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Human gut morphogenesis inotanga crypt-villus features of 3D epithelial microarchitecture and spatial organisation.Ichi chimiro chakasiyana chinodiwa kuchengetedza gut homeostasis nekudzivirira stem cell niche mu basal crypt kubva kune exogenous microbial antigens uye metabolites yavo.Pamusoro pezvo, intestinal villi uye secreting mucussal mucussal mucusal inopesana neaspitelithesis inodzivirira masero aripo. Nokudaro, kudzokorora 3D epithelial structures kwakakosha pakuvakwa kwe in vitro gut models.Notably, iyo organic mimetic gut-on-a-chip inogona kuunza spontaneous 3D morphogenesis ye intestinal epithelium nekuvandudzwa kwehutano hwehupenyu uye biomechanics. fluidic chip pamwe chete neTranswell embedded hybrid chip.Tinotsanangura nzira dzakadzama dzekugadzira mudziyo, kurima kweCaco-2 kana intestinal organoid epithelial masero munzvimbo dzakajairwa pamwe nepapuratifomu yemicrofluidic, induction ye 3D morphogenesis, uye hunhu hweyakagadzika 3D epithelia inoshandiswa nemultiple general inoyerera yeprotocol inoyerera. ye 5 d.Our in vitro morphogenesis nzira inoshandisa physiologically yakakosha shear stress uye mechanical motion uye haidi yakaoma cell engineering kana manipulation, iyo inogona kudarika mamwe maitiro aripo.Tinofungidzira kuti purogiramu yedu yakarongwa inogona kuva nemigumisiro yakawanda kunharaunda yekutsvakurudza yebiomedical, ichipa nzira yekudzorerazve 3D intestinal epithesis, biomedical layer, biomedical epithelical uye kliniki.
Ongororo dzinoratidza kuti intestinal epithelial Caco-2 masero akagadzirwa mugut-on-a-chip1,2,3,4,5 kana bilayer microfluidic device6,7 inogona kungoerekana yaitika 3D morphogenesis in vitro pasina kunzwisisa kwakajeka kweiyo nzira. sis in vitro, iyo yakaratidzwa neCaco-2 uye murwere-inotorwa intestinal organoids.Muchidzidzo ichi, takanyanya kutarisa nezvekugadzirwa kwesero uye kugoverwa kwekusungwa kweanopikisa Wnt ane simba, Dickkopf-1 (DKK-1), mugut-on-a-chip uye yakagadziridzwa michina yemicrofluidic ine Transwell inserts, inonzi "Hybrid Chip" . d-related protein 1, kana Soggy-1) ku-on-chip gut inhibits morphogenesis kana kukanganisa yakagadziriswa 3D epithelial layer, zvichiratidza kuti antagonistic stress panguva yetsika inokonzera intestinal morphogenesis in vitro.Naizvozvo, nzira inobatsira yekuwana robust morphogenesis pane epitheliant inogadzirisa mazinga epithelial inogadzirisa inogadzirisa zvishoma kana kuti inobvisa zvishoma. kugezesa (eg, mugut-on-a-chip kana hybrid-on-a-chip platforms) kana diffusion .Basolateral media (eg, kubva kuTranswell inopinza mumadhamu makuru epasi mumatsime).
Muprotocol iyi, tinopa nzira yakadzama yekugadzira gut-on-a-chip microdevices uye Transwell-insertable hybrid chips (matanho 1-5) kune tsika yemukati epithelial maseru pa polydimethylsiloxane (PDMS) -based porous membranes (matanho 6A, 7A, 8, 9) kana polyesterB inopinza 6 membranes, 9 nhanho, 9 nhanho 3D morphogenesis in vitro (nhanho 10) .Takacherechedzawo ma cellular uye ma molecular features anoratidza tishu-specific histogenesis uye mutsara-inoenderana nemutsara wemasero akasiyana-siyana kuburikidza nekushandisa maitiro akawanda ekufungidzira (matanho 11-24) . , kusikwa kwe2D monolayers, uye intestinal biochemical uye Reproduction of the biomechanical microenvironment.in vitro.Kuita 3D morphogenesis kubva ku2D epithelial monolayers, takabvisa morphogen antagonists mumhando mbiri dzakasimudzwa nekuyerera kwepakati mukati mebasolateral compartment yetsika.Pakupedzisira, regene regene regene rinoshandiswa reD regene regene yemuenzaniso weD regene regene regene regene regene regene regene regene regene regene yeD regene regene regene regene regene regene regene regene regene regene yeD regene regene regene regene regene regene regene regene regene regene regene reD regene regene regene regene regene regene regene yeD regene regene regene regene regene regene. phogen-dependent epithelial growth, longitudinal host-microbiome co-cultures, pathogen infection, kukuvara kwekuputika, epithelial barrier dysfunction, uye probiotic-based therapies Muenzaniso.influences.
Protocol yedu inogona kubatsira kune huwandu hwakawanda hwesainzi mune zvekutanga (semuenzaniso, intestinal mucosal biology, stem cell biology, uye developmental biology) uye kushandisa tsvagiridzo (semuenzaniso, preclinical zvinodhaka kuongororwa, chirwere chemuenzaniso, tishu engineering, uye gastroenterology) yakakura kukanganisa. seminated kune vateereri vachidzidza maitiro emagetsi ekusimbisa masero panguva yekukura kwematumbo, kuberekwa patsva kana homeostasis .Pakuwedzera, chirongwa chedu chinobatsira pakubvunzurudza utachiona pasi pemishonga yakasiyana-siyana inotapukira yakadai seNorovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonellae Salmonellae.Vateereri vechirwere chechirwere uye pathogenesis inobatsirawo.Kushandiswa kwe-on-chip gut microphysiology system inogona kubvumira longitudinal co-culture 10 uye kuongorora kwakatevera kwekudzivirira kwemauto, mhinduro dzezvirwere zvekudzivirira uye kugadziriswa kwekukuvara kwepathogen mumatumbo (GI) turakiti 11 .Zvimwe zvirwere zveGIAcekyl, chirwere cheGrosaky, chirwere cheGroaceky, chirwere cheGroaceky, chirwere cheGroaceky pouchitis, kana kuti irritable bowel syndrome inogona kufananidzwa apo 3D intestinal epithelial layers inogadzirwa uchishandisa murwere 3D intestinal epithelial layers , zvirwere izvi zvinosanganisira villous atrophy, crypt shortening, mucosal damage, kana kukanganisa epithelial barrier.Biopsy kana stem cell-derived, kuti tsinde cell-derived1 muverengi ari nani, muverengi ari nani, muverengi we2, muverengi ari nani, muverengi we2. funga kuwedzera marudzi emasero anoenderana nechirwere, akadai semurwere peripheral blood mononuclear cells (PBMCs), kumhando dzine 3D intestinal villus-crypt microarchitectures.tishu-chaiyo immune masero, 5.
