Na gode don ziyartar Nature.com. Sigar burauzar da kuke amfani da ita tana da iyakataccen tallafi ga CSS. Don mafi kyawun ƙwarewa, muna ba da shawarar ku yi amfani da sabunta burauza (ko kashe yanayin dacewa a cikin Internet Explorer) A halin yanzu, don tabbatar da ci gaba da tallafi, za mu nuna rukunin yanar gizon ba tare da salo da JavaScript ba.
morphogenesis na mutum yana kafa siffofi na crypt-villus na 3D epithelial microarchitecture da kuma sararin samaniya.Wannan tsari na musamman yana buƙatar don kula da gut homeostasis ta hanyar kare kwayoyin halitta a cikin basal crypt daga exogenous microbial antigens da metabolites. Saboda haka, sake ƙirƙirar 3D epithelial Tsarin yana da mahimmanci don gina in vitro gut model. Musamman ma, kwayoyin 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. morphogenesis a cikin gut a kan guntu na microfluidic da kuma a cikin wani nau'i na nau'i na nau'i na nau'i na Transwell.Mun bayyana cikakkun hanyoyin da za a yi amfani da na'ura na na'ura, al'ada na Caco-2 ko intestinal organoid epithelial Kwayoyin a cikin saitunan al'ada da kuma a kan wani microfluidic dandamali, ƙaddamar da 3D morphogenesis, da kuma halayyar da aka kafa ta hanyar yin amfani da 3D da aka kafa ta yin amfani da haɓakar haɓakar haɓakar haɓakawa na 3D. Gut microarchitecture na aiki ta hanyar sarrafa kwararar ruwa na basolateral don 5 d. Hanyarmu ta in vitro morphogenesis tana ɗaukar damuwa mai ƙarfi na physiologically da motsi na inji kuma baya buƙatar hadadden injiniyan tantanin halitta ko magudi, wanda zai iya fin sauran dabarun da ake da su.Muna hasashen cewa ƙa'idar da muka gabatar na iya samun fa'ida mai fa'ida ga al'umman bincike na biomedical don sake haifar da haɓakar tsarin halitta. don nazarin halittu, na asibiti, da aikace-aikacen magunguna.
Gwaje-gwaje sun nuna cewa kwayoyin epithelial na intestinal Caco-2 da aka yi amfani da su 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 fahimtar fahimtar tsarin da ke ciki ba. 3D epithelial 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 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" . frizzled-related protein 1, ko Soggy-1) zuwa ga on-chip gut yana hana morphogenesis ko rushe 3D epithelial Layer da aka riga aka tsara, yana nuna cewa danniya a lokacin al'ada yana da alhakin morphogenesis na hanji a cikin vitro.Saboda haka, hanyar da za a iya amfani da ita don cimma morphogenesis mai ƙarfi a cikin epithelial ba tare da ƙwanƙwasawa ba shine kiyaye matakan da ba a iya gani ba. daki ta hanyar flushing aiki (misali, a cikin gut-on-a-chip ko matasan-on-a-chip dandamali) ko watsawa .Basolateral kafofin watsa labarai (misali, daga Transwell shigar a cikin manyan basolateral reservoirs a rijiyoyin).
