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Mutum gut morphogenesis ya kafa crypt-villus fasali na 3D epithelial microarchitecture da sararin samaniya kungiyar.Wannan musamman tsari ake bukata don kula da gut homeostasis ta hanyar kare kara cell alkuki a cikin basal crypt daga exogenous microbial antigens da su metabolites. Bugu da kari, hanji villi da kuma secreting na hanji villi a kan m surface na banal mucus a halin yanzu a cikin sel mucus mai karewa. Sabili da haka, sake ƙirƙirar sifofin epithelial na 3D yana da mahimmanci don gina samfuran in vitro gut.Mai mahimmanci, ƙwayoyin mimetic gut-on-a-chip na iya haifar da morphogenesis na 3D ba tare da bata lokaci ba na epithelium na intestinal tare da ingantattun ayyukan physiological da biomechanics.A nan, muna samar da wani reproducible producible prostotin induce microorganisms. dic guntu da kuma a cikin wani guntu mai haɗaɗɗen ƙwayar cuta ta Transwell.Mun bayyana cikakkun hanyoyin don ƙirƙira na'urar, al'adar Caco-2 ko intestinal organoid epithelial Kwayoyin a cikin saitunan al'ada da kuma a kan dandamali na microfluidic, ƙaddamar da morphogenesis na 3D, da halayyar kafa 3D epithelia ta amfani da hanyoyin sarrafa hoto da yawa ta hanyar haɓakar haɓakar haɓakar ƙwayoyin cuta. kwarara don 5 d. Our in vitro morphogenesis Hanyar yana amfani da damuwa mai mahimmanci na physiologically da motsi na inji kuma baya buƙatar hadaddun aikin injiniya ko magudi, wanda zai iya wuce sauran fasahohin da ake ciki. Muna tunanin cewa ka'idar da muka gabatar na iya samun tasiri mai yawa ga al'ummar bincike na biomedical, samar da hanyar da za a sake farfado da 3D na hanji, da kayan aiki na kwayoyin halitta, na asibiti da kuma kayan aikin epithelial.
Gwaje-gwaje sun nuna cewa sel Caco-2 na intestinal da aka haɓaka a cikin gut-on-a-chip1,2,3,4,5 ko bilayer microfluidic na'urorin6,7 na iya jurewa 3D morphogenesis a cikin vitro ba tare da cikakkiyar fahimtar tsarin da ke ciki ba. morphogenesis in vitro, wanda Caco-2 ya nuna da kuma marasa lafiya da aka samu na hanji.Kwayoyin Epithelial an inganta su. A cikin wannan binciken, mun mayar da hankali musamman akan samar da kwayar halitta da rarraba rarraba mai karfi na Wnt antagonist, Dickkopf-1 (DKK-1), a cikin gut-on-a-chip da gyare-gyaren microfluidic na'urorin da ke dauke da abubuwan da ake sakawa na Transwell, wanda ake kira "Hybrid Chip" . , ko Soggy-1) zuwa ga gut-chip gut yana hana morphogenesis ko ya rushe 3D epithelial Layer da aka riga aka tsara, yana nuna cewa damuwa a lokacin al'ada yana da alhakin morphogenesis na intestinal a cikin vitro.Saboda haka, hanyar da za a iya amfani da ita don cimma morphogenesis mai ƙarfi a cikin ma'auni na epithelial shine cirewa ko rage matakan da ba a iya amfani da su ba. -on-a-chip ko matasan-on-a-chip dandamali) ko watsawa .Basolateral kafofin watsa labarai (misali, daga Transwell sakawa a cikin manyan basolateral reservoirs a cikin rijiyoyin).
A cikin wannan yarjejeniya, muna samar da cikakken hanyar kirkirar gut-on-a-a-chip na al'ada membranes (Mataki) -bems na al'adu (Matakai 1-5) ko membranes na al'adu (Mataki) ko kuma 6b, 7 B, 8, 9) Kuma da jawo 3D Morphogenis a cikin Vitro (Mataki na 10) .Wannan .WALICALA NA TATTAUNAWAYAR -EDIONS (Mataki na 10-24) .wearfafa Morphogelular da yawa na kayan aiki, kamar yadda ƙungiyoyi masu amfani da su, a cikin tsarin al'ada tare da bayanan fasaha ciki har da Canjin ƙasa na Membranes, halittar 2D na 2D, da kuma tarihin cututtukan ƙwayar cuta na al'adu na 2D, mun cire wakilci na ƙirar gargajiya na gargajiya 3D Epithlial Layer wanda za'a iya amfani dashi don yin kwaikwayon ci gaban morphogen-dogaro da al'adu, cututtukan pathogen, mai lalacewa mai rikicewa, da kuma abubuwan da aka kwantar da hankali.
Yarjejeniyar mu na iya zama da amfani ga masana kimiyya da yawa a cikin asali (misali, ilimin halittar mucosal na hanji, ilmin halitta ta jiki, da ilimin halitta na haɓakawa) da bincike mai amfani (misali, gwajin ƙwayar cuta na preclinical, ƙirar cuta, injiniyan nama, da gastroenterology) tasiri mai fa'ida. Za a iya yada dabarun zuwa ga masu sauraron da ke nazarin motsin siginar kwayar halitta a lokacin ci gaban hanji, sake farfadowa ko homeostasis .Bugu da ƙari, ƙa'idar mu tana da amfani don yin tambayoyi game da kamuwa da cuta a ƙarƙashin nau'o'in cututtuka daban-daban irin su Norovirus 8, Ciwon Cutar Cutar Cutar Cutar Cutar Coronavirus 2 (SARS-CoV-2), Clostridiummumon Difficile.Masu sauraro na cututtukan cututtuka da cututtuka suna da amfani.Yin amfani da tsarin microphysiology na kan-chip gut zai iya ba da izinin haɗin gwiwa na tsawon lokaci 10 da kuma kimantawa na gaba na tsaro na rundunar, amsawar rigakafi da kuma raunin da ya shafi cututtukan cututtuka a cikin gastrointestinal tract (GI) 11 .Wasu cututtuka na GI da ke hade da leaky gutwel syndrome, cututtuka na cututtuka, cututtukan cututtuka na celiac, cututtuka na huhu, cututtuka na celiac, cututtukan cututtuka na huhu, ciwon huhu, cututtuka na celiac, ciwon daji ko ciwon daji. 3D na hanji epithelial yadudduka an shirya ta amfani da haƙuri ta 3D na hanji epithelial yadudduka, wadannan cututtuka sun hada da villous atrophy, crypt shortening, mucosal lalacewa, ko illa epithelial shãmaki.Biopsy ko kara cell-samu hanji organoids12,13.To mafi kyau model da mafi girma da cuta iri-iri iri-iri na marasa lafiya, irin wannan cuta na iya karantawa mafi girma daga cikin cututtuka. Kwayoyin nukiliya (PBMCs), zuwa samfura masu ɗauke da 3D intestinal villus-crypt microarchitectures.Kwayoyin rigakafi na musamman, 5.
