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Human gut morphogenesis imakhazikitsa mawonekedwe a crypt-villus a 3D epithelial microarchitecture ndi spatial organisation. Choncho, kukonzanso mapangidwe a 3D epithelial ndikofunika kwambiri pomanga ma in vitro gut model. Chip fluidic komanso mu Transwell embedded hybrid chip. Timalongosola mwatsatanetsatane njira zopangira chipangizo, kulima kwa Caco-2 kapena maselo a m'mimba otchedwa organoid epithelial m'makonzedwe ochiritsira komanso pa nsanja ya microfluidic, induction ya 3D morphogenesis, ndi maonekedwe a 3D epithelia yokhazikika yokhazikika pogwiritsa ntchito ma generic generic microfluidic. kwa 5 d.Njira yathu ya in vitro morphogenesis imagwiritsa ntchito kumeta ubweya wofunikira komanso kusuntha kwamakina ndipo sikufuna kupanga ma cell ovuta kapena kuwongolera, zomwe zitha kupitilira njira zina zomwe zilipo kale.Tikuwona kuti protocol yathu yomwe tikufuna ikhoza kukhala ndi tanthauzo lalikulu kwa gulu lofufuza zamankhwala, ndikupereka njira yosinthira 3D intestinal layer, biomedical epithesis, biomedical application.
Mayesero amasonyeza kuti m'mimba epithelial Caco-2 maselo opangidwa mu gut-on-a-chip1,2,3,4,5 kapena bilayer microfluidic zipangizo6,7 akhoza kuchitidwa modzidzimutsa 3D morphogenesis mu vitro popanda kumvetsa bwino makina oyambira. sis in vitro, yomwe yawonetsedwa ndi Caco-2 ndi organoids yamatumbo opangidwa ndi odwala.Mu phunziro ili, tidayang'ana makamaka kupanga maselo ndi kugawa kwa ndende kwa wotsutsa wamphamvu wa Wnt, Dickkopf-1 (DKK-1), mu gut-on-a-chip ndi zipangizo zosinthidwa za microfluidic zomwe zili ndi Transwell inserts, zotchedwa "Hybrid Chip". d-zokhudzana ndi mapuloteni 1, kapena Soggy-1) ku chip-chip gut amalepheretsa morphogenesis kapena kusokoneza chigawo chokhazikika cha 3D epithelial layer, kutanthauza kuti kupanikizika kotsutsana pa chikhalidwe kumayambitsa matumbo a morphogenesis mu vitro. kugubuduza (monga m'matumbo-pa-chip kapena hybrid-on-a-chip platforms) kapena kufalitsa .Basolateral media (monga, kuchokera ku Transwell amalowetsa m'madamu akuluakulu apansi pazitsime).
Mu protocol iyi, timapereka njira yatsatanetsatane yopangira ma microdevices a m'matumbo pa-chip ndi ma Transwell-insertable hybrid chips (masitepe 1-5) kuti apangidwe m'matumbo a epithelial cell pa polydimethylsiloxane (PDMS) -based porous nembanemba (masitepe 6A, 7A, 8, 9) kapena ma polyester 6, ma 9 a polyester, 7 3D morphogenesis in vitro (sitepe 10) .Tinazindikiranso mawonekedwe a ma cell ndi ma molekyulu omwe akuwonetsa histogenesis yeniyeni ya minofu ndi kusiyanitsa kwa ma cell amadalira mzere pogwiritsa ntchito njira zingapo zofananira (masitepe 11-24) . , kulengedwa kwa ma monolayers a 2D, ndi matumbo a m'mimba komanso Kubereka kwa biomechanical microenvironment.in vitro.Kuti tipangitse 3D morphogenesis kuchokera ku 2D epithelial monolayers, tinachotsa otsutsa a morphogen mumitundu yonse ya chikhalidwe mwa kuyenda pakati pa chipinda cha basolateral cha chikhalidwe. Kukula kwa epithelial kumadalira phogen, longitudinal host-microbiome co-cultures, matenda a tizilombo toyambitsa matenda, kuvulala kotupa, kuwonongeka kwa epithelial barrier, ndi ma probiotic-based therapies Chitsanzo.influences.
Protocol yathu ikhoza kukhala yothandiza kwa asayansi ambiri pazofunikira (mwachitsanzo, biology yamatumbo am'mimba, stem cell biology, ndi chitukuko cha biology) ndi kafukufuku wogwiritsa ntchito (mwachitsanzo, kuyezetsa mankhwala osokoneza bongo, kufananitsa matenda, uinjiniya wa minofu, ndi gastroenterology) kukhudzidwa kwakukulu. seminated kwa omvera kuphunzira mphamvu ya cell signing pa chitukuko cha matumbo, kubadwanso kapena homeostasis .Kuonjezera apo, ndondomeko yathu ndi yothandiza pofufuza matenda opatsirana pogwiritsa ntchito tizilombo toyambitsa matenda monga Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Salmonella 9.Omvera a matenda a matenda a matenda ndi tizilombo toyambitsa matenda amakhalanso othandiza.Kugwiritsa ntchito pa-chip gut microphysiology system kungalolere longitudinal co-culture 10 ndi kuwunika kotsatira kwa chitetezo cha asilikali, kuyankha kwa chitetezo cha mthupi komanso kukonzanso kuvulala kokhudzana ndi tizilombo toyambitsa matenda m'mimba (GI) thirakiti 11 .Matenda ena a GI Ceaclit, matenda a zilonda zam'mimba, matenda a zilonda zam'mimba, matenda a zilonda zam'mimba, matenda a zilonda zam'mimba, matenda a zilonda zam'mimba, matenda a zilonda zam'mimba, matenda a zilonda zam'mimba, matenda a GI Ceaclit pouchitis, kapena matenda opweteka a m'mimba amatha kutsatiridwa pamene zigawo za 3D za m'mimba za m'mimba zimakonzedwa pogwiritsa ntchito zigawo za 3D matumbo a matumbo a 3D , matendawa akuphatikizapo villous atrophy, crypt shortening, mucosal kuwonongeka, kapena kuwonongeka kwa epithelial barrier. ganizirani kuwonjezera mitundu ya maselo okhudzana ndi matenda, monga ma cell peripheral blood mononuclear cell (PBMCs), ku zitsanzo zomwe zili ndi 3D intestinal villus-crypt microarchitectures.maselo a chitetezo cha mthupi, 5.
