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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 xiidmaha homeostasis iyadoo la ilaalinayo niche unugga stem ee crypt basal ka antigens microbial antigens iyo dheef-shiid kiimikaadkooda. Sidaa darteed, dib-u-soo-celinta qaab-dhismeedka epithelial-ka 3D waxay muhiim u tahay dhismaha moodooyinka mindhicirka ee vitro. Waxaa xusid mudan, mindhicirka mimetic gut-on-a-chip wuxuu keeni karaa morphogenesis 3D iskiis ah ee epithelium mindhicirka oo leh hawlo jireed oo la xoojiyay iyo biomechanics. Halkan, waxaan ku siinaynaa soo saari karo protostroin-guut-gacan-ku-dhalasho dic chip iyo sidoo kale in a Transwell embedded chip hybrid. Waxaan ku tilmaamay hababka faahfaahsan ee qalabaynta qalabka, culturing ee Caco-2 ama mindhicirka organoid unugyada epithelial ee goobaha caadiga ah iyo sidoo kale on madal microfluidic ah, induction of 3D morphogenesis, iyo sifaynta ee epithelia 3D aasaasay iyada oo la isticmaalayo hababka imaging ee nidaamka dib-u-dhaqaajiyaha microflora Tani waxay ku guulaysataa dib-u-dhiska dareeraha. socodka for 5 d. Our in vitro morphogenesis Habka shaqaalaysiinta physiologically khuseeya xiirid stress iyo dhaqdhaqaaqa farsamada oo aan u baahnayn injineernimada unug adag ama manipulation, taas oo laga yaabaa in ka badan farsamooyinka kale ee jira.Waxaan u malaynayaa in nidaamka our soo jeedinta ayaa saameyn ballaaran ku yeelan kara bulshada cilmi-biomedical, bixinta hab lagu cusboonaysiiyo 3D mindhicirka, codsiyada epithelial iyo lakabyada kiliinikada.
Tijaabooyinku waxay muujinayaan in unugyada epithelial ee mindhicirka 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 aasaasiga ah. morphogenesis in vitro, kaas oo lagu muujiyey Caco-2 iyo xubnaha unugyada mindhicirka ee bukaan-socodka.Unugyada Epithelial ayaa la ansixiyay. 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 qalabyada microfluidic ee la beddelay oo ay ku jiraan "Transwell inserts", oo loo yaqaan "Chip Hybrid". , ama Soggy-1) ee qanjidhada-chip-ka waxay joojisaa morphogenesis ama waxay carqaladeysaa lakabka epithelial 3D ee hore loo habeeyay, taasoo soo jeedinaysa in diiqada ka soo horjeeda inta lagu jiro dhaqanku ay mas'uul ka tahay morphogenesis xiidmaha ee vitro. Sidaa darteed, hab wax ku ool ah oo lagu gaaro morphogenesis adag ee isdhexgalka epithelial waa in meesha laga saaro ama hoos u dhigo heerarka gutso ee firfircoon. -on-a-chip ama hybrid-on-a-chip platforms) ama faafin .Warbaahinta aasaasiga ah (tusaale, laga bilaabo Transwell gelinta kaydadka basolateral 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 dhaqanka ee polydimethylsiloxane (PDMS) -xub-dabada ku salaysan (tallaabooyinka 6A, 7A, 8, 9) ama Transwells 6 polyester 3D morphogenesis ayaa keentay in vitro (tallaabada 10) .Waxaan sidoo kale aqoonsannay qaababka unugyada iyo unugyada molecular ee tilmaamaya histogenesis-ka unugyada gaarka ah iyo kala soocida unugyada lineage-ku-tiirsanaanta iyada oo la adeegsanayo hababka sawir-qaadista ee badan (tallaabooyinka 11-24) .Waxaannu kicineynaa morphogenesis iyadoo la adeegsanayo unugyada xiidmaha mindhicirka ee bini'aadamka ee epithelial unugyo, sida qaab-dhismeedka xuubka xuubka ee Caco-2 oo ay ku jiraan unugyo qaab-dhismeed farsamo oo leh qaab-dhismeedka Cacorous abuurista monolayers 2D, iyo biochemical xiidmaha iyo taranka ee microenvironment biomechanical.in vitro. Si loo dhaliyo 3D morphogenesis ka 2D epithelial monolayers, waxaan ka saarnay morphogen antagonists ee labada foomamka culturaled by qulqulaya dhexdhexaad ah galay qaybta aasaasiga ah ee dhaqanka. korriinka epithelial-ku-tiirsanaanta morphogen, dhaqamada-wadaaga martida-microbiome-dheer, caabuqa pathogene, dhaawaca bararka, cilladda xannibaadda epithelial, iyo daawaynta ku saleysan probiotics Tusaalooyinka.
