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Tsarin halittar hanjin ɗan adam yana kafa fasalulluka na crypt-villus na microarchitecture na epithelial 3D da tsarin sarari. Wannan tsari na musamman ana buƙatar don kula da yanayin hanji ta hanyar kare tushen ƙwayoyin halitta a cikin crypt ɗin basal daga ƙwayoyin cuta na waje da metabolites ɗinsu. Bugu da ƙari, villi na hanji da fitar da mucosa suna da ƙwayoyin epithelial daban-daban tare da shinge mai kariya akan saman mucosal na hanji. Saboda haka, sake ƙirƙirar tsarin epithelial na 3D yana da mahimmanci don gina samfuran hanji na in vitro. Abin lura, mimetic gut-on-a-chip na halitta na iya haifar da morphogenesis na 3D na epithelium na hanji tare da ingantattun ayyukan ilimin halittar jiki da biomechanics. A nan, muna samar da wata yarjejeniya mai sake haifuwa don haifar da morphogenesis na hanji mai ƙarfi a cikin hanji akan guntu na microfluidic da kuma a cikin guntu na haɗin gwiwa da aka saka a Transwell. Muna bayyana cikakkun hanyoyin ƙera na'urori, haɓaka ƙwayoyin epithelial na Caco-2 ko na hanji a cikin saitunan gargajiya da kuma akan dandamalin microfluidic, haifar da morphogenesis na 3D, da kuma halayyar 3D da aka kafa. epithelia ta amfani da hanyoyin daukar hoto da yawa. Wannan yarjejeniya tana cimma nasarar sake farfaɗo da tsarin aikin hanji ta hanyar sarrafa kwararar ruwa na basolateral na tsawon kwanaki 5. Hanyarmu ta in vitro morphogenesis tana amfani da matsin lamba na yankewa da motsi na injiniya kuma baya buƙatar injiniyan tantanin halitta mai rikitarwa ko sarrafawa, wanda zai iya yin fice fiye da sauran dabarun da ake da su. Muna hasashen cewa yarjejeniyar da muka gabatar za ta iya samun fa'idodi masu yawa ga al'ummar binciken biomedical, tana samar da hanyar sake farfaɗo da layukan epithelial na hanji na 3D a cikin vitro don aikace-aikacen biomedical, asibiti, da magunguna.
Gwaje-gwaje sun nuna cewa ƙwayoyin Caco-2 na hanji waɗanda aka haɓaka a cikin na'urorin microfluidic guda 1,2,3,4,5 ko bilayer 6,7 na iya fuskantar morphogenesis na 3D ba tare da fahimtar tsarin da ke ƙarƙashinsa ba. A cikin bincikenmu na baya-bayan nan, mun gano cewa cire masu adawa da morphogen da ke fitowa daga na'urorin al'adu ya zama dole kuma ya isa ya haifar da morphogenesis na epithelial na 3D a cikin vitro, wanda Caco-2 da ƙwayoyin hanji da aka samo daga marasa lafiya suka nuna. An tabbatar da ƙwayoyin epithelial. A cikin wannan binciken, mun mayar da hankali musamman kan samar da ƙwayoyin halitta da kuma rarrabawar taro na mai ƙarfi na Wnt antagonist, Dickkopf-1 (DKK-1), a cikin gut-on-a-chip da na'urorin microfluidic da aka gyara waɗanda ke ɗauke da abubuwan da aka saka a cikin Transwell, waɗanda ake kira "Hybrid Chip". Mun nuna cewa ƙara magungunan Wnt na waje (kamar DKK-1, Wnt repressor 1, furotin mai alaƙa da frizzled 1, ko Soggy-1) zuwa cikin hanjin da ke kan guntu yana hana morphogenesis ko kuma yana wargaza Layer na epithelial 3D da aka riga aka tsara, yana nuna cewa damuwa mai adawa yayin al'ada ita ce ke da alhakin morphogenesis na hanji a cikin vitro. Saboda haka, hanya mai amfani don cimma morphogenesis mai ƙarfi a mahaɗin epithelial ita ce cire ko kula da matakan masu adawa da Wnt a cikin sashin basolateral ta hanyar wankewa mai aiki (misali, a cikin dandamalin gut-on-a-chip ko hybrid-on-a-chip) ko yaɗuwa. Kafofin watsa labarai na asali (misali, daga Transwell yana sakawa cikin manyan wuraren ajiyar basolateral a cikin rijiyoyi).
A cikin wannan yarjejeniya, mun samar da cikakken hanya don ƙirƙirar ƙananan na'urori na hanji da kwakwalwan haɗin gwiwa na Transwell-insertable (matakai na 1-5) zuwa haɓaka ƙwayoyin epithelial na hanji akan membranes na polydimethylsiloxane (PDMS) (matakai na 6A, 7A, 8, 9) ko membranes na polyester na Transwell inserts (matakai na 6B, 7B, 8, 9) da kuma haifar da morphogenesis na 3D a cikin vitro (mataki na 10). Mun kuma gano fasalulluka na ƙwayoyin halitta da na kwayoyin halitta waɗanda ke nuna histogenesis na musamman na nama da bambancin ƙwayoyin halitta da suka dogara da layi ta hanyar amfani da hanyoyin daukar hoto da yawa (matakai na 11-24). Muna haifar da morphogenesis ta amfani da ƙwayoyin epithelial na hanji na ɗan adam, kamar Caco-2 ko organoids na hanji, a cikin tsarin al'adu guda biyu tare da cikakkun bayanai na fasaha waɗanda suka haɗa da gyaran saman membranes na ramuka, ƙirƙirar monolayers na 2D, da biochemical na hanji da kuma sake haifar da microenvironment na biomechanical.in vitro. Domin haifar da morphogenesis na 3D daga monolayers na epithelial na 2D, mun cire Masu adawa da morphogen a cikin nau'ikan biyu da aka haɓaka ta hanyar kwararar da matsakaici zuwa cikin ɓangaren basolateral na al'adar. A ƙarshe, muna ba da wakilcin amfanin wani Layer na epithelial na 3D wanda za'a iya amfani da shi don yin kwaikwayon ci gaban epithelial mai dogaro da morphogen, al'adun haɗin gwiwa na masu masaukin baki-microbiome na tsawon lokaci, kamuwa da cuta ta pathogen, raunin kumburi, rashin aikin shinge na epithelial, da kuma hanyoyin magance probiotic. Misali yana tasiri.
