Tinokutendai nekushanyira Nature.com.Shanduro yebrowser yauri kushandisa ine tsigiro shoma yeCSS.Nekuda kwechiitiko chakanakisa, tinokurudzira kuti ushandise bhurawuza yakagadziridzwa (kana kudzima modhi yekuenderana muInternet Explorer).Parizvino, kuti tive nechokwadi chekutsigirwa kunoenderera, ticharatidza saiti isina masitaera uye JavaScript.
Human gut morphogenesis inotanga crypt-villus features of 3D epithelial microarchitecture uye spatial organisation.Ichi chimiro chakasiyana chinodiwa kuchengetedza gut homeostasis nekuchengetedza stem cell niche mu basal crypt kubva kune exogenous microbial antigens uye metabolites yavo. Mucosal surface.Saka, kudzokorora 3D epithelial structures kwakakosha pakuvaka in vitro gut models.Notably, iyo organic mimetic gut-on-a-chip inogona kuunza spontaneous 3D morphogenesis ye intestinal epithelium nekuvandudzwa kwemaitiro ehupenyu uye biomechanics weprotocol inopa reprotestinal reprotestinal. morphogenesis mumatumbu pane microfluidic chip pamwe neTranswell embedded hybrid chip.Tinotsanangura nzira dzakadzama dzekugadzirwa kwechigadzirwa, culturing yeCaco-2 kana intestinal organoid epithelial masero mumagadzirirwo akajairwa pamwe chete nepuratifomu yemicrofluidic, induction ye 3D morphogenesis, uye epitheliation yezvinoitwa maitiro akawanda epithelium3D. of functional gut microarchitecture nekudzora basolateral fluid flow for 5 d.Yedu in vitro morphogenesis nzira inoshandisa physiologically yakakosha shear stress uye mechanical motion uye haidi yakaoma cell engineering kana manipulation, iyo inogona kupfuura mamwe maitiro aripo. ye biomedical, kiriniki, uye mishonga yekushandisa.
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. epithelial morphogenesis 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". yakavanzwa frizzled-inoenderana neprotein 1, kana Soggy-1) kune-chip gut inhibits morphogenesis kana kukanganisa yakagadziridzwa 3D epithelial layer, zvichiratidza kuti antagonistic stress panguva yetsika inokonzera intestinal morphogenesis in vitro.Naizvozvo, nzira inoshanda yekuwana rakasimba morphogenesis mazinga ekugadzirisa zvishoma kana kuti kuchengetedza mazinga ekugadzirisa zvishoma pane epitheliant epithelial yeWorphogenesis. mu basolateral compartment by active flushing (eg, in gut-on-a-chip or hybrid-on-a-chip platforms) kana diffusion .Basolateral media (eg, kubva kuTranswell inopinza muhombe basolateral reservoirs 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 membranes, 8 9) uye yakakonzera 3D morphogenesis in vitro (nhanho 10) .Takaratidzawo maitiro emagetsi uye mashekeri anoratidza maitiro e-tissue-specific histogenesis uye mutsara-unoenderana nemutsara wemasero akasiyana-siyana kuburikidza nekushandisa maitiro akawanda ekufungidzira (matanho 11-24) . ine ruzivo rwehunyanzvi hunosanganisira kugadziridzwa kwepamusoro kweporous membranes, kusikwa kwe2D monolayers, uye intestinal biochemical uye Reproduction of the biomechanical microenvironment.in vitro.Kuita kuti 3D morphogenesis kubva ku2D epithelial monolayers, takabvisa morphogen vanopikisa mumhando mbiri dzakagadzirwa nekuyerera kwepakati mukati meiyo basolateral inopa mumiriri weiyo basolateral inomiririra. regenerable 3D epithelial layer iyo inogona kushandiswa kuenzanisa morphogen-inotsamira epithelial kukura, longitudinal host-microbiome co-cultures, utachiona hwepathogen, kukuvara kwekuputika, epithelial barrier dysfunction, uye probiotic-based therapies Example.influences.
