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morphogenesis mindhicirka bini'aadamku wuxuu dejiyaa sifooyinka crypt-villus ee 3D epithelial microarchitecture iyo hay'adda spatial. Qaabdhismeedkan gaarka ah ayaa loo baahan yahay si loo ilaaliyo homeostasis mindhicirka iyadoo la ilaalinayo niche unugga stem ee crypt basal ka antigens microbial antigens iyo dheef-shiid kiimikaadkooda. Sidaa darteed, dib-u-abuurista qaababka epithelial ee 3D ayaa muhiim u ah dhismaha moodooyinka xiidmaha vitro. Waxaa xusid mudan, mindhicirka mimetic-ka-chip-ka wuxuu keeni karaa morphogenesis 3D ah ee epithelium mindhicirka iyadoo la xoojiyay hawlaha jireed iyo biomechanics. morphogenesis ee mindhicirka ee chip microfluidic ah iyo sidoo kale in a Transwell isku dhafan chip hybrid.Waxaan ku tilmaamay hababka faahfaahsan ee qalabaynta qalabka, culturing of Caco-2 ama unugyada epithelial xiidmaha organoid ee goobaha caadiga ah iyo sidoo kale on madal microfluidic ah, induction of 3D morphogenesis, iyo sifada of 3D dib-u-habaynta qaabaynta qaabaynta qaabaynta 3D. Microarchitecture mindhicirka functional by xakameynaya socodka dareeraha basolateral ee 5 d. Our in vitro morphogenesis Habka shaqaaleeya physiologically ku habboon xiirid stress iyo dhaqdhaqaaqa farsamada oo aan u baahnayn injineernimada unugyada adag ama khalkhalgelinta, kaas oo laga yaabo in ay ka sarreeyaan farsamooyinka kale ee jira loogu talagalay codsiyada bayoolajiga, bukaan-socodka, iyo daawooyinka.
Tijaabooyinku waxay muujinayaan in unugyada epithelial mindhicirka ee Caco-2 ee lagu beeray mindhicirka-on-a-chip1,2,3,4,5 ama aaladaha microfluidic bilayer6,7 ay mari karaan morphogenesis ee 3D ee gudaha vitro iyada oo aan si cad loo fahmin habka asaasiga ah. 3D epithelial morphogenesis in vitro, kaas oo lagu muujiyay Caco-2 iyo xubnaha unugyada mindhicirka ee bukaan-socodka. Unugyada Epithelial ayaa la ansaxiyay. Daraasaddan, waxaan si gaar ah diiradda u saarnay wax soo saarka unugyada iyo qaybinta xoogga saarista Wnt antagonist, Dickkopf-1 (DKK-1), ee gut-on-a-chip iyo qalabka microfluidic ee la beddelay oo ay ku jiraan "Transwell inserts", oo loo yaqaan "Hibrid Chip" . borotiinka frizzled-related 1, ama Soggy-1) ee mindhicirka-chip wuxuu joojiyaa morphogenesis ama wuxuu carqaladeeyaa lakabka epithelial 3D ee hore loo habeeyay, taas oo soo jeedinaysa in cadaadiska ka soo horjeeda inta lagu jiro dhaqanku uu mas'uul ka yahay morphogenesis mindhicirka ee vitro. Sidaa darteed, hab wax ku ool ah si loo gaaro morphogenesis adag ee epithelial interface-ka ugu yar waa in la ilaaliyo heerarka WC. qayb nadiifin firfircoon (tusaale, mindhicirka-on-a-chip-ka ama aaladaha isku-dhafka-on-a-chip) ama fidinta .Warbaahinta aasaasiga ah (tusaale, laga bilaabo Transwell gelinta kaydka hoose ee waaweyn ee ceelasha).
Nidaamkan, waxaanu ku siinaynaa hab faahfaahsan oo loogu talagalay samaynta qalabyada mindhicirka-on-a-chip microdevices iyo Transwell-insertable chips hybrid chips (tallaabooyinka 1-5) ee unugyada epithelial mindhicirka ee dhaqanka ee polydimethylsiloxane (PDMS) -xub-dabada ku salaysan (tallaabooyinka 6A, 7A, 8, 9) ama Transwells Polyester 8, 9) iyo soo jiidashada 3D morphogenesis in vitro (tallaabada 10) .Waxaan sidoo kale aqoonsannay qaababka unugyada iyo unugyada unugyada kuwaas oo tilmaamaya histogenesis-ka gaarka ah ee unugyada iyo kala duwanaanshaha unugyada ku-tiirsanaanta iyada oo la adeegsanayo hababka sawir-qaadista ee badan (tallaabooyinka 11-24) .Waxaannu kicineynaa morphogenesis iyadoo la adeegsanayo habka dheef-shiidka ee xiidmaha bini'aadamka sida unugyada epithelial-2 unugyo qaab-dhismeedka qaab-dhismeedka qaab-dhismeedka Castin-2 sida unugyada epithelial. Faahfaahinta farsamada oo ay ku jiraan wax ka beddelka dusha sare ee xuubka daloolka, abuurista monolayers 2D, iyo biochemical xiidmaha iyo soo saarida microenvironment-ka biomechanical.in vitro. Lakabka epithelial 3D ee dib loo soo celin karo oo loo isticmaali karo in lagu qaabeeyo kobaca epithelial-ku-tiirsan ee morphogen, dhaqamada wadajirka ah ee martida loo yahay-microbiome, caabuqa cudur-sidaha, dhaawaca bararka, cillad la'aanta xannibaadda epithelial, iyo daaweynta probiotic-ku salaysan Tusaalooyinka.