A cikin wannan yarjejeniya, muna ba da cikakkiyar hanya don ƙirƙira microdevices-on-a-chip microdevices da Transwell-insertable matasan kwakwalwan kwamfuta (matakai 1-5) zuwa al'ada na hanji epithelial Kwayoyin a kan polydimethylsiloxane (PDMS) - tushen porous membranes (matakai 6A, 7A, 8, 9) na transwell B, 7 da polyester membranes. 8, 9) da kuma haifar da morphogenesis na 3D a cikin vitro (mataki na 10) .Mun kuma gano sifofin salula da kwayoyin halitta wanda ke nuna alamun histogenesis na musamman na nama da bambance-bambancen salon salula da ke dogara da layi ta hanyar yin amfani da hanyoyi masu yawa (matakai 11-24) .Muna haifar da morphogenesis ta amfani da kwayoyin hanji na hanji kamar yadda tsarin al'ada na hanji-2 irin su Castinal sel a cikin nau'i biyu na kwayoyin halitta, irin su Castinal al'ada. fasaha cikakkun bayanai ciki har da gyaran fuska na porous membranes, halittar 2D monolayers, da hanji biochemical da Haifuwa na biomechanical microenvironment.in vitro.Don haifar da 3D morphogenesis daga 2D epithelial monolayers, mun cire morphogen antagonists a cikin duka al'ada siffofin ta gudãna da matsakaici a cikin basolateral al'adu compartment. regenerative 3D epithelial Layer wanda za a iya amfani da su model morphogen-dogara epithelial girma, tsawo mai watsa shiri-microbiome co-al'adu, pathogen kamuwa da cuta, kumburi rauni, epithelial shãmaki tabarbarewar, da kuma probiotic tushen jiyya Misali.influences.
Yarjejeniyar mu na iya zama da amfani ga masana kimiyya da yawa a cikin asali (misali, ilmin halitta mucosal na hanji, ilimin halittar kwayoyin halitta, da ilmin halitta na ci gaba) da bincike mai amfani (misali, gwajin magungunan preclinical, ƙirar cuta, injiniyan nama, da gastroenterology) tasiri mai fa'ida. yi tunanin cewa dabarun fasahar mu za a iya yadawa 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, Cutar cututtuka na numfashi mai tsanani Coronavirus 2 (SARS-Costridium) ko kuma Salphilemrium Dill, Clofficimum kwalara. Masu sauraro na cututtukan cututtuka da cututtukan cututtuka suna da amfani.Yin amfani da tsarin microphysiology na kan-chip gut zai iya ba da damar haɗin gwiwa na tsayin daka 10 da kuma kima na gaba na tsaro na rundunar, amsawar rigakafi da kuma raunin da ya shafi cututtuka a cikin gastrointestinal tract (GI) 11 .Wasu cututtuka na GI da ke hade da leaky gutwel syndrome, cututtukan cututtuka, cututtukan cututtuka, cututtuka na Crohn, cututtuka na celiac, cututtuka na cututtuka, cututtuka na Crohn's. simulated a lokacin da 3D hanji epithelial yadudduka da aka shirya ta yin 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 organoids. nau'o'in, irin su ƙwayoyin halittar jini guda ɗaya (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 hanya ne musamman m ga masu sauraro nazarin mucosal immunology, gastroenterology, mutum microbiome, culturomics da asibiti microbiology neman noma a baya uncultured gut microbiota a cikin dakin gwaje-gwaje.If mu in vitro morphogenesis yarjejeniya za a iya saba da scalable al'adu Formats, irin su multiwell, reply da kyau a cikin 624. Basolateral compartments, da yarjejeniya kuma za a iya yada zuwa ga wadanda tasowa Pharmaceutical, biomedical Ko high-throughput nunawa ko ingantattun dandamali ga masana'antar abinci.A matsayin hujja-na-ka'ida, mun kwanan nan nuna yiwuwa na multiplex high-samar morphogenesis tsarin scalable zuwa 24-dabawa-da-daba'a-samfurin tsarin da platechi format. commercialized16,17,18.Saboda haka, ingantaccen tsarin mu na in vitro morphogenesis na iya haɓakawa da yuwuwar ɗaukar shi ta yawancin dakunan gwaje-gwaje na bincike, masana'antu ko gwamnati da hukumomin gudanarwa don fahimtar tsarin salon salula na in vitro gut morphogenesis a matakin transcriptomic don gwada magunguna ko biotherapeutics An tantance 'yan takarar ta hanyar yin amfani da gut da safarar miyagun ƙwayoyi organ-on-a-chip model don tantance sake haifuwa na tsarin morphogenesis na gut.
