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Human gut morphogenesis imakhazikitsa mawonekedwe a crypt-villus a 3D epithelial microarchitecture ndi spatial organisation. Chifukwa chake, kukonzanso mapangidwe a epithelial a 3D ndikofunikira kwambiri popanga mitundu ya in vitro gut. morphogenesis m'matumbo pa microfluidic chip komanso mu Transwell embedded hybrid chip. Timalongosola mwatsatanetsatane njira zopangira chipangizo, kulima kwa Caco-2 kapena maselo a matumbo a organoid epithelial m'makonzedwe achizolowezi komanso pa nsanja ya microfluidic, induction ya 3D morphogenesis, ndi maonekedwe a epithelies ovomerezeka a 3D. Njira yathu ya in vitro morphogenesis imagwiritsa ntchito kumeta ubweya wofunikira komanso kusuntha kwamakina ndipo sikufuna uinjiniya wa cell kapena kuwongolera, zomwe zitha kupitilira njira zina zomwe zilipo kale. kwa biomedical, chipatala, ndi mankhwala ntchito.
Mayesero amasonyeza kuti matumbo a 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. epithelial morphogenesis 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". mapuloteni obisika okhudzana ndi frizzled 1, kapena Soggy-1) ku chip-chip gut amalepheretsa morphogenesis kapena kusokoneza chigawo chokhazikika cha 3D epithelial layer, kutanthauza kuti kupsinjika kotsutsana pa chikhalidwe kumayambitsa matumbo a morphogenesis mu vitro. mu basolateral compartment mwa kugubuduza mwachangu (mwachitsanzo, m'matumbo-pa-chip kapena hybrid-on-a-chip platforms) kapena kufalikira .Basolateral media (mwachitsanzo, kuchokera ku Transwell amalowetsa m'masungidwe akuluakulu a basolateral m'zitsime).
Mu protocol iyi, timapereka njira yatsatanetsatane yopangira ma microdevices a m'matumbo pa-chip ndi ma Transwell-insertable hybrid chips (masitepe 1-5) kupita ku ma cell am'mimba a epithelial pa polydimethylsiloxane (PDMS) -opangidwa ndi ma porous nembanemba (masitepe 6A, 7A, 8, 9) kapena ma polyester 6, ma nembanemba a TranswellB, 7 9) ndipo inachititsa 3D morphogenesis mu vitro (sitepe 10) .Tinazindikiranso ma cellular ndi ma molekyulu omwe amasonyeza histogenesis yeniyeni ya minofu ndi kusiyanitsa kwa ma cell odalira mzere pogwiritsa ntchito njira zambiri zowonetsera zithunzi (masitepe 11-24) . ndi tsatanetsatane waukadaulo kuphatikiza kusinthidwa kwapamwamba kwa nembanemba wa porous, kulengedwa kwa 2D monolayers, ndi matumbo am'mimba komanso Kuberekanso kwa biomechanical microenvironment.in vitro. regenerable 3D epithelial wosanjikiza amene angagwiritsidwe ntchito chitsanzo morphogen-odalira epithelial kukula, longitudinal host-microbiome co-cultures, tizilombo toyambitsa matenda, kutupa kuvulala, epithelial barrier kukanika, ndi probiotic-based mankhwala Chitsanzo.influences.