Tun da 3D microstructure na epithelial za a iya gyarawa da kuma gani ba tare da tsarin sashe ba, masu kallo da ke aiki akan fassarar sararin samaniya da babban ƙuduri ko babban ƙuduri na iya zama sha'awar taswirar mu na yanayin sararin samaniya na kwayoyin halitta da sunadaran a kan niches epithelial.Mai sha'awar fasahar.Amsa ga ƙananan ƙwayoyin cuta ko abubuwan motsa jiki na rigakafi.Bugu da ƙari, mai ɗaukar lokaci mai tsawo-microbiome crosstalk 10, 14 wanda ke daidaita gut homeostasis za a iya kafa shi a cikin 3D na hanji mucosal Layer ta hanyar haɓaka nau'ikan nau'ikan microbial daban-daban, al'ummomin microbial ko ƙwayoyin cuta na fecal microbiota-.A cikin dandali.Wannan tsarin yana da ban sha'awa musamman ga masu sauraron da ke nazarin ƙwayoyin cuta na mucosal, gastroenterology, microbiome na mutum, culturomics da microbiology na asibiti da ke neman noma a baya ba a al'adar gut microbiota a cikin dakin gwaje-gwaje. , Hakanan za'a iya watsar da yarjejeniya ga waɗanda ke haɓaka magunguna, biomedical Ko high-throughput screening ko ingantattun dandamali don masana'antar abinci.A matsayin ƙa'idar hujja, kwanan nan mun nuna yiwuwar tsarin morphogenesis na multix wanda zai iya daidaitawa zuwa 24-well plate format. Tabbatar da hanyar in vitro morphogenesis na iya haɓakawa kuma ana iya karbe shi ta yawancin dakunan gwaje-gwaje na bincike, masana'antu ko gwamnati da hukumomin da suka dace don fahimtar tsarin salon salula na in vitro gut morphogenesis a matakin transcriptomic don gwada magunguna ko ilimin halittu An kimanta sha da jigilar 'yan takarar magani ta amfani da samfuran 3D gut surrogates ko ta hanyar yin amfani da tsarin gurɓataccen tsarin kasuwanci ko yin amfani da tsarin gurɓataccen tsarin kasuwanci ko yin amfani da tsarin tsarin kasuwanci tsari.
An yi amfani da ƙayyadaddun ƙididdiga na ƙirar gwaji masu dacewa da ɗan adam don nazarin morphogenesis na intestinal epithelial morphogenesis, musamman saboda rashin ka'idojin aiwatarwa don haifar da morphogenesis na 3D a cikin vitro. cisely ƙayyadaddun tsarin ci gaban ɗan adam.Waɗannan samfuran kuma suna da iyakancewa sosai a cikin ikon su don gwadawa ta hanyar daidaitawa ta hanyoyi da yawa.Saboda haka, ka'idodin mu don sake haɓaka tsarin nama na 3D a cikin vitro ya fi kyau a cikin samfuran dabbobi na vivo da sauran samfuran al'adun gargajiya na 2D na al'ada na al'ada. Kamar yadda aka bayyana a baya, yin amfani da 3D epithelial tsarin da aka ba da izini ga sel daban-daban a cikin sel daban-daban da aka ba mu damar yin la'akari da sifofi daban-daban. daban-daban mucosal ko rigakafi stimuli.3D epithelial yadudduka iya samar da sarari don nazarin yadda ƙananan ƙwayoyin cuta gasa don samar da sararin sarari niches da muhalli juyin halitta a mayar da martani ga rundunar dalilai (misali, ciki da waje gamsai yadudduka, secretion na IgA da antimicrobial peptides) .Bugu da kari, 3D epithelial ilimin halittar jiki iya ba da damar mu microbial ilimin halittar jiki da kuma yadda za a iya ba da damar mu microbial morphology. metabolites (misali, gajeriyar sarkar fatty acids) waɗanda ke siffata ƙungiyar salula da kuma niches cell a cikin basal crypts.Wadannan fasalulluka za a iya nuna su ne kawai lokacin da 3D epithelial layers aka kafa a cikin vitro.
Bugu da ƙari, hanyar mu na samar da 3D intestinal epithelial Tsarin, akwai da dama in vitro hanyoyin.Intestinal organoid al'ada ne na zamani organoid na'ura injiniya fasaha bisa ga namo na hanji kara sel a karkashin takamaiman morphogen yanayi23,24,25.Duk da haka, da yin amfani da 3D organoid organoid host-model da ake amfani da lu'u-lu'u sau da yawa ana gudanar da bincike ko lulubi. an rufe shi a cikin organoid kuma, sabili da haka, ƙaddamar da kayan aikin haske kamar ƙwayoyin microbial ko antigens exogenous yana iyakance.Ana iya inganta samun damar yin amfani da lumen organoid ta hanyar amfani da microinjector,26,27 amma wannan hanya tana da haɗari da kuma aiki mai tsanani kuma yana buƙatar ilimi na musamman don yin aiki.Bugu da ƙari, al'adun gargajiyar gargajiyar gargajiyar da aka kiyaye a cikin ɓangarorin hydrogel a ƙarƙashin yanayi na tsaye ba su nuna daidai da aiki a cikin vivo biomechanics.
