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Kusinthika kwa matumbo a anthu kumakhazikitsa mawonekedwe a crypt-villus a 3D epithelial microarchitecture ndi malo okonzera. Kapangidwe kapadera aka kamafunika kuti matumbo azikhala ndi homeostasis poteteza niche ya stem cell mu basal crypt kuchokera ku ma antigen a microbial akunja ndi ma metabolites awo. Kuphatikiza apo, intestine villi ndi secreting mucus zimakhala ndi maselo a epithelial osiyanitsa bwino ntchito ndi chotchinga choteteza pamwamba pa mucosal yamatumbo. Chifukwa chake, kupanganso mapangidwe a 3D epithelial ndikofunikira kwambiri popanga ma in vitro gut models. Chofunika kwambiri, organic mimetic gut-on-a-chip imatha kuyambitsa 3D morphogenesis yokha ya intestine epithelium yokhala ndi ntchito zabwino za thupi ndi biomechanics. Pano, tikupereka njira yoberekeranso kuti ipangitse matumbo kukhala olimba m'matumbo pa microfluidic chip komanso mu Transwell embedded hybrid chip. Timafotokoza njira zatsatanetsatane zopangira chipangizo, kukulitsa maselo a Caco-2 kapena intestine organoid epithelial m'malo ochiritsira komanso pa microfluidic platform, kuyambitsa 3D morphogenesis, ndi Kufotokozera za epithelia ya 3D yokhazikika pogwiritsa ntchito njira zambiri zojambulira. Ndondomekoyi imapangitsa kuti ma microarchitecture a m'matumbo azitha kugwira ntchito bwino powongolera kuyenda kwa madzi a m'mimba kwa masiku 5. Njira yathu ya in vitro morphogenesis imagwiritsa ntchito kupsinjika kwa shear komwe kumakhudzana ndi thupi komanso kuyenda kwa makina ndipo sikufuna ukadaulo wovuta wa maselo kapena kusintha, zomwe zingapangitse kuti zigwire bwino ntchito kuposa njira zina zomwe zilipo. Tikuganiza kuti njira yathu yomwe tapereka ikhoza kukhala ndi tanthauzo lalikulu kwa gulu lofufuza zamankhwala, kupereka njira yokonzanso zigawo za 3D za m'matumbo mu vitro kuti zigwiritsidwe ntchito pa zamankhwala, zachipatala, komanso zamankhwala.
Kufufuza kwasonyeza kuti maselo a Caco-2 a m'mimba omwe amakula m'matumbo omwe amakula mu zida za m'mimba za 1,2,3,4,5 kapena bilayer microfluidic6,7 amatha kusinthika mwadzidzidzi mu vitro popanda kumvetsetsa bwino momwe zimakhalira. Mu kafukufuku wathu waposachedwa, tapeza kuti kuchotsa ma morphogen antagonists omwe amatulutsidwa m'zida zokulira ndikofunikira komanso kokwanira kuyambitsa 3D epithelial morphogenesis mu vitro, zomwe zawonetsedwa ndi Caco-2 ndi ma organoids am'mimba omwe amachokera kwa odwala. Maselo a epithelial adatsimikiziridwa. Mu kafukufukuyu, tinayang'ana kwambiri pa kupanga ndi kugawa kwa maselo a Wnt antagonist wamphamvu, Dickkopf-1 (DKK-1), mu gut-on-a-chip ndi zida zosinthidwa za microfluidic zomwe zili ndi Transwell inserts, zotchedwa "Hybrid Chip". Tikuwonetsa kuti kuwonjezera kwa Wnt antagonists akunja (monga DKK-1, Wnt repressor 1, protein 1 yokhudzana ndi frizzled, kapena Soggy-1) ku on-chip gut kumaletsa morphogenesis kapena kusokoneza 3D epithelial layer yokonzedwa kale, zomwe zikusonyeza kuti kupsinjika kwa antagonist panthawi yolima ndiko komwe kumayambitsa morphogenesis ya m'mimba mu vitro. Chifukwa chake, njira yothandiza yopezera morphogenesis yolimba pa epithelial interface ndikuchotsa kapena kusunga pang'ono milingo ya Wnt antagonists mu basolateral compartment mwa kutsuka mwachangu (monga, mu gut-on-a-chip kapena hybrid-on-a-chip platforms) kapena kufalikira. Basolateral media (monga, kuchokera ku Transwell inserts mu basolateral reservoirs yayikulu mu zitsime).
Mu ndondomekoyi, tikupereka njira yofotokozera mwatsatanetsatane yopangira ma microdevices a m'mimba ndi Transwell-insertable hybrid chips (masitepe 1-5) kuti tipange maselo a epithelial a m'matumbo pa polydimethylsiloxane (PDMS)-based porous membranes (masitepe 6A, 7A, 8, 9) kapena polyester membranes ya Transwell inserts (masitepe 6B, 7B, 8, 9) ndi kuyambitsa 3D morphogenesis in vitro (sitepe 10). Tinazindikiranso mawonekedwe a maselo ndi mamolekyu omwe akuwonetsa histogenesis yeniyeni ya minofu ndi kusiyana kwa maselo odalira mzere pogwiritsa ntchito njira zambiri zojambula (masitepe 11-24). Timayambitsa morphogenesis pogwiritsa ntchito maselo a epithelial a m'matumbo a anthu, monga Caco-2 kapena matumbo organelles, m'njira ziwiri zophunzitsira ndi tsatanetsatane waukadaulo kuphatikiza kusintha kwa pamwamba pa membranes ya porous, kupanga ma monolayers a 2D, ndi matumbo a biochemical ndi Kuberekanso kwa biomechanical microenvironment.in vitro.Kuyambitsa 3D morphogenesis kuchokera ku 2D epithelial Pomaliza, tikupereka chithunzithunzi cha kufunika kwa gawo la epithelial la 3D lomwe lingagwiritsidwe ntchito kutsanzira kukula kwa epithelial komwe kumadalira morphogen, kufalikira kwa tizilombo toyambitsa matenda m'thupi kwa nthawi yayitali, matenda opatsirana, kuvulala kotupa, kusagwira ntchito bwino kwa epithelial, ndi mankhwala opangidwa ndi probiotic.
Ndondomeko yathu ingakhale yothandiza kwa asayansi ambiri muzoyambira (monga biology ya m'matumbo, biology ya maselo oyambira, ndi biology ya chitukuko) ndi kafukufuku wogwiritsidwa ntchito (monga kuyesa mankhwala asanachire, kupanga zitsanzo za matenda, uinjiniya wa minofu, ndi gastroenterology) zotsatira zazikulu. Chifukwa cha kuberekana komanso kulimba kwa ndondomeko yathu yoyambitsa 3D morphogenesis ya epithelium ya m'matumbo mu vitro, tikuganiza kuti njira yathu yaukadaulo ikhoza kugawidwa kwa omvera omwe akuphunzira momwe maselo amagwirira ntchito panthawi yakukula kwa m'matumbo, kubwezeretsedwa kapena homeostasis. Kuphatikiza apo, ndondomeko yathu ndi yothandiza pofufuza matenda pansi pa zinthu zosiyanasiyana zopatsirana monga Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 kapena Vibrio cholerae. Anthu odziwa za matenda ndi matenda ake ndi othandiza. Kugwiritsa ntchito njira ya on-chip gut microphysiology kungathandize kuti pakhale kukulitsa kwa nthawi yayitali komanso kuwunikanso chitetezo cha m'thupi, mayankho a chitetezo chamthupi komanso kukonza kuvulala komwe kumakhudzana ndi matenda m'mimba (GI) tract 11. Matenda ena a GI okhudzana ndi leaky gut syndrome, matenda a celiac, matenda a Crohn, ulcerative colitis, pouchitis, kapena irritable bowel syndrome amatha kuyerekezeredwa pamene zigawo za 3D intestine epithelial zakonzedwa pogwiritsa ntchito zigawo za 3D intestine epithelial za wodwalayo, matenda awa akuphatikizapo villous atrophy, crypt shortening, mucosal damage, kapena impaired epithelial barrier. Biopsy kapena stem cell-derived intestine organoids12,13. Kuti awonetse bwino zovuta za malo omwe matendawa ali, owerenga angaganizire kuwonjezera mitundu ya maselo okhudzana ndi matenda, monga maselo a m'magazi a wodwala (PBMCs), ku mitundu yokhala ndi 3D intestinal villus-crypt microarchitectures. maselo a chitetezo chamthupi, 5.