Protocol yathu ingakhale yothandiza kwa asayansi ambiri pazofunikira (mwachitsanzo, biology yamatumbo am'mimba, stem cell biology, ndi chitukuko cha biology) ndikugwiritsa ntchito kafukufuku (mwachitsanzo, kuyezetsa mankhwala osokoneza bongo, kufananitsa matenda, uinjiniya wa minofu, ndi gastroenterology) kukhudzidwa kwakukulu. luso njira akhoza kufalitsidwa kwa omvera kuphunzira mphamvu ya chizindikiro cha selo pa chitukuko cha matumbo, kubadwanso kapena homeostasis .Kuonjezera apo, ndondomeko yathu ndi yothandiza pakufunsana ndi matenda opatsirana pogwiritsa ntchito tizilombo toyambitsa matenda monga Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium Salmonelera chotengera 9 kapena Virifila. Omvera a matenda a matenda a matenda ndi tizilombo toyambitsa matenda amakhalanso othandiza.Kugwiritsa ntchito pa-chip gut microphysiology dongosolo kungalolere longitudinal co-culture 10 ndi kuwunika kotsatizana ndi chitetezo cha asilikali, mayankho a chitetezo cha mthupi komanso kukonzanso kuvulala kokhudzana ndi tizilombo toyambitsa matenda m'mimba (GI) thirakiti 11 .Matenda ena a GIceky gut, matenda a Croace, matenda ena a GIceky, matenda a m'mimba, matenda a GI ulcerative colitis, pouchitis, kapena irritable bowel syndrome imatha kuyerekezedwa pamene zigawo za 3D za m'mimba zakonzedwa pogwiritsa ntchito zigawo za 3D zam'mimba zam'mimba, matendawa akuphatikizapo villous atrophy, crypt shortening, mucosal kuwonongeka, kapena kuwonongeka kwa epithelial barrier. Kuchulukirachulukira kwa chilengedwe cha matenda, owerenga angaganizire kuwonjezera mitundu ya maselo okhudzana ndi matenda, monga ma cell peripheral blood mononuclear cell (PBMCs), kumitundu yomwe ili 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 tizilombo toyambitsa matenda.Kuwonjezerapo, longitudinal host-microbiome crosstalk 10, 14 yomwe imagwirizanitsa matumbo a homeostasis ikhoza kukhazikitsidwa mu 3D intestinal mucosal wosanjikiza pogwirizanitsa mitundu yosiyanasiyana ya tizilombo tating'onoting'ono, midzi ya tizilombo toyambitsa matenda kapena fecal microbiota-chip-on-a-gut-a. pa nsanja.Njirayi imakhala yokongola kwambiri kwa omvera omwe amaphunzira mucosal immunology, gastroenterology, human microbiome, culturomics ndi microbiology yachipatala pofuna kulima tizilombo toyambitsa matenda m'mimba mwa labotale. replenish basolateral compartments, protocol ikhoza kufalitsidwanso kwa omwe akupanga mankhwala, biomedical Kapena high-throughput screening kapena kutsimikizira nsanja zamakampani a chakudya.Monga umboni wa mfundo, posachedwapa tawonetsa kuthekera kwa multiplex high-throughput morphogenesis system scalable to a 24-chip-organ products have been multiplex-high-throughput morphogenesis system. commercialized16,17,18.Choncho, kutsimikizika kwa njira yathu ya in vitro morphogenesis kumatha kufulumizitsidwa komanso kuvomerezedwa 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 malonda a organ-on-a-chip kuti awone kuchulukana kwa m'matumbo a morphogenesis.
Chiwerengero chochepa cha zitsanzo zoyesera zokhudzana ndi anthu zakhala zikugwiritsidwa ntchito pofufuza matumbo a epithelial morphogenesis, makamaka chifukwa cha kusowa kwa ndondomeko zomwe zingatheke kuti zipangitse 3D morphogenesis mu vitro. Ndipotu, zambiri zomwe zikudziwika panopa za m'matumbo a morphogenesis zimachokera ku maphunziro a zinyama (mwachitsanzo, zebrafish20, mice21 kapena nkhuku, momwe zimakhalira mtengo wa 2). zokayikitsa, ndipo chofunika kwambiri, musamatchule mwatsatanetsatane njira zachitukuko chaumunthu.Zitsanzozi zimakhalanso zochepa kwambiri kuti athe kuyesedwa m'njira zambiri zowonongeka.Choncho, ndondomeko yathu yokonzanso mapangidwe a 3D mu vitro amapambana mu vivo zinyama komanso zitsanzo zina za chikhalidwe cha 2D cell.Monga momwe tafotokozera kale, kugwiritsira ntchito malo ogwiritsira ntchito malo a 3D amalola kugwiritsa ntchito mawonekedwe amtundu wa 3D. maselo mu crypt-villus axis poyankha zosiyanasiyana mucosal kapena chitetezo cha m'thupi. gut microbiota imapanga midzi yake ndi synergistically imapanga tizilombo tating'onoting'ono tating'ono tating'ono tating'ono (mwachitsanzo, mafuta afupiafupi) omwe amapanga gulu la ma cell ndi stem cell niches mu basal crypts.