Protocol yedu inogona kubatsira kune huwandu hwakawanda hwesainzi mune zvekutanga (semuenzaniso, intestinal mucosal biology, stem cell biology, uye developmental biology) uye yakashandiswa tsvakiridzo (semuenzaniso, preclinical zvinodhaka kuongororwa, chirwere chekuenzanisira, tishu engineering, uye gastroenterology) yakakura kukanganisa. technical strategy inogona kuparadzirwa kune vateereri vanodzidza kusimba kwesero kusaina panguva yekukura kwematumbo, kuberekwa patsva kana homeostasis .Pamusoro pezvo, protocol yedu inobatsira kubvunzurudza hutachiona pasi pemhando dzakasiyana siyana dzinotapukira dzakadai seNorovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium diffilera tym, Salmonera, Vifficilla. Vateereri vechirwere chechirwere uye chirwere chinobatsirawo.Kushandiswa kwe-on-chip gut microphysiology system inogona kubvumira longitudinal co-culture 10 uye kuongorora kunotevera kwekudzivirira kwemauto, mhinduro dzekudzivirira uye kugadziriswa kwekukuvara kwepathogen mumatumbo (GI) turakiti 11 .Zvimwe zvirwere zveGIceky, chirwere cheGroaceky, chirwere cheGroaceky, chirwere cheGroaceky, chirwere cheGroaceky ulcerative colitis, pouchitis, kana irritable bowel syndrome inogona kutevedzerwa kana 3D intestinal epithelial layers yagadzirwa uchishandisa 3D intestinal epithelial layers , zvirwere izvi zvinosanganisira villous atrophy, crypt shortening, mucosal damage, kana kukanganisa epithelial barrier. Kunyanya kuoma kwenzvimbo yechirwere, vaverengi vangafunga kuwedzera marudzi emasero anoenderana nechirwere, akadai semurwere weperipheral 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 inoisa mu 46 zvakanaka zadzisa basolateral compartments, iyo protocol inogonawo kuparadzirwa kune avo vari kugadzira mishonga, biomedical Kana yakakwirira-throughput screening kana kusimbisa mapuratifomu eindasitiri yezvokudya.Sehumbowo-hwe-principle, isu munguva pfupi yapfuura takaratidza kukwanisa kwe multiplex high-throughput kuwedzera morphogenesis system scalable kune a 24-plate format. kutengeserana16,17,18.Naizvozvo, kusimbiswa kwenzira yedu ye in vitro morphogenesis inogona kukurumidzirwa uye inogona kugamuchirwa nevakawanda tsvakurudzo marabhoritari, indasitiri kana hurumende uye masangano anodzora kuti anzwisise cellular reprogramming ye in vitro gut morphogenesis pa transcriptomic level yekuyedza zvinodhaka kana biotherapeutics The absorption was absorption was absorption and the customs ducts using commercial organ-on-a-chip modhi yekuongorora kuberekana kwegut morphogenesis process.
Nhamba shoma yemhando dzekuyedza dzinoenderana nevanhu dzakashandiswa kudzidza intestinal epithelial morphogenesis, zvakanyanya nekuda kwekushaikwa kwezvibvumirano zvinogoneka kuita kuti 3D morphogenesis in vitro.Ichokwadi, yakawanda yeruzivo rwezvino nezve gut morphogenesis yakavakirwa pane zvidzidzo zvemhuka (semuenzaniso, zebrafish20, mice21 kana kuti huku uye huku-inodhura 2). zvine mubvunzo, uye zvinonyanya kukosha, usanyatsoona maitiro ekukura kwevanhu.Mienzaniso iyi inewo zvishoma mukukwanisa kwayo kuongororwa nenzira yakawanda-yakakura.Saka, protocol yedu yekuvandudza 3D tissue structures in vitro outperforms in vivo mhuka dzemhando pamwe chete nedzimwe tsika dzechinyakare 2D cell culture models.Sezvakatsanangurwa kare, zvakabvumira kushandiswa kwenzvimbo yakasiyana-siyana ye3D maitiro ekuongorora nzvimbo dzakasiyana-siyana. masero ari mu crypt-villus axis mukupindura kune zvakasiyana-siyana mucosal kana immune stimuli.3D epithelial layers inogona kupa nzvimbo yekudzidza kuti masero ehutachiona anokwikwidzana sei kuti aite nzvimbo niches uye ecological evolution mukupindura kune mauto zvinhu (somuenzaniso, mukati kurwisana nekunze kwemucus layers, secretion yeIgA uye antimicrobial pepithelies inogona kubvumira kuti morphology yepimore3D inogona kunzwisisa sei morphology ye3D). gut microbiota inogadzirisa nharaunda dzayo uye synergistically inogadzira microbial metabolites (semuenzaniso, pfupi-chain chain fatty acids) inogadzirisa cellular sangano uye stem cell niches mu basal crypts.Zvinhu izvi zvinogona kuratidzwa chete apo 3D epithelial layers inosimbiswa mu vitro.