Popeza kapangidwe ka 3D epithelial kamatha kukonzedwa ndikuwonetsedwa popanda njira yogawa magawo, owonera omwe akugwira ntchito pa spatial transcriptomics ndi high-resolution kapena super-resolution imaging angakonde kudziwa momwe timaonera spatiotemporal dynamics ya majini ndi mapuloteni pa epithelial niches. Okonda ukadaulo. Kuyankha ku microbial kapena immune stimuli. Kuphatikiza apo, longitudinal host-microbiome crosstalk 10, 14 yomwe imagwirizanitsa intestine homeostasis ikhoza kukhazikitsidwa mu 3D intestine mucosal layer pophatikiza mitundu yosiyanasiyana ya microbial, communities kapena fecal microbiota, makamaka mu gut-on-a-chip. mu pulatifomu. Njira imeneyi ndi yokongola kwambiri kwa omvera omwe amaphunzira za mucosal immunology, gastroenterology, human microbiome, culturomics ndi clinical microbiology omwe akufuna kukulitsa ma microbiota a m'mimba omwe sanakulitsidwe kale mu labotale. Ngati protocol yathu ya in vitro morphogenesis ingasinthidwe kuti ikhale ndi mitundu yokulirakulira, monga multiwell inserts mu 24, 96 kapena 384 well plates zomwe zimadzazanso basolateral compartments nthawi zonse, protocol ikhozanso kufalitsidwa kwa iwo omwe akupanga mankhwala, biomedical kapena high-throughput screening kapena ma veritical platforms a makampani azakudya. Monga umboni wa mfundo, posachedwapa tawonetsa kuthekera kwa multiplex high-throughput morphogenesis system yomwe ingakulitsidwe ku 24-well plate format. Kuphatikiza apo, zinthu zambiri za organ-on-a-chip zagulitsidwa16,17,18. Chifukwa chake, kutsimikizika kwa njira yathu ya in vitro morphogenesis kumatha kufulumizitsidwa ndikuvomerezedwa ndi ma laboratories ambiri ofufuza, mafakitale kapena boma ndi mabungwe olamulira kuti amvetsetse kusintha kwa maselo a in vitro gut morphogenesis pamlingo wa transcriptomic kuti ayesere mankhwala kapena Mankhwala Ochiza Matenda a M'mimba Kuyamwa ndi kunyamula mankhwala omwe akufuna kuchiritsidwa kunayesedwa pogwiritsa ntchito njira zosinthira matumbo za 3D kapena kugwiritsa ntchito njira zamakono kapena zamalonda zochizira ziwalo kuti ziwone momwe njira yoberekera m'matumbo imagwirira ntchito.
Pali mitundu yochepa ya zitsanzo zoyesera zogwirizana ndi anthu zomwe zagwiritsidwa ntchito pophunzira za kapangidwe ka epithelial m'matumbo, makamaka chifukwa chosowa njira zomwe zingagwiritsidwe ntchito kuti zipangitse kapangidwe ka 3D mu vitro. Ndipotu, zambiri zomwe zadziwika pano zokhudza kapangidwe ka m'matumbo zimachokera ku maphunziro a nyama (monga zebrafish20, mbewa21 kapena nkhuku22). Komabe, ndi zogwira ntchito komanso zodula, zitha kukhala zokayikitsa pamakhalidwe abwino, ndipo chofunika kwambiri, sizimasankha bwino njira zopitira patsogolo za anthu. Zitsanzozi ndizochepa kwambiri pakutha kwawo kuyesedwa m'njira zambiri zokulira. Chifukwa chake, njira yathu yokonzanso kapangidwe ka minofu ya 3D mu vitro imagwira ntchito bwino kuposa zitsanzo za nyama za vivo komanso zitsanzo zina zachikhalidwe za 2D cell culture. Monga tafotokozera kale, kugwiritsa ntchito kapangidwe ka epithelial ka 3D kunatilola kuti tiwone momwe maselo osiyanasiyana amakhalira mu crypt-villus axis poyankha zinthu zosiyanasiyana za mucosal kapena immune stimuli. Zigawo za epithelial za 3D zitha kupereka malo ophunzirira momwe maselo a tizilombo amapikisana kuti apange malo ozungulira komanso kusintha kwa chilengedwe poyankha zinthu zomwe zimalandira (monga, mkati motsutsana ndi zigawo zakunja za mucus, kutulutsa kwa IgA ndi ma peptide oletsa tizilombo toyambitsa matenda). Kuphatikiza apo, mawonekedwe a epithelial a 3D angatithandize kumvetsetsa momwe microbiota ya m'matumbo imapangira magulu ake ndikupanga ma metabolites a tizilombo toyambitsa matenda (monga, ma acid amafuta afupiafupi) omwe amapanga kapangidwe ka maselo ndi ma niches a maselo oyambira m'ma crypts a basal. Zinthu izi zitha kuwonetsedwa pokhapokha ngati zigawo za epithelial za 3D zakhazikitsidwa mu vitro.
Kuwonjezera pa njira yathu yopangira mapangidwe a epithelial a m'mimba a 3D, pali njira zingapo za mu vitro. Chikhalidwe cha organoid m'matumbo ndi njira yamakono yopangira minofu yozikidwa pa kulima maselo oyambira m'matumbo pansi pa mikhalidwe yeniyeni ya morphogen23,24,25. Komabe, kugwiritsa ntchito mitundu ya organoid ya 3D poyesa mayendedwe kapena kubzala kwa ma microbiome nthawi zambiri kumakhala kovuta chifukwa lumen ya m'matumbo imatsekedwa mkati mwa organoid ndipo, chifukwa chake, kuyambitsa zigawo za luminal monga maselo a microbial kapena ma antigen akunja ndi ochepa. Kupeza ma organoid lumens kumatha kukonzedwa pogwiritsa ntchito microinjector,26,27 koma njira iyi ndi yolowerera komanso yogwira ntchito kwambiri ndipo imafuna chidziwitso chapadera kuti ichite. Kuphatikiza apo, chikhalidwe cha organoid chachikhalidwe chomwe chimasungidwa mu hydrogel scaffolds pansi pa mikhalidwe yosasunthika sichikuwonetsa molondola biomechanics yogwira ntchito mu vivo.