Sauran hanyoyin da ƙungiyoyin bincike da yawa ke amfani da su suna amfani da 3D hydrogel scaffolds da aka riga aka tsara don yin kwaikwayon tsarin epithelial na gut ta hanyar haɓaka ƙwayoyin hanji na mutum a kan gel surface.Fabricate hydrogel scaffolds ta amfani da 3D-buga, micro-milled, ko lithographically ƙirƙira molds. gradients, kafa wani babban al'amari rabo epithelial tsarin da stroma-epithelial crosstalk ta hada da stromal Kwayoyin a cikin scaffold.Amma, da yanayin prestructured scaffolds iya hana nuni na ba da jimawa ba morphogenetic tsari kanta.Waɗannan model kuma ba su samar da tsauri mai ƙarfi ko interstitial shemorphar Kwayoyin da ake bukata a cikin ruwa samuwar physiological kwarara daga cikin ruwa. Babu wani binciken da aka yi kwanan nan da aka yi amfani da ɓangarorin hydrogel a cikin wani dandamali na microfluidic da tsarin tsarin epithelial na hanji ta hanyar amfani da fasaha na laser-etching. daga rukunin guda ɗaya sun sami damar ƙirƙirar ƙananan bututun gut tare da matakan morphogenetic maras lokaci.Duk da haɗaɗɗun ƙirƙira na sassa daban-daban na gut a cikin bututu, wannan ƙirar kuma ba ta da kwararar ruwa mai haske da nakasar injiniya. Bugu da ƙari, ƙirar ƙirar ƙila za a iya iyakancewa, musamman ma bayan aikin bioprinting ya cika, rikitar da yanayin gwaji ko haɓakar haɓakar sel. , physiologically dacewa danniya karfi, biomechanics cewa kwaikwayi gut motility, samun dama ga masu zaman kansu apical da kuma basolateral compartments, da kuma sake haifar da hadaddun microenvironments na halitta kwayoyin halitta.
Yarjejeniyar mu gaba ɗaya ta mayar da hankali kan 3D epithelial morphogenesis, tare da sel epithelial kawai a cikin al'ada kuma babu wasu nau'ikan sel da ke kewaye da su kamar ƙwayoyin mesenchymal, ƙwayoyin endothelial, da ƙwayoyin rigakafi.Kamar yadda aka bayyana a baya, jigon ƙa'idar mu shine ƙaddamar da morphogenesis na epithelial ta hanyar cire morphogen inhibitors da aka ɓoye a gefen mu-gutu. -chip da hybrid-on-a-chip suna ba mu damar sake ƙirƙirar Layer epithelial na 3D mara ƙarfi, ƙarin rikitattun halittu irin su epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 da, a cikin ƙirar mu, crypt-villus fasali waɗanda ke isar da sel niches a cikin basal crypts. a cikin samar da sunadaran ECM da kuma daidaita tsarin morphogenesis na hanji a cikin vivo35,37,38.Ƙarin ƙwayoyin mesenchymal zuwa samfurin mu ya inganta tsarin morphogenetic da ingantaccen haɗewar tantanin halitta.Layin endothelial (watau capillaries ko lymphatics) yana taka muhimmiyar rawa wajen daidaita tsarin jigilar kwayoyin halitta, ƙwayoyin cuta na guguwa 39 da microviculment. wanda za'a iya haɗawa tsakanin nau'in nama shine abin da ake bukata lokacin da aka tsara samfurori na nama don nuna hulɗar hulɗar kwayoyin halitta.Saboda haka, ƙwayoyin endothelial na iya buƙatar a haɗa su don yin samfurin mafi daidaitattun siffofi na ilimin lissafi tare da ƙuduri na matakin gabobin.
Yin amfani da kwakwalwan kwamfuta na matasan ya fi sauƙi fiye da gut-on-a-chip saboda saitin na'urar ya fi sauƙi kuma yin amfani da abubuwan da ake sakawa na Transwell yana ba da damar al'adun scalable na gut epithelium. Duk da haka, tallace-tallace da ake samuwa Transwell abun da ake sakawa tare da polyester membranes ba na roba ba ne kuma ba zai iya yin kama da motsi na peristaltic-kamar motsi ba. Bugu da ƙari kuma, da sheƙar da aka sanya a cikin guntu mai kwakwalwa ba tare da damuwa ba a cikin kwakwalwar da aka sanya a cikin kwakwalwan da aka saka a cikin kwakwalwa tare da haɗin gwiwa tare da guntu mai kwakwalwa. Apical side.A bayyane yake, a tsaye Properties a cikin apical daki da wuya taimaka dogon lokacin da kwayan cuta co-al'adu a matasan chips.Yayin da za mu iya robustly jawo 3D morphogenesis a cikin Transwell abun da ake sakawa a lokacin da amfani da matasan kwakwalwan kwamfuta, da kasawa na physiologically dacewa biomechanics da apical ruwa kwarara iya iyakance aikace-aikace dandali na chipsibility na hybrid.
Cikakkun gyare-gyaren gyare-gyare na crypt-villus axis na ɗan adam a cikin gut-on-a-chip da al'adun matasan-on-a-chip ba a cika su ba.Tun da morphogenesis ya fara daga epithelial monolayer, 3D microarchitectures ba dole ba ne ya ba da kamanceceniya ga crypts a cikin vivo. Lium, crypt da vilous yankuna ba a bayyana a fili ba.Ko da yake mafi girma tashoshi na sama a kan guntu gubar zuwa ƙãra tsawo na microengineered epithelium, matsakaicin tsawo har yanzu yana iyakance zuwa ~ 300-400 µm. Ainihin zurfin crypts na hanji na ɗan adam a cikin ƙananan ƙananan hanji da ƙananan hanji shine ~ 135 µm a cikin girman girman, da kuma ~ 4. 600m41.