Nidaamkeena ayaa laga yaabaa inuu faa'iido u yeesho saynisyahano kala duwan oo aasaasi ah (tusaale, bayoloji 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. Istaraatiijiyad waxaa lagu faafin karaa daawadayaasha baranaya dhaqdhaqaaqa calaamadaha unugga inta lagu jiro koritaanka mindhicirka, dib u soo kabashada ama homeostasis .Intaa waxaa dheer, borotokoolkayaga ayaa faa'iido u leh su'aalo weydiinta caabuqyada hoos yimaada wakiilada kala duwan ee faafa sida Norovirus 8, Syndrome Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium, ama Viostridium Difficile.Dhageystayaasha cudurada cudurada iyo cudurada ayaa sidoo kale faa'iido leh.Isticmaalka nidaamka microphysiology on-chip gut microphysiology 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 . Lakabyada epithelial mindhicirka 3D waxaa lagu diyaariyaa iyadoo la isticmaalayo lakabyada epithelial mindhicirka 3D ee bukaanka, cuduradan waxaa ka mid ah atrophy villous, crypt shortening, xuubka dhaawac, ama cillad epithelial barrier.Biopsy ama stem cell-ka soo jeeda mindhicirka organoids12,13. Si aad si fiican u model ah ee unugyada cudurada kakanaanta ah, sida unug-cudurada kakanaanta, Unugyada nukliyeerka (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 jirkaHabkani wuxuu si gaar ah u soo jiitaa daawadayaasha baranaya difaaca jirka ee xabkaha, gastroenterology, microbiome bini'aadamka, culturomics iyo microbiology Clinical oo doonaya in ay ku beeraan microbiota mindhicirka hore ee aan la dhaqmin ee shaybaarka , Hab-maamuuska ayaa sidoo kale lagu faafin karaa kuwa soo koraya daawooyinka, 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 ee loo yaqaan 'morphogenesis system scalable to a 24-well plates. Ansixinta habkeena in vitro morphogenesis waa la dedejin karaa oo ay suurtogal tahay in ay qaataan shaybaaro cilmi-baaris oo badan, warshadaha ama dawladda iyo wakaaladaha sharciyeynta si ay u fahmaan dib-u-habaynta unugyada ee in vitro gut morphogenesis ee heerka transcriptomic si loo tijaabiyo daawooyinka ama biotherapeutics nuugista iyo gaadiidka musharixiinta daroogada waxaa lagu qiimeeyay iyada oo la adeegsanayo 3D gut surrogates-ama iyadoo la adeegsanayo hab-nololeedka hab-ganacsiyeedka habka.