Njira zina zomwe magulu angapo ofufuza amagwiritsa ntchito amagwiritsa ntchito ma scaffolds a 3D hydrogel okonzedwa kale kuti atsanzire kapangidwe ka epithelial ya m'matumbo mwa kukulitsa maselo a m'mimba a anthu omwe ali pamtunda wa gel. Pangani ma scaffolds a hydrogel pogwiritsa ntchito nkhungu zosindikizidwa mu 3D, micro-milled, kapena lithographically. Njirayi ikuwonetsa dongosolo lodzikonzera la maselo a epithelial odzipatula mu vitro okhala ndi ma morphogen gradients ofunikira pathupi, kukhazikitsa kapangidwe ka epithelial kapamwamba komanso stroma-epithelial crosstalk pophatikiza maselo a stromal mu scaffold. Komabe, mtundu wa ma scaffolds okonzedwa kale ungalepheretse kuwonetsedwa kwa njira yokha ya morphogenetic yokha. Mitundu iyi siyiperekanso kuyenda kwa luminal kapena interstitial, yopanda kupsinjika kwamadzimadzi komwe maselo am'mimba amafunika kuti adutse morphogenesis ndikupeza ntchito ya thupi. Kafukufuku wina waposachedwapa wagwiritsa ntchito ma scaffolds a hydrogel mu nsanja ya microfluidic ndi mapangidwe a epithelial a m'matumbo pogwiritsa ntchito njira za laser-etching. Ma organoids a m'matumbo a mbewa amatsatira mapangidwe ojambulidwa kuti apange mapangidwe a tubular a m'matumbo, ndipo kuyenda kwa madzi mkati mwa luminal kumatha Kubwerezabwereza pogwiritsa ntchito gawo la microfluidics. Komabe, chitsanzochi sichikuwonetsa njira zodziyimira zokha komanso sichikuphatikizapo kayendedwe ka mechanobiological m'matumbo. Njira zosindikizira za 3D kuchokera ku gulu lomwelo zinatha kupanga machubu ang'onoang'ono a m'matumbo okhala ndi njira zodziyimira zokha. Ngakhale kuti mapangidwe a magawo osiyanasiyana a m'matumbo mkati mwa chubucho anali ovuta, chitsanzochi sichimayendetsa madzi a luminal ndi kusintha kwa makina. Kuphatikiza apo, magwiridwe antchito a chitsanzochi akhoza kukhala ochepa, makamaka pambuyo poti njira yosindikizira yatha, zomwe zimasokoneza mikhalidwe yoyesera kapena kuyanjana kwa maselo ndi maselo. M'malo mwake, njira yathu yomwe tapereka imapereka mawonekedwe a m'matumbo odziyimira okha, kupsinjika kwa shear kogwirizana ndi thupi, biomechanics zomwe zimatsanzira kuyenda kwa m'matumbo, kupezeka kwa magawo odziyimira pawokha a apical ndi basolateral, komanso kupanganso malo ovuta achilengedwe a modularity. Chifukwa chake, njira yathu ya in vitro 3D morphogenesis ingapereke njira yowonjezera yothana ndi zovuta za njira zomwe zilipo.
Ndondomeko yathu imayang'ana kwambiri pa 3D epithelial morphogenesis, yokhala ndi maselo a epithelial okha omwe ali m'chilengedwe ndipo palibe mitundu ina ya maselo ozungulira monga maselo a mesenchymal, maselo a endothelial, ndi maselo oteteza thupi. Monga tafotokozera kale, maziko a ndondomeko yathu ndi kuyambitsa morphogenesis ya epithelial mwa kuchotsa zoletsa za morphogen zomwe zimatulutsidwa mbali ya basolateral ya medium yomwe yayambitsidwa. Ngakhale kuti modularity yolimba ya gut-on-a-chip yathu ndi hybrid-on-a-chip imatithandiza kupanganso undulating 3D epithelial layer, zovuta zina zamoyo monga epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35, ndipo, mu chitsanzo chathu, mawonekedwe a crypt-villus omwe amapereka niches za stem cell mu basal crypts akadali kuganiziranso. Maselo a Stromal (monga, fibroblasts) mu mesenchyme amachita gawo lofunika kwambiri pakupanga mapuloteni a ECM ndikuwongolera morphogenesis ya m'mimba mu vivo35,37,38. Kuwonjezera kwa maselo a mesenchymal Ku chitsanzo chathu kunawonjezera njira ya morphogenetic ndi kugwira ntchito bwino kwa maselo. Gawo la endothelial (monga, capillaries kapena lymphatics) limagwira ntchito yofunika kwambiri pakulamulira kayendedwe ka mamolekyulu39 ndi kusonkhanitsa maselo a chitetezo chamthupi40 m'malo ozungulira matumbo. Kuphatikiza apo, zigawo za mitsempha yamagazi zomwe zingalumikizidwe pakati pa zitsanzo za minofu ndizofunikira kwambiri pamene zitsanzo za minofu zapangidwa kuti ziwonetse kuyanjana kwa ziwalo zambiri. Chifukwa chake, maselo a endothelial angafunike kuphatikizidwa kuti awonetse mawonekedwe olondola a thupi ndi kutsimikiza kwa mulingo wa ziwalo. Maselo a chitetezo chamthupi ochokera kwa odwala nawonso ndi ofunikira powonetsa mayankho a chitetezo chamthupi, kuwonekera kwa antigen, kulankhulana kwa chitetezo chamthupi komwe kumachitika mwachibadwa, komanso chitetezo chamthupi chapadera pankhani yotsanzira matenda am'mimba.
Kugwiritsa ntchito ma hybrid chips ndikosavuta kuposa gut-on-a-chip chifukwa makina ogwiritsira ntchito ndi osavuta ndipo kugwiritsa ntchito ma Transwell inserts kumalola kuti epithelium ya m'mimba ikule bwino. Komabe, ma Transwell inserts omwe amapezeka pamsika okhala ndi ma polyester membranes samakhala otanuka ndipo sangafanane ndi kayendedwe ka peristaltic. Kuphatikiza apo, gawo la apical la Transwell insert lomwe lili mu hybrid chip linakhala losasunthika popanda kupsinjika kwa shear kumbali ya apical. Mwachionekere, mawonekedwe osasinthika m'gawo la apical nthawi zambiri samalola kuti mabakiteriya azikhala nthawi yayitali m'ma hybrid chips. Ngakhale titha kuyambitsa 3D morphogenesis mu Transwell inserts pogwiritsa ntchito ma hybrid chips, kusowa kwa biomechanics yofunikira pa physiologically ndi apical fluid flow kungachepetse kuthekera kwa ma hybrid chip platforms omwe angagwiritsidwe ntchito.
Kukonzanso kwathunthu kwa axis ya crypt-villus ya munthu m'matumbo a m'mimba ndi m'matenda a hybrid-on-a-chip sikunakhazikitsidwe mokwanira. Popeza morphogenesis imayamba kuchokera ku monolayer ya epithelial, ma microarchitectures a 3D samapereka kufanana kwa morphological ndi ma crypts mu vivo. Ngakhale tidazindikira kuchuluka kwa maselo omwe akuchulukirachulukira pafupi ndi malo a basal crypt mu epithelium ya microengineered 3D, madera a crypt ndi villous sanapatulidwe bwino. Ngakhale njira zapamwamba zapamwamba pa chip zimapangitsa kuti epithelium ya microengineered ikhale yayitali, kutalika kwakukulu kumakhalabe kochepera ~300–400 µm. Kuzama kwenikweni kwa ma crypts a m'matumbo a munthu m'matumbo ang'onoang'ono ndi akuluakulu ndi ~135 µm ndi ~400 µm, motsatana, ndipo kutalika kwa villi ya m'matumbo ang'onoang'ono ndi ~600 µm41.