Mukuwedzera kune nzira yedu yekugadzira 3D intestinal epithelial structures, kune dzakawanda in vitro nzira.Intestinal organoid tsika inyanzvi yemazuva ano yekugadzirwa kwetishu yakagadzirwa kubva pakurimwa kwemasero emakumbo emukati pasi pemamiriro ezvinhu chaiwo e-morphogen23,24,25.Zvisinei, kushandiswa kwe 3D organoid model for transportation ye-biomicroculture-inonzi cosmic-injiniya inowanzoitwa cosmic-injiniya. lumen yakavharirwa mukati me organoid uye, saka, kuiswa kwe luminal components se microbial cells kana exogenous antigens ishoma. Kuwanikwa kune organoid lumens kunogona kuvandudzwa uchishandisa microinjector, 26,27 asi nzira iyi invasive uye kushanda nesimba uye inoda ruzivo rwakanyanya kuita.Uyezve, tsika dzechinyakare organoid dzakachengetwa mu hydrogel scaffolds pasi pemamiriro e static haaratidzi kunyatsoshanda mu vivo biomechanics.
Dzimwe nzira dzinoshandiswa nemapoka akawanda ekutsvagisa dzinoshandisa yakafanorongwa 3D hydrogel scaffolds kutevedzera iyo gut epithelial chimiro nekugadzira ega ega maseru eura hwemunhu panzvimbo yegel. Gadzira masikafu ehydrogel uchishandisa 3D-yakadhindwa, diki-yakagayiwa, kana lithographically yakagadzirwa nemamolds ega epid. physiologically relevant morphogen gradients, kugadza high aspect ratio epithelial structure uye stroma-epithelial crosstalk nekubatanidza stromal cells mu scaffold.Zvisinei, maitiro ezvakagadzirirwa scaffolds anogona kudzivirira kuratidzwa kwezvinoitika zvemophogenetic process pachayo.Iyi mienzaniso haipiwo simba rinoyerera rinoyerera, rinoyerera rinoyerera pasi pesimba remukati mesero remukati rinoda kuyerera kweshestinal inoyerera pasi pesimba remukati. morphogenesis uye kuwana physiological function.Kumwe kuongorora kwemazuva ano kwakashandisa hydrogel scaffolds mu microfluidic platform uye patterned intestinal epithelial structures using laser-etching techniques.Mouse intestinal organoids inotevedzera etched mapatani kuti iite intestinal tubular structures, uye intraluminal fluid flow can be recapitulated this spoluphoidics procedures using a microfluidics mode. uyewo haisanganisi gut mechanobiological movements.3D nzira dzekudhinda kubva kuboka rimwe chete dzakakwanisa kugadzira miniature gut tubes ne-spontaneous morphogenetic process.Pasinei nekugadzirwa kwakaoma kwezvikamu zvakasiyana-siyana zvegut mukati me tube, iyi modhi inoshayawo luminal fluid flow and mechanical deformation.Uyezve, kushanda kwemuenzaniso kunogona kuderedzwa, kunyanya mushure mokunge bioprinting yesero-inoedza, kuedza kwedu kwe bioprinting-inopedza. proposed protocol inopa zvakangoerekana zvaitika gut morphogenesis, physiologically yakakodzera shear stress, biomechanics inotevedzera gut motility, kusvikika kweapical apical and basolateral compartments, uye kugadzirwazve kwezvakaoma biological microenvironments of modularity.Naizvozvo, yedu in vitro 3D morphogenesis protocol inogona kupa nzira inowirirana yekukunda nzira dziripo.
Protocol yedu yakanyatsotarisana ne 3D epithelial morphogenesis, ine epithelial masero chete mutsika uye pasina mamwe marudzi emasero akakomberedza akadai semesenchymal cells, endothelial cells, uye immune masero.Sezvakatsanangurwa kare, nheyo yeprotocol yedu ndiyo induction ye epithelial morphogenesis nekubvisa morphogen inhibitors yakavanzwa pachikamu chedu che robusodudulati yakaunzwa. gut-on-a-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 ayo anoburitsa stem cellstroes anoramba achionekwa sesero masero. fibroblasts) mu mesenchyme inoita basa rinokosha mukugadzirwa kweECM mapuroteni uye mutemo we intestinal morphogenesis mu vivo35,37,38.Kuwedzerwa kwemasero emesenchymal kumuenzaniso wedu kwakawedzera maitiro e-morphogenetic uye kushandiswa kwesero. microenvironment.Uyezve, zvikamu zvevasculature zvinogona kubatanidzwa pakati pemasikirwo emasikirwo chinhu chinodiwa kana mhando dzetishu dzakagadzirirwa kuratidza kupindirana kwenhengo dzakawanda.Saka, masero endothelial angada kuiswa kuti aenzanise maitiro akanyatsojeka ehupenyu nekugadzirisa kwehutano. kusadzivirirwa mumamiriro ezvinhu ekutevedzera chirwere chemukati.