Tsarin aikinmu na iya zama da amfani ga masana kimiyya iri-iri a fannin asali (misali, ilimin halittar mucosal na hanji, ilimin halittar ƙwayoyin halitta, da ilimin halittar ci gaba) da kuma bincike mai zurfi (misali, gwajin magunguna kafin asibiti, ƙirar cututtuka, injiniyan nama, da kuma ilimin gastroenterology). Saboda sake haifuwa da ƙarfi na tsarin aikinmu don haifar da yanayin 3D na epithelium na hanji a cikin vitro, muna hasashen cewa dabarunmu na fasaha za a iya yaɗa su ga masu sauraro suna nazarin yanayin siginar tantanin halitta yayin ci gaban hanji, sake farfadowa ko homeostasis. Bugu da ƙari, tsarin aikinmu yana da amfani don yin tambayoyi game da kamuwa da cuta a ƙarƙashin wasu cututtukan da ke yaɗuwa kamar Norovirus 8, Ciwon Numfashi Mai Tsanani na Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 ko Vibrio cholerae. Masu sauraron cututtukan cututtuka da kuma cututtukan da ke haifar da su suma suna da amfani. Amfani da tsarin microphysiology na hanji na iya ba da damar yin amfani da dogon lokaci 10 da kuma kimantawa na gaba game da kariyar mai masaukin baki, martanin garkuwar jiki da kuma gyaran raunin da ya shafi ƙwayoyin cuta a cikin hanyar narkewar abinci (GI) 11. Sauran cututtukan GI da ke da alaƙa da ciwon hanji mai zubar jini, cutar celiac, cutar Crohn, ulcerative colitis, pouchitis, ko ciwon hanji mai ban haushi za a iya kwaikwayon su lokacin da aka shirya yadudduka na epithelial na hanji na 3D ta amfani da yadudduka na epithelial na hanji na majiyyaci na 3D, waɗannan cututtukan sun haɗa da atrophy na villous, rage crypt, lalacewar mucosal, ko lalacewar shingen epithelial. Kwayoyin halittar hanji da aka samo daga ƙwayoyin halitta ko ƙwayoyin halitta12,13. Don yin kwaikwayon yanayin cutar mafi girma, masu karatu na iya la'akari da ƙara nau'ikan ƙwayoyin halitta masu alaƙa da cutar, kamar ƙwayoyin mononuclear na jini na marasa lafiya (PBMCs), zuwa samfuran da ke ɗauke da ƙwayoyin microarchitecture na hanji na 3D. ƙwayoyin rigakafi na musamman na nama, 5.
Tunda ana iya gyarawa da kuma nuna tsarin epithelial na 3D ba tare da tsarin rabawa ba, masu kallo da ke aiki akan transcriptomics na sararin samaniya da kuma hoton babban ƙuduri ko babban ƙuduri na iya sha'awar taswirar yanayin yanayin sararin samaniya na kwayoyin halitta da sunadarai akan wuraren epithelial. Sha'awar fasaha. Martani ga abubuwan da ke haifar da ƙwayoyin cuta ko garkuwar jiki. Bugu da ƙari, ana iya kafa haɗin gwiwa na mai masaukin baki na tsawon lokaci 10, 14 wanda ke daidaita yanayin hanji a cikin layin mucosal na hanji na 3D ta hanyar haɓaka nau'ikan ƙwayoyin cuta daban-daban, al'ummomin ƙwayoyin cuta ko ƙwayoyin cuta na hanji, musamman a cikin hanji-on-a-chip. a cikin dandamali. Wannan hanyar tana da kyau musamman ga masu karatu waɗanda ke nazarin ilimin rigakafi na mucosal, gastroenterology, ɗan adam microbiome, culturomics da kuma ilimin ƙwayoyin cuta na asibiti waɗanda ke neman haɓaka ƙwayoyin cuta na hanji da ba a taɓa noma su ba a dakin gwaje-gwaje. Idan za a iya daidaita tsarin morphogenesis ɗinmu na in vitro zuwa tsarin al'adu masu iya canzawa, kamar shigarwar multiwell a cikin faranti 24, 96 ko 384 waɗanda ke ci gaba da cike guraben basolateral, ana iya kuma yaɗa tsarin ga waɗanda ke haɓaka dandamali na tantance magunguna, biomedical ko manyan dandamali na tantancewa ko tabbatarwa ga masana'antar abinci. A matsayin shaida na ƙa'ida, kwanan nan mun nuna yuwuwar tsarin morphogenesis mai yawan amfani da yawa wanda za a iya daidaita shi zuwa tsarin faranti 24. Bugu da ƙari, an sayar da samfuran organ-on-a-chip da yawa16,17,18. Saboda haka, ana iya hanzarta tabbatar da hanyar morphogenesis ɗinmu ta in vitro kuma wataƙila za a iya amfani da ita ta hanyar dakunan gwaje-gwaje da yawa, masana'antu ko gwamnati da hukumomin da ke kula da su don fahimtar sake tsara ƙwayoyin cuta na in vitro morphogenesis a matakin transcriptomic don gwada magunguna ko biotherapeutics Sha. kuma an tantance jigilar masu shan ƙwayoyi ta amfani da magungunan maye gurbin hanji na 3D ko kuma ta amfani da samfuran musamman ko na kasuwanci don tantance yuwuwar sake haɓɓaka tsarin morphogenesis na hanji.
An yi amfani da ƙayyadadden adadin samfuran gwaji masu dacewa da ɗan adam don nazarin yanayin epithelial na hanji, galibi saboda rashin ka'idoji masu aiwatarwa don haifar da yanayin 3D a cikin vitro. A zahiri, yawancin ilimin da ake da shi game da yanayin ƙwai ya dogara ne akan nazarin dabbobi (misali, zebrafish20, beraye21 ko kaji22). Duk da haka, suna da aiki da tsada, ana iya yin tambaya game da ɗabi'a, kuma mafi mahimmanci, ba sa tantance ainihin hanyoyin ci gaban ɗan adam. Waɗannan samfuran kuma suna da iyaka sosai a cikin ikonsu na gwadawa ta hanyoyi da yawa masu iya daidaitawa. Saboda haka, tsarinmu na sake samar da tsarin kyallen 3D a cikin vitro ya fi kyau a cikin samfuran dabbobi a cikin vivo da kuma sauran samfuran al'adun ƙwayoyin 2D na gargajiya. Kamar yadda aka bayyana a baya, amfani da tsarin epithelial 3D ya ba mu damar bincika wurin da aka ware na ƙwayoyin halitta a cikin axis na crypt-villus don mayar da martani ga abubuwan da ke haifar da ƙwayoyin cuta ko garkuwar jiki daban-daban. Layer epithelial 3D na iya samar da sarari don nazarin yadda ƙwayoyin microbial ke fafatawa don samar da wurare na sarari da juyin halittar muhalli don mayar da martani ga abubuwan da ke cikin gida (misali, yadudduka na ciki da na waje, fitar da IgA da peptides na ƙwayoyin cuta). Bugu da ƙari, yanayin epithelial na 3D zai iya ba mu damar fahimtar yadda ƙwayoyin cuta na hanji ke tsara al'ummominsu kuma suna samar da metabolites na ƙwayoyin cuta (misali, fatty acids na gajerun sarƙoƙi) waɗanda ke tsara tsarin ƙwayoyin halitta da kuma ƙwayoyin halitta na tushe a cikin crypts na basal. Waɗannan fasalulluka za a iya nuna su ne kawai lokacin da aka kafa layukan epithelial na 3D a cikin vitro.