Tiro xaddidan oo ka mid ah moodooyinka tijaabada ah ee ku habboon bini'aadamka ayaa loo isticmaalay in lagu barto morphogenesis xiidmaha mindhicirka, inta badan sababtoo ah la'aanta borotokoollada la hirgelin karo si loo kiciyo morphogenesis 3D ee vitro. Dhab ahaantii, inta badan aqoonta hadda jirta ee ku saabsan morphogenesis mindhicirka waxay ku saleysan tahay daraasadaha xayawaanka (tusaale, zebrafish20, mice21 ama digaaga22, si kastaba ha ahaatee, iyo inta ugu badan ee suurtogalka ah ee suurtogalka ah) si cisely go'aamiso hababka korriinka 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 qaababka xayawaanka ee vivo iyo sidoo kale moodooyinka dhaqanka unugyada 2D ee caadiga ah ee caadiga ah. Sida hore loo sharraxay, isticmaalka 3D epithelial qaab-dhismeedyada ayaa loo oggol yahay in lagu baadho qaab-dhismeedka gudaha ee qaab-dhismeedyada kala duwan. Xabxabka kala duwan ama kicinta difaaca jirka dheef-shiid kiimikaad (tusaale, asiidhyada dufanka silsilad-gaaban) ee qaabeeya ururka gacanta iyo niches unugga stem ee crypts-ka asalka ah. Tilmaamahan waxa kaliya oo la muujin karaa 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 unugyada casriga ah ee ku salaysan beerista unugyada tarma xiidmaha hoostooda xaaladaha morphogen gaar ah23,24,25. Si kastaba ha ahaatee, isticmaalka 3D organoid organoid-model-mashruucyada loogu talagalay falanqeynta hufnaanta ee gadiidka inta badan. waxay ku xidhan tahay organoid-ka dhexdiisa, sidaa darteed, soo bandhigida qaybaha iftiinka sida unugyada microbial ama antigens-ka baxsan 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 dhowr kooxood oo cilmi baaris ah ayaa isticmaala 3D hydrogel scaffolds horay loo habeeyay si ay ugu ekaadaan qaab dhismeedka epithelial mindhicirka iyadoo lagu dhaqayo unugyada xiidmaha ee go'doonsan ee dusha jel. gradients, samaynta qaab-dhismeedka sare ee epithelial qaab-dhismeedka iyo stroma-epithelial crosstalk by oo ay ku jiraan unugyada stromal ee scaffold. Si kastaba ha ahaatee, dabeecadda ah ee saqafka hore u habaysan ayaa laga yaabaa in ay ka hortagaan soo bandhigida ee habka morphogenetic iskiis laftiisa. model kuwan sidoo kale ma bixiyaan dynamic luminal ama interstitial socodka unugyada firfircoon ee u baahan hoos u socodka dheecaanka physiological. Daraasad kale oo dhowaan la sameeyay ayaa loo adeegsaday qashinka hydrogel ee madal microfluidic ah iyo qaab-dhismeedka epithelial mindhicirka qaabaysan iyadoo la adeegsanayo farsamooyinka laser-etching. 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 qallafsanaanta qaybaha kala duwan ee mindhicirka gudaha tuubada, qaabkani waxa kale oo uu ka maqan yahay dareeraha dareeraha iftiinka iyo cilladaha farsamada. Intaa waxaa dheer, hawlgalka qaabka ayaa laga yaabaa inuu xaddido, gaar ahaan ka dib marka habka bioprinting uu dhammaado, qallafsanaanta xaaladaha tijaabada ah ama isdhexgalka unugyada. , Cadaadiska jireed ee ku habboon jir ahaan, biomechanics oo u eg dhaqdhaqaaqa mindhicirka, helitaanka qaybo madax-bannaan oo apical iyo basolateral ah, iyo dib-u-abuurista microenvironments bayooloji ee kakan ee modularity.Sidaas darteed, nidaamkayaga morphogenesis ee vitro 3D ayaa laga yaabaa inuu bixiyo hab dhammaystiran si looga gudbo caqabadaha hababka jira.
Nidaamkeena ayaa si buuxda diiradda u saaraya 3D epithelial morphogenesis, oo leh unugyada epithelial oo kaliya ee dhaqanka mana jiraan noocyo kale oo unugyada ku hareeraysan sida unugyada mesenchymal, unugyada endothelial, iyo unugyada difaaca.Sida hore loo sharaxay, xudunta borotokoolkayadu waa soo-saarka morphogenesis epithelial iyadoo laga saarayo morphogen inhibitors-ka-dhex-dhexaadka ah ee lagu soo bandhigo qaabka guga-gacan-basost. -chip iyo hybrid-on-a-chip waxay noo ogolaanaysaa inaan dib u abuurno lakabka epithelial 3D ee undulating, kakanaanta noolaha dheeraadka ah sida epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 iyo, in our model, crypt-villus muuqaalada gudbinaya stem cell niches in basal crypts. Soo saarista borotiinka ECM iyo nidaaminta morphogenesis mindhicirka ee vivo35,37,38.Ku darida unugyada mesenchymal ee qaabkayaga waxay wanaajiyeen habka morphogenetic iyo hufnaanta unugyada. taas oo lagu xidhi karo inta u dhaxaysa moodooyinka unugyadu waa shuruudo marka moodooyinka unugyadu loo qaabeeyey si ay u muujiyaan isdhexgalka xubnaha badan. Sidaa darteed, unugyada endothelial ayaa laga yaabaa inay u baahdaan in lagu daro si ay u qaabeeyaan muuqaalada jireed ee saxda ah oo sax ah oo leh xallinta heerka xubnaha.