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. Mahimmanci, kar a ƙayyade ƙayyadaddun tsarin ci gaban ɗan adam.Waɗannan samfuran kuma suna da iyakancewa sosai a cikin ikon su don gwada su ta hanyar haɓakawa da yawa.Saboda haka, ka'idodin mu don sake haɓaka tsarin nama na 3D a cikin vitro ya fi dacewa a cikin samfuran dabbobi na vivo da sauran samfuran al'adun sel na al'ada na 2D na al'ada. Kamar yadda aka bayyana a baya, yin amfani da 3D da aka yi amfani da su a cikin sel daban-daban da aka ba da izinin nazarin epithei a cikin sel na gida da aka yarda da su a cikin sel na gida. da crypt-villus axis a mayar da martani ga daban-daban mucosal ko rigakafi stimuli.3D epithelial yadudduka iya samar da sarari don nazarin yadda microbial Kwayoyin gasa don samar da 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). microbiota yana tsara al'ummominsa kuma yana samar da kwayoyin halitta metabolites (misali, gajeriyar sarkar fatty acids) waɗanda ke siffanta ƙungiyar salon salula da ƙananan ƙwayoyin ƙwayoyin cuta a cikin basal crypts. Waɗannan fasalulluka za'a iya nuna su ne kawai lokacin da aka kafa yadudduka na epithelial na 3D 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 dabara bisa ga namo na hanji kara Kwayoyin a karkashin takamaiman morphogen yanayi23,24,25.Duk da haka, da yin amfani da 3D organoid host-samfurin da ake amfani da shi sau da yawa ana gudanar da bincike na organoid organoid-model don gudanar da bincike. Lumen intestinal 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 yana da haɗari kuma yana da aiki mai tsanani kuma yana buƙatar ilimin musamman don yin aiki. Bugu da ƙari, al'adun gargajiya na gargajiya 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 ɗan adam a kan gel surface.Fabricate hydrogel scaffolds ta amfani da 3D-buga, micro-milled, ko lithographically ƙirƙira molds. dacewa morphogen gradients, kafa wani babban al'amari rabo epithelial tsarin da stroma-epithelial crosstalk ta hada da stromal Kwayoyin a cikin scaffold.Duk da haka, yanayin prestructured scaffolds na iya hana nuni na maras wata-wata morphogenetic tsari da kanta.These model kuma ba su samar da tsauri luminal ko lacking da danniya da ake bukata a cikin interstitial luminal luminal sel da matsa lamba a cikin jini. morphogenesis da samun aikin physiological.Wani binciken na baya-bayan nan ya yi amfani da matakan hydrogel a cikin wani dandamali na microfluidic da tsarin tsarin epithelial na hanji ta hanyar amfani da fasahar laser-etching. ya haɗa da motsi na mechanobiological na gut. 3D bugu dabaru daga rukuni guda sun sami damar ƙirƙirar ƙananan bututun gut tare da matakan morphogenetic ba tare da bata lokaci ba.Duk da haɗaɗɗen 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 operability na iya zama iyakancewa, musamman bayan tsarin aikin ƙwayoyin cuta-kwayoyin halitta don kammala gwajin-kwayoyin halitta. interactions.Maimakon haka, ka'idar mu da aka ba da shawarar tana ba da morphogenesis na gut ba tare da bata lokaci ba, danniya mai dacewa da yanayin jiki, biomechanics wanda ke kwaikwayon motsin gut, samun dama ga ɗakunan apical da basolateral masu zaman kansu, da sake sake haifar da ƙananan ƙananan ƙwayoyin halitta na modularity.Saboda haka, in vitro 3D morphogenesis na iya ba da ƙalubalen hanyoyin haɓakawa.