Baya ga hanyarmu ta ƙirƙirar tsarin epithelial na hanji na 3D, akwai hanyoyi da yawa a cikin vitro. Al'adar organoid ta hanji wata dabara ce ta zamani ta injiniyan nama bisa ga noman ƙwayoyin hanji a ƙarƙashin takamaiman yanayin morphogen23,24,25. Duk da haka, amfani da samfuran organoid na 3D don nazarin sufuri ko haɗin gwiwar microbiome sau da yawa yana da ƙalubale saboda lumen na hanji yana cikin organoid kuma, saboda haka, gabatar da abubuwan haske kamar ƙwayoyin cuta ko antigens na waje yana da iyaka. Ana iya inganta samun damar organoid lumens ta amfani da microinjector,26,27 amma wannan hanyar tana da ƙarfi kuma tana buƙatar ƙwarewa ta musamman don yin aiki. Bugu da ƙari, al'adun organoid na gargajiya da aka kiyaye a cikin hydrogel scaffolds a ƙarƙashin yanayi mai tsauri ba sa nuna daidai aikin biomechanics na rayuwa.
Sauran hanyoyin da ƙungiyoyin bincike da dama ke amfani da su suna amfani da su wajen ... Tsarin halittar jiki ba tare da wata matsala ba kuma ba ya haɗa da motsin injina na hanji. Dabaru na bugawa na 3D daga wannan rukuni sun sami damar ƙirƙirar ƙananan bututun hanji tare da hanyoyin halittar jiki ba tare da wata matsala ba. Duk da sarkakiyar ƙirƙirar sassa daban-daban na hanji a cikin bututun, wannan samfurin kuma ba shi da kwararar ruwa mai haske da nakasa ta injiniya. Bugu da ƙari, aikin samfurin na iya iyakancewa, musamman bayan an kammala aikin buga bioprinting, yana damun yanayin gwaji ko hulɗar tantanin halitta zuwa tantanin halitta. Madadin haka, tsarin da muka gabatar yana ba da tsarin halittar hanji na bazata, damuwa mai dacewa da ilimin halittar jiki, biomechanics waɗanda ke kwaikwayon motsi na hanji, samun damar sassan apical da basolateral masu zaman kansu, da sake ƙirƙirar hadaddun yanayin halittu na modularity. Saboda haka, tsarin halittar mu na in vitro 3D na iya samar da wata hanya mai dacewa don shawo kan ƙalubalen hanyoyin da ake da su.
Tsarin aikinmu gaba ɗaya yana mai da hankali ne kan tsarin aikin epithelial na 3D, tare da ƙwayoyin epithelial kawai a cikin al'ada kuma babu wasu nau'ikan ƙwayoyin da ke kewaye kamar ƙwayoyin mesenchymal, ƙwayoyin endothelial, da ƙwayoyin rigakafi. Kamar yadda aka bayyana a baya, tushen tsarin aikinmu shine haifar da tsarin aikin epithelial ta hanyar cire masu hana morphogen da aka fitar a gefen basolateral na matsakaiciyar da aka gabatar. Duk da cewa ƙarfin tsarin aikinmu na hanji-kan-a-chip da hybrid-on-a-chip yana ba mu damar sake ƙirƙirar layin epithelial na 3D mai ban sha'awa, ƙarin rikitarwa na halittu kamar hulɗar epithelial-mesenchymal33,34, ajiyar extracellular Matrix (ECM) 35 kuma, a cikin samfurinmu, fasalulluka na crypt-villus waɗanda ke isar da ma'aunin ƙwayoyin tushe a cikin crypts na basal har yanzu ba a yi la'akari da su ba. Kwayoyin Stromal (misali, fibroblasts) a cikin mesenchyme suna taka muhimmiyar rawa wajen samar da sunadaran ECM da kuma daidaita tsarin aikin hanji a cikin vivo35,37,38. Ƙara ƙwayoyin mesenchymal zuwa samfurinmu ya haɓaka tsarin aikin morphogenetic da haɗewar ƙwayoyin halitta Inganci. Tsarin endothelial (watau, capillaries ko lymphatic) yana taka muhimmiyar rawa wajen daidaita jigilar kwayoyin halitta39 da kuma ɗaukar ƙwayoyin rigakafi40 a cikin yanayin hanji. Bugu da ƙari, abubuwan da ke cikin jijiyoyin jini waɗanda za a iya haɗa su tsakanin samfuran nama sune sharadin lokacin da aka tsara samfuran nama don nuna hulɗar gabobin da yawa. Saboda haka, ƙwayoyin endothelial na iya buƙatar a haɗa su don yin ƙirar fasalulluka na ilimin halittar jiki mafi inganci tare da ƙudurin matakin gabobi. Kwayoyin garkuwar jiki da aka samo daga marasa lafiya suma suna da mahimmanci don nuna martanin rigakafi na asali, gabatar da antigen, haɗin gwiwar rigakafi na asali, da rigakafi na musamman na kyallen takarda a cikin mahallin kwaikwayon cututtukan hanji.
Amfani da guntunan haɗin gwiwa ya fi sauƙi fiye da gut-on-a-chip saboda saitin na'urar ya fi sauƙi kuma amfani da guntun Transwell yana ba da damar haɓaka al'adun epithelium na hanji. Duk da haka, guntun Transwell da ake samu a kasuwa tare da membranes na polyester ba su da laushi kuma ba za su iya kwaikwayon motsin peristaltic ba. Bugu da ƙari, ɓangaren haɗin gwiwa na guntun Transwell da aka sanya a cikin guntun haɗin gwiwa ya kasance a tsaye ba tare da wani matsin lamba a gefen haɗin gwiwa ba. A bayyane yake, halayen da ke tsaye a cikin ɓangaren haɗin gwiwa ba sa ba da damar haɗin gwiwa na dogon lokaci na ƙwayoyin cuta a cikin guntun haɗin gwiwa. Duk da cewa za mu iya haifar da morphogenesis na 3D a cikin guntun Transwell lokacin amfani da guntun haɗin gwiwa, ƙarancin biomechanics masu dacewa da ilimin halittar jiki da kwararar ruwa na apical na iya iyakance yuwuwar dandamalin guntun haɗin gwiwa don aikace-aikacen da ake iya yi.
Ba a tabbatar da cikakken tsarin sake gina tsarin crypt-villus na ɗan adam a cikin al'adun gut-on-a-chip da hybrid-on-a-chip ba. Tunda morphogenesis ya fara ne daga wani Layer na epithelial monolayer, ƙananan gine-gine na 3D ba lallai bane su samar da kamanceceniya da crypts a cikin vivo. Kodayake mun siffanta yawan ƙwayoyin halitta da ke yaɗuwa kusa da yankin crypt basal a cikin epithelium na 3D microengineered, ba a bayyana yankunan crypt da villous a sarari ba. Kodayake manyan tashoshi na sama akan guntu suna haifar da ƙaruwar tsayin epithelium na microengineered, matsakaicin tsayi har yanzu yana iyakance zuwa ~300-400 µm. Zurfin ainihin crypts na hanjin ɗan adam a cikin ƙananan hanji da manyan hanji shine ~135 µm da ~400 µm, bi da bi, kuma tsayin ƙaramin hanji shine ~600 µm41.