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 ogolaataa dhaqanka scalable ee epithelium mindhicirka. Si kastaba ha ahaatee, ganacsi ahaan la heli karo Transwell gashto xuubka polyesterka ma aha mid laastik ah oo aan la socon karin dhaqdhaqaaqyada peristaltic u eg Apical side.Clearly, guryaha static in qaybta apical dhif awood muddo dheer bakteeriyada co-dhaqanka in chips hybrid.While aan si xoog leh u keeni kartaa morphogenesis 3D in Transwell geliyaa marka la isticmaalayo chips hybrid, yaraanta ee biomechanics physiologically khuseeya iyo socodka dheecaanka apical xaddido codsiyada suurto gal ah ee chips hybrid.
Dib-u-dhiska buuxa ee dhidibka crypt-villus bini'aadamka ee gut-on-a-chip iyo hybrid-on-a-chip dhaqamada si buuxda looma dhisin. Tan iyo markii morphogenesis uu ka bilaabmayo monolayer epithelial, 3D microarchitectures ma aha qasab inay bixiyaan morphological la mid ah crypts in vivo. Lium, gobollada crypt iyo villous si cad looma kala saarin. Inkasta oo kanaalada sare ee sare ee chip-ka ay keenayaan dhererka sare ee epithelium microengineered, dhererka ugu badan ayaa weli ku xaddidan ~ 300-400 µm qoto dheer ee dhabta ah ee xiidmaha mindhicirka ee xiidmaha yaryar iyo kuwa waaweynba waa ~ 135 µm, dhererka iyo ~40 600 µm41.
Marka laga eego 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 taal nidaamka dhowr milimitir. Si looga gudbo dhibaatadan, ujeeddo fog ayaa loo baahan karaa , Maadaama lakabka-lakabka microfabrication ee mindhicirka ee jajabku uu ku lug leeyahay ku-xidhka joogtada ah ee u dhexeeya lakab kasta, aad ayey u adag tahay in la furo ama laga saaro lakabka sare si loo eego qaabka 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 lagu tixgeliyo qalabka kale ee polymeric u 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 qaadata meel bannaan oo CO2 ah oo ka hortagaysa tijaabooyin ballaaran. Xaddidaaddan ayaa si weyn loo hagaajin karaa iyadoo la adeegsanayo qaab-dhismeedka cusub, qaab-dhismeedka 29, tusaale ahaan, qaab-dhismeed 29-tusaale 4-qaab-dhismeedka Gashado si fiican u daloola oo u oggolaanaya buuxinta joogtada ah iyo ka saarista warbaahinta aasaasiga ah).
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 isbarbar socda iyo xuubka jilicsan ee u dhexeeya si loo abuuro lumen-capillary interface. Waxaan sidoo kale muujineynaa isticmaalka qalab microfluidic ah oo hal-channel ah. morphogenesis ee unugyada epithelial mindhicirka ee kala duwan ayaa lagu muujin karaa iyada oo la adeegsanayo manipulation jihada qulqulka si looga saaro antagonists morphogen oo ka soo jeeda qaybta basolateral. Dhammaan nidaamka tijaabada ah (Jaantus 1) wuxuu ka kooban yahay shan qaybood: (i) microfabrication ee gunta mindhicirka ama Transwell la geliyo chip hybrid chip (tallaabooyin 1-5); organoids stinal;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 loo habeeyey kooxda hoos ku haboon. morphogenesis iyadoo la barbardhigayo morphogenesis epithelial-ka iyo kontaroolada meel bannaan, ku meel gaar ah, shuruudaysan ama habraac.