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, sel endothelial, da ƙwayoyin rigakafi.Kamar yadda aka bayyana a baya, ainihin ƙa'idar mu shine ƙaddamar da morphogenesis na epithelial ta hanyar cire masu hana morphogen da aka ɓoye a gefen mu na robust. gut-on-a-chip da hybrid-on-a-chip suna ba mu damar sake ƙirƙirar Layer epithelial na 3D mara kyau, ƙarin hadaddun halittu kamar su epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 da, a cikin ƙirar mu, crypt-villus fasali wanda ke isar da sifofin sel a cikin sel crypt. fibroblasts) a cikin mesenchyme suna taka muhimmiyar rawa wajen samar da sunadaran ECM da kuma tsara tsarin morphogenesis na hanji a cikin vivo35,37,38. Ƙarin ƙwayoyin ƙwayoyin cuta a cikin samfurinmu ya inganta tsarin morphogenetic da haɓakar haɗin sel. The endothelial Layer (watau capillaries ko lymphatics) yana taka muhimmiyar rawa wajen daukar nauyin kwayar halitta. A cikin gut microenvirment.furthermore, abubuwan haɗin vascurori waɗanda za a iya haɗa su tsakanin ƙa'idodin jiki na iya yin amfani da amsawar ƙwayoyin cuta, ƙwayoyin cuta suna buƙatar nuna amsoshin ƙwaƙwalwar ciki, Antigen Gabatarwa, ƙwaƙwalwar ɗanɗano Croosstalk, kuma ƙayyadaddun rigakafi na nama a cikin mahallin kwaikwayon cututtukan hanji.
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 gut epithelium mai sauƙi. Duk da haka, tallace-tallace da aka samu na Transwell 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, ba a sanya shi a cikin guntu mai kwakwalwa ba tare da haɗin ginin da aka saka a cikin guntu mai kwakwalwa. karfi danniya a kan apical side.A bayyane yake, a tsaye Properties a cikin apical daki da wuya damar 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 dandali kwarara na iya iyakance aikace-aikace na hybrid ruwa kwarara.
Cikakken 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 su kasance cikakke ba.Tun da morphogenesis ya fara ne daga monolayer na epithelial, 3D microarchitectures ba dole ba ne ya ba da kwatankwacin kamanni zuwa crypts a cikin vivo. 3D epithelium, crypt da vilous yankuna ba a bayyana a fili ba.Ko da yake mafi girma tashoshi na sama a kan guntu gubar zuwa tsawo tsawo na microengineered epithelium, matsakaicin tsawo har yanzu iyakance zuwa ~ 300-400 µm. Ainihin zurfin na mutum hanji crypts a cikin kananan da kuma manyan hanji , da girma da girma - 135 µm. Ƙananan villi na hanji shine ~ 600 µm41.
Daga hangen nesa na hoto, a cikin yanayin babban ƙuduri na 3D microarchitectures na iya iyakancewa ga gut akan guntu, tunda nisan aikin da ake buƙata daga ruwan tabarau na haƙiƙa zuwa Layer na epithelial yana kan tsari na 'yan milimita don shawo kan wannan matsalar, ana iya buƙatar manufa mai nisa. Bugu da ƙari, yin sassan bakin ciki don yin hoto mai girma shine babban kalubale. PDMS.Bugu da ƙari, tun da Layer-by-Layer microfabrication na gut a kan guntu ya ƙunshi mannewa na dindindin tsakanin kowane Layer, yana da wuyar gaske don buɗewa ko cire babban Layer don bincika tsarin saman Layer na epithelial. 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 lipophilic kayan 42 glycol ko 4 lipophilic kayan). a yi la'akari da rage 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 29, misali, scalability-6. ko 384- rijiyoyin latsawa da aka saka waɗanda ke ba da damar ci gaba da cikawa da 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 ci gaba da basolar Layer girma epithene. shigarwa.A cikin duka dandamali, ana iya nuna morphogenesis na nau'in kwayoyin epithelial na hanji na mutum daban-daban ta hanyar yin amfani da manipulation na shugabanci na kwarara don cire