Sezvo 3D epithelial microstructure inogona kugadziriswa uye kuoneswa pasina chikamu chechikamu, vataridzi vanoshanda pane spatial transcriptomics uye yakakwirira-resolution kana super-resolution imaging inogona kufarira kumepu yedu ye spatiotemporal dynamics yemajini uye mapuroteni pane epithelial niches.Kufarira teknolojia.Kupindura kune microbial kana immune stimuli.Uyezve, longitudinal host-microbiome crosstalk 10, 14 iyo inobatanidza gut homeostasis inogona kusimbiswa mu 3D intestinal mucosal layer nekubatana-culturing zvakasiyana-siyana zvisikwa zvipenyu, zvipembenene zviduku kana fecal microbiota-kunyanya-mu-gu-gut-a-gut.papuratifomu.Iyi nzira inonyanya kukwezva kune vateereri vanodzidza mucosal immunology, gastroenterology, human microbiome, culturomics uye kliniki microbiology vachitsvaga kurima yaimbove isina kuumbwa gut microbiota murabhoritari.Kana yedu in vitro morphogenesis protocol inogona kuchinjirwa kune scalable tsika mafomati, senge multiwell 43 tsime inoisa 46 tsime re 469 compartments, the protocol inogonawo kuparadzirwa kune avo vanogadzira mishonga, biomedical Kana yakakwirira-kuburikidza kuongorora kana kusimbiswa kwezvigadziro zvekugadzira chikafu.Sehuchapupu-hwe-nheyo, isu munguva pfupi yapfuura takaratidza kukwanisa kwe multiplex high-throughput morphogenesis system scalable to a 24-well-plate format.Pamusoro pe-161-yakawanda, 16-mberi yekutengeserana. Nzira yedu ye in vitro morphogenesis inogona kukwidziridzwa uye inogona kugamuchirwa neakawanda marabhoritari ekutsvagisa, indasitiri kana hurumende uye masangano anodzora kuti anzwisise cellular reprogramming ye in vitro gut morphogenesis pa transcriptomic level yekuyedza zvinodhaka kana biotherapeutics. iyo gut morphogenesis process.
Nhamba shoma yemhando dzekuongorora dzinoshandiswa nevanhu dzakashandiswa kudzidza intestinal epithelial morphogenesis, kunyanya nekuda kwekushayikwa kwezvibvumirano zvinogoneka kuti zvigadzirise 3D morphogenesis in vitro.Kutaura zvazviri, ruzivo rwakawanda rwezvino pamusoro pegut morphogenesis runobva pane zvidzidzo zvemhuka (semuenzaniso, zebrafish20, mice21 kana kuti inodhura zvakanyanya sei, inodhura uye inodhura yakawanda sei, huku uye huku-inodhura2). ly, usanyatsoona maitiro ekukura kwevanhu.Iyi mienzaniso inewo zvishoma mukukwanisa kwayo kuongororwa nenzira yakawanda-yakakura.Naizvozvo, protocol yedu yekuvandudza 3D tissue structures in vitro outperforms in vivo mhuka dzemhando pamwe chete nedzimwe tsika dzechinyakare 2D cell tsika dzemhando.Sezvakatsanangurwa kare, kushandisa 3D spaspitialization yenzvimbo yakasiyana-siyana yakabvumira 3D spaspitialism yenzvimbo yekuongorora masero epithelical. mukupindura kune zvakasiyana-siyana mucosal kana immune stimuli.3D epithelial layers inogona kupa nzvimbo yekudzidza kuti masero e microbial anokwikwidzana sei kuti aite nzvimbo niches uye ecological evolution mukupindura kune zvinhu zvinogamuchira (semuenzaniso, mukati nekunze kwemucus layers, secretion yeIgA uye antimicrobial peptides) . Uyezve, 3D epithelical epitheliation inobvumira kuti micromorphology ikwanise kubudisa maumbirwo ayo epithelical synthesis uye 3D epithelical synthesis inobvumira kuti micromorphology yaro ikwanise kunzwisisa kuti micromorphology ye 3D inobvumira kuti micromorphology yedu ibudise. s microbial metabolites (eg, short-chain fatty acids) iyo inogadzira sangano re cellular uye stem cell niches mu basal crypts.Zvinhu izvi zvinogona kuratidzwa chete apo 3D epithelial layers inosimbiswa in vitro.
Mukuwedzera kune nzira yedu yekugadzira 3D intestinal epithelial structures, kune dzakawanda in vitro nzira.Intestinal organoid tsika inyanzvi yemazuva ano yekugadzirwa kwetishu inyanzvi inobva pakurimwa kwemasero emakumbo emukati pasi pemamiriro ezvinhu chaiwo e-morphogen23,24,25.Zvisinei, kushandiswa kwe 3D organoid model for transportation is chascullomen-cosmetology, organoid-modules ye 3D organoid-module for transportation inchang-en-speeden organoid. d mukati me organoid uye, saka, kuunzwa kwezvinhu zvinopenya senge microbial masero kana exogenous antigens inogumira.Kuwanikwa kune organoid lumens kunogona kuvandudzwa uchishandisa microinjector, 26,27 asi nzira iyi invasive uye basa-yakanyanya uye inoda ruzivo rwakanyanya kuita.Uyezve, tsika dzechinyakare organoid dzakachengetwa mu hydrogel scaffolds pasi pe static mamiriro haaratidzi nemazvo anoshanda mu vivo biomechanics.