Popeza 3D epithelial microstructure imatha kukhazikitsidwa ndikuwonetseredwa popanda kugawa magawo, owonera akugwira ntchito pa spatial transcriptomics ndi malingaliro apamwamba kapena apamwamba kwambiri atha kukhala ndi chidwi ndi mapu athu amphamvu za spatiotemporal za majini ndi mapuloteni pa epithelial niches.Chidwi ndi teknoloji.Kuyankha kwa tizilombo toyambitsa matenda kapena chitetezo cha mthupi.Kuwonjezerapo, longitudinal host-microbiome crosstalk 10, 14 yomwe imagwirizanitsa matumbo a homeostasis ikhoza kukhazikitsidwa mu 3D intestinal mucosal wosanjikiza mwa kugwirizanitsa mitundu yosiyanasiyana ya tizilombo tating'onoting'ono, tizilombo toyambitsa matenda kapena fecal microbiota-chip-on-a-gut-a.pa nsanja.Njirayi imakhala yosangalatsa kwambiri kwa omvera omwe amaphunzira mucosal immunology, gastroenterology, human microbiome, culturomics ndi microbiology yachipatala pofuna kulima tizilombo toyambitsa matenda m'mimba mwa labotale. zipinda, ndondomekoyi ingathenso kufalitsidwa kwa omwe akupanga mankhwala, biomedical Kapena high-throughput screening kapena kutsimikizira nsanja zamakampani a zakudya.Monga umboni wa mfundo, posachedwapa tawonetsa kuthekera kwa multiplex high-throughput morphogenesis system scalable to 24-well plate format. Kusintha kwa njira yathu ya in vitro morphogenesis kutha kufulumizitsidwa ndikuvomerezedwa ndi ma laboratories ambiri ofufuza, mafakitale kapena boma ndi mabungwe owongolera kuti amvetsetse kukonzanso kwa ma cell a in vitro gut morphogenesis pa transcriptomic level kuyesa mankhwala kapena biotherapeutics. njira ya m'matumbo morphogenesis.
Chiwerengero chochepa cha zitsanzo zoyesera zokhudzana ndi anthu zakhala zikugwiritsidwa ntchito pophunzira matumbo a epithelial morphogenesis, makamaka chifukwa cha kusowa kwa ndondomeko zomwe zingagwiritsidwe ntchito kuti zipangitse 3D morphogenesis mu vitro.Zowonadi, zambiri zomwe zikudziwika panopa zokhudzana ndi matumbo a morphogenesis zimachokera ku maphunziro a zinyama (mwachitsanzo, zebrafish20, mice21 kapena mice21 kapena , mtengo wamtengo wapatali, nkhuku ndi zovuta kwambiri2). ly, musadziwe bwino njira zachitukuko zaumunthu.Zitsanzozi zimakhalanso zochepa kwambiri kuti athe kuyesedwa m'njira zambiri zowonongeka.Choncho, ndondomeko yathu yokonzanso mapangidwe a 3D mu m'galasi mu vitro yamtundu wa zinyama komanso mitundu ina ya chikhalidwe cha 2D. poyankha zosiyanasiyana mucosal kapena chitetezo cha m'thupi stimuli.3D epithelial zigawo angapereke danga kuphunzira mmene maselo tizilombo ting'onoting'ono kupikisana kupanga malo niches ndi zamoyo zamoyo poyankha zinthu khamu (mwachitsanzo, mkati ndi kunja ntchofu zigawo, secretion wa IgA ndi antimicrobial peptides) . Komanso, 3D epithelium synthesis tingathe kumvetsa mmene 3D epithelium synthesis tingathe kumvetsa mmene 3D epitheology synthesis angapangire ma micromorphology kuti timvetsetse momwe tingapangire ma micromorphology a 3D. s ma metabolites ang'onoang'ono (mwachitsanzo, mafuta amtundu waufupi) omwe amapanga gulu la ma cell ndi ma stem cell niches mu basal crypts.Zinthuzi zitha kuwonetsedwa pokhapokha zigawo za 3D epithelial zimakhazikitsidwa mu vitro.
Kuphatikiza pa njira yathu yopangira 3D intestinal epithelial structures, pali njira zingapo za in vitro.Chikhalidwe cha organoid cha m'mimba ndi njira yamakono yopangira minofu yochokera ku kulima ma cell a matumbo a m'mimba pansi pa mikhalidwe yeniyeni ya morphogen23,24,25. d mkati mwa organoid ndipo, chifukwa chake, kuyambitsidwa kwa zigawo zounikira monga ma cell a tizilombo toyambitsa matenda kapena ma antigen akunja ndi ochepa.Kufikira kwa lumens ya organoid kungawongoleredwe pogwiritsa ntchito microinjector, 26,27 koma njirayi ndi yosokoneza komanso yogwira ntchito kwambiri ndipo imafuna chidziwitso chapadera kuti chizichita. Komanso, zikhalidwe za chikhalidwe cha organoid zomwe zimasungidwa muzitsulo za hydrogel pansi pazikhalidwe zosasunthika sizikuwonetseratu molondola mu vivo biomechanics.