Kushandiswa kwema hybrid chips kunonyanya kutwasuka kupfuura gut-on-a-chip nokuti kugadzika kwechigadzirwa kuri nyore uye kushandiswa kweTranswell kuisa kunobvumira scalable tsika yegut epithelium.Zvisinei, inotengeswa inotengeswa Transwell inopinza ine polyester membranes haisi elastic uye haigoni kutevedzera peristaltic-kufanana nekufamba. Apical side.Sezviri pachena, iyo static properties muapical compartment haiwanzogonesa kwenguva refu bhakitiriya co-culture mumahybrid chips.Nepo isu tichigona kupinza zvakasimba 3D morphogenesis muTranswell kuisa pakushandisa mahybrid chips, kushomeka kwephysiologically yakakodzera biomechanics uye apical fluid kuyerera kunogona kudzikamisa kugona kweiyo hybrid platforms application.
Magadzirirwo akazara emunhu crypt-villus axis mugut-on-a-chip uye hybrid-on-a-chip tsika haasati anyatsosimbiswa.Sezvo morphogenesis inotanga kubva kune epithelial monolayer, 3D microarchitectures haipei morphological kufanana kune crypts in vivo. 3D epithelium, iyo crypt uye nzvimbo dzakaipa hadzina kuganhurwa zvakajeka.Kunyangwe nzira dzakakwirira dzepamusoro pachipi dzichitungamira kureba kweiyo microengineered epithelium, kureba kunogumira kusvika ~300–400 µm. Kudzika chaiko kwematumbu emunhu mudumbu diki nehombe, ~1mµmµmµmµ40 uye ~5mµmµ4000000 µm. uye kureba kwediki intestinal villi ndeye ~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. PDMS.Uyezve, sezvo iyo layer-by-layer microfabrication yeura pane chip inosanganisira kunamatira kusingaperi pakati pegadziko rega, zvakanyanya kunetsa kuvhura kana kubvisa iyo yepamusoro denga kuti uongorore chimiro chepamusoro cheiyo epithelial layer.Somuenzaniso, nekushandisa scanning electron microscope (SEM).
Iyo hydrophobicity yePDMS yanga iri chinhu chinodzikisira mune microfluidic-based zvidzidzo zvine chekuita nehydrophobic mamorekuru madiki, sezvo PDMS inogona kusanyanya adsorb akadaro hydrophobic mamorekuru.Alternatives kune PDMS inogona kutariswa nemamwe mapolymeric zvinhu.Nerumwewo, pamusoro pekugadzirisa kwePDMS kana polyphilic (eg42polythy) glycol) 43) inogona kutariswa kuderedza adsorption yehydrophobic molecules.
Pakupedzisira, nzira yedu haina kunyatsoratidzwa maererano nekupa kuongorora kwepamusoro-soro kana kuti "imwe-saizi-inokodzera-zvose" kushandiswa-kushamwaridzana kwekuedza platform.Izvino protocol inoda sirinji pombi per microdevice, iyo inotora nzvimbo muCO2 incubator uye inodzivirira kuedza kukuru.Kuderedza uku kunogona kuvandudzwa zvakanyanya ne scalability ye96well, tsika ye24well, tsika ye24well, itsva, tsika ye 96well, tsika ye24well, itsva, tsika ye24well. 384-tsime porous inoisa iyo inobvumira kuenderera kuzere uye kubviswa kweiyo basolateral media).