Dzimwe nzira dzinoshandiswa nemapoka akati wandei ekutsvagisa dzinoshandisa 3D hydrogel scaffolds kutevedzera ura epithelial chimiro nekugadzira ega ega maseru eura hwemunhu panzvimbo yegel. Gadzira masikafu e hydrogel uchishandisa 3D-yakadhindwa, diki-yakagayiwa, kana lithographically yakagadzirwa ega ega epithelial marongero ega ega epithelical masero. zvakakodzera morphogen gradients, kugadza high aspect ratio epithelial structure uye stroma-epithelial crosstalk kuburikidza nekubatanidza stromal cells mu scaffold.Zvisinei, maitiro ezvakagadzirirwa scaffolds anogona kudzivirira kuratidzwa kweiyo spontaneous morphogenetic process pachayo.Idzi mienzaniso haipiwo simba rinopenya kana kuti interstitial kuwana masero emagetsi ekugadzirisa masero emagetsi ekugadzirisa uye interstitial stress inoyerera pasi pemaseru e-morphogenetic inoyerera. basa.Kumwe kuongorora kwemazuva ano kwakashandiswa hydrogel scaffolds mune microfluidic platform uye patterned intestinal epithelial structures kushandisa laser-etching techniques.Mouse intestinal organoids inotevera maitiro ekugadzirisa maitiro ekugadzira intestinal tubular structures, uye intraluminal fluid inoyerera inogona kudzokororwa uchishandisa microfluidics module.Zvisinei, spobinoologic maitiro kana maitiro e-modules e-spobinoologic. ing maitiro kubva kuboka rimwe chete akakwanisa kugadzira miniature gut tubes ne-spontaneous morphogenetic process.Pasinei nekugadzirwa kwakaoma kwezvikamu zvakasiyana-siyana zvehutu mukati mechubhu, iyi modhi inoshayawo luminal fluid flow and mechanical deformation.Uyezve, kushanda kwemuenzaniso kunogona kuderedzwa, kunyanya mushure mokunge bioprinting yapera, inovhiringidza mamiriro ekuedza kana kuti cell-to-cell , cell-to-cell inobatana inopa pfupa. akakodzera shear stress, biomechanics inotevedzera gut motility, kuwanikwa kweapical apical and basolateral compartments, uye kugadzirwa patsva kwezvakaoma biological microenvironments of modularity.Naizvozvo, yedu in vitro 3D morphogenesis protocol inogona kupa nzira inopindirana yekukunda matambudziko enzira dziripo.
Protocol yedu yakanyatsotarisana ne3D epithelial morphogenesis, ine masero epithelial chete mutsika uye pasina mamwe marudzi emasero akakomberedza akadai semesenchymal masero, endothelial masero, uye masero ekudzivirira muviri.Sezvakatsanangurwa kare, nheyo yeprotocol yedu ndiyo induction ye epithelial morphogenesis kuburikidza nekubvisa morphogen inhibitors yakavanzwa pa-robobuso-yakagadzirwa-yakaiswa padivi re-robusod-yakatangwa ne-romobuso-yakatanga. chip uye hybrid-on-a-chip inotibvumira kudzoreredza iyo isingafadzi 3D epithelial layer, yakawedzera zvipenyu zvakaoma senge epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 uye, mumuenzaniso wedu, crypt-villus maficha anoburitsa stem cell niches mu basal cripts, mesenchymal anoramba achionekwa sechikamu chemaseru. mukugadzirwa kweECM mapuroteni uye kurongeka kwematumbo morphogenesis mu vivo35,37,38.Kuwedzerwa kwemasero emesenchymal kumuenzaniso wedu kwakasimudzira morphogenetic process uye cell attachment effective.The endothelial layer (kureva, capillaries kana lymphatics) inoita basa rinokosha pakugadzirisa molecular transport39 uye immune0viculament cell recruit,Fimmune cell cell recruit. yakabatana pakati pemhando dzemasikirwo chinhu chinodiwa kana mhando dzematishu dzakagadzirirwa kuratidza kupindirana kwenhengo dzakasiyana-siyana.Saka, masero ekupedzisira angada kubatanidzwa kuti aenzanise maitiro akanyatsojeka ehupenyu nekugadzirisa kwehutano hwenhengo.Masero ekudzivirira anotorwa nemurwere anokoshawo pakuratidza mhinduro dzekuzvarwa, antigen presentation, innate adaptive immune crosstalk, uye tissue-immunity immunity intestinfi.
Kushandiswa kwema hybrid chips kwakatwasanuka kupfuura gut-on-a-chip nekuti kugadzikwa kwemudziyo kuri nyore uye kushandiswa kweTranswell kuisa kunobvumira scalable tsika yegut epithelium.Zvisinei, inotengeswa inotengeswa Transwell inoisa ine polyester membrane haina elastic uye haigone kutevedzera peristaltic-sekufamba. pakutanga, static properties muapical compartment haigoni kugonesa kwenguva yakareba co-culture yebhakitiriya mumahybrid chips. Nepo isu tichikwanisa kupinza 3D morphogenesis muTranswell kuisa pakushandisa mahybrid chips, kushomeka kwephysiologically anoenderana biomechanics uye apical fluid kuyerera kunogona kudzikisira kugona kweiyo hybrid chip mapuratifomu ekushandisa kweanogona kushandiswa chip mapuratifomu.
Magadzirirwo akazara emunhu crypt-villus axis mugut-on-a-chip uye hybrid-on-a-chip tsika haasati anyatsosimbiswa.Sezvo morphogenesis ichitanga kubva pa epithelial monolayer, 3D microarchitectures haipeiwo morphological kufanana kune crypts in vivo. Lium, iyo crypt uye nharaunda dzakaipa hadzina kuganhurwa zvakajeka.Kunyangwe nzira dzakakwirira dzepamusoro pachipfu dzichitungamira kureba kwemicroengineered epithelium, kureba kwepamusoro kuchiri kugumira ~300–400 µm. Kudzika chaiko kwematumbu emunhu mumatumbu madiki uye mahombe ari ~µm, diki uye diki, diki uye diki diki diki ~ 135 µm uye urefu hwehurefu hwehurefu hwemukati memunhu. lli iri ~600 µm41.
Kubva pakuona kwekufungidzira, in situ super-resolution imaging ye 3D microarchitectures inogona kugumira kudumbu pane chip, sezvo inodiwa yekushanda chinhambwe kubva kune chinangwa lens kusvika kune epithelial layer iri pakurongeka kwemamirimita mashoma. by-layer microfabrication yehutu pane chip inosanganisira kunamatira zvachose pakati pegadzi rega rega, zvakaoma zvikuru kuvhura kana kubvisa chikamu chepamusoro chekuongorora chimiro chepamusoro che epithelial layer.Somuenzaniso, nekushandisa scanning electron microscope (SEM).