Njira zina zogwiritsiridwa ntchito ndi magulu ofufuza angapo amagwiritsira ntchito scaffolds 3D hydrogel scaffolds kutsanzira kapangidwe ka matumbo a epithelial popanga ma cell a m'matumbo amunthu pawokha pa gel osakaniza. Zofunikira za morphogen gradients, kukhazikitsa gawo lalikulu la epithelial kapangidwe kake ndi stroma-epithelial crosstalk mwa kuphatikiza ma cell a stromal mu scaffold. Ntchito.Kafukufuku wina waposachedwapa anagwiritsa ntchito hydrogel scaffolds mu microfluidic platform and patterned intestinal epithelial structures pogwiritsa ntchito njira za laser-etching.Mouse intestinal organoids amatsatira ndondomeko zokhazikika kuti apange matumbo a m'mimba, ndipo intraluminal fluid flow can recapitulated pogwiritsa ntchito microfluidics modules. ing njira kuchokera gulu lomwelo anatha kulenga yaying'ono m'matumbo machubu ndi mowiriza morphogenetic njira.Ngakhale kupanga zovuta zigawo zosiyanasiyana m'matumbo mkati chubu, chitsanzo ichi komanso alibe luminal madzimadzi otaya ndi makina deformation.Kuonjezera apo, chitsanzo operability kungakhale kochepa, makamaka pambuyo bioprinting ndondomeko watha, perturbing mikhalidwe yoyesera kapena cell-to-cells proposedly imapereka ma cell-to-cell. kumeta ubweya wofunikira, ma biomechanics omwe amatsanzira kusuntha kwamatumbo, kupezeka kwa zigawo zodziyimira pawokha za apical ndi basolateral, komanso kupangidwanso kwa zovuta za biological microenvironments of modularity.Choncho, in vitro 3D morphogenesis protocol yathu ingapereke njira yowonjezera yothetsera zovuta za njira zomwe zilipo kale.
Protocol yathu imayang'ana kwambiri pa 3D epithelial morphogenesis, yokhala ndi ma cell a epithelial okha mu chikhalidwe ndipo palibe mitundu ina ya maselo ozungulira monga maselo a mesenchymal, maselo a endothelial, ndi maselo a chitetezo cha mthupi. chip ndi hybrid-on-a-chip imatilola kukonzanso zosanjikiza za 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 stem cell niches mu basal cryptsmeblast cell (mebrosmeblast) amakhalabe gawo lofunikira la ma cell cryptsmeblast. popanga mapuloteni a ECM ndikuwongolera matumbo a morphogenesis mu vivo35,37,38.Kuwonjezera kwa maselo a mesenchymal ku chitsanzo chathu kumapangitsa kuti pakhale ndondomeko ya morphogenetic ndi kugwirizanitsa ma cell. kugwirizana pakati pa zitsanzo za minofu ndizofunikira kwambiri pamene zitsanzo za minofu zimapangidwira kuti ziwonetsere kuyanjana kwamagulu ambiri.Choncho, maselo omaliza angafunike kuphatikizidwa kuti awonetsere zolondola kwambiri za thupi ndi kuthetsa kwa organ-level.
Kugwiritsiridwa ntchito kwa tchipisi wosakanizidwa ndikosavuta kuposa m'matumbo-pa-chip chifukwa kukhazikitsidwa kwa chipangizo ndikosavuta ndipo kugwiritsa ntchito zoyika za Transwell kumapangitsa kuti pakhale chikhalidwe chowopsa chamatumbo a epithelium. Poyambirira, mawonekedwe osasunthika m'chipinda cha apical sichimapangitsa kuti mabakiteriya azigwirizana nthawi yayitali mu tchipisi wosakanizidwa.Ngakhale titha kuyambitsa 3D morphogenesis muzoyika za Transwell tikamagwiritsa ntchito tchipisi wosakanizidwa, kuchepa kwa ma biomechanics okhudzana ndi thupi ndi kutuluka kwamadzimadzi apical kumatha kuchepetsa kuthekera kwa nsanja za hybrid chip zomwe zingagwiritsidwe ntchito.
Kumanganso kwathunthu kwa axis ya human crypt-villus mu gut-on-a-chip ndi hybrid-on-a-chip zikhalidwe sizinakhazikitsidwe kwathunthu. Lium, madera ozungulira ndi oyipa sanasinthidwe bwino. lli ndi ~ 600 µm41.
Kuchokera ku lingaliro la kulingalira, in situ super-resolution imaging ya 3D microarchitectures ikhoza kukhala yochepa m'matumbo pa chip, popeza mtunda wofunikira wogwirira ntchito kuchokera ku lens cholinga kupita ku epithelial wosanjikiza ndi dongosolo la mamilimita angapo.Kugonjetsa vutoli, cholinga chakutali chingafunikire. ndi-wosanjikiza microfabrication ya m'matumbo pa chip imaphatikizapo kumamatira kosatha pakati pa wosanjikiza uliwonse, ndizovuta kwambiri kutsegula kapena kuchotsa pamwamba kuti muwone mawonekedwe a epithelial layer.Mwachitsanzo, pogwiritsa ntchito makina oonera ma electron microscope (SEM).
Hydrophobicity ya PDMS yakhala yolepheretsa maphunziro opangidwa ndi microfluidic okhudzana ndi mamolekyu ang'onoang'ono a hydrophobic, popeza PDMS imatha kutsatsa mosadukiza mamolekyu a hydrophobic. Njira zina za PDMS zitha kuganiziridwa ndi zinthu zina zapolymeric. Kapenanso, kusinthidwa kwapamwamba kwa PDMS kapena glycephilic PDMS (eg4polyphilic) kapena PDMS (eg4polythy) 3) ikhoza kuganiziridwa kuti kuchepetsa kutengeka kwa mamolekyu a hydrophobic.