Nidaamkeena ayaa laga yaabaa inuu faa'iido u yeesho saynisyahano badan oo aasaasi ah (tusaale, bayoolaji xabka xiidmaha, bayoolajiga unugyada stem, iyo bayoolaji korriinka) iyo cilmi baaris la dabaqay (tusaale, tijaabinta daawada preclinical, qaabeynta cudurada, injineernimada unugyada, iyo gastroenterology) saameyn ballaaran. Aragtida in istaraatiijiyadeenna farsamada lagu faafin karo daawadayaasha baranaya dhaqdhaqaaqa calaamadaha unugga inta lagu jiro koritaanka mindhicirka, dib-u-soo-nooleynta ama homeostasis daacuun. Dhageystayaasha cudurada cudurada iyo cudurada ayaa sidoo kale faa'iido leh.Isticmaalka nidaamka microphysiology ee ku-chip mindhicirka ayaa laga yaabaa inuu u ogolaado co-dhaqanka dheer ee 10 iyo qiimeynta xigta ee difaaca martida, jawaabaha difaaca jirka iyo hagaajinta dhaawaca pathogen-ka ee habka dheef-shiid kiimikaadka (GI) 11 . la mid ah marka lakabyada epithelial mindhicirka 3D la diyaariyo iyadoo la isticmaalayo lakabyada epithelial mindhicirka 3D ee bukaanka, cuduradan waxaa ka mid ah atrophy villous, crypt shortening, xuubka dhaawaca, ama caqabada epithelial barrier.Biopsy ama stem cell-ka soo jeeda mindhicirka organoids-ka ku daraya kakanaanta unugyada unugyada laga yaabaa in ay ku daraan kakanaanta deegaanka cudurka. Noocyada, sida unugyada mononuclear-ka ee bukaan-socodka (PBMCs), ee moodooyinka ka kooban 3D mindhicirka villus-crypt microarchitectures. Unugyada difaaca ee unugyada, 5.
Maaddaama 3D qaab-dhismeedka yar-yar ee epithelial la hagaajin karo lana arki karo iyada oo aan la helin habka qaybinta, daawadayaasha ka shaqeynaya transcriptomics spatial transcriptomics iyo sawir-qaadaha sare ama xallinta sare ayaa laga yaabaa inay xiiseynayaan khariidaddayada dhaqdhaqaaqyada spatiotemporal ee hiddo-wadaha iyo borotiinnada ee niches epithelial. Xiiseynaya tignoolajiyada.Jawaabista kicinta microbial ama difaaca jirka Habkani wuxuu si gaar ah u soo jiitaa daawadayaasha baranaya difaaca jirka ee xuubka, gastroenterology, microbiome bini'aadamka, culturomics iyo microbiology kiliinikada ee doonaya in ay ku beeraan microbiota mindhicirka hore ee aan la dhaqmin ee shaybaarka qaybaha aasaasiga ah, borotokoolka ayaa sidoo kale lagu faafin karaa kuwa soo saaraya dawooyinka, biomedical ama baarista sare ee baaritaanka ama goobaha ansaxinta warshadaha cuntada.Sida caddaynta-mabda'a, waxaan dhawaan soo bandhignay suurtagalnimada nidaamka morphogenesis-ka-sare ee multiplex-ka-soo-baxa ah ee la isku dhejin karo 24-sidoo kale qaab-dhismeed saxan ah oo lagu daro qaab-dhismeedka qaab-dhismeed-qaab-dheddig-habboon. commercialized16,17,18.Sidaas darteed, ansaxinta habkeena in vitro morphogenesis waa la dedejin karaa oo ay suurtogal tahay in ay qaataan shaybaaro cilmi baaris oo badan, warshadaha ama dawladda iyo hay'adaha sharciyeynta si ay u fahmaan reprogramming cell of in vitro mindhicirka morphogenesis ee heerka transcriptomic si loo tijaabiyo daawooyinka ama biotherapeutics musharixiinta ama qaadashada daroogada ayaa la qiimeeyay iyada oo la adeegsanayo gut ganacsi moodooyinka organ-on-a-chip si loo qiimeeyo dib u soo saarista habka morphogenesis ee mindhicirka.
Tiro xaddidan oo ka mid ah moodooyinka tijaabada ah ee bini'aadamka ku habboon ayaa loo isticmaalay in lagu barto morphogenesis xiidmaha mindhicirka, inta badan sababtoo ah la'aanta borotokoolka la fulin karo si loo kiciyo morphogenesis 3D ee vitro. Dhab ahaantii, inta badan aqoonta hadda jirta ee ku saabsan morphogenesis mindhicirka waxay ku salaysan tahay daraasado xayawaan ah (tusaale, zebrafish20, mice21 ama digaaga22) -Si kastaba ha ahaatee, iyo inta badan waa su'aal macquul ah. Muhiimad ahaan, si sax ah ha u go'aamin hababka koritaanka bini'aadamka. Moodooyinkaani waxay sidoo kale aad u xaddidan yihiin awoodda ay u leeyihiin in lagu tijaabiyo hab isku dhafan oo kala duwan. Sidaa darteed, borotokoolkayaga dib-u-soo-celinta unugyada unugyada 3D ee vitro ayaa ka sarreeya moodooyinka xayawaanka ee vivo iyo sidoo kale qaababka kale ee dhaqanka unugyada 2D ee caadiga ah. dhidibka crypt-villus ee ka jawaabaya xuubka xuubka kala duwan ama kicinta difaaca.3D lakabyada epithelial waxay ku siin karaan meel bannaan oo lagu barto sida unugyada microbial-ku u tartamayaan si ay u sameeyaan niches spatial iyo evolution ecological ee ka jawaab celinta arrimaha martida loo yahay (tusaale, gudaha gudaha iyo lakabyada xabka dibadda, qarsoodiga IgA iyo peptides antimicrobial antimicrobial). Microbiota waxay dhistaa bulshooyinkeeda waxayna si isku mid ah u soo saartaa dheef-shiid kiimikaad yar yar (tusaale, asiidhyada dufanka silsiladda gaaban) ee qaabeeya ururka gacanta iyo niches unugga stem ee crypts-ka asalka ah. Tilmaamahaan waxaa la muujin karaa oo keliya marka lakabyada epithelial 3D lagu sameeyo vitro.