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 zinthu zenizeni za morphogen23,24,25. lumen imatsekedwa mkati mwa organoid ndipo, chifukwa chake, kukhazikitsidwa kwa zigawo zounikira monga ma cell a microbial 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 angapo ofufuza amagwiritsa ntchito scaffolds zokonzedweratu za 3D hydrogel kutsanzira kapangidwe ka m'matumbo a epithelial popanga ma cell a m'matumbo amunthu pawokha pa gel osakaniza. Physiologically relevant morphogen gradients, kukhazikitsa chiwerengero chapamwamba cha epithelial structure ndi stroma-epithelial crosstalk mwa kuphatikizapo maselo a stromal mu scaffold. 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, komanso kutuluka kwa intraluminal fluid kungakhalenso recapitulated pogwiritsa ntchito microfluidics moduli. komanso imaphatikizapo kayendedwe ka matumbo a mechanobiological.Njira zosindikizira za 3D kuchokera ku gulu lomwelo zinatha kupanga machubu ang'onoang'ono a m'matumbo ndi njira zodziwikiratu za morphogenetic.Ngakhale kuti mapangidwe ovuta a zigawo zosiyana za m'matumbo mkati mwa chubu, chitsanzochi chimakhalanso ndi kutuluka kwa luminal fluid ndi kusinthika kwa makina. Proposed protocol amapereka modzidzimutsa m'matumbo morphogenesis, physiologically zogwirizana kukameta ubweya kupsyinjika, biomechanics kuti amatsanzira matumbo motility, kupezeka kwa odziimira apical apical ndi basolateral zigawo, ndi kulenganso zovuta zamoyo microenvironments modularity.Choncho, mu vitro 3D morphogenesis protocol athu angapereke njira yowonjezereka ya 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 otsiriza, ndi maselo a chitetezo cha mthupi. gut-on-a-chip ndi hybrid-on-a-chip imatilola kukonzanso zosanjikiza za 3D epithelial layer, zovuta zowonjezera zachilengedwe monga epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 ndipo, m'chitsanzo chathu, mawonekedwe a crypt-villus omwe amapereka tsinde cell niches, kukhalabe ma cell a cell. fibroblasts) mu mesenchyme imagwira ntchito yofunika kwambiri popanga mapuloteni a ECM komanso kuwongolera matumbo a morphogenesis mu vivo35,37,38.Kuwonjezeredwa kwa maselo a mesenchymal ku chitsanzo chathu kumapangitsa kuti ma morphogenetic apangidwe bwino komanso kugwirizanitsa maselo. microenvironment.Kuwonjezera apo, zigawo za vasculature zomwe zingathe kugwirizanitsidwa pakati pa zitsanzo za minofu ndizofunikira kwambiri pamene zitsanzo za minofu zimapangidwira kuti ziwonetsere kuyanjana kwamagulu ambiri.Choncho, maselo a endothelial angafunike kuphatikizidwa kuti apange mawonekedwe olondola kwambiri a thupi ndi kuthetsa kwa organ. chitetezo chokwanira potengera matenda a m'mimba.
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. Mwachiwonekere, mawonekedwe osasunthika m'chipinda cha apical sichimapangitsa kuti chikhalidwe cha bakiteriya chikhale nthawi yayitali mu tchipisi wosakanizidwa. Ngakhale titha kuyambitsa 3D morphogenesis muzoyika za Transwell tikamagwiritsa ntchito tchipisi wosakanizidwa, kuchepa kwa ma biomechanics okhudzana ndi thupi komanso kutuluka kwamadzimadzi apical kumatha kuchepetsa kuthekera kwa nsanja ya haibridi.