Daga mahangar hoto, a cikin yanayin babban ƙuduri na 3D microarchitectures na iya iyakance ga gut akan guntu, tun da nisan aiki da ake buƙata daga ruwan tabarau na haƙiƙa zuwa Layer na epithelial yana kan tsari na 'yan milimita. Don shawo kan wannan matsala, ana iya buƙatar manufa mai nisa. Bugu da ƙari, yin sassan bakin ciki don yin hoto yana da girma. , Tun da Layer-by-Layer microfabrication na gut a kan guntu ya haɗa da mannewa na dindindin tsakanin kowane Layer, yana da wuyar gaske don buɗewa ko cire babban Layer don bincika tsarin farfajiya na epithelial Layer. Misali, ta amfani da microscope na lantarki (SEM).
A hydrophobicity na PDMS ya kasance mai iyakance factor a cikin microfluidic-tushen karatu da ake hulda da hydrophobic kananan kwayoyin halitta, tun PDMS iya nonspecifically adsorb irin hydrophobic kwayoyin.Alternatives zuwa PDMS za a iya la'akari da sauran polymeric kayan.A madadin, surface gyare-gyare na PDMS (misali, shafi tare da lip lip polym43) kayan da aka yi la'akari. hana adsorption na hydrophobic kwayoyin.
A ƙarshe, hanyarmu ba ta kasance da kyau ba dangane da samar da babban aikin nunawa ko "ɗaya-size-daidai-duk" mai amfani-abokin gwaji dandamali.Ka'idar ta yanzu tana buƙatar fam ɗin sirinji ta microdevice, wanda ke ɗaukar sararin samaniya a cikin incubator CO2 kuma yana hana manyan gwaje-gwajen.Wannan iyakancewa za a iya inganta shi sosai ta hanyar scalability na al'ada, 6 ko 4 (misali 3, 6, scalability, 3) ko 6. -Maɗaukaki masu kyau waɗanda ke ba da damar ci gaba da cikawa da kuma kawar da kafofin watsa labarai na basolateral).
Don haifar da 3D morphogenesis na ɗan adam na hanji epithelium a cikin vitro, mun yi amfani da microfluidic guntu hanji na'urar dauke da biyu layi daya microchannels da wani roba porous membrane a tsakanin don ƙirƙirar lumen-capillary interface.We kuma nuna amfani da guda-tashar microfluidic na'urar (a matasan guntu) da za a samar da ci gaba da duka biyu dandali girma dandali. morphogenesis na daban-daban na kwayoyin epithelial na hanji na mutum za a iya nunawa ta hanyar yin amfani da manipulations na shugabanci na kwarara don cire antagonists na morphogen daga sashin basolateral. Dukkanin gwajin gwaji (Figure 1) ya ƙunshi sassa biyar: (i) microfabrication na guntu gut ko Transwell shigar da guntun matasan (matakai 1-5; 2) Akwatin sel a cikin sel na sel a cikin sel. kwayoyin kwayoyin halitta;kwalaye 2-5), (iii) al'ada na intestinal epithelial Kwayoyin a kan hanji kwakwalwan kwamfuta ko matasan kwakwalwan kwamfuta (matakai 6-9), (iv) shigar da 3D morphogenesis a cikin vitro (mataki 10) da (v) )) don kwatanta 3D epithelial microstructure (matakai 11-24) da aka tsara wani tasiri a ƙasa (mataki 11-24) da aka tsara a ƙasa mai inganci. morphogenesis ta hanyar kwatanta morphogenesis na epithelial zuwa sararin samaniya, na ɗan lokaci, sharadi, ko sarrafawar tsari.
Mun yi amfani da dandamali na al'adu daban-daban guda biyu: gut-on-a-chip tare da madaidaiciyar tashoshi ko tashoshi marasa daidaituwa, ko kwakwalwan kwakwalwan kwamfuta da ke dauke da abubuwan da ake sakawa na Transwell (TW) a cikin na'urar microfluidic, wanda aka ƙirƙira kamar yadda aka bayyana a cikin Akwatin 1, da mataki na 1 -5. In vitro morphogenesis" yana nuna matakan gaba ɗaya wanda Caco-2 ko organoid-samu epithelial Kwayoyin da aka samu a kan guntu na hanji ko a kan Transwell abun da ake sakawa na guntu na matasan, sannan shigar da 3D morphogenesis da samuwar tsari na lambar da aka nuna a ƙasa. stinal epithelial layers za a iya amfani da, misali, a cikin cell bambance-bambancen hali, gut physiology nazarin, kafa rundunar-microbiome ecosystems, da cuta modeling.Immunofluorescence Hotuna a cikin "Cell Bambanci" nuna nuclei, F-actin da MUC2 bayyana a cikin 3D Caco-2 epithelial epithelial siginar da aka yi a cikin sel mucosal mucosa da aka yi a cikin sel mucosal. Hotuna masu haske a cikin Gut Physiology suna nuna ƙwayar da aka samar ta hanyar lalatawa ga sialic acid da kuma N-acetylglucosamine sharan gona ta amfani da ƙwayar alkama mai fluorescent agglutinin. Hotunan biyu masu ɗaukar hoto a cikin "Mai watsa shiri-Microbe Co-Cultures" suna nuna wakilin mai watsa shiri-microbiome co-al'adu a cikin gut a kan gut a kan guntu na micro-cultures na hagu. gineered 3D Caco-2 epithelial cell.The dama panel yana nuna yanayin GFP E. coli tare da 3D Caco-2 epithelial Kwayoyin, biye da immunofluorescence tabo tare da F-actin (ja) da kuma nuclei (blue) .Cutar yin samfuri yana kwatanta lafiya tare da leaky gut a cikin ƙwayoyin cuta na ƙwayoyin cuta, ƙwayoyin cuta na ƙwayoyin cuta na ƙwayoyin cuta, ƙwayoyin cuta na ƙwayoyin cuta da ƙwayoyin cuta na ƙwayoyin cuta da ƙwayoyin cuta na L. da ƙwayoyin rigakafi (misali, PBMC;kore) .Caco-2 Kwayoyin An al'ada don kafa 3D epithelial Layer.Scale bar, 50 µm. Hotuna a kasa jere: "Bambancin Kwayoyin" daidaita tare da izini daga tunani.2.Jami'ar Oxford Press;Sake bugawa tare da izini daga Ref.5.NAS;"Co-Culture Mai watsa shiri-Microbe" wanda aka daidaita tare da izini daga ref.3.NAS;“Tsarin Cuta” wanda aka daidaita tare da izini daga tunani.5.NAS.