Kuti tipe 3D morphogenesis yehutumbu hwemunhu epithelium in vitro, takashandisa microfluidic chip intestinal device ine ma microchannels maviri anoenderana uye elastic porous membrane iri pakati kugadzira lumen-capillary interface. kuisa.Mumapuratifomu ose maviri, morphogenesis yemasero akasiyana-siyana emudumbu epithelial anogona kuratidzwa nekushandisa nzira yekushandura nzira yekuyerera kuti ibvise morphogen vanopikisa kubva muchikamu chebasolateral.Iyo yose yekuedza nzira (Mufananidzo 1) ine zvikamu zvishanu: (i) microfabrication yegut chip kana Transwellable chip (5step chip) chips-1 kugadzirira chip. intestinal epithelial masero (Caco-2 masero) kana organoids yemukati wemunhu; mabhokisi 2-5), (iii) tsika yemukati epithelial masero pamakumbo emukati kana hybrid chips (matanho 6-9), (iv) induction ye 3D morphogenesis in vitro (nhanho 10) uye (v)) kuratidza 3D epithelial microstructure (matanho 11-24) . in vitro morphogenesis 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 sezvakatsanangurwa muBhokisi 1, uye nhanho 1 -5. (Caco-2 kana human intestinal organoids) uye maitiro etsika anoshandiswa muprotocol."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 kwe3D morphogenesis uye epithe yenhamba dhizaini nhamba pazasi dhizaini nhamba. Museve.Chishandiso chinopa mienzaniso yekuti zvakamiswa sei intestinal epithelial layers inogona kushandiswa, semuenzaniso, mu cell differentiation characterization, gut physiology studies, kugadzwa kwehost-microbiome ecosystems, nechirwere modelling.Immunofluorescence mifananidzo mu "Cell Differentiation" inoratidza nuclei, F-actin uye MUC2D the Capilicoal kubudisa 3D inoratidzwa mu e-ecoal-2 yakaratidzwa mu-ecosystem. chip.MUC2 chiratidzo chiripo mumasero egoblet uye mucus yakavanzwa kubva mucosal surfaces.Mifananidzo yeFluorescent muGut Physiology inoratidza mucus inogadzirwa nekusvibisa sialic acid uye N-acetylglucosamine zvakasara zvichishandisa fluorescent wheat germ agglutinin.Mifananidzo miviri inopindirana mu "Host-Culture-Microbe" inoratidza mumiriri-Culture-Microbe ura pane chip.Paneti yekuruboshwe inoratidza co-culture yeE. coli inoratidzira green fluorescent protein (GFP) ine microengineered 3D Caco-2 epithelial masero.Paneti yekurudyi inoratidza nzvimbo yeGFP E. coli co-cultured ne 3D Caco-2 epithelial cells, inoteverwa ne immunofluored neFDiincleaseicle stain blue (FDiin-blue) kuenzanisira kunoratidza hutano hunopesana nehutu hunoputika muhutu huputi huputi pasi pedambudziko rehupenyu nebhakitiriya antigens (eg, lipopolysaccharide, LPS) uye masero emumune (eg, PBMC; green) . Masero eCaco-2 akagadzirwa kuti agadzire 3D epithelial layer.Scale bar, 50 µmage magedhi kubva kuzasi kwesero: chirevo.2. Oxford University Press; Rakabudiswa 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 zvakaitwa uchishandisa PDMS replicas iyo yakagadziriswa kubva kusilicon molds ne soft lithography1,44 uye yakafananidzwa neSU-8.Kugadzirwa kwema microchannels mu chip imwe neimwe kunotarirwa nekufungisisa hydrodynamics yakadai seshear stress uye hydrodynamic pressure1,4,12.The original gut-on-design yeFig-on-yakatanga maviri juxtaposed parallel straight microchannels, yakashanduka ikava yakaoma gut-on-a-chip (Extended Data Fig. 1b) iyo inosanganisira peya yakakomberedzwa microchannels kuti iite Kuwedzera kwemvura yekugara nguva, nonlinear kuyerera maitiro, uye multiaxial deformation ye cultured masero (Fig. 2a-f) 12. gut-on-a-chips inogona kusarudzwa.Takaratidza kuti convoluted Gut-Chip inokonzerawo zvakasimba 3D morphogenesis mune imwe nguva yakafanana ine dhigirii yakafanana yekukura kwe epithelial kana ichienzaniswa neyekutanga Gut-Chip, pasinei nerudzi rwesero yakagadzirwa.Nokudaro, kuunza 3D morphogenesis, linear uye yakaoma pa-silicone yakagadziriswa pa-chipplild ye-silicone inogadziriswa. ine SU-8 mapatani yakapa maitiro asina kunaka mushure mekudhirowa (Fig. 2a) .Kugadzira ura pane chip, iyo yakagadzirwa yepamusoro PDMS layer yakasungirirwa sequentially kune porous PDMS firimu uye yobva yaenderana nechepasi PDMS layer nekusingachinjiki bonding uchishandisa corona treater (Fig. 2b-f) .Kugadzira hybriddes bondedMS machipisi kuti agadzire ma hybrid de bonded machipisi, kuumba ma hybrid de bonde. single-channel microfluidic zvishandiso zvinogona kugadzirisa Transwell inserts (Fig. 2h uye Extended Data Fig. 2) .Kubatana kunoitwa nekurapa nzvimbo dzePDMS replica uye girazi neoxygen plasma kana corona treatment.Mushure mekuita sterilization ye microfabricated device yakanamatirwa kusilicone tube, chigadzirwa che 3D chigadziro che mororiphostine chakagadzirira kuita 3D chigadziriso che mororiphos (Mufananidzo 2g).