Kuchokera pamalingaliro ojambulira, kujambula zithunzi za 3D microarchitectures mu situ kungakhale kokha m'matumbo pa chip, popeza mtunda wofunikira wogwirira ntchito kuchokera pa lens yolunjika kupita ku epithelial layer ndi pafupifupi mamilimita angapo. Kuti vutoli lithe, cholinga chakutali chingafunike. Kuphatikiza apo, kupanga magawo oonda kuti mukonzekere zitsanzo za kujambula ndikovuta chifukwa cha kulimba kwa PDMS. Kuphatikiza apo, popeza kupanga zinthu zazing'ono m'matumbo pa chip kumaphatikizapo kumamatira kosatha pakati pa gawo lililonse, zimakhala zovuta kwambiri kutsegula kapena kuchotsa gawo lapamwamba kuti muwone kapangidwe ka pamwamba pa epithelial layer. Mwachitsanzo, pogwiritsa ntchito scanning electron microscope (SEM).
Kusagwira bwino ntchito kwa PDMS kwakhala chinthu cholepheretsa maphunziro ozikidwa pa microfluidic okhudza mamolekyu ang'onoang'ono osagwirizana ndi madzi, popeza PDMS imatha kunyamula mamolekyu osagwirizana ndi madzi oterewa. Njira zina zosinthira PDMS zitha kuganiziridwa ndi zinthu zina za polymeric. Kapenanso, kusintha kwa pamwamba pa PDMS (monga, kuphimba ndi zinthu za lipophilic 42 kapena poly(ethylene glycol) 43) kungaganizidwe kuti kuchepetsa kulowerera kwa mamolekyu osagwirizana ndi madzi.
Pomaliza, njira yathu sinafotokozedwe bwino pankhani yopereka njira yowunikira bwino kwambiri kapena nsanja yoyesera "yofanana ndi yonse" yosavuta kugwiritsa ntchito. Njira yomwe ilipo pano imafuna pampu ya syringe pa chipangizo chilichonse, chomwe chimatenga malo mu chosungira cha CO2 ndikuletsa kuyesa kwakukulu. Kuchepetsa kumeneku kumatha kusinthidwa kwambiri ndi kukula kwa mitundu yatsopano ya chikhalidwe (monga, 24-well, 96-well, kapena 384-well porous inserts zomwe zimalola kubwezeretsanso kosalekeza ndikuchotsa basolateral media).
Pofuna kuyambitsa kusintha kwa 3D kwa epithelium ya m'mimba mwa munthu mu vitro, tinagwiritsa ntchito chipangizo cha m'mimba cha microfluidic chip chokhala ndi njira ziwiri zofanana ndi nembanemba yosalala pakati kuti tipange mawonekedwe a lumen-capillary. Tikuwonetsanso kugwiritsa ntchito chipangizo cha microfluidic cha single-channel (hybrid chip) chomwe chimapereka kuyenda kosalekeza kwa basolateral pansi pa zigawo za epithelial zozungulira zomwe zimamera pa Transwell inserts. M'mapulatifomu onse awiri, kusintha kwa maselo osiyanasiyana a m'mimba mwa munthu kumatha kuwonetsedwa pogwiritsa ntchito njira yowongolera kayendedwe ka kayendedwe ka madzi kuti achotse otsutsana ndi morphogen kuchokera ku basolateral compartment. Njira yonse yoyesera (Chithunzi 1) ili ndi magawo asanu: (i) microfabrication ya gut chip kapena Transwell insertable hybrid chip (masitepe 1-5; Bokosi 1), (ii) kukonzekera maselo a m'mimba (maselo a Caco-2) kapena ma organoids a m'mimba mwa munthu; mabokosi 2-5), (iii) kukulitsa maselo a epithelial m'matumbo pa matumba a m'mimba kapena matumba osakanizidwa (masitepe 6-9), (iv) kuyambitsa morphogenesis ya 3D mu vitro (sitepe 10) ndi (v) ) kuti afotokoze kapangidwe ka 3D epithelial (masitepe 11-24). Pomaliza, gulu loyenera lolamulira (lomwe lakambidwa pansipa) linapangidwa kuti litsimikizire kugwira ntchito kwa morphogenesis mu vitro poyerekeza morphogenesis ya epithelial ndi zowongolera za malo, nthawi, zokhazikika, kapena njira.
Tinagwiritsa ntchito nsanja ziwiri zosiyana za chikhalidwe: gut-on-a-chip yokhala ndi njira zowongoka kapena njira zosakanikirana zosalunjika, kapena ma hybrid chips okhala ndi Transwell (TW) inserts mu chipangizo cha microfluidic, chopangidwa monga tafotokozera mu Bokosi 1, ndi gawo 1 -5. "Kupanga Chipangizo" chikuwonetsa njira zazikulu zopangira chip imodzi kapena hybrid chip. "Chikhalidwe cha Maselo a Epithelial a M'mimba mwa Munthu" chikufotokoza gwero la maselo (Caco-2 kapena ma organoids a m'matumbo a munthu) ndi njira yolerera yomwe imagwiritsidwa ntchito mu protocol iyi. "In vitro morphogenesis" ikuwonetsa njira zonse zomwe Caco-2 kapena maselo a epithelial ochokera ku organoid amaleredwa pa matumbo kapena pa Transwell inserts ya hybrid chip, kutsatiridwa ndi kuyambitsa 3D morphogenesis ndi kupanga kapangidwe ka epithelial kodziwika bwino. Nambala ya sitepe ya pulogalamuyo kapena nambala ya bokosi ikuwonetsedwa pansi pa muvi uliwonse. Ntchitoyi imapereka zitsanzo za momwe zigawo za epithelial za m'matumbo zokhazikika zingagwiritsidwe ntchito, mwachitsanzo, pakusiyanitsa maselo, maphunziro a physiology ya m'matumbo, kukhazikitsa zachilengedwe za host-microbiome, ndi matenda. Kujambula zithunzi. Zithunzi za immunofluorescence mu "Cell Differentiation" zomwe zikuwonetsa ma nuclei, F-actin ndi MUC2 zomwe zimawonetsedwa mu 3D Caco-2 epithelial layer yopangidwa pa gut chip. MUC2 signaling ilipo m'maselo a goblet ndi ma mucus otulutsidwa kuchokera ku mucosal surfaces. Zithunzi za fluorescent mu Gut Physiology zikuwonetsa ma mucus opangidwa ndi utoto wa sialic acid ndi N-acetylglucosamine residues pogwiritsa ntchito fluorescent wheat germ agglutinin. Zithunzi ziwiri zomwe zimagwirizana mu "Host-Microbe Co-Cultures" zikuwonetsa ma co-cultures oyimira host-microbiome m'matumbo pa chip. Gulu lakumanzere likuwonetsa co-culture ya E. coli yomwe ikuwonetsa green fluorescent protein (GFP) yokhala ndi microengineered 3D Caco-2 epithelial cells. Gulu lakumanja likuwonetsa komwe GFP E. coli imakulitsidwa ndi ma cell a 3D Caco-2 epithelial, kutsatiridwa ndi immunofluorescence staining ndi F-actin (wofiira) ndi ma nuclei (buluu). Kujambula zithunzi za matenda kukuwonetsa matumbo abwino komanso otuluka m'matumbo. Zidutswa za kutupa zomwe zili pansi pa vuto la thupi ndi ma antigen a bakiteriya (monga lipopolysaccharide, LPS) ndi maselo oteteza thupi (monga PBMC; wobiriwira). Ma cell a Caco-2 adakulitsidwa kuti apange gawo la epithelial la 3D. Scale bar, 50 µm. Zithunzi pamzere wapansi: "Kusiyana kwa maselo" komwe kwasinthidwa ndi chilolezo kuchokera ku reference.2. Oxford University Press; Yopangidwanso ndi chilolezo kuchokera ku Ref.5. NAS; "Host-Microbe Co-Culture" komwe kwasinthidwa ndi chilolezo kuchokera ku ref.3. NAS; "Disease Modeling" komwe kwasinthidwa ndi chilolezo kuchokera ku reference.5. NAS.