Daga mahangar hoto, hoton ƙananan gine-gine na 3D na iya zama iyakance ga hanji a kan guntu, tunda nisan aiki da ake buƙata daga ruwan tabarau na zahiri zuwa layin epithelial yana kan tsari na milimita kaɗan. Don shawo kan wannan matsalar, ana iya buƙatar manufa mai nisa. Bugu da ƙari, yin ƙananan sassa don shirya samfurin daukar hoto yana da ƙalubale saboda yawan sassaucin PDMS. Bugu da ƙari, tunda ƙirar hanji a kan guntu-da-layi ta ƙunshi mannewa na dindindin tsakanin kowane layi, yana da matuƙar wahala a buɗe ko cire saman Layer don bincika tsarin saman Layer na epithelial. Misali, ta amfani da na'urar duba electron microscope (SEM).
Rashin kyawun sinadarin PDMS ya kasance wani abu mai iyakancewa a cikin nazarin da aka yi kan ƙananan ƙwayoyin hydrophobic, tunda PDMS na iya ɗaukar irin waɗannan ƙwayoyin hydrophobic ba tare da takamaiman takamaiman ba. Ana iya la'akari da madadin PDMS tare da wasu kayan polymeric. A madadin haka, ana iya ɗaukar gyaran saman PDMS (misali, shafa kayan lipophilic 42 ko poly(ethylene glycol) 43) don rage shaye-shayen ƙwayoyin hydrophobic.
A ƙarshe, hanyarmu ba ta da wata siffa mai kyau dangane da samar da na'urar tantancewa mai ƙarfi ko kuma dandamalin gwaji mai sauƙin amfani da "girma ɗaya-ya dace da kowa". Tsarin aikin na yanzu yana buƙatar famfon sirinji a kowace na'urar microdevice, wanda ke ɗaukar sarari a cikin incubator na CO2 kuma yana hana gwaje-gwaje masu girma. Wannan iyakancewa za a iya inganta shi sosai ta hanyar haɓaka tsarin al'adu masu ƙirƙira (misali, abubuwan da aka saka a cikin rijiya 24, rijiya 96, ko ramuka masu ramuka 384 waɗanda ke ba da damar ci gaba da cikewa da cire kafofin watsa labarai na basolateral).
Domin haifar da yanayin 3D na epithelium na hanjin ɗan adam a cikin vitro, mun yi amfani da na'urar hanji mai siffar microfluidic chip wacce ke ɗauke da ƙananan tashoshi guda biyu masu layi ɗaya da membrane mai laushi a tsakanin don ƙirƙirar hanyar haɗin lumen-capillary. Haka nan mun nuna amfani da na'urar microfluidic mai tashoshi ɗaya (guntu mai haɗaka) wanda ke samar da ci gaba da kwararar basolateral a ƙarƙashin yadudduka na epithelial masu rarrabuwar polarized da aka girma akan abubuwan da aka saka a Transwell. A cikin dandamali biyu, ana iya nuna yanayin morphogenesis na ƙwayoyin epithelial na hanjin ɗan adam ta hanyar amfani da sarrafa kwararar hanya don cire masu adawa da morphogen daga sashin basolateral. Duk hanyar gwaji (Hoto na 1) ta ƙunshi sassa biyar: (i) ƙirar microcirculation na guntu na hanji ko guntu mai haɗaka da Transwell za a iya sakawa (matakai na 1-5; Akwati na 1), (ii) shirya ƙwayoyin epithelial na hanji (ƙwayoyin Caco-2) ko ƙwayoyin hanji na ɗan adam; akwatuna 2-5), (iii) al'adar ƙwayoyin epithelial na hanji akan kwakwalwan hanji ko kwakwalwan haɗin gwiwa (matakai 6-9), (iv) haifar da morphogenesis na 3D a cikin vitro (mataki na 10) da (v)) don siffanta ƙananan tsarin epithelial na 3D (matakai na 11-24). A ƙarshe, an tsara ƙungiyar kulawa mai dacewa (wanda aka tattauna a ƙasa) don tabbatar da ingancin morphogenesis na in vitro ta hanyar kwatanta morphogenesis na epithelial zuwa iko na sarari, na ɗan lokaci, na yanayi, ko na tsari.
Mun yi amfani da dandamali daban-daban na al'adu guda biyu: gut-on-a-chip tare da tashoshi madaidaiciya ko tashoshi marasa layi, ko kwakwalwan haɗin gwiwa waɗanda ke ɗauke da abubuwan da aka saka na Transwell (TW) a cikin na'urar microfluidic, wanda aka ƙera kamar yadda aka bayyana a cikin Akwati na 1, da kuma mataki na 1 -5. "Na'urar Ƙirƙirar" yana nuna manyan matakai wajen yin guntu ɗaya ko guntu na haɗin gwiwa." Al'adar Kwayoyin Epithelial na Hanjin Ɗan Adam" ya bayyana tushen tantanin halitta (Caco-2 ko ƙwayoyin hanji na ɗan adam) da kuma hanyar al'ada da aka yi amfani da ita a cikin wannan yarjejeniya. "In vitro morphogenesis" yana nuna matakan gabaɗaya inda aka haɓaka ƙwayoyin epithelial na Caco-2 ko organoid da aka samo daga organoid akan guntu na hanji ko akan abubuwan da aka saka na Transwell na guntu na haɗin gwiwa, sannan aka biyo baya da haifar da morphogenesis na 3D da kuma ƙirƙirar tsarin epithelial mai siffantawa. Lambar matakin shirin ko lambar akwatin an nuna ta a ƙasan kowace kibiya. Aikace-aikacen yana ba da misalai na yadda za a iya amfani da yadudduka na epithelial na hanji da aka kafa, misali, a cikin halayyar bambance-bambancen tantanin halitta, nazarin ilimin halittar hanji, kafa tsarin halittu masu masaukin baki-microbiome, da kuma ƙirar cuta. Hotunan immunofluorescence a cikin "Tantanin halitta" Bambancin da ke nuna nuclei, F-actin da MUC2 da aka bayyana a cikin layin epithelial na Caco-2 na 3D wanda aka samar a kan guntun hanji. Siginar MUC2 tana nan a cikin ƙwayoyin goblet da majina da aka fitar daga saman mucosal. Hotunan fluorescent a cikin Gut Physiology suna nuna majina da aka samar ta hanyar yin tabo don sialic acid da ragowar N-acetylglucosamine ta amfani da ƙwayar alkama mai haske agglutinin. Hotunan biyu da suka haɗu a cikin "Host-Microbe Co-Cultures" suna nuna wakilcin haɗin gwiwar microbiome a cikin hanji akan guntu. Bangon hagu yana nuna haɗin gwiwar E. coli yana bayyana furotin mai haske kore (GFP) tare da ƙwayoyin epithelial na Caco-2 microengineered. Bangon dama yana nuna inda GFP E. coli ke haɓaka tare da ƙwayoyin epithelial na Caco-2 na 3D, sannan kuma yana maye gurbin immunofluorescence tare da F-actin (ja) da nuclei (shuɗi). Tsarin cututtuka yana nuna lafiya da zubar hanji a cikin guntuwar kumburi a ƙarƙashin ƙalubalen ilimin halittar jiki tare da antigens na ƙwayoyin cuta (misali, lipopolysaccharide, LPS) da ƙwayoyin rigakafi (misali, PBMC; kore). An haɓaka ƙwayoyin Caco-2 don kafa layin epithelial na 3D. Maƙallin sikelin, 50 µm. Hotuna a layi na ƙasa: "Bambance-bambancen ƙwayoyin halitta" an daidaita shi da izini daga tunani.2. Oxford University Press; An sake bugawa da izini daga Ref.5. NAS; "Mai watsa shirye-shirye-microbe Co-Culture" an daidaita shi da izini daga ref.3. NAS; "Tsarin Cuta" an daidaita shi da izini daga tunani.5. NAS.