Kumanganso kwathunthu kwa axis ya crypt-villus axis m'matumbo-pa-chip ndi hybrid-on-a-chip zikhalidwe sizinakhazikitsidwe kwathunthu. 3D epithelium, madera a crypt ndi oyipa sanasinthidwe momveka bwino. ndipo kutalika kwa matumbo aang'ono ndi ~ 600 µm41.
Kuchokera ku lingaliro la kulingalira, in situ super-resolution imaging ya 3D microarchitectures ikhoza kungokhala 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 chingafunike. PDMS.Kuphatikiza apo, popeza kusanjikiza-ndi-wosanjikiza microfabrication m'matumbo pa chip kumaphatikizapo kumamatira kosatha pakati pa gawo lililonse, zimakhala zovuta kwambiri kutsegula kapena kuchotsa pamwamba kuti muwone mawonekedwe a pamwamba a epithelial layer.Mwachitsanzo, pogwiritsa ntchito makina oonera ma electron microscope (SEM).
Hydrophobicity ya PDMS yakhala yolepheretsa maphunziro a microfluidic okhudzana ndi mamolekyu ang'onoang'ono a hydrophobic, popeza PDMS imatha kutsatsa mamolekyu otere a hydrophobic. Njira zina za PDMS zitha kuganiziridwa ndi zinthu zina za polymeric. glycol) 43 ) akhoza kuganiziridwa kuti achepetse 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.Kuchepetsa uku kungathe kusinthidwa kwambiri ndi scalability ya 94well, chikhalidwe cha 94well, chikhalidwe cha 94well, chosinthika, chosinthika, kapena chosinthika. 384-olowetsa bwino porous omwe amalola kubwezeredwa kosalekeza ndikuchotsa media media).
Kuti tisunge 3d morphogeneis ya epithelilium ya anthu a epithelium ya anthu mu vitro, tidagwiritsa ntchito chipya cha microfluidic chomwe chili ndi mawonekedwe a microfchel zigawo zomwe zimakula M'mapulatifomu onsewa, morphogenesis ya maselo osiyanasiyana a m'matumbo a munthu amatha kuwonetsedwa pogwiritsa ntchito njira yowonongeka kuti achotse otsutsa a morphogen kuchokera ku chipinda chotsatira. matumbo epithelial maselo (Caco-2 maselo) kapena anthu matumbo organoids; mabokosi 2-5), (iii) chikhalidwe cha maselo a m'mimba 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) . mu vitro morphogenesis poyerekezera epithelial morphogenesis ndi malo, temporal, conditional, kapena procedural controls.
Tidagwiritsa ntchito nsanja ziwiri zachikhalidwe: gut-on-a-chip yokhala ndi njira zowongoka kapena njira zolumikizirana zosagwirizana, kapena tchipisi zosakanizidwa zomwe zili ndi zoyika za Transwell (TW) mu chipangizo cha microfluidic, chopangidwa monga momwe tafotokozera mu Box 1, ndi sitepe 1 -5. (Caco-2 kapena human intestinal organoids) ndi ndondomeko ya chikhalidwe yomwe imagwiritsidwa ntchito mu ndondomekoyi."In vitro morphogenesis" imasonyeza masitepe onse omwe ma cell a Caco-2 kapena organoid-derived epithelial cell amapangidwira pamatumbo a m'matumbo kapena pa Transwell amaikapo chip hybrid, ndikutsatiridwa ndi kulowetsedwa kwa 3D morphogenesis ndi mapangidwe a nambala ya epithelial yomwe ili pansipa. Ntchitoyi imapereka zitsanzo za momwe zigawo za epithelial zokhazikika zingagwiritsire ntchito, mwachitsanzo, mu kusiyanitsa kwa maselo, maphunziro a matumbo a physiology, kukhazikitsidwa kwa tizilombo toyambitsa matenda tizilombo toyambitsa matenda, ndi njira za matenda. chip.MUC2 signing imapezeka m'maselo a goblet ndi ntchofu zotulutsidwa kuchokera ku mucosal surfaces. Zithunzi za fluorescent mu Gut Physiology zimasonyeza ntchofu yopangidwa ndi madontho a sialic acid ndi zotsalira za N-acetylglucosamine pogwiritsa ntchito fulorosenti ya tirigu germ agglutinin. matumbo pa tchipisi kutsanzira kumasonyeza thanzi labwino motsutsana ndi matumbo otupa m'matumbo otupa m'matumbo pansi pa zovuta za thupi ndi ma antigen a bakiteriya (mwachitsanzo, lipopolysaccharide, LPS) ndi maselo a chitetezo cha mthupi (mwachitsanzo, PBMC; green) Maselo a Caco-2 adapangidwa kuti akhazikitse 3D epithelial layer. umboni.