antagonists na morphogen daga sashin basolateral. Dukan hanyar gwaji (Figure 1) ya ƙunshi sassa biyar: (i) microfabrication na guntu gut ko Transwell insertable matasan), (1-5 guntu ; Kwayoyin epithelial (Caco-2 Kwayoyin) ko ƙwayoyin hanji na mutum; 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 kasa (matakai 11-24) da aka tsara a ƙasa. na in vitro 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 shigar da Transwell (TW) a cikin na'urar microfluidic, wanda aka ƙirƙira kamar yadda aka bayyana a cikin Akwatin 1, da mataki na 1 -5. (Caco-2 ko organoids na hanji na mutum) da tsarin al'ada da aka yi amfani da su a cikin wannan yarjejeniya. "In vitro morphogenesis" yana nuna matakan gabaɗaya wanda Caco-2 ko organoid-samu epithelial Kwayoyin suna al'ada a kan guntu na hanji ko a kan abubuwan da ake sakawa na guntu na matasan, biye da shigar da 3D morphogenesis da lambar ƙirar kowane tsarin da aka nuna a ƙasa. arrow.A aikace-aikace na ba da misalai na yadda za a iya amfani da kafa na intestinal epithelial yadudduka, misali, a cikin cell bambance-bambancen halayen, gut physiology nazarin, kafa rundunar-microbiome ecosystems, da cuta modeling.Immunofluorescence Hotuna a cikin "Cell Bambanci" nuna nuclei, F-actin da MUC2 da aka bayyana a cikin guntu-2D Layer Layer epicotlid. sigina yana samuwa a cikin sel kwalabe da gamsai da aka ɓoye daga saman mucosal. Hotuna masu haske a cikin Gut Physiology suna nuna ƙwayar da aka samar ta hanyar yin lalata ga sialic acid da kuma N-acetylglucosamine ragowar ta amfani da ƙwayar alkama mai fluorescent agglutinin. Hotunan biyu masu haɗuwa a cikin "Mai watsa shiri-Microbe Co-Cultures" ya nuna guntu mai masaukin baki-mic co-al'adu na E. coli bayyana kore mai kyalli furotin (GFP) tare da microengineered 3D Caco-2 epithelial cell.The dama panel nuna localization na GFP E. coli co-al'adu tare da 3D Caco-2 epithelial Kwayoyin, bi da immunofluorescence tabo tare da F-actin (ja) da kuma nucleiaset guguwar rashin lafiya model. kwakwalwan kwamfuta a ƙarƙashin ƙalubalen ilimin lissafi tare da antigens na kwayan cuta (misali, lipopolysaccharide, LPS) da ƙwayoyin rigakafi (misali, PBMC; kore) .Caco-2 Kwayoyin sun kasance masu al'ada don kafa 3D epithelial Layer. Scale bar, 50 µm. Hotuna a cikin layi na kasa: "Bambancin kwayoyin halitta" 2. An daidaita tare da izini daga tunani. 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.Asali na gut-on-a-chip na ƙira na Fig. juxtaposed layi daya madaidaiciya microchannels, ya samo asali a cikin wani hadadden gut-on-a-chip (Extended Data Fig. 1b) wanda ya hada da biyu na lankwasa microchannels don haifar da Increasingara yawan lokacin zama na ruwa, yanayin kwarara mara kyau, da nakasar multiaxial na ƙwayoyin al'ada (Fig. 2a-f) 12.Lokacin da ƙarin hadaddun gut-creat-creat-buƙatar 12. za a iya zaba.Mun nuna cewa convoluted Gut-Chip Har ila yau, da karfi induces 3D morphogenesis a cikin irin wannan lokaci frame tare da irin wannan mataki na epithelial girma idan aka kwatanta da na asali Gut-Chip, ko da kuwa da al'ada cell type.Saboda haka, don haifar da 3D morphogenesis, mikakke da hadaddun on-chip gut kayayyaki suna musanya canje-canje a kan silicon-siffofi m fasali a kan PD8 m fasali. demolding (Fig. 2a) .Don ƙirƙira gut a kan guntu, shirin PDMS na sama da aka shirya an binne shi zuwa fim ɗin PDMS mai ƙyalli sannan kuma ya daidaita tare da ƙananan PDMS Layer ta hanyar haɗin da ba za a iya jurewa ba ta amfani da mai maganin corona (Fig. 2b-f) . Abubuwan da ake sakawa na Transwell (Fig. 2h da 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.