Waxaan isticmaalnay laba nooc oo dhaqameed oo kala duwan: Gut-on-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. In vitro morphogenesis" waxay muujinaysaa tallaabooyinka guud ee Caco-2 ama organoid-ka-soo-baxa unugyada epithelial-ka ee lagu beeray jajab mindhicirrada mindhicirrada ama gudaha Transwell ee chip hybrid, oo ay ku xigto soo-gelinta 3D morphogenesis iyo samaynta lambar tirade ah ee qaab dhismeedka hoos ku qoran. lakabyada epithelial stinal waxaa loo isticmaali karaa, tusaale ahaan, sifada kala duwanaanshaha unugyada, cilmi-nafsi physiology mindhicirka, samaynta ee host-microbiome ecosystems, iyo cudurka modeling.Immunofluorescence images in "Cell Kala duwanaansho" oo muujinaya nuclei, F-actin iyo MUC2 ee lagu muujiyay 3D Caco-2 epithelial epithelial ee unugyada mucosal ee soo bandhigay unugyada mucosal gocus. Sagxadaha. Sawirada fluorescent ee Gut Physiology waxay muujinayaan xab ay soo saartay wasakhaynta sialic acid iyo N-acetylglucosamine hadhaaga iyadoo la adeegsanayo jeermiska sarreenka ah ee agglutinin.Labada sawir ee isku dhafan ee "Host-Microbe Co-Cultures" waxay muujinayaan wakiilka martida-microbiome co-cultures ee mindhicirka ku yaala borootiinka bidix ee EFP gineered 3D Caco-2 unugyada epithelial. Gudida saxda ah waxay muujinaysaa degaanka ee GFP E. coli co-cultured with 3D Caco-2 epithelial cells, oo ay ku xigto immunofluorescence wasakhaynta F-actin (casaan) iyo nuklei (buluug) iyo unugyada difaaca (tusaale, PBMC;cagaar) .Caco-2 unugyada ayaa la dhaqan galiyay si loo dhiso lakabka epithelial 3D. Miisaanka, 50 µm. Sawirada safka hoose: "Ka duwanaanshaha unugyada" oo la waafajiyay ogolaanshaha tixraaca.2.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 Cudurka" 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 kuwaas oo laga burburiyay caaryada silikoon by lithography jilicsan1,44 iyo qaabaysan SU-8. Naqshadeynta microchannels ee chip kasta waxaa lagu go'aamiyaa iyadoo la tixgelinayo hydrodynamics sida cadaadiska xiiridda iyo cadaadiska hydrodynamic1,4,12.The asalka mindhicirka-on-a-chip design ka kooban yahay Fig. microchannels, ayaa u kobcay mindhicirka adag ee-on-a-chip (Extended Data Fig. 1b) oo ay ku jiraan labo ka mid ah microchannels qalooca si ay u kiciyaan korodhka wakhtiga joogitaanka dareeraha, hababka socodka aan tooska ahayn, iyo qallafsanaanta multiaxial unugyada dhaqameed (Jaantus. 2a-f) 12. Marka aad u adag tahay biomechanics-ka adag, waxay u baahan tahay dib u soo celinta gutchilu. ted Gut-Chip ayaa sidoo kale si xooggan u kicinaysa morphogenesis 3D waqti isku mid ah oo leh heer la mid ah koritaanka epithelial marka la barbar dhigo Gut-Chip asalka ah, iyada oo aan loo eegin nooca unugyada dhaqanka.Sidaas darteed, si loo soo saaro 3D morphogenesis, linear iyo kakan naqshadaha mindhicirka-chip-ka ayaa is-beddeli kara.2a) . Si loo sameeyo mindhicirka jilibka, 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 la adeegsanayo isku-xidhka aan la bedeli karin iyadoo la adeegsanayo daaweeyaha korona (Jaantus. 2b-f) . Xogta la dheereeyey sawirka 2) . Habka isku xidhka waxaa lagu sameeyaa iyada oo lagu daaweynayo dusha sare ee PDMS nuqulka iyo muraayadda oksijiinta plasma ama daaweynta corona. Ka dib markii la nadiifiyo qalabka microfabricated ee ku xiran tuubada silikoon, qalabka qalabka ayaa diyaar u ah inuu sameeyo morphogenesis 3D ee epithelium mindhicirka (Jaantus 2g).