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 zinapangidwa pogwiritsa ntchito PDMS replicas zomwe zinapangidwa kuchokera ku silicon molds ndi soft lithography1,44 ndi zojambula 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-chip-on-design (Expressed Data) awiri juxtaposed kufanana molunjika microchannels, zasintha kukhala zovuta m'matumbo-on-a-chip (Extended Data Fig. 1b) yomwe imaphatikizapo ma microchannels okhotakhota kuti apangitse Kuchulukitsa kwamadzi okhalamo nthawi, machitidwe osakanikirana osakanikirana, ndi matembenuzidwe ochuluka a maselo otukuka (mkuyu 2a-f) 12. Tawonetsa kuti Gut-Chip convoluted imapangitsanso kwambiri 3D morphogenesis mu nthawi yofanana ndi kukula kwa epithelial poyerekeza ndi chiyambi cha Gut-Chip, mosasamala kanthu za mtundu wa selo. ndi machitidwe a SU-8 anapereka zinthu zoipa pambuyo pobowola (mkuyu 2a) .Kupanga matumbo pa chip, chokonzekera chapamwamba cha PDMS chosanjikiza chinagwirizanitsidwa motsatizana ndi filimu ya porous PDMS ndiyeno imagwirizana ndi PDMS yapansi ya PDMS ndi kugwirizanitsa kosasinthika pogwiritsa ntchito mankhwala a corona (mkuyu 2b-f) . makina ang'onoang'ono a microfluidic omwe angagwirizane ndi zoikamo za Transwell (mkuyu 2h ndi Deta Yowonjezereka Mkuyu. 2) . Njira yolumikizira imachitidwa pochiza malo a PDMS replica ndi galasi ndi plasma ya okosijeni kapena corona treatment.Atamaliza kutseketsa kwa chipangizo chopangidwa ndi microfabricated chogwiritsidwa ntchito ku chubu la silicone, chipangizo cha 3D mu epiliforum chokonzekera chokonzekera cha epiliphoum cha 3D chinali chokonzeka kuchita (Chithunzi 2 g).
a, Schematic illustration of the preparation of PDMS parts from SU-8 patterned silicon molds.The uncureed PDMS solution anatsanuliridwa pa silicon nkhungu (kumanzere), kuchiritsidwa pa 60 °C (pakati) ndi kugwetsedwa (kumanja).The demolded PDMS anadulidwa mzidutswa ndi kutsukidwa kuti agwiritse ntchito.b, Chithunzi chogwiritsidwa ntchito pa silicon mold. silicon mold yomwe imagwiritsidwa ntchito popanga PDMS porous membrane.d, Zithunzi zingapo za PDMS zam'mwamba ndi zam'munsi ndi chipangizo cham'mimba chosonkhanitsidwa.e, Schematic of the alignment of the chapper, membrane, and lower PDMS components. convoluted microchannels ndi vacuum chambers.g, Kukonzekera kwa gut-on-a-chip kwa microfluidic cell culture.M'matumbo opangidwa pa chip wosonkhanitsidwa ndi chubu la silikoni ndi syringe anayikidwa pa coverslip.Chip chipangizo chinayikidwa pa chivindikiro cha 150 mm Petri mbale kuti processing.The wosakanizidwa binder silikoni ntchito kutseka Silicone chubu ntchito. Kupanga ndi 3D morphogenesis pogwiritsa ntchito tchipisi chosakanizidwa.Zoyika za Transwell zokonzedwa modziyimira pawokha ku chikhalidwe cha 2D ma monolayers a m'matumbo a epithelial cell adalowetsedwa mu chip hybrid kuti apangitse matumbo a 3D morphogenesis.Sing'anga amapangidwa kudzera mu ma microchannels pansi pa cell layer yomwe idakhazikitsidwa pa Transwell insert.Scale bar, 4 cm. Elsevier.