Iyo hydrophobicity yePDMS yave inomisa chinhu mumicrofluidic-based zvidzidzo zvinobata nehydrophobic mamorekuru madiki, sezvo PDMS inogona kusanyanya adsorb akadaro hydrophobic molecules.Alternatives kune PDMS inogona kutariswa nemamwe mapolymeric materials.Nerimwewo, pamusoro pekugadzirisa kwePDMS kana glycephilic (eg4polyethylene) kana PDMS (eg42polythy) ne PDMS (eg42polythy) 3) inogona kutariswa kuderedza adsorption yehydrophobic mamorekuru.
Pakupedzisira, nzira yedu haina kunyatsoratidzwa maererano nekupa kuongorora kwepamusoro-soro kana kuti "imwe-saizi-inokodzera-yose" kushandiswa-kushamwaridzana kwekuedza platform.Izvino protocol inoda syringe pombi per microdevice, iyo inotora nzvimbo muCO2 incubator uye inodzivirira kuedza kukuru.Kupera uku kunogona kuvandudzwa zvakanyanya ne scalability ye 94well, tsika ye24well, tsika ye24well-innovative, 96well, tsika ye24well, tsika ye24well-innovative, 34well, tsika ye24well-innovative. zvakanaka porous inopinza inobvumira kuenderera kuzere uye kubviswa kwebasolateral media).
Kuti tigadzirise 3D morphogenesis yedumbu remunhu epithelium in vitro, takashandisa microfluidic chip intestinal device ine microchannels mbiri dzakafanana uye elastic porous membrane pakati kuti tigadzire lumen-capillary interface. s, morphogenesis yemasero akasiyana-siyana ematumbo evanhu epithelial anogona kuratidzirwa nekushandisa kutungamira kwekugadzirisa kuyerera kuti abvise morphogen vanopikisa kubva muchikamu chebasolateral.Iyo yose yekuedza nzira (Mufananidzo 1) ine zvikamu zvishanu: (i) microfabrication yegut chip kana Transwellable hybrid chip 1-5lix (stepinal insertable chip ye1-5lix masero estinal) co-2 masero) kana munhu intestinal organoids;mabhokisi 2-5), (iii) tsika yemukati epithelial masero pamakumbo emukati kana ma hybrid chips (matanho 6-9), (iv) kuiswa kwe 3D morphogenesis in vitro (nhanho 10) uye (v)) kuratidza 3D epithelial microstructure (matanho 11-24) . genesis nekuenzanisa epithelial morphogenesis kune nzvimbo, temporal, conditional, kana procedural controls.
Takashandisa mapuratifomu maviri akasiyana etsika: gut-on-a-chip ane nzira dzakatwasuka kana nonlinear convoluted channels, kana mahybrid chips ane Transwell (TW) anoisa mumicrofluidic device, yakagadzirwa sezvinotsanangurwa muBhokisi 1, uye nhanho 1 -5. stinal organoids) uye maitiro etsika anoshandiswa muprotocol iyi."In vitro morphogenesis" inoratidza nhanho dzese umo Caco-2 kana organoid-inotorwa epithelial masero anogadzirwa pane intestinal chip kana paTranswell kuiswa kweiyo hybrid chip, inoteverwa nekuiswa kwe 3D morphogenesis uye kuumbwa kweiyo ine mavara epithelial nhamba inotaridzwa nhamba yepithelial nhamba iri pasi peiyo nhanho yemaitiro epithelial bhokisi. epithelial layers inogona kushandiswa, semuenzaniso, mu cell differentiation characterization, gut physiology zvidzidzo, kugadzwa kwehost-microbiome ecosystems, uye chirwere chekufananidzira.Immunofluorescence mifananidzo mu "Cell Differentiation" inoratidza nuclei, F-actin uye MUC2 inoratidzwa mu 3D Caco-2 epithelial inobudisa masero epithelial muC2 masero epithelial epithelial epithelial. mucosal surfaces.Mifananidzo yeFluorescent muGut Physiology inoratidza mucus inogadzirwa nekusvibisa sialic acid uye N-acetylglucosamine residues vachishandisa fluorescent wheat germ agglutinin.Mifananidzo miviri inopindirana mu "Host-Microbe Co-Cultures" inoratidza mumiririri host-microbiome gut-cultural panels yefluo-culture yakasvibirira mu co-culture yefluo. rescent protein (GFP) ine microengineered 3D Caco-2 epithelial masero.Panera yakarurama inoratidza nzvimbo yeGFP E. coli yakasanganiswa ne 3D Caco-2 epithelial cells, inoteverwa ne immunofluorescence staining neF-actin (tsvuku) uye nuclei (blue) .Chirwere chemuenzaniso chinoratidza hutano hwehutachiona hunopesana nehutachiona hwehutachiona hunofananidzira kupikisa kwechirwere chechirwere chinopesana nehutachiona hwehutachiona. s (semuenzaniso, lipopolysaccharide, LPS) uye masero ekudzivirira (semuenzaniso, PBMC;green).Masero eCaco-2 akagadzirwa kuti agadzire 3D epithelial layer.Scale bar, 50 µm.Mifananidzo iri mumutsara wezasi: "Kusiyanisa kwemasero" akagadziriswa nemvumo kubva kune referensi.2.Oxford University Press;Yakadhirowa nemvumo kubva kuRef.5.NAS;"Host-Microbe Co-Culture" yakagadziriswa nemvumo kubva kune ref.3.NAS;"Chirwere Modelling" yakagadziridzwa nemvumo kubva pareferensi.5.NAS.