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) PDMS da rushewa da aka yanka a cikin guda da kuma tsabtace don kara amfani.b, Hoton na silicon PDMS da aka yi amfani da shi don shirya moldc na silicon . da PDMS porous membrane.d, Jerin hotuna na manya da ƙananan sassan PDMS da na'urar haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe. chambers.g, Saitin gut-on-a-chip don al'adun cell microfluidic.An ƙirƙira gut a kan guntu da aka haɗa tare da bututun silicone da sirinji an sanya shi a kan murfin murfin. chips.Transwell abubuwan da aka shirya da kansa zuwa al'ada 2D monolayers na hanji epithelial Kwayoyin an saka a cikin matasan guntu don jawo hanji 3D morphogenesis.Matsakaici yana perfused ta microchannels karkashin cell Layer kafa a kan Transwell saka.Scale bar, 1 cm.h Sake buga tare da izini daga tunani.4. Elsevier.
A cikin wannan yarjejeniya, Caco-2 cell line da intestinal organoids da aka yi amfani da matsayin epithelial tushen (Fig. 3a) .Dukansu nau'in sel sun kasance masu al'ada da kansu (Box 2 da Box 5) kuma an yi amfani da su don zur da ECM-mai rufi microchannels na on-chip gut ko Transwell sakawa.Lokacin da sel suke confluent (> sk 95% Cabet sel na yau da kullum) sel sel na flabet, ɗaukar hoto na yau da kullun. 10 da 50) a cikin T-flasks ana girbe su don shirya abubuwan dakatarwar cell da aka rabu da ruwa ta hanyar trypsinization ruwa (akwatin 2) .Human intestinal organoids daga intestinal biopsies ko tiyata resections an al'ada a cikin Matrigel scaffold domes a cikin 24-riji faranti don tallafawa tsarin tsarin, morphntogens mai mahimmanci na microenvironment. R-spondin, da Noggin) da kuma abubuwan haɓaka da aka shirya kamar yadda aka bayyana a cikin Akwatin 3 an ƙara su kowace rana har sai kwayoyin halitta sun girma zuwa ~ 500 µm a diamita. An girbe organoids masu girma da yawa kuma an rarraba su cikin sel guda ɗaya don shuka a kan gut ko Transwell sakawa a kan guntu (Box 5) . Kamar yadda muka riga muka ruwaito a baya, yana iya zama daban-daban nau'in cututtuka na Crown 1, cututtuka na 1. cuta, ciwon daji mai launi, ko mai ba da gudummawa na al'ada), wurin rauni (misali, rauni da yanki mara lahani) da wurin gastrointestinal a cikin fili (misali, duodenum, jejunum, ileum, cecum, colon, ko dubura).Muna samar da ingantaccen tsari a cikin Akwatin 5 don culturing colonic organoids (coloids) wanda yawanci yana buƙatar mafi girma na ƙwayoyin cuta.