An ƙera duka guntu-kan-chip da na haɗaka ta amfani da kwafi na PDMS waɗanda aka cire daga molds na silicon ta hanyar lithography mai laushi1,44 kuma aka tsara su da SU-8. Tsarin ƙananan tashoshi a cikin kowane guntu ana ƙaddara shi ta hanyar la'akari da hydrodynamics kamar matsin lamba na shear da matsin lamba na hydrodynamic1,4,12. Tsarin asali na gut-on-a-chip (Extended Data Hoto na 1a), wanda ya ƙunshi manyan tashoshi guda biyu masu layi ɗaya, ya canza zuwa wani hadadden gut-on-a-chip (Extended Data Hoto na 1b) wanda ya haɗa da ƙananan tashoshi biyu masu lanƙwasa don haifar da ƙaruwar lokacin zama na ruwa, tsarin kwararar da ba ta layi ba, da nakasa mai yawa na ƙwayoyin da aka ƙera (Hoto na 2a-f) 12. Lokacin da ake buƙatar sake ƙirƙirar ƙwayoyin halitta masu rikitarwa na hanji, ana iya zaɓar ƙwayoyin cuta masu rikitarwa na gut-on-a-chip. Mun nuna cewa Gut-Chip mai rikitarwa shima yana haifar da morphogenesis na 3D a cikin lokaci iri ɗaya tare da matakin girma na epithelial iri ɗaya idan aka kwatanta da na asali Gut-Chip, ba tare da la'akari da nau'in ƙwayar halitta da aka haɓaka ba. Saboda haka, don haifar da morphogenesis na 3D, ƙirar hanji mai layi da rikitarwa ana iya musanya su. Kwafi na PDMS da aka warke akan molds na silicon tare da tsarin SU-8 sun ba da siffofi mara kyau bayan rushewa (Hoto na 2a). Don ƙera hanji akan guntu, an haɗa Layer na PDMS na sama da aka shirya a jere zuwa fim ɗin PDMS mai ramuka sannan aka daidaita shi da Layer na PDMS na ƙasa ta hanyar haɗin gwiwa mara canzawa ta amfani da maganin corona (Hoto na 2b-f). Don ƙera kwakwalwan haɗin gwiwa, an haɗa kwafi na PDMS da aka warke zuwa zamewar gilashi don ƙirƙirar na'urorin microfluidic guda ɗaya waɗanda zasu iya ɗaukar abubuwan da aka saka na Transwell (Hoto na 2h da Extended Data Hoto na 2). Ana yin tsarin haɗin gwiwa ta hanyar magance saman kwafi na PDMS da gilashi tare da oxygen plasma ko maganin corona. Bayan an tsaftace na'urar microƙera da aka haɗa da bututun silicone, saitin na'urar ya shirya don yin morphogenesis na 3D na epithelium na hanji (Hoto na 2g).
a, Zane-zanen shirye-shiryen sassan PDMS daga molds silicon masu siffar SU-8. An zuba maganin PDMS da ba a warke ba a kan mold silicon (hagu), an warke a 60 °C (tsakiya) sannan aka rushe shi (dama). An yanke PDMS da aka rushe shi guda-guda kuma an tsaftace shi don ƙarin amfani.b, Hoton mold silicon da aka yi amfani da shi don shirya saman Layer na PDMS.c, Hoton mold silicon da aka yi amfani da shi don ƙera membrane mai ramuka na PDMS.d, Jerin hotunan abubuwan PDMS na sama da ƙasa da na ƙasa da na'urar hanji da aka haɗa a kan guntu.e, Zane-zanen daidaita abubuwan PDMS na sama, membrane, da na ƙasa. Kowane Layer yana da alaƙa da plasma ko maganin corona ba tare da juyawa ba.f, Zane-zanen na'urar hanji da aka ƙera tare da ƙananan tashoshi masu rikitarwa da ɗakunan vacuum.g, Saita gut-on-a-chip don al'adar ƙwayoyin microfluidic. An sanya hanjin da aka ƙera a kan guntu da aka haɗa tare da bututun silicon da sirinji a kan murfin murfin. An sanya na'urar guntu a kan murfin Kayan Petri mai tsawon mm 150 don sarrafawa. Ana amfani da maƙallin don rufe bututun silicone.h, Hotunan gani na ƙirƙirar guntu na haɗin gwiwa da kuma morphogenesis na 3D ta amfani da guntu na haɗin gwiwa. Abubuwan da aka saka na Transwell waɗanda aka shirya daban-daban don haɓaka 2D monolayer na ƙwayoyin epithelial na hanji an saka su cikin guntu na haɗin gwiwa don haifar da morphogenesis na 3D na hanji. Ana tura matsakaicin ta cikin ƙananan tashoshi a ƙarƙashin layin tantanin halitta da aka kafa akan insert ɗin Transwell. Sandar sikelin, 1 cm.h An sake bugawa tare da izini daga tunani.4. Elsevier.