a, Sawirka Schematic ee diyaarinta qaybaha PDMS oo ka soo SU-8 caaryada silikoon qaabaysan.Xalka PDMS ee aan la dawayn ayaa lagu shubay caaryada silikoon ah (bidix), lagu daaweeyay 60 °C (dhexe) iyo dumiyey (midig). rous membrane.d, Sawirro taxane ah oo ka kooban qaybaha sare iyo hoose ee PDMS iyo qalabka mindhicirka on-chip ee la ururiyey.e, Schematic of alignment ee sare, xuub, iyo qaybaha PDMS hoose on-a-chip for microfluidic cell culture.Xidhiyaha la sameeyay ee chip lagu soo ururiyay tuubo silikoon ah iyo sirinji ayaa lagu dhejiyay daboolka. ers ee unugyada epithelial mindhicirka ayaa la geliyey chip hybrid si ay u kiciyaan morphogenesis mindhicirka 3D. Dhexdhexaadiyaha waxaa lagu shubay iyada oo microchannels ka hooseeya lakabka unug aasaasay on Transwell gelin. Miisaanka bar, 1 cm.h Dib loo daabacay ogolaansho ka 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 dhaqan (Sanduuqa 2 iyo Sanduuqa 5) waxaana loo isticmaalay in lagu beero microchannels-ka ECM-dahaarka leh ee mindhicirka-chip ama Transwell.Marka unugyadu isku dhafan yihiin (> sk00) unugyada caadiga ah ee Cabet 1, daboolka caadiga ah ee Cabet2. 50) ee T-flasks ayaa la goostay si ay u diyaariyaan joojinta unugyada kala-baxsan ee dareeraha trypsinization (sanduuqa 2) . Sida ku qeexan sanduuqa 3 ayaa la kabay maalin kasta oo kale ilaa organoids ay koraan ilaa ~ 500 µm dhexroor. oo ka soo horjeeda aagga aan dhaawaca lahayn) iyo meesha caloosha ee marinka (tusaale, duodenum, jejunum, ileum, cecum, colon, ama malawadka) .Waxaan ku bixinaa hab-maamuuska la hagaajiyay ee sanduuqa 5 ee loogu talagalay xayndaabka xuubka xuubka (coloid) kaas oo sida caadiga ah u baahan qadar sare oo morphogens ah marka loo eego organoids xiidmaha yar yar.
a, Workflow for induction of morphogenesis mindhicirka ee jilibka xiidmaha.Caco-2 bini'aadamka epithelium mindhicirka iyo xiidmaha organoids ayaa loo isticmaalaa hab maamuuska this si ay u muujiyaan morphogenesis 3D. 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 la raacayo dhaqdhaqaaqyada fidinta wareegga wareegga (fidinta, socodka, AP iyo BL) marka monolayer 2D dhamaystiran la sameeyay. unugyada co-2 ee tallaabo kasta oo tijaabo ah ama barta wakhtiga (garaafka bar, 100 µm) .Afar jaantusyo jaantusyo muujinaya jaantusyada u dhigma ee morphogenesis mindhicirka (midig sare) villi on lakabka 3D Caco-2 (midig) .c, Horizontal aragtida hore ee aasaasay Caco-2 3D, claudin (ZO-1, cas) iyo xuub xuub joogto ah buraashka calaamadeeyay F-actin (cagaaran) iyo nuclei (buluug) Immunofluorescence confocal visualization ee unugyada epithelial ee muuqaal kasta oo xiidmaha ee chips ee xiidmaha. .d, Waqtiga koorsada isbeddellada morphological ee organoid-ka lagu dhaqay jajabka laga helay microscopy waji ka duwan maalmaha 3, 7, 9, 11, iyo 13. Galitaanka (midigta sare) waxay muujinaysaa weyneynta sare ee sawirka la bixiyay.magenta), unugyada goblet (MUC2; cagaaran), F-actin (cawlan) iyo nuclei (cyan) koray on chips mindhicirka 3 maalmood, siday u kala horreeyaan (Bidix) iyo 13-maalin (dhexe) organoids on lakabka epithelial. Eeg sidoo kale Extended Xogta Jaantus 3, taas oo muujinaysa LGR5 signaling iyada oo aan MUC2 signaling images Fc3. organoid epithelium oo lagu asaasay mindhicirka jilibka iyadoo lagu dheehdo xuubka balaasmaha dheeha CellMask (midig) maalinta 13 ee dhaqanka. Bar miisaankeedu waa 50 μm haddii aan si kale loo sheegin.b Dib loo daabacay iyadoo ogolaansho laga haysto.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 nidaamka this, waxaan codsan laba hab oo kala duwan si ay u bedelaan hydrophobicity ee PDMS. , 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.Ka dib markii dusha wax ka beddelka, borotiinada ECM ayaa lagu shubay si ay u daboosho functionalized PDMS dusha sare iyo ka dibna la soo bandhigay unuggu waxaa lagu dhejiyaa unugyada epifludic. s by cadar kaliya dhexdhexaad ah galay microchannel sare ilaa unugyadu ay ka samaysan yihiin monolayer dhamaystiran, halka microchannel hoose ilaalinayaan xaaladaha taagan. Habkan optimized ee firfircoonida dusha iyo daahan ECM awood lifaaqa ee epithelium organoid in ay keento 3D morphogenesis dusha PDMS.