Mu protocol iyi, mzere wa cell wa Caco-2 ndi organoids wa m'mimba unagwiritsidwa ntchito ngati magwero a epithelial (mkuyu 3a) . Mitundu yonse iwiri ya maselo inakula paokha (Bokosi 2 ndi Bokosi 5) ndipo amagwiritsidwa ntchito kubzala ma microchannels opangidwa ndi ECM a pa-chip gut kapena Transwell insert. (pakati pa ndime 10 ndi 50) mu T-flasks amakololedwa kukonzekera dissociated cell suspensions ndi trypsinization madzimadzi (bokosi 2) . Human intestinal organoids kuchokera m'matumbo biopsies kapena opaleshoni resections anali otukuka mu Matrigel scaffold domes mu 24-chitsime mbale kuthandizira structural W. R-spondin, ndi Noggin) ndi zinthu za kukula zomwe zakonzedwa monga momwe zafotokozedwera mu Bokosi 3 zinawonjezeredwa tsiku lililonse mpaka ma organoids anakula kufika ~ 500 µm m'mimba mwake. Ma organoids okula bwino amakololedwa ndikugawidwa m'maselo amodzi kuti abzalire m'matumbo kapena Transwell amaika pa chip (Bokosi 5) . Matenda a Crohn, khansa yapakhungu, kapena wopereka wamba), malo otupa (mwachitsanzo, zilonda motsutsana ndi malo omwe alibe zotupa) komanso malo am'mimba m'matumbo (monga duodenum, jejunum, ileum, cecum, colon, kapena rectum). matumbo organoids.
a, Workflow for induction of gut morphogenesis in the gut chip.Caco-2 human intestinal epithelium ndi intestinal organoids amagwiritsidwa ntchito mu protocol iyi kuti asonyeze 3D morphogenesis.Maselo a epithelial akutali adayikidwa mu chipangizo chokonzekera m'matumbo-pa-chip (chip-chip kukonzekera) .Kamodzi ma cell amaikidwa pa seedased (chipMS) atayikidwa pa membrane) pa tsiku la 0 (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 chikhalidwe cha morphogenesis pambuyo pa masiku 5 chinachitika, chikhalidwe cha micromorphogenesis cha 5 chinachitika. D5) . Zithunzi zosiyana za gawo zimasonyeza maonekedwe oimira maselo a Caco-2 pa sitepe iliyonse yoyesera kapena nthawi (bar graph, 100 µm) . Zithunzi zinayi zowonetsera zojambulajambula zomwe zikuwonetseratu ma cascades a m'matumbo a morphogenesis (pamwamba kumanja) . chithunzi chosonyeza malo okulirapo (bokosi loyera) likuwonetsa microvilli yosinthidwanso pa 3D Caco-2 wosanjikiza (kumanja).c, Kuyang'ana kutsogolo kwa Caco-2 3D yokhazikitsidwa, claudin (ZO-1, red) ndi nembanemba yopitilira m'malire ya burashi yotchedwa F-actin (yobiriwira) ndi nuclei (buluu) yowoneka bwino ya ma cell owoneka bwino a Caco-2 3D. tchipisi.Mivi yolozera ku schematic yapakati imasonyeza malo a ndege yapakatikati pa kawonedwe kalikonse.d, Nthawi ya kusintha kwa ma morphological mu organoids otukulidwa pa chip chopezedwa ndi gawo losiyanitsa maikulosikopu pa masiku 3, 7, 9, 11, ndi 13. Pamwambapa (pamwamba kumanja) akuwonetsa kukulitsa kwapamwamba kwa chithunzi choperekedwa cha 3D chojambula chojambulidwa cha 3D chojambula chopangidwa ndi guIC kapena Dganoid chojambula cha 3D chojambulidwa. pa kagawo kotengedwa pa tsiku la 7.f, Zithunzi zophimbidwa za immunofluorescence zowonetsa zolembera zama cell stem (LGR5; magenta), ma cell a goblet (MUC2; wobiriwira), F-actin (imvi) ndi ma nuclei (cyan) okulira pa tchipisi m'matumbo kwa masiku a 3, motsatana (Kumanzere) ndi masiku 13 (pakati) organolitended wosanjikiza3. LGR5 chizindikiro popanda chizindikiro cha MUC2. Zithunzi za Fluorescence zosonyeza epithelial microstructure (kumanja) ya 3D 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 mosiyana. 