Dukansu gut-on-chip da matasan kwakwalwan kwamfuta an ƙirƙira su ta hanyar amfani da kwafin PDMS waɗanda aka lalatar da su daga ƙirar silicon ta hanyar lithography mai laushi1,44 kuma an tsara su tare da SU-8.An ƙaddamar da ƙirar microchannels a cikin kowane guntu ta hanyar yin la’akari da abubuwan da ke tattare da ruwa kamar damuwa mai ƙarfi da matsin lamba na hydrodynamic1,4,12.Ainihin gut-on-a-chip ɗin da aka yi daidai da Fig. microchannels, ya samo asali a cikin wani hadadden gut-on-a-chip (Extended Data Fig. 1b) wanda ya hada da nau'i-nau'i na microchannels mai lankwasa don haifar da Ƙara yawan lokacin zama na ruwa, tsarin tafiyar da ba tare da layi ba, da kuma nakasar multiaxial na ƙwayoyin al'ada (Fig. 2a-f) 12. Lokacin da ƙarin hadaddun gut biomechanted, za a iya zabar gutchilu-gutcreaon. ted Gut-Chip kuma yana da karfi ya haifar da 3D morphogenesis a cikin lokaci mai kama da irin wannan nau'i na girma na epithelial idan aka kwatanta da Gut-Chip na asali, ba tare da la'akari da nau'in kwayar halitta ba.Saboda haka, don haifar da 3D morphogenesis, linzamin kwamfuta da hadaddun kan-chip gut kayayyaki suna canzawa. PDMS kwafi da aka ba da magani a kan-siffar siliki 8 (Figures) da aka ba da su a kan sifofin siliki 8.2a) .Don ƙirƙira gut a kan guntu, shirin PDMS na sama da aka shirya an binne shi zuwa fim ɗin PDMS mai laushi sannan kuma ya daidaita tare da ƙananan PDMS Layer ta hanyar haɗin da ba za a iya jurewa ba ta amfani da ma'aunin corona (Fig. 2b-f) . Don ƙirƙirar kwakwalwan kwakwalwan kwamfuta, PDMS da aka warkar da kwafi an haɗa su zuwa gilashin nunin faifai wanda zai iya haifar da na'urori na transcodic Fig. Extended Data Fig. 2) Ana aiwatar da tsarin haɗin kai ta hanyar kula da saman PDMS replica da gilashi tare da oxygen plasma ko corona magani.Bayan haifuwa na microfabricated na'urar da aka haɗe zuwa tube na silicone, saitin na'urar ya shirya don yin 3D morphogenesis na epithelium na hanji (Figure 2g).
a, Schematic kwatanci na shirye-shiryen PDMS sassa daga SU-8 alamu silicon molds.The uncured PDMS bayani da aka zuba a kan wani silicon mold (hagu), warke a 60 °C (tsakiyar) da kuma rushe (dama).The PDMS da rushewa da aka yanka a cikin guda da kuma tsabtace don kara amfani.b, Hoton da silicon PDMS da aka yi amfani da Layer na silicon molds da aka yi amfani da shi don shirya mold na silicon PDMS. rous membrane.d, Jerin hotuna na manyan PDMS na sama da na ƙasa da na'urar haɗe-haɗe-haɗe-haɗe. on-a-chip don microfluidic cell al'ada.The ƙirƙira gut a kan guntu taro tare da silicone tube da sirinji da aka sanya a kan cover.The guntu na'urar da aka sanya a kan murfi na 150 mm Petri tasa don aiki.The dauri da ake amfani da su rufe da silicone tube.h, Kayayyakin gani hoto na matasan chipsmorphs al'adu shirye-shirye da 3D hybrid mai zaman kansa 3D. An saka ers of intestinal sel epithelial a cikin guntu matasan don haifar da morphogenesis na 3D na hanji.Matsakaici yana ruɗar ta hanyar microchannel a ƙarƙashin layin tantanin halitta da aka kafa akan mashigin Transwell.Scale bar, 1 cm.h An sake bugawa tare da izini daga tunani.4.Elsevier.
A cikin wannan yarjejeniya, Caco-2 cell line da intestinal organoids an yi amfani da su azaman tushen epithelial (Fig. 3a) .Dukansu nau'ikan kwayoyin halitta sun kasance masu al'ada da kansu (Box 2 da Box 5) kuma an yi amfani da su don zuriyar microchannels na ECM-coated na on-chip gut ko Transwell.Lokacin da kwayoyin halitta suke confluent (> 95% Cabet na yau da kullum da kuma 95% na sel na flabet 1) , sel 95% flabet sel na yau da kullun. 50) a cikin T-flasks ana girbe su don shirya rarrabuwa da dakatarwar cell ta hanyar ruwa na trypsinization (akwatin 2) .Human intestinal organoids daga intestinal biopsies ko tiyata resections an al'adance a cikin Matrigel scaffold domes a cikin 24-riji faranti don tallafawa tsarin tsarin microenvironment.Matsakaici da abubuwan da ba su da mahimmanci na R. Kamar yadda aka bayyana a cikin Akwatin 3 an ƙara shi kowace rana har sai kwayoyin halitta sun girma zuwa ~ 500 µm a diamita. Ana girbe kwayoyin da aka girma da kuma rarraba su cikin sel guda ɗaya don shuka a kan gut ko Transwell a kan guntu (Box 5) . Kamar yadda muka fada a baya, ana iya bambanta bisa ga cututtuka irin12,13, cututtuka na al'ada, ciwon daji ko ciwon daji, ko ciwon daji na al'ada , ko ciwon daji na al'ada , ko ciwon daji na al'ada. tare da yankin da ba shi da lahani) da kuma wurin gastrointestinal a cikin fili (misali, duodenum, jejunum, ileum, cecum, colon, ko dubura) .Muna samar da ingantacciyar yarjejeniya a cikin Akwatin 5 don culturing colonic organoids (coloids) wanda yawanci yana buƙatar ƙananan ƙwayoyin morphogens fiye da ƙananan ƙwayoyin hanji.