A cikin wannan yarjejeniya, an yi amfani da layin ƙwayoyin Caco-2 da ƙwayoyin hanji a matsayin tushen epithelial (Hoto na 3a). An yi amfani da nau'ikan ƙwayoyin guda biyu daban-daban (Akwati na 2 da Akwati na 5) kuma an yi amfani da su don shuka ƙananan hanyoyin ECM na hanji ko abubuwan da aka saka a cikin Transwell. Lokacin da ƙwayoyin suka haɗu (fiye da kashi 95% na rufewa a cikin kwalba), ana girbe ƙwayoyin Caco-2 da aka noma akai-akai (tsakanin sassa na 10 da 50) a cikin kwalbar T don shirya dakatarwar ƙwayoyin da aka raba ta hanyar ruwan trypsinization (akwati na 2). An girbe ƙwayoyin hanji na ɗan adam daga biopsies na hanji ko yankewar tiyata a cikin ɗakunan Matrigel a cikin faranti 24 don tallafawa yanayin tsarin. Matsakaici mai ɗauke da mahimman morphogens (kamar Wnt, R-spondin, da Noggin) kuma ana ƙara abubuwan girma da aka shirya kamar yadda aka bayyana a cikin Akwati na 3 kowace rana har sai ƙwayoyin organoids sun girma zuwa ~500 µm a diamita. Ana girbe ƙwayoyin organoids da aka girma gaba ɗaya kuma ana raba su cikin Kwayoyin halitta guda ɗaya don shukawa a cikin hanji ko Transwell a kan guntu (Akwati na 5). Kamar yadda muka ruwaito a baya, ana iya bambanta shi bisa ga nau'in cuta na 12,13 (misali ulcerative colitis, cutar Crohn, ciwon daji na hanji, ko mai bayarwa na yau da kullun), wurin rauni (misali, rauni idan aka kwatanta da yankin da ba shi da rauni) da wurin hanji a cikin hanyar (misali, duodenum, jejunum, ileum, cecum, hanji, ko dubura). Muna samar da ingantaccen tsari a cikin Akwati na 5 don haɓaka ƙwayoyin halitta na hanji (coloids) waɗanda yawanci ke buƙatar yawan ƙwayoyin halitta fiye da ƙananan ƙwayoyin halitta na hanji.
a, Tsarin aiki don haifar da morphogenesis na hanji a cikin guntun hanji. Ana amfani da Caco-2 na hanjin ɗan adam da ƙwayoyin hanji a cikin wannan tsari don nuna morphogenesis na 3D. An shuka ƙwayoyin epithelial da aka ware a cikin na'urar da aka shirya ta hanyar gut-on-a-chip (shirin guntu). Da zarar an shuka (an shuka) kuma an haɗa su (an haɗa su) zuwa membrane mai laushi na PDMS a rana ta 0 (D0), ana fara kwararar apical (AP) kuma ana kiyaye ta na tsawon kwanaki 2 na farko (guda, AP, D0-D2). Haka kuma ana fara kwararar basolateral (BL) tare da motsin miƙewa na cyclic (stretch, flow, AP da BL) lokacin da aka samar da cikakken Layer na 2D. Tsarin 3D na hanji ya faru ba zato ba tsammani bayan kwanaki 5 na al'adar microfluidic (morphogenesis, D5). Hotunan bambanci na mataki suna nuna wakilcin yanayin ƙwayoyin Caco-2 a kowane mataki na gwaji ko lokacin lokaci (jadawali na mashaya, 100 µm). Zane-zane huɗu na zane-zane waɗanda ke nuna matakan da suka dace na hanji morphogenesis (saman dama). Kibiyoyi masu lanƙwasa a cikin tsarin suna wakiltar alkiblar kwararar ruwa.b, Hoton SEM yana nuna yanayin saman epithelium na Caco-2 3D (hagu). Tsarin da ke nuna yankin da aka ƙawata (farin akwatin da aka lanƙwasa) yana nuna microvilli da aka sake farfaɗowa akan layin Caco-2 3D (dama).c, Hoton gaba na kwance na Caco-2 3D da aka kafa, claudin (ZO-1, ja) da membranes na gefe mai ci gaba da goga mai lakabin F-actin (kore) da nuclei (shuɗi) Immunofluorescence confocal na ƙwayoyin epithelial akan kwakwalwan hanji. Kibiyoyi da ke nuna tsarin tsakiya suna nuna wurin da aka sanya ido don kowane kallon confocal.d, Lokaci na canje-canje na morphological a cikin organoids da aka haɓaka akan guntu da aka samu ta hanyar microscopy na bambanci na mataki a ranakun 3, 7, 9, 11, da 13. Tsarin (saman dama) yana nuna babban girman hoton da aka bayar.e, DIC photomicrograph na organoid 3D epithelium da aka kafa a cikin hanji a kan yanki da aka ɗauka a rana ta 7.f, Hotunan immunofluorescence masu rufewa waɗanda ke nuna alamun ƙwayoyin tushe (LGR5; magenta), ƙwayoyin goblet (MUC2; kore), F-actin (launin toka) da nuclei (cyan) da aka girma a kan guntun hanji na tsawon kwanaki 3, bi da bi (Hagu) da organoids na kwanaki 13 (tsakiya) akan layin epithelial. Duba kuma Ƙarin Bayani Hoto na 3, wanda ke nuna siginar LGR5 ba tare da siginar MUC2 ba. Hotunan haske suna nuna tsarin epithelial (dama) na epithelium organoid 3D da aka kafa a cikin hanji akan guntu ta hanyar yin fenti da membrane na plasma da fenti na CellMask (dama) a rana ta 13 ta al'ada. Madaurin sikelin shine 50 μm sai dai idan an faɗi akasin haka.b An sake bugawa tare da izini daga tunani.2. Oxford University Press; c An daidaita shi tare da izini daga tunani.2. Oxford University Press; e da f an daidaita shi tare da izini ta tunani.12 A ƙarƙashin Creative Commons Lasisi CC BY 4.0.
A cikin hanji a kan guntu, ya zama dole a gyara saman hydrophobic na membrane mai ramuka na PDMS don nasarar rufewar ECM. A cikin wannan yarjejeniya, muna amfani da hanyoyi guda biyu daban-daban don gyara hydrophobic na membranes na PDMS. Don haɓaka ƙwayoyin Caco-2, kunna saman ta hanyar maganin UV/ozone kawai ya isa ya rage hydrophobic na saman PDMS, shafa ECM da haɗa ƙwayoyin Caco-2 zuwa membrane na PDMS. Duk da haka, al'adar microfluidic na organoid epithelium yana buƙatar aikin saman da aka dogara da sinadarai don cimma ingantaccen ajiyar sunadaran ECM ta hanyar amfani da polyethyleneimine (PEI) da glutaraldehyde a jere zuwa ƙananan tashoshi na PDMS. Bayan gyaran saman, an ajiye sunadaran ECM don rufe saman PDMS mai aiki sannan a shigar da su cikin epithelium na organoid da aka ware. Bayan an haɗa ƙwayoyin, al'adar microfluidic tantanin halitta tana farawa ta hanyar tura turare kawai zuwa babban tashar microchannel har sai ƙwayoyin sun samar da cikakken monolayer, yayin da ƙananan tashar microchannel ke kula da yanayi mai tsauri. Wannan hanyar da aka inganta don kunna saman da murfin ECM yana ba da damar haɗawa da organoid. epithelium don haifar da morphogenesis na 3D akan saman PDMS.