Zvose zviri zviviri gut-on-chip uye hybrid chips zvakagadzirwa zvichishandisa PDMS replicas iyo yakagadziriswa kubva kune silicon molds ne soft lithography1,44 uye yakafananidzwa neSU-8.Kugadzirwa kwema microchannels mune imwe neimwe chip inotarirwa nekufungisisa nezve hydrodynamics yakadai seshear stress uye hydrodynamic pressure1,4,12.The original gut-on-tapad-design (Expressed Data) iyo yepakutanga yeData-chip-on-taasted parallel straight microchannels, yakashanduka kuva yakaoma gut-on-a-chip (Extended Data Fig. 1b) iyo inosanganisira peya ye curved microchannels kuti iite Kuwedzera kwemvura yekugara nguva, nonlinear kuyerera maitiro, uye multiaxial deformation yemaseru akagadzirwa (Fig. 2a-f) 12.Kana biochip-yakaoma-inoda kuva yakaoma. Isu takaratidza kuti iyo convoluted Gut-Chip inokonzerawo zvakasimba 3D morphogenesis mune imwe nguva yakafanana ine dhigirii yekukura kwe epithelial kana ichienzaniswa neyekutanga Gut-Chip, pasinei nekugadzirwa kwesero rudzi.Naizvozvo, kuti iite 3D morphogenesis, mutsara uye yakaoma pa-chip gut designs inoshandurwa.PDMS yakapiwa maitiro e-silicone-demo-demo.2a) .Kugadzira ura pachip, iyo yakagadzirwa yepamusoro yePDMS layer yakabatanidzwa sequentially kune porous PDMS firimu uye yakazoenderana nechepasi PDMS layer nekusingadzoreki bonding uchishandisa corona treater (Fig. 2b-f) Kugadzira hybrid chips, akapora PDMS replicas vakanga bonded kuti girasi slidec slidec slidec slidec slidec slid Fig. 2h uye Extended Data Fig. 2) .Kubatanidza kunoitwa nekurapa nzvimbo dzePDMS replica uye girasi neoxygen plasma kana corona treatment.Mushure mekuita sterilization ye microfabricated device yakanamatirwa nesilicone tube, chigadziro chekugadzirisa chakanga chakagadzirira kuita 3D morphogenesis ye intestinal epithelium 2g (Figure).
a, Schematic illustration of the preparation of PDMS parts from SU-8 patterned silicon molds.Mushonga usina kugadziriswa wePDMS wakadirwa pasilicon mold (kuruboshwe), wakaporeswa pa60 °C (pakati) uye wakaderedzwa (kurudyi).The demolded PDMS yakachekwa kuita zvidimbu uye yakacheneswa kuti ishandiswezve.b,Mufananidzo wakashandiswa kusilicone mold. gadzira iyo PDMS ine porous membrane.d, Mutsara wemifananidzo yekumusoro uye yakaderera PDMS zvikamu uye yakaunganidzwa pa-chip intestinal mudziyo.e, Schematic yekurongeka kwekumusoro, membrane, uye yakaderera PDMS zvikamu.Chimwe nechimwe chinosungirirwa chisingachinjiki neplasma kana corona treatment.f, Schematic-yakaitwa supercumber-yakagadzirwa gum-channel-gum-channel yakagadzirwa ne-contactum s.g, Setup of gut-on-a-chip for microfluidic cell culture.Ura hwakagadzirwa pachipu chakasanganiswa nesilicone chubhu nesirinji yakaiswa pa coverslip.Chip device yakaiswa pachivharo che 150 mm Petri dish for processing.Binder inoshandiswa kuvhara silicone tube.h, Visual chipshots chipsd genesis chips genesis. yakagadzirirwa yakazvimiririra kune tsika 2D monolayers ye intestinal epithelial masero akaiswa mu hybrid chip kuti aite intestinal 3D morphogenesis.Iyo svikiro inoshandiswa kuburikidza ne microchannels pasi pesero layer yakagadzwa paTranswell insert.Scale bar, 1 cm.h Yakadhindwazve nemvumo kubva kune referensi.4.Elsevier.
Muchirongwa ichi, Caco-2 cell line uye intestinal organoids yakashandiswa se epithelial sources (Fig. 3a) . Mhando dzose dzemasero dzakagadzirwa zvakasununguka (Bhokisi 2 uye Bhokisi 5) uye yakashandiswa kugadzira mbeu dzeECM-coated microchannels ye-chip gut kana Transwell inserts.Apo masero ari confluent mu-flaskly5% caverage masero (> 95 % cavecod caverage masero, 95% Cave coated masero) 0 uye 50) muT-flasks inokohwewa kuti igadzirire dissociated cell suspensions ne trypsinization fluid (bhokisi 2) .Human intestinal organoids kubva mumatumbo emukati kana kuvhiyiwa kubviswa kwakagadzirwa muMatrigel scaffold domes mu 24-well plates kuti itsigire structural structural organoid organoids from intestinal biopsies kana surgical resections were cultured in Matrigel scaffold domes in 24-well plates kuti itsigire structural structural organoids, ine spophogennt microenvironment, uye inokosha Asgginnt, Aspophondiuch Anokosha, uye Anokosha morspogennmenoch (muspophondiuch) inokosha. zvinhu zvinokura zvakagadzirirwa sezvinotsanangurwa muBhokisi rechitatu zvakawedzerwa zuva rega rega kusvikira ma organoids akura kusvika ~ 500 µm muhupamhi. Organoids yakakura zvakakwana inokohwewa uye inoparadzaniswa kuva masero mamwechete nokuda kwekudyara mumatumbu kana Transwell kuisa pane chip (Bhokisi 5) . Sezvatakambotaura kare, inogona kusiyaniswa maererano nechirwere type12,13, semuenzaniso, ulcerative colitis, chirwere chegomarara, chirwere cheronda, chirwere chegomarara, chirwere chegomarara, chirwere chegomarara, chirwere chegomarara, chirwere chegomarara. saiti (semuenzaniso, chironda chinopesana nenzvimbo isina maronda) uye nzvimbo yedumbu mukati meturakiti (eg, duodenum, jejunum, ileum, cecum, colon, kana rectum) .Tinopa optimized protocol muBhokisi 5 yekurima colonic organoids (coloids) iyo inowanzoda huwandu hwepamusoro hwemofogens kana madiki madiki.