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.Pa culturing Caco-2 maselo, yambitsa pamwamba ndi UV / ozoni mankhwala yekha kunali kokwanira kuchepetsa hydrophobicity ECM ndi kulumikiza pamwamba ECM hydrophobicity ndi kulumikiza pamwamba Caatco ma cell a Caatco PDMS. PDMS nembanemba. maselo amamangiriridwa, ndi microfluidic selo chikhalidwe amayamba perfusing sing'anga kumtunda microchannel mpaka maselo kupanga monolayer wathunthu, pamene m'munsi microchannel amakhalabe malo amodzi.Njira 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 zochitirapo kale kuti ziyambitse pamwamba pa zoyikapo porous.Pa kukula kwa maselo a Caco-2 pa zoikamo za Transwell, ECM ❖ kuyanika pa zoikapo porous imathandizira kumangirira kwa ma cell a Caco-2 osagwirizana (<1 ora) ndi mapangidwe olimba otchinga (<1-2 masiku) . ku nembanemba pamwamba (<3 h) ndi kusungidwa mpaka organoids kupanga monolayer wathunthu ndi chotchinga umphumphu .Transwell zikhalidwe zimachitidwa mu 24-chitsime mbale popanda kugwiritsa ntchito tchipisi wosakanizidwa.
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 microchannels (mkuyu 3a) .Monga tafotokozera kale, ndikofunika kuti tidziwitse kutuluka kwamadzimadzi mu continent continuous fluid (mkuyu 3a) . Kuti tipereke zakudya zokwanira ndi seramu ku maselo omangidwa pazitsulo za porous ndikupanga kupsinjika kwa kumeta ubweya wonyezimira, timagwiritsa ntchito maulendo apawiri m'matumbo pa chip. pambuyo poyambitsa kayendedwe ka basolateral mumagulu onse a chikhalidwe.
Maonekedwe a morphological a microengineered 3D epithelial layers akhoza kufufuzidwa pogwiritsa ntchito njira zosiyanasiyana zoganizira, kuphatikizapo gawo la kusiyana kwa microscopy, differential interference difference (DIC) microscopy, SEM, kapena immunofluorescence confocal microscopy (Zithunzi 3 ndi 4) . Chifukwa cha kuwala kwa kuwala kwa PDMS ndi mafilimu a polyester, mapulaneti onse a gut-on-a-chip ndi hybrid chip angapereke nthawi yeniyeni yojambula zithunzi popanda kufunikira kwa kugawa kapena kupasuka kwa chipangizocho. (wt / vol) paraformaldehyde (PFA), yotsatiridwa ndi Triton X-100 ndi 2% (wt / vol) ) bovine serum albumin (BSA), mu dongosolo.Malingana ndi mtundu wa selo, ma fixatives osiyanasiyana, permeabilizers, ndi otsekereza othandizira angagwiritsidwe ntchito.Ma antibodies oyambirira omwe akulozera ku mzere wodalira mzere wa cell kapena chip markers amatsatiridwa ndi maselo amtundu wa imtuze amatsatiridwa pamodzi ndi utoto wa counterstain womwe umaloza nyukiliya (mwachitsanzo, 4′, 6-diamidino-2-phenylene) indole, DAPI) kapena F-actin (mwachitsanzo, fluorescently labeled phalloidin) .Fluorescence-based live imaging ingathenso kuchitidwa mu situ kuti azindikire kutulutsa kwa ntchentche (Mkuyu wosiyana ndi "Cell" koloni, "Gulu 1", "Cell" mosasintha. ma cell microbial (mkuyu 1, "Host-microbe co-culture") , kulembedwa kwa maselo a chitetezo cha mthupi (mkuyu 1, 'Matenda Modelling') kapena ma contours a 3D epithelial morphology (mkuyu 3c, f ndi 4b, c) .Posintha matumbo pa chip kuti alekanitse gawo laling'ono la microf. 2, 3D epithelial morphology komanso microvilli pamalire a apical burashi amatha kuwonedwa ndi SEM (mkuyu 3b) kusanthula chibadwa.