Ma chips onse a gut-on-chip ndi hybrid adapangidwa pogwiritsa ntchito ma replicas a PDMS omwe adachotsedwa ku silicon molds ndi soft lithography1,44 ndipo adapangidwa ndi SU-8. Kapangidwe ka ma microchannels mu chip chilichonse kamatsimikiziridwa poganizira za hydrodynamics monga shear stress ndi hydrodynamic pressure1,4,12. Kapangidwe koyambirira ka gut-on-a-chip (Extended Data Fig. 1a), komwe kanali ndi ma microchannels awiri ofanana olunjika, kasanduka gut-on-a-chip yovuta (Extended Data Fig. 1b) yomwe imaphatikizapo ma microchannels awiri opindika kuti apangitse nthawi yowonjezera yamadzimadzi kukhalamo, njira zoyendera zosalunjika, komanso kusintha kwa ma cell ambiri okulira (Chithunzi 2a–f) 12. Pamene biomechanics yovuta kwambiri ya m'mimba ikufunika kupangidwanso, ma gut-on-a-chips ovuta amatha kusankhidwa. Tawonetsa kuti Gut-Chip yolumikizidwa imalimbikitsanso kwambiri 3D morphogenesis munthawi yofanana ndi kukula kofanana kwa epithelial poyerekeza ndi yoyambirira. Gut-Chip, mosasamala kanthu za mtundu wa maselo obzalidwa. Chifukwa chake, kuti apangitse mawonekedwe a 3D, mapangidwe a m'mimba okhala ndi mzere komanso ovuta amatha kusinthana. Ma replica a PDMS ochiritsidwa pa silicon molds okhala ndi mawonekedwe a SU-8 amapereka mawonekedwe oipa pambuyo pochotsa (Chithunzi 2a). Kuti apange matumbo pa chip, gawo lapamwamba la PDMS lokonzedwa linalumikizidwa motsatizana ku filimu ya PDMS yokhala ndi mabowo kenako n’kugwirizana ndi gawo lotsika la PDMS mwa kulumikiza kosasinthika pogwiritsa ntchito chothandizira korona (Chithunzi 2b–f). Kuti apange ma hybrid chips, ma replica a PDMS ochiritsidwa adalumikizidwa ku magalasi kuti apange zida za microfluidic za single-channel zomwe zitha kukhala ndi Transwell inserts (Chithunzi 2h ndi Extended Data Fig. 2). Njira yolumikizira imachitika pochiza pamwamba pa replica ya PDMS ndi galasi ndi oxygen plasma kapena chithandizo cha korona. Pambuyo poyeretsa chipangizo cha microfabricated chomwe chili pa chubu cha silicone, kukhazikitsa chipangizocho kunali kokonzeka kuchita 3D morphogenesis ya epithelium ya m'mimba (Chithunzi 2g).
a, Chithunzi chojambulidwa cha kukonzekera kwa zigawo za PDMS kuchokera ku zinyalala za silicon zopangidwa ndi SU-8. Yankho la PDMS losachiritsidwa linathiridwa pa chinyalala cha silicon (kumanzere), lokonzedwa pa 60 °C (pakati) ndikuchotsedwa (kumanja). PDMS yochotsedwayo inadulidwa mzidutswa ndikutsukidwa kuti igwiritsidwe ntchito mobwerezabwereza.b, Chithunzi cha chinyalala cha silicon chomwe chinagwiritsidwa ntchito kukonzekera gawo lapamwamba la PDMS.c, Chithunzi cha chinyalala cha silicon chomwe chinagwiritsidwa ntchito popanga nembanemba ya PDMS yokhala ndi porous membrane.d, Zithunzi zingapo za zigawo za PDMS zapamwamba ndi zapansi ndi chipangizo cham'mimba chosonkhanitsidwa pa chip.e., Chithunzi chojambulidwa cha kulinganiza kwa zigawo za PDMS zapamwamba, nembanemba, ndi zapansi. Gawo lililonse limalumikizidwa mosasinthika ndi chithandizo cha plasma kapena corona.f, Chithunzi cha chipangizo chopangidwa cha gut-on-a-chip chokhala ndi njira zazing'ono zolumikizirana ndi zipinda zotsukira.g, Kukhazikitsa gut-on-a-chip kuti maselo a microfluidic akhazikitsidwe. Matumbo opangidwa pa chipu chosonkhanitsidwa ndi chubu cha silicone ndi syringe adayikidwa pa coverlip. Chipangizo cha chipu chinayikidwa pa chivindikiro cha mbale ya Petri ya 150 mm yokonzedwa. Chomangiracho chimagwiritsidwa ntchito kutseka chubu cha silicone.h, Zithunzi zowoneka bwino za kupanga ma chip osakanizidwa ndi 3D morphogenesis pogwiritsa ntchito ma chip osakanizidwa. Ma Transwell inserts okonzedwa paokha kuti azitha kukulitsa Ma monolayer a 2D a maselo a epithelial am'mimba adayikidwa mu hybrid chip kuti apangitse 3D morphogenesis yam'mimba. Cholumikiziracho chimalowetsedwa kudzera m'njira zazing'ono pansi pa gawo la selo lomwe limakhazikitsidwa pa Transwell insert. Scale bar, 1 cm.h Yosindikizidwanso ndi chilolezo kuchokera ku reference.4. Elsevier.