Marka lagu daro habkayaga abuurista qaababka epithelial mindhicirka mindhicirka 3D, waxaa jira dhowr habab in vitro. Dhaqanka organoid mindhicirka waa farsamo injineernimada nudaha casriga ah ee ku salaysan beerista unugyada tarma xiidmaha hoostooda xaaladaha morphogen gaar ah23,24,25. Si kastaba ha ahaatee, isticmaalka 3D organoid organoid-modelka loogu talagalay falanqeynta chamomile inta badan, lumen mindhicirka ayaa ku xidhan organoid gudahooda, sidaas darteed, soo bandhigida qaybaha iftiinka sida unugyada microbial ama antigens exogenous waa xaddidan tahay. Helitaanka lumen organoid waa la wanaajin karaa iyadoo la adeegsanayo microinjector,26,27 laakiin habkani waa mid soo jiidasho leh oo xoog leh wuxuuna u baahan yahay aqoon gaar ah si loo fuliyo. Intaa waxaa dheer, dhaqamada organoid-ka ee dhaqameed ee lagu ilaaliyo qashinka hydrogel ee hoos yimaada xaaladaha taagan si sax ah uma muujiyaan firfircoonida biomechanics vivo.
Hababka kale ee ay adeegsadaan kooxo badan oo cilmi baaris ah ayaa isticmaala 3D hydrogel scaffolds si ay u ekaysiiyaan qaab dhismeedka epithelial mindhicirka iyada oo la abuurayo unugyada xiidmaha ee go'doonsan ee dusha jel. morphogen gradients ee khuseeya, samaynta qaab-dhismeedka epithelial ratio dhinaca sare ah iyo stroma-epithelial crosstalk by oo ay ku jiraan unugyada stromal ee scaffolds. Si kastaba ha ahaatee, dabeecadda saffolds hore u habaysan ayaa laga yaabaa in ay ka hortagaan soo bandhigida habka morphogenetic iskiis laftiisa. Moodooyinkan sidoo kale ma bixiyaan fir-fircoon ee sheyga dareere ah ama qulqulka interstitial unugyo u baahan dareere dareere ah. morphogenesis iyo helitaanka function physiological. Daraasad kale oo dhawaan la isticmaalay hydrogel scaffolds in madal microfluidic ah iyo qaab-dhismeedka epithelial mindhicirka qaabaysan isticmaalaya laser-etching farsamooyinka. waxaa ka mid ah dhaqdhaqaaqa mindhicirka mechanobiological. Farsamooyinka daabacaadda 3D ee isla koox isku mid ah ayaa awooday in ay abuuraan tuubooyinka mindhicirka yar yar oo leh hababka morphogenetic ee iskood ah.In kasta oo ay ka samaysan yihiin qaybo kakan oo ah qaybaha kala duwan ee mindhicirka gudaha tuubada, qaabkani waxa kale oo uu ka maqan yahay qulqulka dareeraha iftiinka iyo cilladaha farsamada. Intaa waxaa dheer, shaqeynta moodeelka ayaa laga yaabaa in la xaddido, gaar ahaan ka dib markii habka tijaabada ah ee unugyada dhexgalka. Halkii, borotokoolkayaga la soo jeediyay wuxuu bixiyaa morphogenesis ee mindhicirka iskiis ah, diiqad jireed oo ku habboon xiirta, biomechanics ee u ekaanaysa dhaqdhaqaaqa mindhicirka, helitaanka qaybo madax-bannaan oo apical iyo basolateral, iyo dib-u-abuurista microenvironments bayooloji adag ee modularity. Sidaa darteed, in vitro 3D morphogenesis ayaa laga yaabaa inay ka adkaato hababka hore ee morphogenesis.
Nidaamkeenu wuxuu si buuxda diiradda u saarayaa 3D epithelial morphogenesis, oo leh unugyada epithelial oo kaliya ee dhaqanka mana jiraan noocyo kale oo unugyada ku wareegsan sida unugyada mesenchymal, unugyada endothelial, iyo unugyada difaaca.Sida hore loo sharaxay, xudunta borotokoolkayadu waa soo-saarka morphogenesis epithelial iyadoo laga saarayo morphogen inhibitors ee dhexdhexaadka ah ee lagu soo bandhigay qaab-dhismeedka robust. gut-on-a-chip iyo hybrid-on-a-chip waxay noo ogolaanaysaa in aan dib u abuurno lakabka epithelial 3D undulating, kakanaanta noole dheeraad ah sida epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 iyo, in our model, crypt-villus muuqaalada gudbinaya unugyada stem in basal . Fibroblasts) ee mesenchyme waxay door muhiim ah ka ciyaaraan soo saarista borotiinka ECM iyo nidaaminta morphogenesis xiidmaha ee vivo35,37,38. Ku darida unugyada mesenchymal ee qaabkayaga waxay kor u qaadeen habka morphogenetic iyo hufnaanta unugyada unugyada. Intaa waxaa dheer, qaybaha vasculature ee lagu xiri karo inta u dhaxaysa moodooyinka unugyada ayaa ah shuruudo marka moodooyinka unugyada loogu talagalay inay muujiyaan isdhexgalka xubnaha badan. Sidaa darteed, unugyada endothelial ayaa laga yaabaa inay u baahdaan in lagu daro si ay u qaabeeyaan sifooyin jireed oo sax ah oo leh xallinta heerka xubinta. difaac gaar ah oo nudaha marka la eego la jaanqaadida cudurrada mindhicirka.