Dhaqamada Transwell waxay sidoo kale u baahan yihiin daahan ECM ka hor abuuritaanka unugyada;si kastaba ha ahaatee, dhaqamada Transwell uma baahna tillaabooyin ka hortag ah oo adag si ay u dhaqaajiyaan dusha sare ee gelinta daloolada.For koritaanka unugyada Caco-2 on gelisyada Transwell, daahan ECM on gelinno dalool dardargelinta lifaaqa ee unugyada Caco-2 kala go'ay (<1-saac) iyo formation xannibaad xannibaad adag (<1-2 maalmood) . 3 h) waxaana la ilaaliyaa ilaa organoids-yadu ay ka sameeyaan monolayer dhamaystiran oo leh daacadnimo .Dhaqannada Transwell waxaa lagu sameeyaa saxannada 24-ceel 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 dhufto microchannels sare iyo hoose (Jaantus. 3a) .Sida hore loo sharraxay, waa muhiim in la soo bandhigo qulqulka dareeraha ee hooseeya (basolateral) si loo bixiyo jihada sirta ah ee nafaqeeyayaalka hooseeya. serum in unugyada ku xidhan xuubka dalool iyo abuuraan stress luminal xiirtay, 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 gelinka dhex microchannel ka dib Intetitin ee dhaqanka ee 5 maalmood gudahood.
Tilmaamaha morphological ee lakabyada epithelial 3D microengineered waxaa lagu falanqeyn karaa iyada oo la adeegsanayo habab sawireed oo kala duwan, oo ay ku jiraan mikroscopy-ga duwanaanshaha wajiga, farqiga faragelinta kala duwan (DIC) microscopy, SEM, ama immunofluorescence confocal microscopy (Jaantus 3 iyo 4) .Wejiga isbarbardhigga ama sawir-qaadista DIC si fudud ayaa loo samayn karaa si loo kormeero qaabka iyo qaabka 3. Daah-furnaanta PDMS iyo filimada polyester-ka, labadaba gut-on-a-chip iyo chips chip hybrid waxay ku siin karaan waqtiga-dhabta ah sawirka goobta iyada oo aan loo baahnayn qaybta ama kala-baxa qalabka 2% (wt / vol) ) Serum albumin (BSA), si ay u kala horreeyaan. Iyadoo ku xiran nooca unugga, hagaajinta kala duwan, permeabilizers, iyo wakiilada xannibaya ayaa la isticmaali karaa. Unugyada asaasiga ah ee lagu beegsanayo unug ku-tiirsan unugyada ama calaamadaha gobolka ayaa loo isticmaalaa si loo muujiyo unugyada aan dhaqdhaqaaqa lahayn ee goobta ku yaal chip, oo ay ku xigto unugyada difaaca sare oo ay weheliyaan counterstain-nucleus. indole, DAPI) ama F-actin (tusaale, phalloidin si fluorescently calaamadeysan) .1, "Cell differentiation" iyo "Gut physiology"), gumeysi random unugyada microbial (Jaantus. 1, "Host-microbe co-culture"), qorista unugyada difaaca jirka (Jaantus. 1, 'Cudurka Modeling') ama contours of 3D epithelial morphology (Jaantus. 3c. 4. Marka ay ka soocaan lakabka hoose, fc). lakabka nel, sida lagu sharaxay ref.Sida ku cad sawirka 2, 3D epithelial morphology iyo sidoo kale microvilli ee xudduudaha buraashka apical waxaa lagu arki karaa by SEM (Jaantus. 3b) muujinta calaamadaha kala duwanaansho waxaa lagu qiimeyn karaa iyadoo la fulinayo PCR5 tirada ama hal unug RNA isku xigxiga. trypsinization ka dibna loo isticmaalo falanqaynta molecular ama genetic.