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 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 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 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 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 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 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 ta Caco-2 . 0 (D0), 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 bayan morphigenesis na al'ada bayan kwanaki 5. D5) Hotunan bambance-bambancen lokaci suna nuna nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan Caco-2 a kowane mataki na gwaji ko lokacin lokaci (hoton mashaya, 100 µm) .Haɗin ƙirar ƙira huɗu waɗanda ke kwatanta madaidaicin cascades na gut morphogenesis (saman dama) .Ƙaƙƙarfan kibau a cikin tsari suna wakiltar jagorancin ruwa mai gudana.b, Hoton SEM yana nuna 2 surface topology. Nuna girman yanki (akwatin da aka dasa fari) yana nuna microvilli da aka sabunta akan 3D Caco-2 Layer (dama) .c, Horizontal frontal view of kafa Caco-2 3D, claudin (ZO-1, ja) da ci gaba da goga kan iyakar membranes mai lakabi F-actin (kore) da kuma nuclei (blue) na rigakafi da ke haifar da haɓakar sel na gani na epithelium. chips.Kibiyoyi masu nuni zuwa ga tsarin tsakiya suna nuna wurin da jirgin sama mai da hankali ga kowane ra'ayi na confocal.d, Tsarin lokaci na morphological canje-canje a cikin kwayoyin halitta a kan guntu da aka samu ta hanyar microscopy na lokaci-lokaci a kwanakin 3, 7, 9, 11, da 13. Ƙirar (saman dama) yana nuna girman girman girman da aka bayar a kan hoton da aka ba da shi a cikin hoto na Epiomicroum 3 DIC da aka kafa a cikin hoto na Epicroum. yanki da aka ɗauka a rana 7.f, Hotunan da aka rufe da immunofluorescence da ke nuna alamomi don ƙwayoyin sel (LGR5; magenta), sel goblet (MUC2; kore), F-actin (launin toka) da nuclei (cyan) waɗanda aka girma akan guntun gut na tsawon kwanaki 3, bi da bi (Hagu) da 13-day (tsakiyar) gabobin, bayanan LG5 da aka fitar da su. sigina ba tare da siginar MUC2 ba. Hotunan fuka-fukan da ke nuna ƙananan ƙwayoyin cuta na epithelial (dama) na 3D organoid epithelium da aka kafa a cikin gut a kan guntu ta hanyar lalata ƙwayar plasma tare da launi na CellMask (dama) a ranar 13 na al'ada. Matsakaicin sikelin shine 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 gyara 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 Caco-CM. PDMS membrane.However, microfluidic al'adu na organoid epithelium bukatar sinadaran tushen surface functionalization cimma m jigo na ECM sunadaran da sequentially da ake ji polyethyleneimine (PEI) da glutaraldehyde zuwa PDMS microchannels.Bayan surface gyara, ECM sunadaran da aka ajiye don rufe da functionalized PDMS da aka gabatar da sunadaran ECM a cikin epithelium. Ana haɗe sel, al'adun ƙwayoyin microfluidic yana farawa ne ta hanyar turawa kawai matsakaici a cikin microchannel na sama har sai sel sun samar da cikakkiyar monolayer, yayin da ƙananan microchannel ke kula da yanayin da ba daidai ba.Wannan hanyar da aka inganta don kunnawa saman da kuma murfin ECM yana ba da damar haɗuwa 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 m junction shãmaki samuwar (<1-2 days) .To al'ada organoids a kan Transwell organoid, an haɗe da ECM shigar da aka sanya a kan Canje-canje da aka shigar da ECM. da membrane surface (<3 h) da kuma kiyaye har sai organoids samar da cikakken monolayer tare da shãmaki mutunci .Transwell al'adu da aka yi a cikin 24-riji faranti ba tare da amfani da matasan kwakwalwan kwamfuta.
In vitro 3D morphogenesis za a iya fara 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). samar da isasshen abinci mai gina jiki da jini ga sel da ke daure a kan membranes mai laushi kuma suna haifar da danniya mai laushi, yawanci muna amfani da dual kwarara a cikin gut a kan guntu.A cikin kwakwalwan kwakwalwan kwamfuta, an shigar da abubuwan da ke dauke da epithelial monolayers a cikin kwakwalwan matasan. Sa'an nan, an yi amfani da matsakaici a ƙarƙashin gefen basolateral na porous Transwell sakawa ta hanyar microchannel na kwanaki 3. gudana a cikin dandamalin al'adu biyu.