Isticmaalka chips hybrid waa ka toosan badan yahay mindhicirka-on-a-chip sababtoo ah qalabaynta qalabku waa ka sahlan yahay isticmaalka Transwell gelisyada waxay u oggolaanaysaa dhaqanka scalable ee epithelium mindhicirka. Si kastaba ha ahaatee, ganacsi ahaan la heli karo Transwell gelisyada xuubka polyesterka ma aha kuwo laastik ah oo aan la socon karin dhaqdhaqaaqyada peristaltic u eg Cadaadiska xiirid ee dhinaca apical. Si cad, hantida maguurtada ah ee qolka apical dhif ayaa awood u leh wada-shaqeynta bakteeriyada muddada-dheer ee chips-ka isku-dhafka ah
Dib-u-dhiska buuxa ee dhidibka crypt-villus bini'aadamka ee gunta-on-a-chip iyo dhaqamada hybrid-on-a-chip si buuxda looma aasaasin. Tan iyo markii morphogenesis uu ka bilaabmayo monolayer epithelial, 3D microarchitectures qasab maaha inay bixiyaan isku mid ahaanshaha morphological si ay u crypts in vivo. 3D epithelium, crypt iyo villous gobollada si cad looma kala saarin. Inkasta oo kanaalada sare ee sare ee jajabku ay keenaan dhererka sare ee epithelium microengineered, dhererka ugu badan ayaa weli ku xaddidan ~ 300-400 µm qoto dheer ee dhabta ah ee xiidmaha xiidmaha ee xiidmaha yaryar iyo kuwa waaweyn ee xiidmaha waa ~ 135 µm. Fiilada mindhicirka yar waa ~ 600 µm41.
Laga soo bilaabo aragtida sawir-qaadista, sawirka sare-xalinta ee goobta 3D microarchitectures ayaa laga yaabaa inay ku xaddidan tahay mindhicirka jilibka, maaddaama masaafada shaqada ee loo baahan yahay ee lenska ujeeddada ilaa lakabka epithelial ay ku jirto amarka dhowr milimitir. Si looga gudbo dhibaatadan, ujeedo fog ayaa loo baahan karaa PDMS.Intaa waxaa dheer, maadaama lakabka-lakabka microfabrication ee mindhicirka ee jajabku uu ku lug leeyahay ku-xidhka joogtada ah ee u dhexeeya lakab kasta, aad bay u adag tahay in la furo ama laga saaro lakabka sare si loo eego qaab-dhismeedka dusha sare ee lakabka epithelial. Tusaale ahaan, adoo isticmaalaya mikroskoob elektaroonig ah (SEM).
Hydrophobicity ee PDMS waxay ahayd arrin xaddidaysa daraasadaha ku salaysan microfluidic ee la tacaalaya molecules yar yar ee hydrophobic, tan iyo PDMS waxay si aan kala sooc lahayn u xayeysiin kartaa molecules hydrophobic sida. Beddelka PDMS waxaa laga yaabaa in loo tixgeliyo qalabyo kale oo polymeric ah loo tixgeliyo in la yareeyo adsorption of molecules hydrophobic.
Ugu dambeyntii, habkayaga si fiican looma sifeyn marka la eego bixinta baarista sare-sare ama "hal-cabbir-ku habboon-dhan" madal tijaabo ah oo isticmaale-saaxiibtinimo ah. Nidaamka hadda jira wuxuu u baahan yahay bamka saliingaha halkii microdevice, kaas oo qaada booska CO2 incubator wuxuuna ka hortagayaa tijaabooyin ballaaran. Xaddidaaddan si weyn ayaa loo hagaajin karaa, qaab-cusbooneysii qaab-dhismeedka 29-tusaale ahaan, qaab-dhismeed 29-tusaale. ama 384-si fiican la gelinayo daloolo taasoo u oggolaanaysa buuxinta joogtada ah iyo ka saarida warbaahinta salka hoose).
Si loo kiciyo morphogenesis 3D ee epithelium mindhicirka bini'aadamka ee vitro, waxaanu isticmaalnay qalab mindhicir ah oo microfluidic ah oo ka kooban laba microchannels oo siman iyo xuub jilicsan oo u dhexeeya si loo abuuro lumen-capillary interface. Waxaan sidoo kale muujineynaa isticmaalka qalab microfluidic ah oo hal-channel ah (chibrid chip) kaas oo si joogto ah u koraya lakabka hoose ee lakabka ah. Labada qayboodba, morphogenesis ee unugyada epithelial mindhicirka ee kala duwan ayaa lagu muujin karaa iyada oo la adeegsanayo khalkhalgelinta jihada ee socodka si looga saaro antagonists morphogen oo ka soo jeeda qaybta basolateral. Dhammaan habka tijaabada ah (Jaantus 1) wuxuu ka kooban yahay shan qaybood: (i) microfabrication ee gunta mindhicirka ama Transwell la geli karo hybrid), (1-5) sanduuqa sanduuqa Unugyada epithelial (unugyada Caco-2) ama organoids xiidmaha aadanaha; sanduuqyada 2-5), (iii) dhaqanka unugyada epithelial mindhicirka ee chips mindhicirka ama chips hybrid (tallaabooyinka 6-9), (iv) induction of 3D morphogenesis in vitro (tallaabada 10) iyo (v)) si ay u tilmaamaan 3D epithelial microstructure (tallaabooyin 11-24) si wax ku ool ah ayaa loo qaabeeyey kooxda hoose. ee morphogenesis ee in vitro iyadoo la barbardhigayo morphogenesis epithelial ilaa boos, ku meel gaar, shuruudo, ama kontaroolada nidaamka.