Al'adun Transwell kuma suna buƙatar rufin ECM kafin a shuka ƙwayoyin halitta; duk da haka, al'adun Transwell ba sa buƙatar matakai masu rikitarwa kafin a fara aiki don kunna saman abubuwan da aka saka a ramuka. Don haɓaka ƙwayoyin Caco-2 akan abubuwan da aka saka a Transwell, rufin ECM akan abubuwan da aka saka a ramuka yana hanzarta haɗewar ƙwayoyin Caco-2 da aka raba (
Ana iya yin nazarin siffofin siffofi na yadudduka na epithelial 3D na microengineered ta hanyar amfani da hanyoyi daban-daban na daukar hoto, gami da microscopy na bambanci na lokaci, microscopy na bambanci na tsangwama (DIC), SEM, ko immunofluorescence confocal microscopy (Figures 3 da 4). Ana iya yin sauƙin yin hoton mataki ko DIC a kowane lokaci yayin al'ada don sa ido kan siffa da fitowar yadudduka na epithelial na 3D. Saboda bayyananniyar gani na PDMS da fina-finan polyester, duka dandamali na gut-on-a-chip da na hybrid chip na iya samar da hoton a ainihin lokaci ba tare da buƙatar rabawa ko wargaza na'urar ba. Lokacin yin hoton immunofluorescence (Figures 1, 3c, f da 4b, c), yawanci ana gyara ƙwayoyin halitta da paraformaldehyde 4% (wt/vol) (PFA), sannan Triton X-100 da 2% (wt/vol) ) bovine serum albumin (BSA), a cikin tsari. Dangane da nau'in tantanin halitta, gyara daban-daban, permeabilizers, da toshewa Ana iya amfani da magunguna. Ana amfani da manyan ƙwayoyin rigakafi da ke kai hari ga ƙwayoyin halitta ko alamun yanki don haskaka ƙwayoyin da ba su da motsi a wuri a kan guntu, sannan sai a biyo baya da ƙwayoyin rigakafi na biyu tare da rini mai hana tabo wanda ke kai hari ga ko dai tsakiya (misali, 4′, 6-diamidino-2-phenylene) indole, DAPI) ko F-actin (misali, phalloidin mai suna fluorescent). Hakanan ana iya yin hoton kai tsaye na tushen fluorescent a wuri don gano samar da majina (Hoto na 1, "Bambance-bambancen ƙwayoyin halitta" da "Tsarin Gut"), mamaye ƙwayoyin cuta bazuwar (Hoto na 1, "Mai masaukin baki-ƙwayoyin cuta"), ɗaukar ƙwayoyin rigakafi (Hoto na 1, 'Tsarin Cututtuka') ko kuma yanayin siffar epithelial na 3D (Hoto na 3c, f da 4b, c). Lokacin da ake gyara hanjin da ke kan guntu don raba saman Layer daga ƙananan microchannel Layer, kamar yadda aka bayyana a cikin ref. Kamar yadda aka nuna a Hoto na 2, yanayin epithelial na 3D da kuma microvilli akan Ana iya ganin iyakar goga ta hanyar SEM (Hoto na 3b). Ana iya tantance bayyanar alamun bambance-bambance ta hanyar yin jerin PCR5 ko RNA na tantanin halitta guda ɗaya. A wannan yanayin, ana girbe yadudduka 3D na ƙwayoyin epithelial da aka girma a cikin guntun hanji ko guntun haɗin gwiwa ta hanyar trypsinization sannan a yi amfani da su don nazarin kwayoyin halitta ko kwayoyin halitta.
a, Tsarin aiki don haifar da morphogenesis na hanji a cikin guntu na haɗin gwiwa. Ana amfani da Caco-2 da ƙwayoyin hanji a cikin wannan tsari don nuna morphogenesis na 3D a cikin dandamalin guntu na haɗin gwiwa. An shuka ƙwayoyin epithelial da aka raba a cikin abubuwan da aka shirya na Transwell (TW prepare; duba hoton da ke ƙasa). Da zarar an shuka (tsaba) kuma an haɗa su da membranes na polyester a cikin abubuwan da aka saka na Transwell, an haɓaka dukkan ƙwayoyin a ƙarƙashin yanayi mai tsauri (TW culture). Bayan kwana 7, an haɗa wani abun Transwell guda ɗaya wanda ke ɗauke da Layer ɗaya na 2D na ƙwayoyin epithelial a cikin guntu na haɗin gwiwa don gabatar da kwararar basolateral (Flow, BL), wanda a ƙarshe ya haifar da samar da Layer ɗin epithelial na 3D (morphogenesis). Micrographs na bambanci na mataki suna nuna fasalulluka na ƙwayoyin epithelial na gabobin ɗan adam waɗanda aka samo daga mai bayarwa na al'ada (layin C103) suna hawa a kowane mataki na gwaji ko lokaci. Tsarin da ke cikin saman yadudduka yana nuna tsarin gwaji don kowane mataki.b, Chips masu haɗin gwiwa (tsaba na hagu) na iya haifar da morphogenesis na 3D na ƙwayoyin epithelial na organoid tare da confocal na sama-ƙasa An ɗauki hotunan microscopy a wurare daban-daban na Z (na sama, na tsakiya, da na ƙasa; duba layukan zane na dama da masu digo-digo masu dacewa). sun nuna halayen siffa bayyanannu. F-actin (cyan), nucleus (launin toka).c, Micrographs masu siffar fluorescence confocal (kallon kusurwa 3D) na ƙwayoyin epithelial da aka samo daga organoid waɗanda aka haɓaka a cikin Transwell mai tsauri (TW; saka a cikin akwatin farin da aka yi wa laƙabi) idan aka kwatanta da guntu na haɗin gwiwa (mafi girman cikakken hoto) idan aka kwatanta da siffa ta 2D idan aka kwatanta da 3D, bi da bi. Biyu daga cikin ra'ayoyi masu tsayi na 2D (sashi a kusurwar dama ta sama; "XZ") suma suna nuna fasalulluka na 2D da 3D. Sandunan sikelin, 100 µm.c An sake bugawa tare da izini daga nassoshi.4. Elsevier.
Ana iya shirya abubuwan sarrafawa ta hanyar haɓaka ƙwayoyin halitta iri ɗaya (Caco-2 ko ƙwayoyin epithelial na hanji) zuwa layuka masu girma biyu a ƙarƙashin yanayin al'ada mai tsauri. Abin lura shi ne, ƙarancin abinci mai gina jiki na iya faruwa saboda ƙarancin ƙarfin girma na ƙananan tashoshi (watau ~4 µL a cikin babban tashar akan ƙirar gut-chip na asali). Saboda haka, ana iya kwatanta yanayin epithelial kafin da bayan amfani da kwararar basolateral.
Ya kamata a yi aikin lithography mai laushi a cikin ɗaki mai tsabta. Ga kowane layi akan guntu (sama da ƙasan yadudduka da membranes) da guntu masu haɗaka, an yi amfani da fuskokin hoto daban-daban kuma an ƙera su akan wafers na silicon daban-daban saboda tsayin ƙananan tashoshi sun bambanta. Tsayin da aka nufa na manyan ƙananan hanyoyin hanji a kan guntu shine 500 µm da 200 µm, bi da bi. Tsayin da aka nufa na tashar guntu masu haɗaka shine 200 µm.
Sanya wafer silicon mai inci 3 a cikin kwano mai acetone. Juya farantin a hankali na daƙiƙa 30, sannan a busar da wafer ɗin a iska. A mayar da wafer ɗin zuwa farantin da IPA, sannan a juya farantin na daƙiƙa 30 don ya tsaftace.