Za'a iya bincikar sifofin sifofi na 3D microengineered epithelial yadudduka ta hanyar yin amfani da hanyoyi daban-daban na hoto, ciki har da microscopy bambanci na lokaci, bambancin tsangwama (DIC) microscopy, SEM, ko immunofluorescence confocal microscopy (Figures 3 da 4) .Za'a iya yin sauƙin saka idanu ko yanayin DIC a kowane lokaci na al'ada na al'ada. Yadudduka.Sakamakon bayyananniyar gani na PDMS da fina-finai na polyester, duka gut-on-a-chip da dandamali na guntu na matasan na iya samar da ainihin lokacin a cikin hoto ba tare da buƙatar sashe ko rarraba na'urar ba.Lokacin yin hoto na immunofluorescence (Figures 1, 3c, f da 4b) yawanci suna daidaitawa tare da sel, c. (PFA), biye da Triton X-100 da 2% (wt / vol) ) na bovine serum albumin (BSA), a cikin tsari.Ya danganta da nau'in tantanin halitta, ana iya amfani da gyare-gyare daban-daban, masu lalata, da kuma masu hanawa. (misali, 4′,6-diamidino-2-phenylene) indole, DAPI) ko F-actin (misali, phalloidin mai suna fluorescently). Hakanan za'a iya yin hoto mai rai na tushen fluorescence a wurin don gano ƙwayar ƙwayar cuta (Fig. 1, "Bambancin Kwayoyin Halitta" da "Gut physiology"), bazuwar ƙwayoyin cuta na microbi. co-al'adu"), da daukar ma'aikata na rigakafi Kwayoyin (Fig. 1, 'Cutar Modeling') ko contours na 3D epithelial ilimin halittar jiki (Fig. 3c, f da 4b, c) .Lokacin da gyaggyarawa gut a kan guntu don raba babba Layer daga ƙananan microchannel Layer, kamar yadda aka bayyana a cikin ref.As2, kamar yadda aka nuna a cikin Fig. a kan iyakar goga na apical za a iya gani ta hanyar SEM (Fig. 3b) . Za'a iya kimanta ma'anar alamar bambance-bambance ta hanyar yin PCR5 mai ƙididdigewa ko tsarin RNA guda ɗaya.
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 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 ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta. al'ada a karkashin yanayi na tsaye (TW al'ada) .Bayan 7 kwanaki, wani guda Transwell shigarwa dauke da 2D monolayer na epithelial Kwayoyin da aka haɗa 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 na nuna bambanci micrographs nuna morphological fasali na al'ada kwayoyin don sel epitheli 3. colon a kowane mataki na gwaji ko lokaci.Tsarin da ke cikin manyan yadudduka suna kwatanta tsarin gwajin 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'ayi na microscopy na sama-sau da aka dauka a wurare daban-daban na Z (na sama, tsakiya, da ƙasa; duba lambobi masu dacewa da daidaitattun layi). 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 versus versus 2D. ra'ayoyin giciye (saka a kusurwar dama na sama; "XZ") kuma yana nuna fasalin 2D da 3D. Matsakaicin girman, 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 cell) a cikin nau'i-nau'i biyu na monolayers a ƙarƙashin yanayin al'ada na al'ada. Musamman, raguwar abinci mai gina jiki na iya haifar da ƙananan ƙananan ƙananan ƙwayoyin microchannels (watau ~ 4 µL a cikin babban tashar a kan asalin gut-chip na zane-zane na asali, da kuma zane-zane na asali). kwarara kuma za a iya kwatanta.
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 500 µm na tsayin daka. Hybrid guntu ne 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 a 1,500 rpm, ko cimma kauri na 1,500 rpm Tsawon µm don 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 gwargwadon maƙasudin kauri 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 haɗa shi da akwatin UV5µ. babba.
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. Za a iya sanya wafers da nunin faifai a gefe da gefe.Idan an raba vacuum desiccator zuwa yadudduka da yawa ta faranti, sanya nunin faifai a cikin ƙananan ɗakin da wafers a cikin babban ɗakin. injin don silanization.
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.