Waxaan isticmaalnay laba goobood oo dhaqameed oo kala duwan: Gut-on-a-chip oo leh kanaalo toosan ama kanaalo isku dhafan, ama jajabyo isku dhafan oo ay ku jiraan Transwell (TW) gelinta aaladda microfluidic, oo la sameeyay sida lagu qeexay sanduuqa 1, iyo tallaabada 1 -5. (Caco-2 ama organoids xiidmaha bini'aadamka) iyo habka dhaqameed ee loo isticmaalo nidaamkan. "In vitro morphogenesis" waxay muujineysaa tillaabooyinka guud ee Caco-2 ama unugyada epithelial-ka-soo-baxa ay ku dhaqan yihiin jajab mindhicirka mindhicirka ama Transwell gelisyada chip hybrid, oo ay ku xigto soo-gelinta 3D morphogenesis iyo lambarka qaabdhismeedka qaab-dhismeedka mid kasta oo ka hooseeya qaab-dhismeedka. Arrow. Codsigu wuxuu bixiyaa tusaalooyin ku saabsan sida lakabyada epithelial mindhicirka ee la aasaasay loo isticmaali karo, tusaale ahaan, sifeynta kala duwanaanta unugyada, daraasaadka physiology mindhicirka, samaynta nidaamyada deegaanka ee martida-microbiome, iyo qaabaynta cudurrada. Sawirada Immunofluorescence ee "Cell Kala duwanaanta" oo muujinaya nuclei, F-actin iyo MUC2 ayaa lagu muujiyay lakabka gutli-2D. Calaamadaynta waxay ku jirtaa unugyada goblet iyo xabka laga soo saaro dusha sare ee xuubka. Sawirada fluorescent ee Gut Physiology waxay muujinayaan xab-xab ah oo ay soo saarto wasakhaynta sialic acid iyo N-acetylglucosamine oo isticmaalaya jeermiska qamadiga ah ee agglutinin. Labada sawir ee isku dhafan ee "Host-Microbe Co-Cultures" waxay muujinayaan guddiga martida loo yahay ee gut-mic co-culture of E. coli oo muujinaya borotiinka fluorescent cagaaran (GFP) oo leh microengineered 3D Caco-2 unugyada epithelial.Guddiga saxda ah wuxuu muujinayaa meelaynta GFP E. coli co-cultured with 3D Caco-2 epithelial cells, oo ay ku xigto immunofluorescence wasakhaynta F-actin (casaan) iyo nustrates gulutky gulutuse gulutuse. chips under physiological challenge with antigens bakteeriyada (tusaale, lipopolysaccharide, LPS) iyo unugyada difaaca (tusaale, PBMC; cagaar) .Caco-2 Unugyada ayaa la dhaqan si ay u abuuraan lakabka epithelial 3D. Miisaanka bar, 50 µm.Sawirada safka hoose: "Ka duwanaanshaha unugyada"2 waafajiyey ogolaansho ka tixraac. Jaamacadda Oxford Press; Dib loo soo saaray iyadoo ogolaansho laga haysto Tixraaca 5. NAS; "Dhaqan-Dhaqan-Microbe Host-Microbe" oo la waafajiyay ogolaanshaha tix.3. NAS; "Modeling Cudur" oo lagu habeeyey ogolaansho tixraaceed.5. NAS.
Labada jibbaar-chip-ka iyo chips-ga isku-dhafka ah ayaa la sameeyay iyadoo la adeegsanayo nuqullada PDMS oo laga burburiyay caaryada silikoon by lithography jilicsan1,44 iyo qaabka SU-8. Naqshadeynta microchannels ee jajab kasta waxaa lagu go'aamiyaa iyada oo la tixgelinayo hydrodynamics sida cadaadiska xiiridda iyo cadaadiska hydrodynamic1,4,12. The asalka mindhicirka-on-a-chip design ka kooban yahay. isku-dhafka microchannels-ka tooska ah ee isku midka ah, ayaa u kobcay mindhicirka-ka-kan-a-chip-ka adag (Jaantuska Xogta Dheeraadka ah. 1b) oo ay ku jiraan labo ka mid ah microchannels qaloocsan si ay u kiciyaan korodhka wakhtiga joogitaanka dareeraha, hababka socodka aan tooska ahayn, iyo qallafsanaanta multiaxial unugyada dhaqanka (Jaantus. 2a-f) waa la dooran karaa dumin (Sawir 2a) .Si loo sameeyo mindhicirka jajabka, lakabka sare ee PDMS ee la diyaariyey ayaa si isdaba joog ah loogu xidhay filim PDMS ah oo jilicsan ka dibna la jaanqaadaya lakabka hoose ee PDMS iyada oo xidhid aan la beddeli karin iyadoo la adeegsanayo daaweeyaha korona (Jaantus. 2b-f) . Galinta Transwell (Sawir 2h iyo Xogta Dheeraadka ah ee Sawirka 2) . Habka isku-xidhka waxaa lagu sameeyaa iyadoo lagu daaweynayo sagxadaha PDMS replica iyo galaaska leh plasma oksijiinta ama daaweynta corona. Ka dib markii la nadiifiyo qalabka microfabricated ee ku xiran tuubada silikoon, qalabka qalabku wuxuu diyaar u ahaa inuu sameeyo morphogenesis 3D ee epithelium mindhicirka.
a, Sawirka naqshadaynta ee diyaarinta qaybaha PDMS ee SU-8 caaryada silikoon qaabaysan.Xalka PDMS ee aan la daweynin ayaa lagu shubay caaryar silikoon (bidix), lagu daaweeyay 60 ° C (dhexe) iyo burburay (midig). xuubka daloolsan ee PDMS chambers.g, Setup of gut-on-a-chip for microfluidic cell culture.Xidhiyaha la sameeyay ee jajabka lagu soo ururiyay tuubo silikoon iyo silinge ayaa la saaray daboolka daboolka Chips.Transwell gelis si madax banaan loo diyaariyey si dhaqanka monolayers 2D monolayers unugyada epithelial mindhicirka ah ayaa la geliyey chip hybrid si ay u kiciyaan morphogenesis xiidmaha 3D. Dhexdhexaadiyaha waxaa lagu shubay iyada oo microchannels ka hooseeya lakabka gacanta ee lagu aasaasay dhex Transwell ah. Miisaanka bar, 1 cm.h Dib loo daabacay iyadoo ogolaansho laga helo tixraac.4. Elsevier.