Potsirizira pake, njira yathu sinadziwike bwino popereka kuwonetsetsa kwapamwamba kwambiri kapena "kukula-kufanana-zonse" njira yoyesera yogwiritsira ntchito.Protocol yamakono imafuna pampu ya syringe pa microdevice, yomwe imatenga malo mu CO2 incubator ndikuletsa kuyesa kwakukulu.Kuchepetsaku kungathe kusinthidwa kwambiri ndi scalability ya 94well, chikhalidwe cha 94well, chikhalidwe cha 94well, 34well, chikhalidwe cha 94well, 34well-innovative, zoyikapo porous zomwe zimalola kubwezeredwa kosalekeza ndikuchotsa media media).
Kuti tipangitse 3D morphogenesis ya epithelium yamatumbo amunthu mu vitro, tidagwiritsa ntchito chipangizo cham'mimba cha microfluidic chomwe chili ndi tinthu tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'onoting'ono komanso zotanuka porous nembanemba pakati kuti tipange lumen-capillary interface. s, morphogenesis ya maselo osiyanasiyana a m'matumbo a m'mimba a anthu amatha kuwonetsedwa pogwiritsa ntchito njira yowonongeka kuti achotse otsutsa a morphogen kuchokera ku chipinda chotsatira. co-2 maselo) kapena organoids m'matumbo a munthu;mabokosi 2-5), (iii) chikhalidwe cha maselo a m'matumbo a epithelial pamatumbo a m'mimba kapena tchipisi ta hybrid (masitepe 6-9), (iv) kulowetsedwa kwa 3D morphogenesis mu vitro (sitepe 10) ndi (v)) kuti awonetsere 3D epithelial microstructure (masitepe 11-24) . genesis pofanizira epithelial morphogenesis ndi kuwongolera kwa malo, kwakanthawi, kokhazikika, kapena kachitidwe.
Tidagwiritsa ntchito nsanja ziwiri zachikhalidwe: gut-on-a-chip yokhala ndi njira zowongoka kapena masitayilo osakanikirana, kapena tchipisi zosakanizidwa zomwe zimakhala ndi Transwell (TW) zoyika mu chipangizo cha microfluidic, chopangidwa monga momwe tafotokozera mu Box 1, ndi sitepe 1 -5. stinal organoids) ndi ndondomeko ya chikhalidwe yomwe imagwiritsidwa ntchito mu protocol iyi." In vitro morphogenesis "imasonyeza masitepe onse omwe ma cell a Caco-2 kapena organoid-derived-derived epithelial cell amapangidwa pa chip cha matumbo kapena pa Transwell amaika a chip hybrid chip, kutsatiridwa ndi kulowetsedwa kwa 3D morphogenesis ndi mapangidwe a chiwerengero chodziwika cha epithelial nambala yomwe ili m'munsiyi. zigawo za epithelial zingagwiritsidwe ntchito, mwachitsanzo, mu kusiyanitsa kwa maselo, maphunziro a physiology ya m'matumbo, kukhazikitsidwa kwa tizilombo toyambitsa matenda tizilombo toyambitsa matenda, ndi njira za matenda. mucosal surfaces.Zithunzi za fluorescent mu Gut Physiology zikuwonetsa mamina opangidwa ndi kuipitsidwa kwa sialic acid ndi zotsalira za N-acetylglucosamine pogwiritsa ntchito fulorosenti yatirigu agglutinin. rescent protein (GFP) with microengineered 3D Caco-2 epithelial cells.Panja lakumanja likuwonetsa kukhazikika kwa GFP E. coli co-cultured ndi 3D Caco-2 epithelial cell, kutsatiridwa ndi immunofluorescence staining ndi F-actin (red) ndi nuclei (blue) .Disease modeling inllustological guversus antibacterial chips illustological antiphysical chips. s (mwachitsanzo, lipopolysaccharide, LPS) ndi maselo a chitetezo cha mthupi (mwachitsanzo, PBMC;green).Maselo a Caco-2 adakonzedwa kuti akhazikitse 3D epithelial layer.Scale bar, 50 µm. Zithunzi zomwe zili m'munsimu: "Kusiyana kwa maselo" kusinthidwa ndi chilolezo chochokera kuzinthu.2.Oxford University Press;Kujambulanso ndi chilolezo chochokera pa Ref.5.NAS;"Host-Microbe Co-Culture" idasinthidwa ndi chilolezo kuchokera ku ref.3.NAS;"Matenda Achitsanzo" adasinthidwa ndi chilolezo chochokera ku chilankhulo.5.NAS.
Zonse ziwiri za gut-on-chip ndi hybrid chips zidapangidwa pogwiritsa ntchito PDMS replicas zomwe zidapangidwa kuchokera ku silicon molds ndi soft lithography1,44 ndikujambula ndi SU-8.Mapangidwe a ma microchannels mu chip chilichonse amatsimikiziridwa poganizira za hydrodynamics monga shear stress ndi hydrodynamic pressure1,4,12.The original gut-on-tastad12.The original gut-on. ma microchannels ofanana molunjika, asintha kukhala zovuta zam'matumbo-pa-a-chip (Extended Data Fig. 1b) yomwe imaphatikizapo ma microchannels okhotakhota kuti apangitse Kuchulukitsa kwamadzi okhalamo nthawi, machitidwe osakanikirana osagwirizana, ndi ma multiaxial deformation a maselo otukuka (mkuyu 2a-f) Tawonetsa kuti convoluted Gut-Chip imapangitsanso kwambiri 3D morphogenesis mu nthawi yofanana ndi kukula kwa epithelial poyerekeza ndi chiyambi cha Gut-Chip, mosasamala kanthu za mtundu wa selo lopangidwa.2a) .Kupanga matumbo pa chip, okonzeka chapamwamba PDMS wosanjikiza anali sequentially zomangika kwa porous PDMS filimu ndiyeno limagwirizana ndi m'munsi PDMS wosanjikiza ndi zosasinthika kugwirizana ntchito corona treater (mkuyu. 2b-f) .Kupanga tchipisi wosakanizidwa, anachiritsa PDMS replicas anali bonded kwa magalasi ang'onoang'ono ang'onoang'ono ang'onoang'ono kulenga magalasi ang'onoang'ono a Transfluodec (Mkuyu 2b-f). Chithunzi cha 2h ndi Extended Data Fig. 2) .Njira yolumikizira imachitidwa pochiza malo a PDMS replica ndi galasi ndi plasma ya okosijeni kapena chithandizo cha corona.Pambuyo pa kutseketsa kwa chipangizo chopangidwa ndi microfabricated chophatikizidwa ndi chubu la silicone, kukhazikitsidwa kwa chipangizocho kunali kokonzeka kuchita 3D morphogenesis ya matumbo a epithelium 2g (Chithunzi).