a, Workflow for induction of morphogenesis mindhicirka ee chip hybrid ah.Caco-2 iyo mindhicirka organoids ayaa loo isticmaalaa in hab maamuuska 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). (Dhaqan TW) .Ka dib 7 maalmood, gelin hal Transwell ah oo ka kooban monolayer 2D ee unugyada epithelial ayaa la dhexgaliyay chip hybrid si ay u soo bandhigaan socodka basolateral (Flow, BL), taas oo ugu dambeyntii keentay in jiilka lakabka 3D epithelial (morphogenesis) .Waji ka soo horjeeda micrographs muujinaya muuqaalada morphological ee xubnaha bini'aadamka epithelial ama xuubka mindhicirka waqti kasta oo tijaabo ah. schematics ee lakabyada sare waxay muujinayaan qaabka tijaabada ah ee tallaabo kasta.b, Chibrid chips (qaabka bidix) waxay u horseedi kartaa morphogenesis 3D ee unugyada epithelial organoid oo leh aragtiyo konfocal oo kor-hoos ah oo laga qaaday meelo kala duwan oo Z ah (sare, dhexe, iyo hoose;eeg jaantus sax ah iyo xariiqyo dhibco leh oo u dhigma).waxay muujisay astaamo muuqaal ah oo muuqda.F-actin (cyan), nucleus (cawlan) gelinta koonaha midig ee sare, “XZ”) waxa kale oo ay muujinayaan muuqaalada 2D iyo 3D. Baararka 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. .
Habka lithography ee jilicsan waa in lagu sameeyaa qol nadiif ah. Lakab kasta oo ku yaal chips (lakabyada sare iyo kan hoose iyo xuubka) iyo chips hybrid, sawir-qaadyo kala duwan ayaa la isticmaalay oo lagu sameeyay wafer silikoon oo kala duwan sababtoo ah dhererka microchannels way kala duwanaayeen. Bartilmaameedka dhererka microchannels sare iyo hoose ee mindhicirka ee chip waa 500 µm dhererka iyo 500 µm 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 si siman ayaa loogu qaybiyey waferka iyadoo la ordaya dahaarka lafdhabarta.Barnaamijka wareegtada soo socota ee SU-8 ee 5-10 s si loo faafiyo 500 rpm dardargelinta 100 rpm/s lakabka sare ee mindhicirka ee jilibka; eeg "Tillaabooyinka muhiimka ah" hoos) oo lagu dejiyay dardar 300 rpm/s 30 ilbiriqsi at 1,200 rpm.
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 jilibka, dahaarka lafdhabarta iyo tillaabooyinka 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 250 µm Lakab qaro ah oo SU-8 ah, kaas oo lagu dhejin karo oo lagu biirin karo sanduuqa 0 µ.
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 su'aasha ku filan SU-8 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 ah. Haddii qalajiyaha vacuum-ka loo qaybiyo dhowr lakab oo saxan ah, dhig slides qolka hoose iyo wafers gudaha qolka sare.Drop 100 μL ee trichloro (1H, 1H, 2Hroo) galaas silfluum ah kicin.
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-darka, marka hore diirran Caco-2 dhexdhexaad ah, PBS, iyo 0.25% trypsin/1 mM EDTA gudaha qubeyska 37°C ee biyaha.
Ku dhaq unugyada laba jeer 5 mL oo diirran PBS adiga oo ku celcelinaya hamiga faakuumka oo ku dar PBS cusub.