaaraha wareegga ugu weyn waxaa lagu hagaajin karaa iyadoo loo eegayo dhumucda bartilmaameedka ee qaabka SU-8 ee ku yaal wafer silicon ah.
Si loo sameeyo qaababka SU-8 oo dhererkoodu yahay 500 µm lakabka sare ee mindhicirka ee ku yaal jajabka, dahaarka wareegga iyo tallaabooyinka dubista jilicsan ee Sanduuqan (tallaabooyinka 7 iyo 8) ayaa si isdaba joog ah loogu celceliyay (fiiri tallaabada 9) si loo soo saaro laba lakab oo ah lakab qaro weyn oo 250 µm ah oo SU-8 ah, kaas oo lakab lagu dhejin karo oo lagu dari karo soo-gaadhista UV ee tallaabada 12 ee sanduuqan, taasoo ka dhigaysa lakab dhererkiisu yahay 500 µm.
Si jilicsan u dub buskudka dahaaran ee SU-8 adigoo si taxaddar leh u saaraya buskudka saxan kulul oo heerkulkiisu yahay 65 °C muddo 5 daqiiqo ah, ka dibna u beddel dejinta 95 °C oo ku rid 40 daqiiqo oo dheeraad ah.
Si aad u gaarto dherer dhan 500 μm oo qaabka SU-8 ah oo ku yaal kanaalka sare ee microchannel, ku celi tallaabooyinka 7 iyo 8 si aad u soo saarto laba lakab oo SU-8 ah oo dhumucdiisu tahay 250 μm.
Adigoo isticmaalaya Qalabka Maaskarada UV, samee tijaabo nal ah sida ku cad tilmaamaha soo saaraha si aad u xisaabiso wakhtiga soo-gaadhista ee wafer-ka. (waqtiga soo-gaadhista, ms) = (qiyaasta soo-gaadhista, mJ/cm2)/(awoodda laambadda, mW/cm2).
Ka dib marka aad go'aamiso wakhtiga soo-gaadhista, dhig maaskarada sawirka ee ku dheggan meesha maaskarada ee qalabka maaskarada UV-ga oo dhig maaskarada sawirka ee ku dheggan wafer-ka dahaaran SU-8.
Dusha daabacan ee maaskarada sawirka si toos ah ugu dheji dhinaca dahaarka leh ee SU-8 ee wafer silicon si loo yareeyo faafitaanka UV.
Wafer-ka dahaaran ee SU-8 iyo maaskaro sawireed si toosan ugu soo bandhig 260 mJ/cm2 oo iftiin UV ah wakhtiga la go'aamiyay ee soo-gaadhista (fiiri tallaabada 10 ee sanduuqan).
Ka dib markii la soo bandhigo UV-ga, buskudka silicon-ka ee SU-8-dahaarka leh waxaa lagu dubay heerkul ah 65°C muddo 5 daqiiqo ah iyo 95°C muddo 15 daqiiqo ah saxan kasta oo kulul si loo sameeyo qaabab leh dherer ah 200 μm. Ku kordhi wakhtiga dubista ka dib 95°C ilaa 30 daqiiqo si aad u samayso qaabab leh dherer ah 500 µm.
Horumariyaha waxaa lagu shubaa saxan galaas ah, ka dibna saxanka la dubay ayaa la dhigayaa saxanka. Mugga horumariyaha SU-8 wuu kala duwanaan karaa iyadoo ku xiran cabbirka saxanka galaaska. Hubi inaad isticmaasho horumariye ku filan oo SU-8 ah si uu gebi ahaanba uga saaro SU-8 aan la daboolin. Tusaale ahaan, marka aad isticmaaleyso saxan galaas ah oo dhexroorkiisu yahay 150 mm oo leh awood 1 L ah, isticmaal ~300 mL oo horumariye SU-8 ah. Samee qaabka 25 daqiiqo iyadoo marmar la wareejinayo si tartiib ah.
Ku raaci caaryada la sameeyay ~10 mL oo horumariye cusub ah ka dibna IPA adigoo ku buufinaya xalka adigoo isticmaalaya pipette.
Ku rid buskudka nadiifiyaha balaasmaha oo ku dabool balaasmaha oksijiinta (gaaska hawada, cadaadiska bartilmaameedka 1 × 10−5 Torr, awoodda 125 W) muddo 1.5 daqiiqo ah.
Wafer-ka geli qalajiye faakiyuum ah oo gudaha galaas ku jira. Wafer-ka iyo slide-ka waxaa la dhigi karaa dhinac-is-dhaafsi. Haddii qalajiyaha faakiyuumku uu dhowr lakab u qaybiyo saxan, slide-ka dhig qolka hoose, wafer-kana dhig qolka sare. Ku rid 100 μL oo ah xalka trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane muraayadda oo mari faakiyuum si aad u hesho silanization.
Dhalaal dhalo ah oo unugyada Caco-2 ah oo barafaysan ku shub biyo kulul oo 37°C ah, ka dibna unugyada dhalaalay u wareeji weel T75 ah oo ka kooban 15 mL oo ah 37°C oo hore loo diiriyay Caco-2.
Si aad unugyada Caco-2 ugu gudubto ~90% isku-darka, marka hore diirran Caco-2 dhexdhexaad ah, PBS, iyo 0.25% trypsin/1 mM EDTA ku dhex qubeys biyo ah oo 37°C ah.
Ku neefso daawada adigoo isticmaalaya nuugid faakiyuum ah. Ku dhaq unugyada laba jeer 5 mL oo PBS diirran ah adigoo ku celcelinaya nuugid faakiyuum ah oo ku daraya PBS cusub.