a, Ayyukan aiki don ƙaddamar da ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta.Caco-2 ana amfani da epithelium na intestinal na mutum da kuma intestinal organoids a cikin wannan ka'ida don nuna morphogenesis na 3D. Kwayoyin epithelial da aka keɓe sun kasance a cikin shirye-shiryen gut-on-a-chip na'urar (shiri na guntu) .Da zarar an yi amfani da kwayoyin halitta (seeded) MS (seeded) a kan sel PD (seeded). Apical (AP) ya fara gudana da kuma kiyaye shi don kwanakin 2 na farko (gudanarwa, AP, D0-D2) .Basolateral (BL) kwarara kuma an fara farawa tare da motsi na motsa jiki na cyclic (mike, gudana, AP da BL) lokacin da aka kafa cikakken 2D monolayer. Intestinal 3D morphogenesis ya faru kwatsam na morphogenesis na al'ada bayan 5 days nunawa. Kwayoyin co-2 a kowane mataki na gwaji ko lokacin lokaci (ginfin mashaya, 100 µm) .Hudu zane-zane na zane-zane da ke kwatanta daidaitattun cascades na gut morphogenesis (saman dama) .The dashed kiban a cikin makirci suna wakiltar jagorancin ruwa mai gudana.b, Hoton SEM yana nuna saman topology na kafa 3D Caco-2 epithelirated a cikin akwatin da aka sake gyarawa (wanda aka sake yin amfani da shi) a cikin akwati. villi a kan 3D Caco-2 Layer (dama).c, A tsaye gaban view kafa Caco-2 3D, claudin (ZO-1, ja) da ci gaba da goga iyakan membranes labeled F-actin (kore) da kuma nuclei (blue) Immunofluorescence confocal hangen nesa na epithelial Kwayoyin nuna na tsakiya na hanji ra'ayi na hanji. .d, Tsarin lokaci na sauye-sauyen yanayi a cikin kwayoyin halitta da aka yi a kan guntu da aka samu ta hanyar microscopy na lokaci-lokaci a kan kwanakin 3, 7, 9, 11, da 13. The inset (saman dama) yana nuna girman girman girman hoton da aka bayar.magenta), Kwayoyin goblet (MUC2; kore), F-actin (launin toka) da kuma nuclei (cyan) girma a kan kwakwalwan gut don kwanaki 3, bi da bi (Hagu) da 13-day (tsakiyar) organoids a kan epithelial Layer. Dubi kuma Extended Data Figure 3, wanda ya nuna LGR5 sigina ba tare da nuna alama na LGR5 ba tare da siginar MUC2 ba. organoid epithelium da aka kafa a cikin hanji akan guntu ta hanyar lalata membrane na plasma tare da rini na CellMask (dama) a ranar 13 na al'ada. Girman ma'auni yana da 50 μm sai dai in ba haka ba.b An sake bugawa tare da izini daga tunani.2.Jami'ar Oxford Press;c An kar~a da izini daga Reference.2.Jami'ar Oxford Press;e da f daidaitawa tare da izini ta hanyar tunani.12 Karkashin lasisin Creative Commons CC BY 4.0.
A cikin gut a kan guntu, ya zama dole don canza yanayin hydrophobic na PDMS porous membrane don cin nasara ECM shafi.A cikin wannan yarjejeniya, muna amfani da hanyoyi daban-daban guda biyu don canza hydrophobicity na PDMS membranes.Domin culturing Caco-2 Kwayoyin, surface kunnawa ta UV / ozone magani kadai ya isa ya rage da hydrophobicity na ECM sel da kuma hašawa da ECM. , microfluidic al'ada na organoid epithelium bukatar sinadaran tushen surface functionalization cimma m jijiya na ECM sunadaran ta sequentially da ake ji polyethyleneimine (PEI) da glutaraldehyde zuwa PDMS microchannels.Bayan surface gyara, ECM sunadaran da aka ajiye don rufe da functionalized PDMS surface da aka gabatar da kwayoyin halitta a cikin kwayoyin halitta da aka haɗe da al'ada. s ta hanyar turare kawai matsakaici a cikin microchannel na sama har sai sel sun samar da cikakkiyar monolayer, yayin da ƙananan microchannel ke kula da yanayin tsaye.Wannan ingantacciyar hanya don kunnawa saman da kuma murfin ECM yana ba da damar haɗakar da epithelium na organoid don haifar da morphogenesis na 3D akan saman PDMS.
Al'adun Transwell kuma suna buƙatar suturar ECM kafin shuka tantanin halitta;duk da haka, Transwell al'adu ba sa bukatar hadaddun pretreatment matakai don kunna surface na porous abun da ake sakawa.Domin girma Caco-2 Kwayoyin a kan Transwell abun da ake sakawa, ECM shafi a kan m abun da ake sakawa accelerates da abin da aka makala na dissociated Caco-2 Kwayoyin (<1 hour) da kuma m junction shãmaki samuwar (<1-2 days) .To al'ada organoids a kan Transwell kwayoyin da aka haɗe da ECM da aka sanya a kan Transwell kwayoyin, an haɗe su. 3 h) kuma ana kiyaye su har sai organoids sun samar da cikakkiyar monolayer tare da mutuncin shinge .An yi al'adun transwell a cikin faranti 24-riji ba tare da amfani da kwakwalwan kwamfuta ba.