a, Schematic illustration of the preparation of PDMS parts from SU-8 patterned silicon molds.The uncurred PDMS solution anatsanuliridwa pa silicon nkhungu (kumanzere), kuchiritsidwa pa 60 °C (pakati) ndi kugwetsedwa (kumanja).The demolded PDMS anadulidwa mu zidutswa ndi kutsukidwa kuti agwiritse ntchito.b, Photograph of the silicon mold to the silicon Photograph of the silicon mold. kupanga PDMS porous membrane.d, Zithunzi zingapo za PDMS zapamwamba ndi zapansi komanso chipangizo cham'mimba chosonkhanitsidwa.e, Schematic of the alignment of the chapper, membrane, and lower PDMS components.Chirichonse chimamangidwa mosagwirizana ndi plasma kapena coronatherapy s.g, Kukonzekera kwa gut-on-a-chip kwa microfluidic cell culture.Mmatumbo opangidwa pa chip wosonkhanitsidwa ndi chubu la silikoni ndi syringe anaikidwa pa coverslip.Chipangizo cha chip chinayikidwa pa chivindikiro cha mbale ya Petri ya 150 mm kuti ikonzedwe.Chomangiracho chimagwiritsidwa ntchito kutseka silicone chubu.h, Visual chipsD chips chipsgenesis chipsD chips genesis. okonzekera paokha ku chikhalidwe 2D monolayers a m'matumbo epithelial maselo analowetsedwa mu chip hybrid kulimbikitsa matumbo 3D morphogenesis.Sing'anga ndi perfused kudzera microchannels pansi selo wosanjikiza anakhazikitsa pa Transwell insert.Scale bar, 1 cm.h Kusindikizidwanso ndi chilolezo kuchokera ku reference.4.Elsevier.
Mu protocol iyi, ma cell a Caco-2 cell ndi organoids a m'mimba adagwiritsidwa ntchito ngati magwero a epithelial (mkuyu 3a) . Mitundu yonse iwiri ya maselo idapangidwa mwaokha (Bokosi 2 ndi Bokosi 5) ndipo amagwiritsidwa ntchito kubzala ma microchannels opangidwa ndi ECM a pa-chip gut kapena Transwell insert. 0 ndi 50) mu T-flasks kukololedwa kukonzekera dissociated cell suspensions ndi trypsinization madzimadzi (bokosi 2) .Human intestinal organoids ku matumbo biopsies kapena opaleshoni resections anali otukulidwa mu Matrigel scaffold domes mu 24-chitsime mbale kuthandizira structural structural microenvironment, ndi microenvironment yofunikira ya Rpogennt, ndi Wpogennt Angspogennt, ndi Agnspogennt Angspogennt, ndi Agspogennt Angspogennt. Zinthu zakukula zomwe zakonzedwa monga momwe zafotokozedwera mu Bokosi 3 zinawonjezeredwa tsiku lililonse mpaka ma organoids atakula kufika ~ 500 µm m'mimba mwake. Ma organoids okhwima mokwanira amakololedwa ndikugawidwa kukhala maselo amodzi kuti abzalire m'matumbo kapena oyikapo Transwell pa chip (Bokosi 5) . malo (mwachitsanzo, zilonda motsutsana ndi malo omwe sali otupa) ndi malo a m'mimba m'matumbo (monga duodenum, jejunum, ileum, cecum, colon, kapena rectum). Timapereka ndondomeko yowonjezereka mu Bokosi 5 ya kulima ma colonic organoids (coloids) omwe nthawi zambiri amafuna kuchuluka kwa ma morphogens ang'onoang'ono kapena ang'onoang'ono.