a, Workflow for induction of gut morphogenesis in the gut chip.Caco-2 epithelium yemunhu uye intestinal organoids inoshandiswa muprotocol kuratidzira 3D morphogenesis.Masero epithelial ari oga akaiswa mbeu mumucheka wakagadzirirwa gut-on-a-chip device (chip gadziriro) .Kamwe masero akaiswa seeda (yakaiswa paPD) pazuva (yakaiswa seeda) membrane (yakaiswa paPD) zuva (yakabatanidzwa) membrane. D0), apical (AP) kuyerera kunotangwa uye kuchengetwa kwemazuva ekutanga e2 (kuyerera, AP, D0-D2) .Basolateral (BL) kuyerera kunotangwawo pamwe chete ne cyclic kutambanudza mafambiro (kutambanudza, kuyerera, AP uye BL) apo 2D yakakwana monolayer inoumbwa.Itestinal 3D morphogenesis yakaitika tsika yemazuva emumiririri we micromorphogenesis 5, mumiriri we 5 morphogenesis, mumiriri we 5 morphogenesis anoratidza kusiyana kwemazuva emumiririri we micromorphogenesis mushure me 5 morphgenesis. yeCaco-2 masero pane imwe neimwe nhanho yekuedza kana nguva yenguva (bar graph, 100 µm) .Mana madhiyamu ezvirongwa anoratidza anowirirana cascades of gut morphogenesis (kumusoro kurudyi) .Miseve yakanyungudutswa muchirongwa inomiririra kutungamirirwa kwemvura inoyerera.b, SEM mufananidzo unoratidza topology yepamusoro yeiyo yakasimbiswa 3D Cacolisheding bhokisi (white magn2ft). inoratidza yakagadziridzwa microvilli pa 3D Caco-2 layer (kurudyi).c, Yakatwasuka kumberi kwekuona kwakasimbiswa Caco-2 3D, claudin (ZO-1, tsvuku) uye inoenderera bhurashi bhodha membranes yakanyorwa F-actin (girinhi) uye nuclei (blue) Immunofluorescence confocal visualization ye epithelial inonongedza machipisi kune imwe neimwe nzvimbo inonongedza machipisi pamukati memukati mesero. confocal view.d, Time course of morphological change in organoids cultured on a chip yakawanikwa ne phase difference microscopy pamazuva 3, 7, 9, 11, uye 13. The inset (kumusoro kurudyi) inoratidza kukwidziridzwa kwepamusoro kwemufananidzo wakapiwa.e, DIC photomicrograph ye organoid 3D epithelium yakasimbiswa mugut restem 7 masero, inoratidzira overrescence masero. GR5;magenta), masero egoblet (MUC2; girini), F-actin (grey) uye nuclei (cyan) akakurira pagut chips kwemazuva 3, zvichiteerana (Kuruboshwe) uye 13-zuva (pakati) organoids pane epithelial layer.Onawo Yakawedzerwa Data Figure 3, iyo inosimbisa LGR5 siginecha isina MUCruoredhi ye3 microstructures chiratidzo cheMUCF2 chiratidziro cheMUC1D chiratidziro. organoid epithelium yakasimbiswa muura pane chip nekusvibisa plasma membrane neCellMask dhayi (kurudyi) pazuva rechigumi nematatu retsika.Scale bar ndeye 50 μm kunze kwekunge zvataurwa neimwe nzira.b Yakadhindwazve nemvumo kubva pareferensi.2.Oxford University Press;c Yakagadziridzwa nemvumo kubva kuChiratidzo.2.Oxford University Press;e uye f zvakagadziridzwa nemvumo nereferensi.12 Under Creative Commons License CC BY 4.0.
Mumudumbu pane chip, zvakakosha kugadzirisa hydrophobic pamusoro pePDMS porous membrane yekubudirira kweECM coating.Muiyi protocol, tinoshandisa nzira mbiri dzakasiyana dzekugadzirisa hydrophobicity yePDMS membranes.Nekugadzira masero eCaco-2, kushandiswa kwepamusoro neUV / ozone kurapwa chete kwakanga kwakakwana kuderedza hydrophobicity yeECMMS uye kubatanidza masero e-hydrophobicity yeCaCMMS uye Caco-2. zvachose, microfluidic tsika ye organoid epithelium inoda kemikari-based surface functionalization kuti iwane kunyatsogadziriswa kwemapuroteni eECM kuburikidza nekushandisa sequentially polyethyleneimine (PEI) uye glutaraldehyde kune PDMS microchannels.Mushure mekugadziriswa kwepamusoro, mapuroteni eECM akaiswa kuti avhare iyo inoshandiswa PDMS pamusoro pepamusoro kana kuti inoiswa mumasero eganodi ega ega ega anoiswa mumasero eganodi ega ega ega ega anoiswa mumasero egano. tsika inotanga nekunhuhwirira kwepakati chete mukati me microchannel yepamusoro kusvikira masero aita monolayer yakakwana, nepo microchannel yakaderera inochengetedza static mamiriro.Iyi nzira yakagadziridzwa yekushanda kwepamusoro uye ECM coating inogonesa kusungirirwa kwe organoid epithelium kuti iite 3D morphogenesis pamusoro pePDMS pamusoro.
Tsika dzeTranswell dzinodawo kupfekedzwa kweECM isati yadyarwa sero;zvisinei, tsika dzeTranswell hadzidi matanho akaoma ekugadzirisa kuti aite pamusoro pekuisa porous.Kukura kweCaco-2 masero paTranswell inserts, ECM coating pane porous inserts inokurumidzira kusungirirwa kwemaseru akaparadzaniswa eCaco-2 (<1 awa) uye yakasimba junction barrier formation (<1-2 mazuva) . <3 h) uye inochengetwa kusvikira ma organoids aita monolayer yakakwana ine barrier integrity .Transwell tsika dzinoitwa mu 24-well plates pasina kushandiswa kwe hybrid chips.
In vitro 3D morphogenesis inogona kutangwa nekushandisa kuyerera kwemvura kune basolateral chikamu cheiyo yakasimbiswa epithelial layer.Mudumbu pane chip, epithelial morphogenesis yakatanga apo iyo svikiro yakasvibiswa mukati mepamusoro uye yakadzika microchannels (Fig. 3a) . Sezvakatsanangurwa kare, zvakakosha kuunza introduate or morphogen kuyerera kwepachivande kuyerera kwepachivande morphogenment yemvura yakavanzika (continuous flow of the secret or morphogenesis). Kupa kudya kwakakwana uye serum kumaseru akasungwa pamaporous membranes uye kuunza luminal shear stress, tinowanzo shandisa kuyerera kwehuviri muhutu pane chip.Muma hybrid chips, Transwell inserts ine epithelial monolayers yakaiswa mukati me hybrid chips.Zvino, iyo yakashandiswa pasi pe basolateral side ye porous Transwell inoyerera. zvose tsika platforms.