In vitro 3D morphogenesis za a iya farawa ta hanyar yin amfani da ruwa mai gudana zuwa ga basolateral al'amari na kafa epithelial Layer.A cikin gut a kan guntu, epithelial morphogenesis fara a lokacin da matsakaici da aka perfused a cikin manya da ƙananan microchannels (Fig. 3a) . Kamar yadda aka bayyana a baya, yana da mahimmanci don gabatar da ruwa mai gudana a cikin ƙananan ƙananan (basolateral inhibited directions). serum zuwa sel da aka ɗaure a kan membranes porous kuma suna haifar da danniya mai ƙarfi, yawanci muna amfani da kwararar dual a cikin gut akan guntu.A cikin kwakwalwan kwamfuta na matasan, an shigar da abubuwan da ake sakawa waɗanda ke ɗauke da epithelial monolayers a cikin chips ɗin matasan.Sa'an nan, an yi amfani da matsakaici a ƙarƙashin gefen basolateral na porous Transwell sakawa ta hanyar microchannelogenesis a cikin al'adun morphso a cikin kwanaki 3 na al'ada.
Za'a iya bincika fasahar cututtukan cututtukan cututtukan micrethelial 3D (DIC) Mossivencerics, Sem, ko rigakafi na musamman da keɓaɓɓen ƙwayar cuta ta Microsocal (DIC) Mossivalencesce, SEL, ko imprussial Vissioncececes, SEC, ko immunsory ko kuma dipruse na yadudduka na 3D. Sakamakon bayyanar pdms da finafinan pdes na pdester, duka biyu na na'urar. ta Triton X-100 da 2% (WT / Vol)) Bovine Serum Album (BSA), a cikin tsari na musamman da aka yi amfani da shi a cikin guntu, daban-daban gyare-gyare da aka yi niyya a matsayin nucleus (misali , 4 ', 6-diamidino-2-Phenylene) Indole, DAPI) ko F-Actincently1, "Bambancin Kwayoyin Halitta" da "Gut Physiology"), bazuwar mulkin mallaka na ƙananan ƙwayoyin cuta (Fig. 1, "Mai watsa shiri-microbe co-al'adu"), da daukar ma'aikata na rigakafi Kwayoyin (Fig. 1, 'Cutar Modeling') ko contours na 3D epithelial ilimin halittar jiki (Fig. 4bifying da babba Layer, da kuma microbes). nel Layer, kamar yadda aka bayyana a cikin ref.Kamar yadda aka nuna a cikin siffa 2, da 3D epithelial ilimin halittar jiki da kuma microvilli a kan apical goga iyaka za a iya gani ta hanyar SEM (Fig. 3b) Maganar bambance-bambancen alamomi za a iya tantance ta yin adadi PCR5 ko guda-cell RNA sequencing. trypsinization sannan a yi amfani dashi don nazarin kwayoyin halitta ko kwayoyin halitta.
a, Gudun aiki don ƙaddamar da morphogenesis na intestinal a cikin guntu na matasan.Caco-2 da intestinal organoids ana amfani da su a cikin wannan yarjejeniya don nuna morphogenesis na 3D a cikin tsarin guntu na matasan. (TW al'ada) .Bayan 7 kwanaki, wani guda Transwell shigar dauke da wani 2D monolayer na epithelial Kwayoyin da aka hadedde a cikin wani matasan guntu don gabatar da wani basolateral kwarara (Flow, BL), wanda kyakkyawan ya haifar da ƙarni na 3D epithelial Layer (morphogenesis) .Phase bambanci micrographs nuna morphological fasali na mutum sashin jiki epithelial donor lokaci ko wani gwajin gwajin lokaci daga sel. schematics a cikin manyan yadudduka suna kwatanta tsarin gwaji don kowane mataki.b, Ƙwararren kwakwalwan kwamfuta (hagu na hagu) na iya haifar da morphogenesis na 3D na kwayoyin epithelial na organoid tare da ra'ayoyin microscopy na sama-ƙasa da aka ɗauka a wurare daban-daban na Z (na sama, tsakiya, da ƙananan;duba daidai tsari da layukan dige masu dacewa).ya nuna halaye na zahiri.F-actin (cyan), tsakiya (launin toka).c, Fluorescence confocal micrographs (3D angled view) na organoid-derived epithelial cells cultured in static Transwell (TW; inset within white dashed box) versus hybrid guntu (mafi girman cikakken harbi) kwatanta 3D madaidaiciya madaidaiciya. shigar a kusurwar dama ta sama; “XZ”) kuma yana nuna fasalulluka na 2D da 3D. Matsakaicin sikelin, 100 µm.c An sake bugawa tare da izini daga tunani.4.Elsevier.
Ana iya shirya sarrafawa ta hanyar al'ada iri ɗaya (Caco-2 ko intestinal organoid epithelial sel) zuwa cikin monolayers masu girma biyu a ƙarƙashin yanayin al'ada na al'ada. Musamman, raguwar abinci mai gina jiki na iya haifar da ƙananan ƙarfin ƙarar microchannels (watau ~ 4 µL a cikin tashar sama a kan asalin gut-chip na zane-zane na iya zama ba tare da morphology ba). .
Dole ne a aiwatar da tsarin lithography mai laushi a cikin ɗaki mai tsabta. Ga kowane Layer akan guntu (na sama da ƙananan yadudduka da membranes) da kwakwalwan kwamfuta, an yi amfani da hotuna daban-daban kuma an ƙirƙira su akan wafer siliki daban-daban saboda tsayin microchannels sun bambanta. Matsakaicin tsayin daka na sama da ƙananan microchannels na gut a kan guntu suna da tsayin 500 µm na guntu. 200 µm.
Sanya wafer silicon 3-inch a cikin tasa tare da acetone. A hankali juya farantin don 30 seconds, sa'an nan kuma iska bushe wafer. Canja wurin wafer zuwa faranti tare da IPA, sa'an nan kuma juya farantin don 30 s don tsaftacewa.