a, Workflow for induction of gut morphogenesis in the gut chip.Caco-2 epithelium ya m'mimba ya munthu ndi organoids ya m'mimba imagwiritsidwa ntchito mu protocol iyi kuti iwonetsere 3D morphogenesis.Maselo a epithelial omwe amadzipatula adayikidwa mu chipangizo chokonzekera cha m'matumbo (chip-chip) (chip-chip) .Kamodzi ma cell amaikidwa pa PD (chidutswa cha chip). D0), kutuluka kwa apical (AP) kumayambika ndikusungidwa kwa masiku oyambirira a 2 (kuthamanga, AP, D0-D2) .Basolateral (BL) kutuluka kumayambikanso pamodzi ndi maulendo otambasula a cyclic (kutambasula, kutuluka, AP ndi BL) pamene 2D monolayer wathunthu amapangidwa.M'mimba 3D morphogenesis inachitika masiku a chikhalidwe cha micromorphogenesis 5 (5) kusonyeza kusiyana kwa micromorphogenesis 5 pambuyo pa micromorphogenesis 5. ya ma cell a Caco-2 pa sitepe iliyonse yoyesera kapena nthawi (bar graph, 100 µm) . Zithunzi zinayi zowonetsera ma cascades a m'matumbo a morphogenesis (pamwamba kumanja) .Mivi yophwanyidwa mu schematic imayimira mayendedwe a madzi otuluka.b, chithunzi cha SEM chosonyeza topology ya pamwamba pa 3D Cacolisheding bokosi (yoyera) imasonyeza malo opangidwa ndi 3D Cacolisheding (yoyera). ikuwonetsa ma microvilli opangidwanso pa 3D Caco-2 wosanjikiza (kumanja).c, Kuyang'ana kutsogolo kwa Caco-2 3D yokhazikitsidwa, claudin (ZO-1, yofiira) ndi nembanemba zam'malire za burashi zomwe zimatchedwa F-actin (zobiriwira) ndi nuclei (buluu) Immunofluorescence confocal visualization ya epithelial tchipisi tomwe timayang'ana mkati mwa ma cell amkati amkati. confocal view.d, Nthawi ya kusintha kwa morphological mu organoids otukuka pa chip chopezedwa ndi gawo losiyanitsa maikulosikopu pamasiku 3, 7, 9, 11, ndi 13. Choyikapo (pamwamba kumanja) chikuwonetsa kukulitsa kwakukulu kwa chithunzi choperekedwa. GR5;magenta), ma cell a goblet (MUC2; obiriwira), F-actin (imvi) ndi nuclei (cyan) okulira pa tchipisi m'matumbo kwa masiku atatu, motsatana (Kumanzere) ndi masiku 13 (pakati) organoids pa epithelial layer. organoid epithelium yokhazikitsidwa m'matumbo pa chip podetsa nembanemba ya plasma ndi utoto wa CellMask (kumanja) pa tsiku la 13 la chikhalidwe. Scale bar ndi 50 μm pokhapokha atanenedwa mwanjira ina.b Kusindikizidwanso ndi chilolezo chochokera pazambiri.2.Oxford University Press;c Kusinthidwa ndi chilolezo kuchokera ku Buku.2.Oxford University Press;e ndi f adasinthidwa ndi chilolezo potengera.12 Under Creative Commons License CC BY 4.0.
M'matumbo pa chip, m'pofunika kusintha hydrophobic pamwamba pa PDMS porous nembanemba kuti bwino ECM zokutira.Mu protocol iyi, timagwiritsa ntchito njira ziwiri zosiyana kusintha hydrophobicity wa PDMS nembanemba.Kwa culturing Caco-2 maselo, kutsegulira pamwamba ndi UV / ozoni mankhwala okha kunali kokwanira kuchepetsa hydrophobicity ya ECM ndi kulumikiza ma cell a hydrophobicity a CaCMPD nthawi zonse, microfluidic chikhalidwe cha organoid epithelium amafuna mankhwala ofotokoza pamwamba functionalization kukwaniritsa imayenera mafunsidwe a ECM mapuloteni ndi sequentially ntchito polyethyleneimine (PEI) ndi glutaraldehyde kuti PDMS microchannels. Pambuyo kusinthidwa pamwamba, mapuloteni ECM anali waikamo kuti kuphimba functionalized PDMS pamwamba kapena kulowetsedwa ndi cellophatikizidwe micropifluid mu cellophatikizidwe ndi microfluide cell. chikhalidwe chimayamba ndi perfusing okha sing'anga kumtunda microchannel mpaka maselo kupanga monolayer wathunthu, pamene m'munsi microchannel amasunga malo amodzi.Njira iyi wokometsedwa kwa kutsegula pamwamba ndi ECM ❖ kuyanika chimathandiza ubwenzi wa organoid epithelium kukopa 3D morphogenesis pa PDMS pamwamba.
Zikhalidwe za Transwell zimafunanso zokutira za ECM musanayambe kubzala ma cell;Komabe, zikhalidwe za Transwell sizifuna njira zovuta zowonetsera kuti zitsegule pamwamba pa zolowetsa porous.Pa kukula kwa maselo a Caco-2 pa zoikamo za Transwell, ECM ❖ kuyanika pa zoyikapo porous imafulumizitsa kumangirizidwa kwa maselo a Caco-2 osagwirizana (<1 ora) ndi mapangidwe olimba otchinga (<1-2 masiku) . <3 h) ndikusungidwa mpaka ma organoids amapanga monolayer wathunthu wokhala ndi chotchinga chokhazikika .Zikhalidwe za Transwell zimachitidwa mu mbale za 24-chitsime popanda kugwiritsa ntchito tchipisi cha hybrid.
In vitro 3D morphogenesis ikhoza kuyambitsidwa pogwiritsa ntchito madzi otuluka ku gawo la basolateral la epithelial layer yokhazikika. M'matumbo pa chip, epithelial morphogenesis inayamba pamene sing'angayo inkagwiritsidwa ntchito kumtunda ndi kumunsi kwa ma microchannels (Mkuyu 3a) .Monga tafotokozera kale, ndikofunika kuti tidziwitse kutuluka kwachinsinsi kwa compolateral kuchotsedwa kwa continual morphogenesis (mkuyu 3a) . Kuti tipereke zakudya zokwanira ndi seramu ku maselo omangidwa pa nembanemba wa porous ndi kupanga luminal kukameta ubweya kupsyinjika, ife timakonda kugwiritsa ntchito maulendo awiri m'matumbo pa chip. onse chikhalidwe nsanja.