Ana iya amfani da maganin piranha (cakuda hydrogen peroxide da sinadarin sulfuric acid mai ƙarfi, 1:3 (vol/vol)) don rage yawan ragowar sinadarai daga saman silicon wafer.
Maganin Piranha yana da matuƙar lalata kuma yana haifar da zafi. Akwai buƙatar ƙarin matakan tsaro. Don zubar da shara, a bar maganin ya huce ya kuma koma cikin akwati mai tsabta da busasshe. Yi amfani da kwantena na biyu kuma a sanya wa kwantena na shara alama yadda ya kamata. Da fatan za a bi jagororin aminci na wurin don ƙarin cikakkun bayanai.
A bar wafers ɗin su jiƙa ta hanyar sanya su a kan faranti mai zafi na 200°C na minti 10. Bayan bushewar ruwan, an girgiza wafer ɗin sau biyar a cikin iska don ya huce.
Zuba kimanin gram 10 na mai hana haske a cikin SU-8 2100 a tsakiyar wafer ɗin silicon da aka tsaftace. Yi amfani da tweezers don yada mai hana haske a kan wafer ɗin. Lokaci-lokaci sanya wafer ɗin a kan farantin zafi mai zafi na 65°C don hana haske a cikin hasken da kuma sauƙin yaɗuwa. Kada a sanya wafer ɗin kai tsaye a kan farantin zafi.
An rarraba SU-8 daidai gwargwado a kan wafer ta hanyar amfani da murfin juyawa. Shirya juyawa mai shigowa na SU-8 na daƙiƙa 5-10 don yaɗuwa a rpm 500 a hanzarin rpm 100/s. Saita babban juyawa don tsarin kauri µm 200 a rpm 1,500, ko cimma kauri µm 250 (yin tsayi µm 500 ga saman Layer na hanji akan guntu; duba "Matakai masu mahimmanci" a ƙasa) wanda aka saita a hanzarin rpm 300/s daƙiƙa 30 a rpm 1,200.
Ana iya daidaita babban saurin juyawa bisa ga kauri da aka yi niyya na tsarin SU-8 akan wafer ɗin silicon.
Domin ƙera tsarin SU-8 mai tsawon µm 500 don saman Layer na hanji a kan guntu, an maimaita murfin juyawa da matakan gasa mai laushi na wannan Akwatin (matakai na 7 da 8) a jere (duba mataki na 9) don samar da layuka biyu na Layer mai kauri 250 µm na SU-8, wanda za'a iya haɗa shi da haɗuwa ta hanyar fallasa UV a mataki na 12 na wannan akwatin, wanda ke sa Layer mai tsayi 500 µm.
Gasa wafers ɗin da aka lulluɓe da SU-8 a hankali ta hanyar sanya wafers ɗin a kan faranti mai zafi a zafin digiri 65 na Celsius na tsawon minti 5, sannan a canza wurin zuwa zafin digiri 95 na Celsius sannan a saka a cikin injin daskarewa na ƙarin minti 40.
Domin cimma tsayin tsarin SU-8 mai girman μm 500 a cikin babban tashar microchannel, maimaita matakai na 7 da 8 don samar da layuka biyu na SU-8 mai kauri μm 250.
Ta amfani da UV Mask Aligner, yi gwajin fitila bisa ga umarnin masana'anta don ƙididdige lokacin fallasa na wafer. (lokacin fallasa, ms) = (kashi na fallasa, mJ/cm2)/(ƙarfin fitila, mW/cm2).
Bayan ƙayyade lokacin fallasa, sanya abin rufe fuska a kan mariƙin abin rufe fuska na mai daidaita abin rufe fuska na UV sannan a sanya abin rufe fuska a kan wafer mai rufi na SU-8.
Sanya saman da aka buga na abin rufe fuska kai tsaye a kan gefen SU-8 mai rufi na wafer silicon don rage yaduwar UV.
A fallasa wafer mai rufi da abin rufe fuska na SU-8 a tsaye zuwa 260 mJ/cm2 na hasken UV don lokacin da aka ƙayyade (duba mataki na 10 na wannan akwatin).
Bayan fallasa UV, an gasa wafers ɗin silicon mai rufi da SU-8 a zafin 65°C na minti 5 da kuma zafin 95°C na minti 15 a kan kowane farantin zafi don ƙera alamu tare da tsayin μm 200. A tsawaita lokacin bayan gasawa a 95°C zuwa minti 30 don ƙera alamu tare da tsayin µm 500.
Ana zuba mai haɓaka kayan a cikin kwano na gilashi, sannan a sanya wafer ɗin da aka gasa a cikin kwano. Girman mai haɓaka kayan SU-8 na iya bambanta dangane da girman farantin gilashin. Tabbatar da amfani da isasshen mai haɓaka kayan SU-8 don cire SU-8 mara fallasa gaba ɗaya. Misali, lokacin amfani da kwano na gilashi mai diamita 150 mm tare da ƙarfin L 1, yi amfani da ~ 300 ml na mai haɓaka kayan SU-8. Haɓaka kayan a cikin na tsawon mintuna 25 tare da juyawa mai laushi lokaci-lokaci.
Kurkura mold ɗin da aka samar da shi da kimanin milimita 10 na sabon mai haɓaka, sannan a shafa IPA ta hanyar fesa maganin ta amfani da pipette.
Sanya wafer ɗin a cikin injin tsabtace plasma sannan a fallasa shi ga iskar oxygen a cikin plasma (iskar iskar gas, matsin lamba na 1 × 10−5 Torr, ƙarfin 125 W) na minti 1.5.
Sanya wafer a cikin injin cirewa mai cirewa mai cirewa tare da gilashin zamiya a ciki. Ana iya sanya wafers da zamiya a gefe da gefe. Idan an raba na'urar cirewa mai cirewa zuwa yadudduka da yawa ta faranti, sanya zamiya a cikin ɗakin ƙasa da wafers a cikin ɗakin sama. A sauke 100 μL na maganin trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane a kan zamiya a gilashi sannan a shafa injin cirewa don silanization.
A narke kwalbar ƙwayoyin Caco-2 daskararre a cikin ruwan wanka mai zafin digiri 37, sannan a mayar da ƙwayoyin da suka narke zuwa kwalbar T75 mai ɗauke da matsakaicin Caco-2 mai zafin digiri 37 na Celsius wanda aka riga aka dumama shi.
Domin wucewar ƙwayoyin Caco-2 a ~90% na haɗuwa, da farko Caco-2 mai ɗumi, PBS, da 0.25% trypsin/1 mM EDTA a cikin ruwan wanka mai zafin digiri 37 na Celsius.
A yi amfani da injin tsotsar ruwa ta hanyar tsotsar ruwa. A wanke ƙwayoyin halitta sau biyu da 5 mL na PBS mai dumi ta hanyar maimaita tsotsar ruwa da kuma ƙara sabbin PBS.
Lokacin Saƙo: Yuli-16-2022