Maganin piranha (cakuda na hydrogen peroxide da sulfuric acid mai tattarawa, 1:3 (vol/vol)) za a iya amfani da zaɓin zaɓi don ƙara yawan cire ragowar kwayoyin halitta daga saman siliki wafer.
Maganin Piranha yana da lalata sosai kuma yana haifar da zafi.Ƙarin matakan tsaro suna da mahimmanci.Don zubar da sharar gida, ba da izinin bayani don kwantar da hankali da kuma canjawa wuri mai tsabta, busassun busassun busassun.
Dehydrate wafers ta hanyar sanya su a kan farantin zafi na 200 ° C na minti 10. Bayan bushewa, an girgiza wafer sau biyar a cikin iska don kwantar da hankali.
Zuba ~ 10 g na photoresist SU-8 2100 a tsakiyar tsakiyar wafer siliki mai tsabta. Yi amfani da tweezers don yada photoresist a ko'ina a kan wafer. Lokaci-lokaci sanya wafer a kan farantin zafi na 65 ° C don sa photoresist ya zama mai laushi da sauƙi don yadawa.Kada ku sanya wafer kai tsaye a kan farantin zafi.
SU-8 an rarraba a ko'ina a kan wafer ta hanyar yin amfani da sutura. Shirye-shiryen juyawa mai shigowa na SU-8 don 5-10 s don yaduwa a 500 rpm a hanzari na 100 rpm / s. Sanya babban juyi don ƙirar kauri na 200 µm don ƙirar kauri a 1,500 rpm, ko cimma tsayin 1,500 rpm. babban Layer na gut akan guntu; duba "Mahimman matakai" a ƙasa) saita a hanzari na 300 rpm/s 30 seconds a 1,200 rpm.
Ana iya daidaita babban saurin juzu'i bisa ga kauri da aka yi niyya na tsarin SU-8 akan wafer silicon.
Don ƙirƙira tsarin SU-8 na tsayin 500 µm don babban Layer na gut akan guntu, murfin juzu'i da matakan gasa mai laushi na wannan Akwatin (matakai na 7 da 8) an sake maimaita su akai-akai (duba mataki na 9) don samar da yadudduka biyu na 250 µm A lokacin farin ciki Layer na SU-8, wanda za'a iya sanyawa kuma ya shiga cikin akwatin UV05.
Gasa mai laushi SU-8 wafers mai rufi ta hanyar sanya wafers a hankali a kan farantin zafi a 65 ° C na minti 5, sannan canza saitin zuwa 95 ° C kuma a sanya shi don ƙarin 40 min.
Don cimma tsayin μm 500 μm na tsarin SU-8 a cikin microchannel na sama, maimaita matakai 7 da 8 don samar da yadudduka SU-8 mai kauri 250 μm biyu.
Amfani da UV Mask Alligner, yi gwajin fitila bisa ga umarnin masana'anta don ƙididdige lokacin bayyanar wafer.(lokacin fallasa, ms) = (kashin fallasa, mJ/cm2)/(ikon fitila, mW/cm2).
Bayan kayyade lokacin bayyanarwa, sanya hoton hoto akan abin rufe fuska na UV mask aligner kuma sanya hoton hoto akan wafer mai rufi SU-8.
Sanya saman bugu na hoto kai tsaye a gefen SU-8 mai rufi na wafer silicon don rage tarwatsewar UV.
Nuna wafer ɗin SU-8 mai rufi da mashin hoto a tsaye zuwa 260mJ/cm2 na hasken UV don ƙayyadadden lokacin fallasa (duba mataki na 10 na wannan akwatin).
Bayan bayyanar UV, SU-8-coated silicon wafers an gasa a 65 ° C don 5 min da 95 ° C na 15 min akan kowane farantin zafi don ƙirƙirar alamu tare da tsayin 200 μm. Ƙara lokacin bayan gasa a 95 ° C zuwa 30 min don ƙirƙirar alamu tare da tsawo na 500 µm.
An zuba mai haɓakawa a cikin gilashin gilashi, kuma an sanya wafer gasa a cikin tasa. Ƙarfin SU-8 mai haɓakawa na iya bambanta dangane da girman gilashin gilashin. Tabbatar yin amfani da isasshen SU-8 mai haɓakawa don cire gaba ɗaya SU-8 wanda ba a bayyana ba. Misali, lokacin amfani da gilashin gilashin diamita na 150 mm tare da iyawar 1 L, yi amfani da ~ 300 ml na SU-8 mL na SU-8 m lokaci mai tsawo.
Kurkura da gyare-gyaren da aka haɓaka tare da ~ 10 ml na sabo mai haɓakawa wanda IPA ya biyo baya ta hanyar fesa maganin ta amfani da pipette.
Sanya wafer a cikin mai tsabtace plasma kuma fallasa zuwa plasma oxygen (gas na yanayi, matsa lamba 1 × 10−5 Torr, iko 125 W) na 1.5 min.
Sanya wafer a cikin injin daskarewa tare da gilashin gilashi a ciki. Ana iya sanya wafers da nunin faifai a gefe da gefe.Idan an raba vacuum desiccator zuwa yawancin yadudduka ta faranti, sanya zane-zane a cikin ƙananan ɗakin da kuma wafers a cikin ɗakin babba.Drop 100 μL na trichloro (1H, 1Hroo-Hroo) gilashin siliki da sildi 2H don maganin sildi. ization.
Narke vial na sel Caco-2 daskararre a cikin wankan ruwa na 37°C, sannan a tura sel ɗin da aka narke zuwa flask T75 mai ɗauke da 15 ml na 37°C prewarmed Caco-2 matsakaici.
Don wuce sel Caco-2 a ~90% confluency, farko dumi Caco-2 matsakaici, PBS, da 0.25% trypsin/1 mM EDTA a cikin wani 37°C ruwa wanka.
Nuna matsakaita ta hanyar buri. A wanke sel sau biyu tare da 5 ml na PBS mai dumi ta maimaita buri da ƙara sabon PBS.