The morphological features of microengineered 3D epithelial layers can reviewed by put into various iging modalities, including phase difference microscopy, differential interference difference (DIC) microscopy, SEM, kapena immunofluorescence confocal microscopy (Figures 3 ndi 4) .Phase Kusiyana kapena DIC kulingalira nthawi ndi protrupial mawonekedwe a DIC akhoza kuchitidwa mosavuta pa mawonekedwe a DIC. e kwa kuwala kwa kuwala kwa PDMS ndi mafilimu a poliyesitala, onse m'matumbo-pa-chip ndi hybrid chip nsanja angapereke zenizeni zenizeni mu situ kulingalira popanda kufunika kwa magawo kapena disassembly wa chipangizo. Pamene akuchita immunofluorescence imaging (Zizindikiro 1, 3c, f ndi 4b, ndi 4b), zotsatiridwa ndi ma cell amtundu wa 4 (cF) amatsatiridwa ndi triform / hyde ton X-100 ndi 2% (wt/vol) ) bovine serum albumin (BSA), mwadongosolo.Malingana ndi mtundu wa selo, zokonza zosiyanasiyana, permeabilizers, ndi zotchinga zingagwiritsidwe ntchito.Ma antibodies oyambirira omwe akutsata maselo odalira mzere kapena zizindikiro za chigawo amagwiritsidwa ntchito kuwunikira maselo osasunthika mu situ pa chip, chigawo chachiwiri, chigawo cha 6, anti-anti-anti-6. diamidino-2-phenylene) indole, DAPI) kapena F-actin (mwachitsanzo, fluorescently labeled phalloidin) .Fluorescence-based live imaging ingathenso kuchitidwa mu situ kuti azindikire kupanga kwa ntchentche (Chith.1, "Kusiyanitsa kwa ma cell" ndi "Gut physiology"), kusakhazikika kwachisawawa kwa ma cell a tizilombo tating'onoting'ono (mkuyu 1, "Host-microbe co-culture"), kulembedwa kwa maselo a chitetezo chamthupi (mkuyu 1, 'Matenda Modelling') kapena ma contours a 3D epithelial morphology (mkuyu 4bc pa chip, fc ndi kupatukana ndi wosanjikiza). M'munsi mwa microchannel wosanjikiza, monga tafotokozera mu ref.Monga momwe tawonetsera mu mkuyu 2, 3D epithelial morphology komanso microvilli pa apical burashi malire akhoza visualized ndi SEM (mkuyu 3b) .Mafotokozedwe a zizindikiro zosiyana akhoza kuyesedwa pochita kuchuluka kwa PCR5 kapena single-cell RNA chipsal sequencing 3 cell RNA sequencing. Amakololedwa ndi trypsinization kenako amagwiritsidwa ntchito posanthula ma cell kapena ma genetic.
a, Workflow kwa kupatsidwa ulemu wa matumbo morphogenesis mu hybrid chip.Caco-2 ndi organoids m'mimba ntchito ndondomeko kusonyeza 3D morphogenesis mu wosakanizidwa Chip nsanja.Dissociated epithelial maselo anali seeded mu okonzeka Transwell amaika (TW prep; onani chithunzi pansipa).Kamodzi maselo anali mbewu chikhalidwe anaikapo ma cell achilengedwe (seedseed cell) zonse zolumikizidwa ndi ma cell a polystatic) (T chikhalidwe cha TW) .Pambuyo pa masiku a 7, choyikapo chimodzi cha Transwell chokhala ndi 2D monolayer wa maselo a epithelial chinaphatikizidwa mu chip hybrid kuti adziwitse kayendedwe ka basolateral (Flow, BL), komwe pamapeto pake kunayambitsa kubadwa kwa 3D epithelial wosanjikiza (morphogenesis) . sitepe kapena nthawi.The schematics mu zigawo kumtunda kusonyeza kuyesera kasinthidwe pa sitepe iliyonse.b, tchipisi Hybrid (kumanzere schematic) zingachititse 3D morphogenesis ma cell organoid epithelial ndi pamwamba-pansi confocal maikroskopu mawonedwe otengedwa pa osiyana Z malo (pamwamba, pakati, ndi pansi;onani mizere yolondola ndi madontho ofanana).adawonetsa mawonekedwe achilengedwe.F-actin (cyan), nucleus (imvi).c, Fluorescence confocal micrographs (3D angled view) ya organoid-derived epithelial cell otukuka mu static Transwell (TW; inset mkati mwa white dashed box) motsutsana ndi hybrid chip (largest full shot) kuyerekeza 3D respectively morphus2 morphology 2D mawonedwe (omwe ali pakona yakumanja yakumanja; "XZ") amawonetsanso mawonekedwe a 2D ndi 3D. Scale bar, 100 µm.c Yosindikizidwanso ndi chilolezo kuchokera ku zolembera.4.Elsevier.
Ulamuliro ukhoza kukonzedwa ndi culturing maselo ofanana (Caco-2 kapena matumbo organoid epithelial maselo) mu awiri-dimensional monolayers pansi ochiritsira malo amodzi chikhalidwe.
Njira yofewa ya lithography iyenera kuchitidwa m'chipinda choyera. Pagawo lililonse pa chip (zigawo zakumwamba ndi zapansi ndi nembanemba) ndi tchipisi wosakanizidwa, ma photomasks osiyanasiyana anagwiritsidwa ntchito ndikupangidwira pazitsulo zosiyana za silicon chifukwa kutalika kwa ma microchannels kunali kosiyana. Kutalikira kwa ma microchannel apamwamba ndi otsika a m'matumbo pa chip ndi 500 chip µm µm kutalika kwa chip 200µm.
Ikani chowotcha cha silicon cha 3-inch mu mbale yokhala ndi acetone.Pezani mbaleyo pang'onopang'ono kwa masekondi 30, kenaka muwumitse chophatikizikacho mpweya.Tsitsani chowotchacho ku mbale yokhala ndi IPA, kenaka zungulirani mbaleyo kwa masekondi 30 kuti muyeretse.
Mankhwala a piranha (kusakaniza kwa hydrogen peroxide ndi sulfuric acid, 1:3 (vol/vol)) angagwiritsidwe ntchito mwachisawawa kuti achulukitse kuchotsa zotsalira za organic kuchokera pamwamba pa silicon wafer.
Njira ya Piranha ndiyowononga kwambiri ndipo imatulutsa kutentha. Njira zina zodzitetezera ndizofunikira. Potaya zinyalala, lolani kuti madziwo azizire ndikusamutsidwa ku chidebe cha zinyalala chaukhondo, chowuma.