The morphological features of microengineered 3D epithelial layers inogona kuongororwa nekushandisa nzira dzakasiyana-siyana dzekufungidzira, kusanganisira chikamu chekusiyanisa microscopy, differential interference difference (DIC) microscopy, SEM, kana immunofluorescence confocal microscopy (Figures 3 uye 4) .Phase kusiyana kana DIC imaging panguva yekuongorora kweDIC inogona kuitwa nyore nyore pane chero nguva yeDIC yekufungidzira. e kune kujeka kwemaziso ePDMS uye mafirimu epolyester, zvose gut-on-a-chip uye hybrid chip platforms zvinogona kupa chaiyo-nguva in situ imaging pasina kudiwa kwechikamu kana kupatsanurwa kwechigadzirwa.Paunenge uchiita immunofluorescence imaging (Figures 1, 3c, f uye 4b, c) inotevedzwa neTrixw / volde, masero anoteverwa neTrixw / volde) toni X-100 uye 2% (wt/vol) ) bovine serum albumin (BSA), mukurongeka.Zvichienderana nerudzi rwesero, zvigadziriso zvakasiyana-siyana, permeabilizers, uye blocking agents zvinogona kushandiswa.Masoja ekutanga anonangidzira mutsara-anotsamira sero kana nharaunda marker anoshandiswa kuratidza masero immobilized in situ pa chip, chechipiri-dhayi , inotevedzwa ne nyucleus dhayi 6, inotevedzwa ne-anti-dayi diamidino-2-phenylene) indole, DAPI) kana F-actin (eg, fluorescently labeled phalloidin) .Fluorescence-based live imaging inogonawo kuitwa in situ kuti ione kugadzirwa kwemucus (Fig.1, "Kusiyanisa Cell" uye "Gut physiology"), randomization yemasero ehutachiona (Fig. 1, "Host-microbe co-culture"), kutorwa kwemasero ekudzivirira muviri (Fig. 1, 'Chirwere Modelling') kana kuti contours ye 3D epithelial morphology (Fig. 4bt pakugadzirisa, f kuparadzanisa , 3D, kana kupatsanurwa kwe chip, f kuparadzanisa , kana 3D chip, kuparadzanisa). iyo yakaderera microchannel layer, sezvinotsanangurwa muRef.Sezvinoratidzwa muFig. 2, 3D epithelial morphology pamwe chete ne microvilli pamuganhu weapical brush inogona kuonekwa neSEM (Fig. 3b) .Kuratidzwa kwezviratidzo zvekusiyanisa kunogona kuongororwa nekuita quantitative PCR5 kana single-cell RNA inokura sequencing masero e-RNA e-hybride-sequencing. inokohwewa ne trypsinization uye yobva yashandiswa pakuongorora mamorekuru kana genetic.
a, Workflow for induction of intestinal morphogenesis in a hybrid chip.Caco-2 uye intestinal organoids inoshandiswa muprotocol kuratidzira 3D morphogenesis mu hybrid chip platform.Dissociated epithelial cells dzakasimwa mukugadzirwa kweTranswell inserts (TW prep; ona mufananidzo pazasi) (TW tsika) .Mushure memazuva manomwe, imwe Transwell insert ine 2D monolayer ye epithelial cells yakabatanidzwa mu hybrid chip kuti ibudise basolateral flow (Flow, BL), iyo yakazopedzisira yaita kuti kuve nekugadzirwa kwe 3D epithelial layer (morphogenesis) .Phase kusiyana micrographs inoratidza morphological features of normal organisation of human organ epithelium mutsara weCaledoral masero emumuviri wemuviri wemuviri wemuviri womunhu. nhanho kana nguva yenguva.Zvirongwa zviri muzvikamu zvepamusoro zvinoratidza maitiro ekuedza kune imwe neimwe nhanho.b, Hybrid chips (kuruboshwe schematic) inogona kutungamirira ku 3D morphogenesis ye organoid epithelial masero ane kumusoro-pasi confocal microscopy maonero akatorwa panzvimbo dzakasiyana dzeZ (kumusoro, pakati, uye pasi;ona hurongwa hwekurudyi uye mitsara ine madoti inoenderana).yakaratidza morphological maitiro akajeka.F-actin (cyan), nucleus (grey) c, Fluorescence confocal micrographs (3D angled view) ye organoid-derived epithelial cells akagadzirwa mu static Transwell (TW; inset mukati me white dashed box) maringe ne hybrid chip (yakakura yakazara pfuti) tichienzanisa pairivevertical morphus 2D morphology 2D maonero (akaiswa mukona yepamusoro yekurudyi; “XZ”) anoratidzawo 2D uye 3D zvinhu.Scale bar, 100 µm.c Yakadhindwazve nemvumo kubva pareferensi.4.Elsevier.
Kudzora kunogona kugadzirirwa nekugadzira masero mamwechete (Caco-2 kana intestinal organoid epithelial cells) muzvikamu zviviri-dimensional monolayers pasi pechinyakare static culture conditions.Notably, kupera kwekudya kunogona kuguma nekuda kwehuwandu hwehuwandu hwehuwandu hwe microchannels (kureva ~ 4 µL mumugero wepamusoro pane yepakutanga gut-chippiso yekugadzirwa kwechipiso uye kuenzaniswa kwekutanga kwechipiso chekugadzirisa mushure mekugadzirwa kwe emorphology.
The soft lithography process inofanira kuitwa muimba yakachena.Pane imwe neimwe layer pa chip (yekumusoro nezasi ma layers and membranes) uye mahybrid chips, akasiyana photomasks akashandiswa uye akagadzirwa paasiyana silicon wafers nekuti hurefu hwema microchannels hwaive hwakasiyana.The target heights of the top and low low microchannels of the gut on the chip are 500 chip µm hrib 200 µm.
Isa 3-inch silicon wafer mundiro ine acetone.Zvinyorovesa ndiro yacho kwemasekonzi makumi matatu, wobva waomesa mhepo.Dzorera mupurete mundiro ine IPA, wozopepeta ndiro ye30 s kuti uchenese.