Nidaamkan, xariiqda unugyada Caco-2 iyo organoids-ka mindhicirka ayaa loo isticmaalay ilaha epithelial (Jaantus. 3a) .Labada nooc ee unugyada ayaa si madax-bannaan u dhaqantay (Sanduuqa 2 iyo Sanduuqa 5) waxaana loo adeegsaday in lagu beero microchannels-ka ECM-dahaarka leh ee mindhicirka-chip ama Transwell.Marka unugyadu isku dhafan yihiin (> 95% Ceymiska Cabet ee caadiga ah ee unugyada skwee) 10 iyo 50) ee T-flasks ayaa la goostay si ay u diyaariyaan joojinta unugyada kala-baxsan ee dareeraha trypsinization (sanduuqa 2). R-spondin, iyo Noggin) iyo qodobbada koritaanka ee loo diyaariyey sida lagu qeexay sanduuqa 3 ayaa la kabay maalin kasta oo kale ilaa organoids-ka koray ~ 500 µm dhexroorka. cudur, kansarka mindhicirka, ama ku deeqaha caadiga ah), goobta dhaawaca (tusaale, nabar ka soo horjeeda aagga aan dhaawaca lahayn) iyo meesha caloosha iyo mindhicirka ee mareenka (tusaale, duodenum, jejunum, ileum, cecum, mindhicirka, ama malawadka) .Waxaan ku siin hab maamuuska optimized in Box 5 for culturing colonic organoids (coloid) kuwaas oo sida caadiga ah u baahan in ka badan uruurinta organic yar.
a, Workflow for induction of morphogenesis mindhicirka ee gunta hoose 0 (D0), qulqulka apical (AP) ayaa la bilaabay oo la ilaaliyaa maalmaha 2 ee ugu horreeya (qulqulka, AP, D0-D2) . socodka Basolateral (BL) ayaa sidoo kale la bilaabay iyada oo ay weheliso dhaqdhaqaaqyada fidinta wareegga wareegga (fidinta, qulqulka, AP iyo BL) marka monolayer 2D dhamaystiran la sameeyay. Intestinal 3D morphogenesis ka dib morphogenesis ee 5 maalmood ka dib morphogenesis. D5) .Sawirada ka soo horjeeda wejigu waxay muujinayaan qaab-dhismeedka wakiillada unugyada Caco-2 ee tallaabo kasta oo tijaabo ah ama barta waqtiga (garaafka bar, 100 µm) muujinta aagga la weyneeyey (sanduuqa cad cad) wuxuu muujinayaa microvilli-ka dib loo soo celiyay ee lakabka 3D Caco-2 (midig) chips.Falaaraha tilmaamaya in schematic dhexe waxay tilmaamaysaa meesha diyaaradda focal ee view kasta confocal.d, Koorsada Time ee isbeddellada morphological ee organoids cultured on chip helay by wejiga liddi ku microscopy maalmaha 3, 7, 9, 11, iyo 13. Inset (midig sare) waxay muujinaysaa weyneynta sare ee image ah bixinta ee image ah, DIC ee sawiro guga ee la aasaasay. jeex la qaaday maalintii 7.f, Sawirada immunofluorescence ee daboolan oo muujinaya calaamadaha unugyada asliga ah (LGR5; magenta), unugyada goblet (MUC2; cagaar), F-actin (cawlan) iyo nuclei (cyan) oo ku koray chips mindhicirka 3 maalmood, siday u kala horreeyaan (Bidix) iyo 13-maalmood (dhexda) organoids, oo sidoo kale iftiimiyay lakabka LGR5 calaamadeyn la'aan MUC2. Sawirada fluorescence oo muujinaya qaab-dhismeedka epithelial microstructure (midig) ee 3D organoid epithelium oo lagu aasaasay mindhicirka jilibka iyadoo la miirayo xuubka balaasmaha dheeha CellMask (midig) maalinta 13 ee dhaqanka. Miisaanka bar waa 50 μm haddii aan si kale loo sheegin.b Dib loo daabacay iyadoo ogolaansho laga helay tixraac.2. Jaamacadda Oxford Press; c Lagu soo qaatay ogolaanshaha tixraaca.2. Jaamacadda Oxford Press; e iyo f la waafajiyay ogolaanshaha tixraac ahaan.12 Sida hoos timaada shatiga Creative Commons CC BY 4.0.
In mindhicirka on chip a, waxaa lagama maarmaan ah in la beddelo dusha hydrophobic ee PDMS xuubka dalool ee guusha ECM daahan.In hab maamuuska this, waxaan codsan laba hab oo kala duwan si ay u beddelaan hydrophobicity ee PDMS. Waayo, dhaqanka Caco-2 unugyada, firfircoonida dusha by daawaynta UV / ozone oo keliya ahaa ku filan si loo yareeyo hydrophobicity ee unugyada ECM ah, PDMS membrane.Si kastaba ha ahaatee, dhaqanka microfluidic ee epithelium organoid wuxuu u baahan yahay functionalization dusha kiimiko ku salaysan si loo gaaro dhigista hufan ee borotiinada ECM by sequentially codsanaya polyethyleneimine (PEI) iyo glutaraldehyde in microchannels PDMS.Kadib wax ka beddelka dusha sare, borotiinada ECM ayaa lagu shubay si ay u daboosho ee functionalized PDMS ah. Unugyada ayaa ku xiran, dhaqanka unugyada microfluidic wuxuu ku bilaabmaa kaliya dhexdhexaadinta microchannel sare ilaa unugyadu ay sameeyaan monolayer dhamaystiran, halka microchannel hoose ay ilaalinayaan xaaladaha static. Habkan la wanaajiyey ee loogu talagalay firfircoonida dusha sare iyo daahan ECM wuxuu awood u leeyahay in lagu xiro epithelium organoid si uu u keeno morphogenesis 3D dusha PDMS.
Dhaqamada Transwell waxay sidoo kale u baahan yihiin daahan ECM ka hor abuuritaanka unugyada; si kastaba ha ahaatee, dhaqamada Transwell uma baahna tillaabooyinka pretreatment adag si ay u dhaqaajiso dusha sare ee gelinno dalool ah.For koritaanka unugyada Caco-2 on gelis Transwell, daahan ECM on gelinno dalool dardaro lifaaqa ee unugyada Caco-2 kala go'ay (<1-saac) iyo isku xidhka adag formation caqabad adag (<1-2 maalmood) . dusha xuubka (<3 h) oo la ilaaliyo ilaa organoids ay sameeyaan monolayer dhamaystiran oo leh daacadnimo .Dhaqamada Transwell waxaa lagu sameeyaa taarikada 24-well iyada oo aan la isticmaalin chips hybrid.