Thirani madzi ophatikizika powayika pa mbale yotentha ya 200 ° C kwa mphindi 10. Pambuyo pa kutaya madzi m'thupi, chophikacho chinagwedezeka kasanu mumlengalenga kuti chizizizira.
Thirani ~ 10 g ya photoresist SU-8 2100 pakatikati pa chofufumitsa cha silicon choyeretsedwa. Gwiritsani ntchito ma tweezers kuti mufalitse chithunzithunzi mofanana pa wafer. Nthawi zina ikani chophatikizira pa mbale yotentha ya 65 ° C kuti photoresist isakhale yomamatira komanso yosavuta kufalitsa.Musayike chophatikiziracho molunjika pa mbale yotentha.
SU-8 inagawidwa mofanana pa wafer poyendetsa zokutira zozungulira. Pulogalamu yozungulira yolowera ya SU-8 kwa 5–10 s kuti ifalikire pa 500 rpm pa mathamangitsidwe 100 rpm/s. Khazikitsani kupota kwakukulu kwa 200 µm makulidwe patterning pa 1,500mµmµ0000mµmµ kapena kukwaniritsa 500mµmµ kutalika kwa m'mimba kumtunda kwa matumbo pa chip; onani "Masitepe Ofunikira" pansipa) akhazikitsidwa pa liwiro la 300 rpm/s masekondi 30 pa 1,200 rpm.
Kuthamanga kwakukulu kwa spin kumatha kusinthidwa molingana ndi makulidwe a chandamale cha SU-8 patani ya silicon.
Kuti apange mawonekedwe a SU-8 a kutalika kwa 500 µm kumtunda wapamwamba wa matumbo pa chip, kupopera kozungulira ndi masitepe ofewa a Bokosi ili (masitepe 7 ndi 8) adabwerezedwa motsatizana (onani sitepe 9) kuti apange zigawo ziwiri za 250 µm A wandiweyani wosanjikiza wa SU-8, womwe ukhoza kuwululidwa ndi 0 µ wowunjika µ µ µ wophatikizidwa ndi 0 µ mu bokosi la 0. .
Kuphika mofewa zophika zophika za SU-8 poyika mosamalitsa zophika pa mbale yotentha pa 65 ° C kwa mphindi 5, kenaka sinthani zoikamozo kukhala 95 ° C ndikuumirira kwa mphindi 40 zina.
Kuti mukwaniritse kutalika kwa 500 μm kwa chitsanzo cha SU-8 kumtunda wa microchannel, bwerezani masitepe 7 ndi 8 kuti mupange zigawo ziwiri za 250 μm za SU-8.
Pogwiritsa ntchito UV Mask Aligner, chitani chiyeso cha nyali molingana ndi malangizo a wopanga kuti muwerenge nthawi yowonekera ya chophatikizira.
Pambuyo pozindikira nthawi yowonekera, ikani chithunzithunzi pa chotengera chigoba cha UV chigoba aligner ndikuyika chithunzithunzi pa chophimbidwa ndi SU-8 chophimbidwa.
Ikani malo osindikizidwa a photomask molunjika kumbali ya SU-8 yokutidwa ndi silicon wafer kuti muchepetse kubalalitsidwa kwa UV.
Onetsani chowotcha chokhala ndi SU-8 chophimbidwa ndi chithunzithunzi cha 260 mJ/cm2 cha kuwala kwa UV kwa nthawi yodziwikiratu (onani sitepe 10 ya bokosi ili).
Pambuyo pa kuwonekera kwa UV, SU-8-wokutidwa ndi silicon wafers amawotcha 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 30 min kuti mupange mapatani okhala ndi kutalika kwa 500 µm.
Wopangayo amatsanuliridwa mu mbale yagalasi, ndipo chophika chophika chimayikidwa mu mbale.Voliyumu ya SU-8 wopanga akhoza kusiyana malingana ndi kukula kwa mbale ya galasi. Onetsetsani kuti mugwiritse ntchito makina okwana SU-8 kuti muchotseretu SU-8.Mwachitsanzo, mukamagwiritsa ntchito galasi la galasi la 150 mm ndi mphamvu ya 1 L, gwiritsani ntchito ~ 300 mold rovelopment mphindi 300 mld. .
Tsukani nkhungu yopangidwa ndi ~ 10 mL ya makina atsopano otsatiridwa ndi IPA popopera mankhwala pogwiritsa ntchito pipette.
Ikani chofufumitsa mu plasma chotsuka ndikuwonetsa mpweya wa plasma (gasi wa mumlengalenga, kuthamanga kwa cholinga 1 × 10-5 Torr, mphamvu 125 W) kwa 1.5 min.
Ikani chowotcha mu vacuum desiccator ndi galasi slide mkati.Wafers ndi slide akhoza kuikidwa mbali ndi mbali.Ngati vacuum desiccator amagawidwa mu zigawo zingapo ndi mbale, ikani slides mu chipinda chapansi ndi zopyapyala mu chipinda chapamwamba.Drop 100 μL wa trichloro,H 2H,H,H,H,H,L,L,L,,H,H,H,L. tsitsani ndikuyika vacuum kuti musalanize.
Thirani mbale yachisanu ya Caco-2 mumtsuko wamadzi wa 37 ° C, kenaka tumizani maselo osungunuka mu botolo la T75 lomwe lili ndi 15 mL ya 37 ° C yotenthedwa kale Caco-2 sing'anga.
Kudutsa ma cell a Caco-2 pa ~ 90% confluency, yoyamba yotentha ya Caco-2 sing'anga, PBS, ndi 0.25% trypsin/1 mM EDTA mu 37 ° C osamba m'madzi.
Sambani ma cell kawiri ndi 5mL ya PBS yofunda pobwereza vacuum aspiration ndikuwonjezera PBS yatsopano.