Mushonga wepiranha (musanganiswa wehydrogen peroxide uye concentrated sulfuric acid, 1:3 (vol/vol)) unogona kusarudza kushandiswa kuwedzera kubviswa kwezvisaririra kubva pasilicon wafer pamusoro.
Mushonga wePiranha unoodza zvakanyanya uye unopa kupisa.Kumwe kuzvidzivirira kunodiwa.Pakurasa marara, bvumira mushonga kuti utonhore uye woendeswa mumudziyo wakachena, wakaoma wemarara.Shandisai midziyo yechipiri uye nyorai zvakanaka midziyo inorasa.Ndokumbirawo mutevere nhungamiro dzekuchengetedzwa kwenzvimbo iyi kuti muwane mamwe maitiro akadzama.
Dehydrate the wafers nekuaisa pa 200 ° C inopisa ndiro ye 10 min. Mushure mekupera mvura, chidimbu chakazununguswa kashanu mumhepo kuti chitonhorere.
Dururira ~ 10 g ye photoresist SU-8 2100 pakati pesilicon wafer yakacheneswa.Shandisa tweezers kuparadzira photoresist zvakaenzana pawafer. Dzimwe nguva isa chidimbu pane 65 ° C inopisa ndiro kuitira kuti photoresist isanyanya kunamira uye nyore kupararira.
SU-8 yakagovaniswa zvakaenzana pawafer nekumhanyisa spin coating.Program irikuuya kutenderedzwa kweSU-8 kwema5–10 s kupararira pa500 rpm nekukasira kwe100 rpm/s.Seta main spin ye200 µm ukobvu patterning pa1,500mµ0mµ0m 2pm m kureba kwechikamu chepamusoro chehura pane chip; ona "Matanho Akakosha" pazasi) set at a acceleration of 300 rpm/s 30 seconds at 1,200 rpm.
Iyo huru spin yekumhanyisa inogona kugadziridzwa zvinoenderana nekwakavavarira ukobvu hweSU-8 patani pane silicon wafer.
Kugadzira SU-8 mapatani e500 µm kureba kwepamusoro pehumbu pane chip, iyo spin yekubikira uye yakapfava yekubheka nhanho dzeBhokisi iri (nhanho 7 uye 8) dzakadzokororwa sequentially (ona nhanho 9) kugadzira maviri akaturikidzana e250 µm Iyo gobvu layer yeSU-8, iyo inogona kuvharidzirwa ye25m nhanho ichiita 1 nhanho mubhokisi re0, UV 250 nhanho inobatanidzwa mubhokisi re0. .
Bika zvitete zvakapfava zveSU-8 nekunyatsoisa mawafer pandiro inopisa pa65 °C kwemaminetsi mashanu, wobva wachinja kuseta kuenda ku95 °C uye incubate kweimwe 40 min.
Kuti uwane 500 μm kureba kweSU-8 pateni mune yepamusoro microchannel, dzokorora nhanho 7 uye 8 kugadzira maviri 250 μm akakora SU-8 akaturikidza.
Uchishandisa UV Mask Aligner, ita mwenje bvunzo maererano nemirairo yemugadziri kuti uverenge nguva yekuratidzwa yewaferi.(exposure time, ms) = (exposure dose, mJ/cm2)/(lamp power, mW/cm2).
Mushure mekutarisa nguva yekuratidzwa, isa iyo fotomask pane mask inobata yeUV mask aligner uye isa iyo photomask pane SU-8 yakavharwa wafer.
Isa iyo yakadhindwa pamusoro peiyo photomask yakananga paSU-8 yakavharwa padivi resilicon wafer kuti uderedze kupararira kweUV.
Ratidza SU-8 yakavharwa wafer uye photomask yakatwasuka kusvika ku260 mJ/cm2 yeUV mwenje kune yakafanotarwa nguva yekuratidzwa (ona danho rechigumi rebhokisi rino).
Mushure mekuratidzwa kweUV, SU-8-yakavharwa nesilicon wafers yakabikwa pa65 ° C kwemaminetsi mashanu uye 95 ° C kwemaminetsi gumi nemashanu pandiro yega yega inopisa kugadzira mapatani nehurefu hwe200 μm.
Mugadziri anodururwa mundiro yegirazi, uye yakabikwa yakabikwa inoiswa mundiro.Kuwanda kweSU-8 developer kunogona kusiyana zvichienderana nehukuru hwegirazi ndiro.Iva nechokwadi chekushandisa zvakakwana SU-8 developer kubvisa zvachose isina kuvharwa SU-8.Somuenzaniso, paunenge uchishandisa 150 mm dhayamita yegirazi ndiro ine 1 L kukwanisa, shandisa ~ 300 dhizaini yekuvandudza . .
Sukurudza mold yakagadziridzwa ne ~ 10 mL yemugadziri mutsva ichiteverwa neIPA nekupfapfaidza mushonga uchishandisa pipette.
Isa chidimbu mu plasma cleaner uye uise pachena kune oxygen plasma (gasi remuchadenga, chinangwa chekumanikidza 1 × 10-5 Torr, simba 125 W) ye1.5 min.
Isa chidimbu muvacuum desiccator ine girazi slide mukati.Wafers uye masiraidhi anogona kuiswa parutivi.Kana vacuum desiccator yakakamurwa muzvikamu zvakawanda nendiro, isa masiraidhi mukamuri rezasi uye zvimedu mukamuri yepamusoro.Drop 100 μL ye trichloro,H 2H, 2H, girazi re chloroctrooct,H 2H, 2H, 100 μL. tsvedza uye shandisa vacuum ye silanization.
Nyura bhodhoro remasero eCaco-2 akaomeswa nechando mumvura yekugezesa ye37°C, wozoendesa maseru akanyungudutswa kune T75 flask ine 15 mL ye37°C prewarmed Caco-2 medium.
Kupfuura Caco-2 masero pa ~ 90% confluency, kutanga inodziya Caco-2 yepakati, PBS, uye 0.25% trypsin/1 mM EDTA mune 37 ° C yekugezesa mvura.
Sviridzira zvepakati nevacuum aspiration. Geza maseru kaviri ne5 mL yePBS inodziya nekudzokorora vacuum aspiration uye nekuwedzera nyowani PBS.