In vitro 3D morphogenesis waxaa lagu bilaabi karaa iyada oo la adeegsanayo qulqulka dareeraha ee dhinaca hoose ee lakabka epithelial ee la aasaasay.In mindhicirka jilibka, morphogenesis epithelial wuxuu bilaabmay markii dhexdhexaadinta lagu dhajiyay microchannels sare iyo hoose (Sawir 3a) Sida hore loo sharaxay, waa muhiim in la soo bandhigo qulqulka dareeraha ee hoose ee hoose (basolateral inhibited morphoge). siiya nafaqo ku filan iyo serum unugyada ku xidhan xuubka daloolka iyo dhalin stress xiirto luminal, waxaan sida caadiga ah codsan socodka dual ee mindhicirka on chip.In chips hybrid, gelinno Transwell ka kooban monolayers epithelial ayaa la geliyey chips hybrid. Markaas, dhexdhexaad ah ayaa lagu dabaqay ka hooseeya dhinaca basolateral ee xiiran Transwell dhexda ka dib markii morphititane 5 maalmood gudahood ka dhacay microchannel 3. socodka labada dhufto ee dhaqanka.
Tilmaamaha morphological ee lakabyada epithelial 3D ee microengineered waxaa lagu falanqeyn karaa iyada oo la adeegsanayo habab sawireed oo kala duwan, oo ay ku jiraan mikroskoopy isbarbardhiga wajiga, farqiga faragelinta kala duwan (DIC) microscopy, SEM, ama immunofluorescence confocal microscopy (Jaantus 3 iyo 4) ). lakabyada.Sababtoo ah hufnaanta indhaha ee PDMS iyo filimada polyester-ka, labadaba mindhicirka-on-a-chip iyo chips chip hybrid waxay ku siin karaan sawir-qaadista-waqtiga dhabta ah ee goobta iyada oo aan loo baahnayn qaybta ama kala-baxa qalabka. (PFA), oo ay ku xigto Triton X-100 iyo 2% (wt / vol) ) Serum albumin (BSA), si ay u kala horreeyaan. Iyadoo ku xiran nooca unugyada, hagaajinta kala duwan, permeabilizers, iyo wakiilada xannibaya ayaa loo isticmaali karaa (tusaale, 4′,6-diamidino-2-phenylene) indole, DAPI) ama F-actin (tusaale, fluorescently calamadeeyay phalloidin) . Sawirka tooska ah ee fluorescence ayaa sidoo kale lagu samayn karaa goobta si loo ogaado wax soo saarka xab-xabeedka (Jaantus. 1, "Ka duwanaanshaha unugyada" iyo "Gut physiology"), unugyada random-ka ee microbiology (sawir). co-culture"), qorista unugyada difaaca jirka (Sawir 1, 'Modeling Disease') ama jaangooyooyinka 3D epithelial morphology (Sawir 3c,f iyo 4b,c) . Marka la beddelo mindhicirka chip si loo kala saaro lakabka sare ee lakabka hoose ee microchannel, sida lagu qeexay ref.As2, si fiican Fig.3. on xuduudda buraashka apical waxaa lagu arki karaa by SEM (Jaantus. 3b) . Muujinta calaamadaha kala duwanaanta waxaa lagu qiimeyn karaa iyada oo la fulinayo tirada PCR5 ama hal unug ee RNA. Xaaladdan oo kale, lakabyada 3D ee unugyada epithelial ee ku koray chips mindhicirka ama chips hybrid ayaa la goostay by trypsinization ka dibna loo isticmaalo falanqaynta molecular.
a, Workflow for induction of morphogenesis mindhicirka ee chip hybrid ah.Caco-2 iyo organoids mindhicirka ayaa loo isticmaalaa in nidaamka this si ay u muujiyaan 3D morphogenesis ee platform chip hybrid. Unugyada epithelial kala qaybsan ayaa lagu beeray in la diyaariyey Transwell gelis (TW prep; eeg sawirka hoose). 7 maalmood ka dib, hal gelin Transwell ah oo ka kooban monolayer 2D ee unugyada epithelial ayaa lagu dhex daray chip hybrid si ay u soo bandhigaan socodka basolateral (Flow, BL), taas oo ugu dambeyntii keentay in jiilka lakabka epithelial 3D ah (morphogenesis) .Wejiga isbarbardhigga micrographs muujinaya muuqaalada morphological ee unugyada donor unugyada epitheli1 ee unugyada epitheli1. mindhicirka marxalad kasta oo tijaabo ah ama wakhti kasta. Shakhsiyaadka lakabyada sare waxay muujinayaan qaabka tijaabada ah ee tallaabo kasta.b, Chibrid chips (qaabka bidix) waxay u horseedi kartaa 3D morphogenesis ee unugyada epithelial organoid oo leh aragtiyo kor-hoos ah oo konfocal microscopy ah oo laga qaaday meelo kala duwan oo Z ah (sare, dhexe, iyo hoose; eeg khadadka saxda ah ee la sameeyay iyo kuwa u dhigma). waxay muujisay astaamo muuqaal ah oo muuqda.F-actin (cyan), nucleus (cawlan) Aragtida la gooyey (lagu dhejiyay koonaha midig ee sare; "XZ") waxay sidoo kale muujinayaan sifooyinka 2D iyo 3D. Baarka cabbirka, 100 µm.c Dib loogu daabacay ogolaansho tixraaceed.4. Elsevier.
Xakamaynta waxaa lagu diyaarin karaa iyada oo lagu dhaqo isla unugyo isku mid ah (Caco-2 ama unugyada organoid xiidmaha ee unugyada epithelial) ee monolayers laba-geesoodka ah ee hoos yimaada xaaladaha dhaqanka caadiga ah. socodka sidoo kale waa la barbar dhigi karaa.
Habka lithography jilicsan waa in lagu sameeyaa qol nadiif ah.Lakab kasta oo ku yaala chips (lakabyada sare iyo hoose iyo xuubka) iyo chips hybrid, sawiro sawiro kala duwan ayaa la isticmaalay oo lagu sameeyay wafer silikoon oo kala duwan sababtoo ah dhererka microchannels way ka duwanaayeen. Chip hybrid waa 200 µm.