Mu ndondomekoyi, mzere wa maselo a Caco-2 ndi ma organoids a m'mimba adagwiritsidwa ntchito ngati magwero a epithelial (Chithunzi 3a). Mitundu yonse iwiri ya maselo idalimidwa payokha (Bokosi 2 ndi Bokosi 5) ndipo idagwiritsidwa ntchito kubzala mbewu za microchannels zophimbidwa ndi ECM za on-chip gut kapena Transwell inserts. Pamene maselo ali pamodzi (>95% m'mabotolo), maselo a Caco-2 omwe amalimidwa nthawi zonse (pakati pa magawo 10 ndi 50) mu T-flasks amakololedwa kuti akonze ma cell suspensions olekanitsidwa ndi trypsinization fluid (bokosi 2). Ma organoids a m'matumbo a anthu ochokera ku matumbo a biopsies kapena opaleshoni adalimidwa mu Matrigel scaffold domes m'mabolo 24 kuti athandizire chilengedwe. Ma morphogens ofunikira apakati (monga Wnt, R-spondin, ndi Noggin) ndi zinthu zokulira zomwe zakonzedwa monga tafotokozera mu Bokosi 3 zidawonjezeredwa tsiku lililonse mpaka ma organoids atakula kufika pa ~500 µm m'mimba mwake. Ma organoids okhwima mokwanira amakololedwa ndipo Amagawanika kukhala maselo amodzi kuti amere m'matumbo kapena Transwell inserts pa chip (Bokosi 5). Monga tanenera kale, amatha kusiyanitsidwa malinga ndi mtundu wa matenda 12,13 (monga ulcerative colitis, matenda a Crohn, khansa ya m'matumbo, kapena wopereka wabwinobwino), malo otupa (monga malo otupa poyerekeza ndi malo osavulala) ndi malo am'mimba m'njira yodulira (monga duodenum, jejunum, ileum, cecum, colon, kapena rectum). Timapereka njira yabwino kwambiri mu Bokosi 5 yopangira ma organoids a m'matumbo (coloids) omwe nthawi zambiri amafunikira kuchuluka kwa morphogens kuposa ma organoids ang'onoang'ono am'mimba.
a, Kayendedwe ka ntchito yoyambitsa kusintha kwa matumbo mu chipu cha m'mimba. Caco-2 intestine epithelium ndi matumbo organelles amagwiritsidwa ntchito mu ndondomekoyi kusonyeza kusintha kwa 3D. Maselo a epithelial olekanitsidwa adabzalidwa mu chipangizo chokonzedwa cha m'mimba (kukonzekera chipu). Maselo akabzalidwa (kubzalidwa) ndi kulumikizidwa (kulumikizidwa) ku nembanemba ya PDMS yoboola pa tsiku la 0 (D0), kuyenda kwa apical (AP) kumayambitsidwa ndikusungidwa kwa masiku awiri oyamba (kuyenda, AP, D0-D2). Kuyenda kwa basolateral (BL) kumayambitsidwanso limodzi ndi mayendedwe otambasula (kutambasula, kuyenda, AP ndi BL) pamene monolayer yonse ya 2D imapangidwa. Kusintha kwa 3D m'matumbo kunachitika mwadzidzidzi patatha masiku 5 a microfluidic culture (morphogenesis, D5). Zithunzi zosiyana za phase zikuwonetsa mawonekedwe oyimira a maselo a Caco-2 pa sitepe iliyonse yoyesera kapena nthawi (bar graph, 100 µm). Zithunzi zinayi zosonyeza ma cascades ofanana za kapangidwe ka m'matumbo (pamwamba kumanja). Mivi yodulidwa mu chithunzicho ikuyimira njira ya madzi oyenda.b, chithunzi cha SEM chosonyeza pamwamba pa epithelium ya 3D Caco-2 yokhazikika (kumanzere). Chithunzi chowunikira malo okulitsidwa (bokosi loyera lodulidwa) chikuwonetsa microvilli yobwezeretsedwanso pa 3D Caco-2 layer (kumanja).c, Mawonekedwe olunjika a Caco-2 3D yokhazikika, claudin (ZO-1, wofiira) ndi nembanemba yopingasa ya burashi yolembedwa F-actin (wobiriwira) ndi nuclei (buluu) Kuwonetsa kwa Immunofluorescence confocal kwa maselo a epithelial pa matumbo. Mivi yolozera ku chithunzi chapakati imasonyeza malo a focal plane pa confocal view iliyonse.d, Nthawi ya kusintha kwa morphological mu organoids yomwe yakula pa chip yomwe yapezeka ndi phase contrast microscopy pa masiku 3, 7, 9, 11, ndi 13. Chithunzi chowonjezera (pamwamba kumanja) chikuwonetsa kukula kwakukulu kwa chithunzicho.e, DIC photomicrograph ya organoid 3D epithelium yokhazikika m'matumbo pa chidutswa chojambulidwa pa tsiku la 7.f, Zithunzi zophimba za immunofluorescence zomwe zikuwonetsa zizindikiro za maselo oyambira (LGR5; magenta), maselo a goblet (MUC2; wobiriwira), F-actin (imvi) ndi nuclei (cyan) zomwe zimakula pa tchipisi ta m'mimba kwa masiku atatu, motsatana (Kumanzere) ndi organoids ya masiku 13 (yapakati) pa epithelial layer. Onaninso Extended Data Figure 3, yomwe ikuwonetsa chizindikiro cha LGR5 popanda chizindikiro cha MUC2. Zithunzi za fluorescence zomwe zikuwonetsa kapangidwe ka epithelial (kumanja) ka 3D organoid epithelium yomwe idakhazikitsidwa m'matumbo pa chip popaka utoto wa plasma membrane ndi CellMask dye (kumanja) pa tsiku la 13 la culture. Scale bar ndi 50 μm pokhapokha ngati tanena mwanjira ina.b Yosindikizidwanso ndi chilolezo chochokera ku reference.2. Oxford University Press; c Yosinthidwa ndi chilolezo chochokera ku Reference.2. Oxford University Press; e ndi f yosinthidwa ndi chilolezo chochokera ku reference.12 Pansi pa Creative Commons License CC BY 4.0.
M'matumbo pa chip, ndikofunikira kusintha pamwamba pa hydrophobic ya nembanemba ya PDMS kuti ECM ipambane. Mu protocol iyi, timagwiritsa ntchito njira ziwiri zosiyana kuti tisinthe hydrophobic ya nembanemba ya PDMS. Pakukula maselo a Caco-2, kuyambitsa pamwamba pogwiritsa ntchito UV/ozone kokha kunali kokwanira kuchepetsa hydrophobic ya pamwamba pa PDMS, kuphimba ECM ndikulumikiza maselo a Caco-2 ku nembanemba ya PDMS. Komabe, chikhalidwe cha microfluidic cha organoid epithelium chimafuna ntchito yochokera ku mankhwala kuti ikwaniritse bwino kuyika mapuloteni a ECM mwa kugwiritsa ntchito polyethyleneimine (PEI) ndi glutaraldehyde ku ma microchannels a PDMS. Pambuyo pa kusintha kwa pamwamba, mapuloteni a ECM adayikidwa kuti aphimbe pamwamba pa PDMS yogwira ntchito kenako nkulowetsedwa mu epithelium ya organoid yodzipatula. Maselo atalumikizidwa, chitukuko cha microfluidic cell chimayamba potulutsa madzi okha mu microchannel yapamwamba mpaka maselo atapanga monolayer yonse, pomwe microchannel yotsika imasunga mikhalidwe yosasunthika. Njira yabwinoyi yoyendetsera pamwamba ndi ECM coating imalola kuti organoid iphatikizidwe bwino. epithelium kuti ipangitse 3D morphogenesis pamwamba pa PDMS.