a, Workflow for induction of intestinal morphogenesis in a hybrid chip.Caco-2 ndi intestinal organoids amagwiritsidwa ntchito mu protocol iyi kusonyeza 3D morphogenesis mu hybrid chip platform.Maselo osakanikirana a epithelial adayikidwa m'malo okonzekera a Transwell (TW prep; onani chithunzi pansipa) .Nthawi imodzi maselo adayikidwa ndi ma cell a polyseed (ma cell a transwell) chikhalidwe pansi pa malo amodzi (TW chikhalidwe) .Pambuyo pa masiku 7, mmodzi Transwell Ikani munali 2D monolayer wa maselo epithelial anali Integrated mu hybrid Chip kuyambitsa basolateral otaya (Flow, BL), amene potsiriza anatsogolera m'badwo wa 3D epithelial wosanjikiza (morphogenesis). (C103 mzere) wokwera m'matumbo pa sitepe iliyonse yoyesera kapena nthawi.Mapangidwe apamwamba akuwonetserako kuyesa kwa sitepe iliyonse.b, tchipisi cha Hybrid (chitsanzo chakumanzere) chingapangitse 3D morphogenesis ya maselo a organoid epithelial okhala ndi pamwamba-pansi confocal microscopy mawonedwe otengedwa pazigawo zosiyana siyana, mawonedwe apakati a Z, ndi mawonedwe apakati apakati; 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) poyerekeza 3D morphology2D morphology. mawonedwe opingasa ofukula (omwe ali pakona yakumanja yakumanja; “XZ”) amawonetsanso mawonekedwe a 2D ndi 3D. Scale bar, 100 µm.c Yosindikizidwanso ndi chilolezo chochokera muzolemba.4. Elsevier.
Ulamuliro ukhoza kukonzedwa mwa kukulitsa maselo omwewo (Caco-2 kapena maselo a matumbo a organoid epithelial) kukhala ma monolayers awiri-dimensional pansi pa chikhalidwe chokhazikika cha chikhalidwe. kufananizidwanso.
Njira yofewa ya lithography iyenera kuchitidwa m'chipinda choyera. Pagawo lililonse pa chip (zigawo zakumtunda ndi zapansi ndi nembanemba) ndi tchipisi zosakanizidwa, ma photomasks osiyanasiyana anagwiritsidwa ntchito ndikupangidwira pazitsulo zosiyana za silicon chifukwa kutalika kwa ma microchannels kunali kosiyana.Kutalikirana kwa ma microchannel apamwamba ndi otsika a m'matumbo pa chip ndi 500 µmµm kutalika kwa 500 µmµm hybrid chip ndi 200 µm.
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 pakati pa chofufumitsa cha silicon chotsukidwa. 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 a 100 rpm / s. 500 µm kutalika kwa matumbo akumtunda pa chip;
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, yemwe amatha kuwululidwa ndi 0 wosanjikiza ndi 0 mubokosi la 0. µm mkulu.
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 choyimirira mpaka 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 kusiyanasiyana malinga ndi kukula kwa mbale ya galasi. Onetsetsani kuti mugwiritse ntchito makina okwana SU-8 kuti muchotseretu SU-8.Mwachitsanzo, mukamagwiritsa ntchito galasi la 150 mm m'mimba mwake ndi mphamvu ya 1 L, gwiritsani ntchito ~ 300 mold mold . kusinthasintha kwapang'onopang'ono.
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,1H,2H,H trichloro 2H-perfluorooctyl) silane yankho pagalasi slide ndikuyika vacuum kuti silanization.
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.