Ku rid weel silikoon ah oo 3-inch ah saxan leh acetone. Si tartiib ah u leexi saxanka ilaa 30 ilbiriqsi, ka dibna hawo engeji maraqa.
Xalka piranha (isku-darka hydrogen peroxide iyo sulfuric acid oo xoogaysan, 1:3 (vol/vol)) ayaa si ikhtiyaari ah loo isticmaali karaa si loo kordhiyo ka-saarista hadhaaga dabiiciga ah ee dusha sare ee silikoon.
Xalka Piranha aad buu u daxaleeyaa wuxuuna dhaliyaa kuleyl
Fuuqbaxa maraqa adigoo saaraya saxan kulul oo ah 200 °C 10 daqiiqo. fuuqbaxa ka dib, maraqa ayaa la ruxay shan jeer hawada si uu u qaboojiyo.
Ku shub ~ 10 g oo ah photoresist SU-8 2100 bartamaha wafer silikoon la nadiifiyey.Isticmaal tweezers si aad ugu faafiso sawir-qaadista si siman wafer-ka. Mararka qaarkood ku rid waferka saxan kulul 65°C si aad sawir-qaadaha uga dhigto mid aan dhegdheg lahayn oo sahlanaato in la faafiyo.Ha dhigin maraqa si toos ah saxanka kulul.
SU-8 waxaa si siman loogu qaybiyey waferka iyadoo la ordaya dahaarka lafdhabarta ah Dhererka µm ee lakabka sare ee mindhicirka ee jilibka;
Xawaaraha lafdhabarta ugu weyn waxaa lagu hagaajin karaa iyadoo loo eegayo dhumucda bartilmaameedka ee qaabka SU-8 ee wafer silikoon.
Si loo sameeyo qaababka SU-8 ee dhererka 500 µm ee lakabka sare ee mindhicirka ee jajabka, dahaarka lafdhabarta iyo tillaabooyinka dubista jilicsan ee Sanduuqan (tallaabooyinka 7 iyo 8) ayaa si isdaba joog ah loo soo celiyay (eeg tallaabada 9) si loo soo saaro laba lakab oo ah 250 µm Lakab qaro ah oo SU-8 ah, kaas oo lagu dhejin karo oo lagu dari karo lakabka UV 0 ee tallaabadan 5. sare
Dubo jilicsan SU-8 maraqa dahaarka leh adigoo si taxadar leh u saaraya maraqa saxan kulul 65 °C 5 min, ka dibna u beddel goobta 95 ° C oo ku dheji 40 daqiiqo oo dheeraad ah.
Si aad u gaadho dhererka 500 μm ee qaabka SU-8 ee microchannelka sare, ku celi tillaabooyinka 7 iyo 8 si aad u dhaliso laba lakab oo SU-8 ah oo dhumucdiisu tahay 250 μm.
Adigoo isticmaalaya Aligner-ka Maaskarada UV, samee tijaabada laambada si waafaqsan tilmaamaha soo saaraha si aad u xisaabiso wakhtiga soo-gaadhista ee wafer-ka
Kadib go'aaminta wakhtiga soo-gaadhista, dulsaar sawir-qaadista maaskaro-haystaha maaskarada UV oo dhig sawir-qaadista maraqa SU-8.
Si toos ah u dhig dusha daabacan ee sawir-qaadista dhinaca SU-8 ee dahaarka leh ee wafer silikoon si loo yareeyo faafitaanka UV.
U soo bandhig maraqa dahaarka leh ee SU-8 iyo sawir-qaadista si toos ah 260 mJ/cm2 ee iftiinka UV ee wakhtiga la go'aamiyay (eeg tallaabada 10 ee sanduuqan).
Ka dib soo-gaadhista UV, suugo silikoon ah oo SU-8 ah ayaa lagu dubay 65 ° C ee 5 min iyo 95 ° C ee 15 min saxan kasta oo kulul si loo sameeyo qaababka leh dhererka 200 μm. Kordhi wakhtiga dubista ka dib 95 ° C ilaa 30 min si aad u sameyso qaabab leh dhererka 500 µm.
Horumariyaha waxaa lagu shubaa saxan galaas ah, iyo maraqa la dubay ayaa lagu dhejiyaa saxanka.Mugga SU-8 horumariye wuu kala duwanaan karaa iyadoo ku xiran cabbirka saxanka dhalada. Hubi inaad isticmaasho suu-8 ku filan si aad si buuxda uga saarto SU-8.
Ku raaci caaryada la sameeyay ~ 10 mL ee horumariye cusub oo ay ku xigto IPA adiga oo ku buufinaya xalka adigoo isticmaalaya pipette.
Waferka dhig balaasmaha nadiifiyaha oo u bandhig balasmaha oksijiinta (gaaska atmospheric, cadaadiska bartilmaameedka 1 × 10−5 Torr, awoodda 125 W) 1.5 min.
Wafer-da ku rid qallajiye vacuum ah oo leh muraayad muraayad ah oo gudaha ah. Wafers iyo slides ayaa la dhigi karaa dhinac dhinac dhinac ah. Haddii qalajiyaha vacuum-ka loo qaybiyo dhowr lakab oo saxan ah, dhig slides qolka hoose iyo maraqyada gudaha qolka sare.Drop 100 μL ee trichloro (1H, 1H, 2Hroo) galaas silfluen ah mari, vacuum for silanization.
Ku dhalaali fiil ka mid ah unugyada Caco-2 ee barafaysan ee qubeyska 37°C, ka dibna unugyadii dhalaalay ku wareeji weel T75 ka kooban 15 mL oo 37°C ah oo hore loo sii direy Caco-2 dhexdhexaad ah.
Si aad uga gudubto unugyada Caco-2 ee ~ 90% isku-dhafka, marka hore diirran Caco-2 dhexdhexaad ah, PBS, iyo 0.25% trypsin/1 mM EDTA ee qubeyska 37°C ee biyaha.
Ku dhaq dhexdhexaadka adigoo isticmaalaya vacuum hami.