Kubzala kwa Transwell kumafunikanso kuphimba kwa ECM musanayambe kubzala; komabe, kubzala kwa Transwell sikufunikira njira zovuta zochizira kuti ziyambe kugwira ntchito pamwamba pa ma inserto okhala ndi mabowo. Pakukula kwa maselo a Caco-2 pa ma inserto a Transwell, kuphimba kwa ECM pa ma inserto okhala ndi mabowo kumathandizira kuti maselo a Caco-2 olekanitsidwa azigwira ntchito (
Mawonekedwe a mawonekedwe a zigawo za 3D epithelial zopangidwa ndi microengineered zitha kusanthulidwa pogwiritsa ntchito njira zosiyanasiyana zojambulira, kuphatikiza phase contrast microscopy, differential interference contrast (DIC) microscopy, SEM, kapena immunofluorescence confocal microscopy (Zithunzi 3 ndi 4). Phase contrast kapena DIC imaging zitha kuchitika mosavuta nthawi iliyonse panthawi yolima kuti ziwunikire mawonekedwe ndi kutuluka kwa zigawo za 3D epithelial. Chifukwa cha kuwala kwa PDMS ndi mafilimu a polyester, nsanja zonse za gut-on-a-chip ndi hybrid chip zimatha kupereka kujambula kwa nthawi yeniyeni popanda kufunikira kugawa kapena kusokoneza chipangizocho. Pochita kujambula kwa immunofluorescence (Zithunzi 1, 3c, f ndi 4b, c), maselo nthawi zambiri amakhala okhazikika ndi 4% (wt/vol) paraformaldehyde (PFA), kutsatiridwa ndi Triton X-100 ndi 2% (wt/vol)) bovine serum albumin (BSA), motsatira. Kutengera mtundu wa selo, ma fixatives osiyanasiyana, Ma permeabilizer, ndi zinthu zoletsa zitha kugwiritsidwa ntchito. Ma antibodies oyambira omwe amalimbana ndi mzere wa maselo kapena zizindikiro za dera amagwiritsidwa ntchito kuwonetsa maselo omwe sakuyenda bwino pa chip, kutsatiridwa ndi ma antibodies ena pamodzi ndi utoto wotsutsana womwe umalimbana ndi nucleus (monga 4′,6-diamidino-2-phenylene) indole, DAPI) kapena F-actin (monga phalloidin wolembedwa ndi fluorescently). Kujambula zithunzi zamoyo zochokera ku fluorescence kumathanso kuchitika pa chitu kuti azindikire kupanga kwa mucus (Chithunzi 1, “Kusiyanitsa kwa maselo” ndi “physiology ya m'matumbo”), kukhazikika kwa maselo a microbial mwachisawawa (Chithunzi 1, “Host-microbe co-culture”), kusonkhanitsa maselo a chitetezo chamthupi (Chithunzi 1, ‘Disease Modeling’) kapena mawonekedwe a 3D epithelial morphology (Chithunzi 3c,f ndi 4b,c). Mukasintha matumbo pa chip kuti mulekanitse layer yapamwamba ndi layer yotsika ya microchannel, monga tafotokozera mu ref. Monga tawonetsera mu Chithunzi 2, 3D epithelial Kapangidwe ka thupi komanso microvilli yomwe ili pamalire a burashi ya apical ikhoza kuwonetsedwa ndi SEM (Chithunzi 3b). Kuwonetsera kwa zizindikiro zosiyanitsa kumatha kuyesedwa pochita kuchuluka kwa PCR5 kapena sequencing ya RNA ya selo limodzi. Pankhaniyi, zigawo za 3D za maselo a epithelial omwe amakula mu ma chips a m'mimba kapena ma chips osakanizidwa zimakololedwa ndi trypsinization kenako zimagwiritsidwa ntchito pofufuza ma molecular kapena majini.
a, Kayendedwe ka ntchito yoyambitsa kusintha kwa matumbo mu chip chosakanizidwa. Caco-2 ndi ma organoids a m'matumbo amagwiritsidwa ntchito mu ndondomekoyi kuwonetsa kusintha kwa 3D mu nsanja ya chip chosakanizidwa. Maselo a epithelial olekanitsidwa adabzalidwa mu Transwell inserts zokonzedwa (TW prep; onani chithunzi pansipa). Maselo akabzalidwa (kubzalidwa) ndikulumikizidwa ku nembanemba ya polyester mu Transwell inserts, maselo onse adakulitsidwa pansi pa mikhalidwe yosasunthika (TW culture). Patatha masiku 7, Transwell insert imodzi yokhala ndi monolayer ya 2D ya maselo a epithelial idalumikizidwa mu chip chosakanizidwa kuti ipange kuyenda kwa basolateral (Flow, BL), zomwe pamapeto pake zidapangitsa kuti pakhale gawo la 3D epithelial (morphogenesis). Ma micrographs osiyanitsa magawo omwe akuwonetsa mawonekedwe a maselo a epithelial a ziwalo za anthu ochokera ku donor wamba (C103 line) akukwera m'matumbo pa sitepe iliyonse yoyesera kapena nthawi. Ma schematics omwe ali m'magawo apamwamba akuwonetsa kasinthidwe koyesera pa sitepe iliyonse.b, Ma hybrid chips (left schematic) amatha kutsogolera ku kusintha kwa 3D kwa epithelial ya organoid maselo okhala ndi mawonedwe a pamwamba-pansi a confocal microscopy otengedwa m'malo osiyanasiyana a Z (pamwamba, pakati, ndi pansi; onani mizere yolunjika kumanja ndi yofanana ndi madontho). adawonetsa mawonekedwe omveka bwino a mawonekedwe. F-actin (cyan), nucleus (imvi).c, Ma micrograph a confocal a Fluorescence (mawonekedwe a 3D angled) a maselo a epithelial ochokera ku organoid omwe adakulitsidwa mu static Transwell (TW; mkati mwa bokosi loyera lokhala ndi mipata) motsutsana ndi hybrid chip (chithunzi chachikulu kwambiri) poyerekeza mawonekedwe a 2D motsutsana ndi 3D, motsatana. Mawonedwe awiri a 2D vertical cross-cut (mkati mwa ngodya yakumanja yakumtunda; "XZ") akuwonetsanso mawonekedwe a 2D ndi 3D. Scale bar, 100 µm.c Yosindikizidwanso ndi chilolezo kuchokera ku reference.4. Elsevier.
Zowongolera zitha kukonzedwa mwa kukulitsa maselo omwewo (maselo a Caco-2 kapena a m'matumbo a organoid epithelial) kukhala ma monolayer awiri-dimensional pansi pa mikhalidwe yachikhalidwe yokhazikika. Chodziwika bwino ndichakuti, kuchepa kwa michere kumatha kuchitika chifukwa cha mphamvu yochepa ya microchannels (monga ~4 µL mu njira yapamwamba pa kapangidwe koyambirira ka gut-chip). Chifukwa chake, mawonekedwe a epithelial asanayambe komanso atagwiritsa ntchito basolateral flow akhoza kuyerekezeredwanso.
Njira yofewa yojambulira mawu iyenera kuchitikira m'chipinda choyera. Pa gawo lililonse pa chip (zigawo zapamwamba ndi zapansi ndi nembanemba) ndi ma chips osakanizidwa, ma photomasks osiyanasiyana adagwiritsidwa ntchito ndikupangidwa pa ma wafer osiyana a silicon chifukwa kutalika kwa ma microchannels kunali kosiyana. Kutalika kwa ma microchannels apamwamba ndi apansi a m'mimba pa chip ndi 500 µm ndi 200 µm, motsatana. Kutalika kwa channel channel ya hybrid chip ndi 200 µm.
Ikani wafer wa silicon wa mainchesi atatu mu mbale yokhala ndi acetone. Pukutani mbaleyo pang'onopang'ono kwa masekondi 30, kenako muumitse waferyo ndi mpweya. Tumizani waferyo ku mbale yokhala ndi IPA, kenako zungulizani mbaleyo kwa masekondi 30 kuti muyeretse.
Njira yothetsera piranha (chisakanizo cha hydrogen peroxide ndi sulfuric acid yokhazikika, 1:3 (vol/vol)) ingagwiritsidwe ntchito mwanjira ina kuti ichotse kwambiri zotsalira za organic pamwamba pa silicon wafer.
Mankhwala a Piranha ndi owononga kwambiri ndipo amapangitsa kutentha. Njira zina zodzitetezera ndizofunikira. Pofuna kutaya zinyalala, lolani kuti mankhwalawa azizire ndikusamutsira ku chidebe choyera komanso chouma. Gwiritsani ntchito zidebe zina ndikulemba zilembo zoyenera pa zidebe zotayira zinyalala. Chonde tsatirani malangizo achitetezo a malowa kuti mudziwe zambiri.
Sungunulani ma wafers mwa kuwayika pa mbale yotentha ya 200 °C kwa mphindi 10. Pambuyo pochotsa madzi m'thupi, waferyo inagwedezeka kasanu mumlengalenga kuti izizire.
Thirani ~10 g ya photoresist SU-8 2100 pakati pa wafer yotsukidwa ya silicon. Gwiritsani ntchito ma tweezers kuti mufalikire photoresist mofanana pa wafer. Nthawi zina ikani wafer pa mbale yotentha ya 65°C kuti photoresist isamamatire komanso kuti isafalikire mosavuta. Musayike wafer mwachindunji pa mbale yotentha.
SU-8 inagawidwa mofanana pa wafer pogwiritsa ntchito chophimba cha spin. Konzani kuzungulira komwe kukubwera kwa SU-8 kwa masekondi 5-10 kuti ifalikire pa 500 rpm pa liwiro la 100 rpm/s. Khazikitsani kuzungulira kwakukulu kwa makulidwe a 200 µm pa 1,500 rpm, kapena pangani makulidwe a 250 µm (kupanga kutalika kwa 500 µm kwa gawo lapamwamba la m'mimba pa chip; onani "Masitepe Ofunika" pansipa) okhazikika pa liwiro la 300 rpm/s masekondi 30 pa 1,200 rpm.
Liwiro lalikulu la kuzungulira lingasinthidwe malinga ndi makulidwe a SU-8 pa silicon wafer.
Kuti apange mapatani a SU-8 a kutalika kwa 500 µm pa gawo lapamwamba la matumbo pa chip, chophimba chozungulira ndi masitepe ophika ofewa a Bokosi ili (masitepe 7 ndi 8) adabwerezedwa motsatizana (onani gawo 9) kuti apange zigawo ziwiri za 250 µm. Gawo lokhuthala la SU-8, lomwe lingathe kuyikidwa ndikuphatikizidwa ndi kuwala kwa UV mu gawo 12 la bokosi ili, ndikupanga gawo lokwera la 500 µm.
Phikani ma wafers okhala ndi SU-8 mofewa powayika mosamala ma waferswo pa mbale yotentha pa 65 °C kwa mphindi 5, kenako sinthani kutentha kufika pa 95 °C ndikuyikamo kwa mphindi zina 40.
Kuti mukwaniritse kutalika kwa 500 μm kwa chitsanzo cha SU-8 mu microchannel yapamwamba, bwerezani masitepe 7 ndi 8 kuti mupange zigawo ziwiri za SU-8 zokhuthala za 250 μm.
Pogwiritsa ntchito UV Mask Aligner, yesani nyali motsatira malangizo a wopanga kuti muwerengere nthawi yowonekera pa wafer. (nthawi yowonekera, ms) = (mlingo wowonekera, mJ/cm2)/(mphamvu ya nyali, mW/cm2).
Mukatha kudziwa nthawi yowonekera, ikani chigoba cha zithunzi pa chogwirira chigoba cha aligner ya chigoba cha UV ndikuyika chigoba cha zithunzi pa wafer yokutidwa ndi SU-8.
Ikani pamwamba pa chigoba chosindikizidwa cha photomask mwachindunji kumbali yophimbidwa ndi SU-8 ya silicon wafer kuti muchepetse kufalikira kwa UV.
Ikani chivundikiro cha SU-8 chophimbidwa ndi chigoba cha photomask molunjika ku 260 mJ/cm2 ya kuwala kwa UV kwa nthawi yodziwikiratu (onani gawo 10 la bokosi ili).
Pambuyo powunikira kuwala kwa dzuwa, ma wafer a silicon okhala ndi SU-8 anaphikidwa pa 65°C kwa mphindi 5 ndi 95°C kwa mphindi 15 pa mbale iliyonse yotentha kuti apange mapatani okhala ndi kutalika kwa 200 μm. Wonjezerani nthawi yophika pambuyo pa 95°C mpaka mphindi 30 kuti apange mapatani okhala ndi kutalika kwa 500 µm.
Chopangira chimathiridwa mu mbale yagalasi, ndipo chophikira chophikidwacho chimayikidwa mu mbale. Kuchuluka kwa chopangira cha SU-8 kumatha kusiyana kutengera kukula kwa mbale yagalasi. Onetsetsani kuti mwagwiritsa ntchito chopangira cha SU-8 chokwanira kuti muchotse SU-8 yonse yomwe sinawonekere. Mwachitsanzo, mukagwiritsa ntchito mbale yagalasi ya mainchesi 150 yokhala ndi mphamvu ya 1 L, gwiritsani ntchito ~300 mL ya chopangira cha SU-8. Pangani chikombolecho kwa mphindi 25 ndi kuzungulira pang'onopang'ono nthawi zina.
Tsukani nkhungu yomwe yapangidwa ndi ~10 mL ya developer yatsopano kenako ndi IPA popopera yankho pogwiritsa ntchito pipette.
Ikani wafer mu chotsukira plasma ndipo ikani plasma ya okosijeni (mpweya wa mlengalenga, kuthamanga kwa 1 × 10−5 Torr, mphamvu ya 125 W) kwa mphindi 1.5.
Ikani wafer mu vacuum desiccator yokhala ndi galasi slide mkati. Ma wafer ndi ma slide akhoza kuyikidwa mbali ndi mbali. Ngati vacuum desiccator yagawidwa m'zigawo zingapo ndi mbale, ikani ma slide m'chipinda chapansi ndi ma wafer m'chipinda chapamwamba. Thirani 100 μL ya yankho la trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane pa galasi slide ndikugwiritsa ntchito vacuum kuti silanization.
Sungunulani botolo la maselo oundana a Caco-2 m'madzi otentha madigiri 37, kenako sungani maselo osungunukawo ku botolo la T75 lomwe lili ndi 15 mL ya 37°C yotenthedwa kale ya Caco-2.
Kuti maselo a Caco-2 adutse pa malo okwana 90%, choyamba sakanizani Caco-2 medium yofunda, PBS, ndi 0.25% trypsin/1 mM EDTA mu bafa la madzi lotentha 37°C.
Sambani maselo kawiri ndi 5 mL ya PBS yofunda pobwerezanso kusamba ndi kuwonjezera PBS yatsopano.
Nthawi yotumizira: Julayi-16-2022