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) PDMS yakadhindwa yakachekwa kuita zvidimbu uye yakacheneswa kuti ishandiswe zvakare.b, Mufananidzo wakashandiswa kumucheka wesilicon. silicon mold inoshandiswa kugadzira iyo PDMS porous membrane.d, Yakatevedzana mapikicha ekumusoro uye ezasi PDMS zvikamu uye yakaungana pa-chip intestinal mudziyo.e, Schematic yekurongeka kwekumusoro, membrane, uye yepasi PDMS zvikamu. Imwe neimwe layer inosungirirwa zvisingadzoreki neplasma kana korona-yakagadzirwa nemucheka-chip-chip-chip-chigadzirwa cheSchematic convoluted microchannels and vacuum chambers.g, Setup of gut-on-a-chip for microfluidic cell culture.Ura hwakagadzirwa pachipu chakasanganiswa nesilicone chubhu nesirinji yakaiswa pacoverslip.Chip device yakaiswa pachivharo che 150 mm Petri dish for processing.Chip hybrids silicones chinoshandiswa kuvhara binder yesilicone yebhinder. kugadzirwa uye 3D morphogenesis vachishandisa hybrid chips.Transwell inoiswa yakagadzirirwa yakazvimiririra kune tsika 2D monolayers ye intestinal epithelial masero akaiswa mukati meiyo hybrid chip kuti iite intestinal 3D morphogenesis.Iyo yepakati inonhuhwirira kuburikidza ne microchannels iri pasi pesero layer yakagadzwa paTranswell referensi.Scale bar, yakadhindwa 4 cm. 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 kudyara ECM-yakavharidzirwa microchannels ye-chip gut kana Transwell inserts.Apo masero ari confluent mu-flaskly 95% masero eCacocoid, 95%. (pakati pendima 10 ne50) muT-flasks inokohwewa kuti igadzirire dissociated cell suspensions ne trypsinization fluid (bhokisi 2) .Human intestinal organoids kubva mumatumbo emakumbo kana kuvhiyiwa kwemaitiro akagadzirwa muMatrigel scaffold domes mu 24-well plates kuti atsigire structural microenvironment, microenvironment inokosha se W. R-spondin, uye Noggin) uye zvinhu zvekukura zvakagadzirirwa sezvinotsanangurwa muBhokisi 3 zvakawedzerwa zuva rimwe nerimwe kusvikira organoids yakakura kusvika ~ 500 µm muhupamhi. Organoids yakanyatsokura inokohwewa uye inoparadzaniswa kuva masero mamwechete ekudyara pamatumbo kana Transwell kuisa pane chip (Bhokisi 5) .Sezvatakambotaura, maererano ne3, chirwere chinokonzerwa ne1, chinogona kuva che3, chironda che1. Chirwere cheCrohn, colorectal cancer, or normal donor), lesion site (eg, lesion against non-lesioned area) uye gastrointestinal nzvimbo muturakiti (eg, duodenum, jejunum, ileum, cecum, colon, or rectum) intestinal organoids.
a, Workflow for induction of gut morphogenesis in the gut chip.Caco-2 yemunhu intestinal epithelium 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 seedased) membrane (yakabatanidzwa) membrane (PDD) yakasungirirwa. pazuva 0 (D0), apical (AP) kuyerera kunotanga 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 tsika ye micromorphogenesis mushure memazuva e5 yakaitika spontaneous morphogenesis yakaitika 5 D5) .Pase kusiyana kwemifananidzo inoratidza mumiririri morphology yeCaco-2 masero pane imwe neimwe yekuedza nhanho kana nguva yenguva (bar graph, 100 µm) .Mana schematic diagrams inoratidzira inowirirana cascades ye gut morphogenesis (kumusoro kurudyi).Miseve yakadhindwa muchirongwa inomiririra kutungamirirwa kwemvura inoyerera.b, SEM yeCacoli yemufananidzo wakagadzwa pamusoro pe2D yakasimbiswa kumusoro. inset inoratidzira nzvimbo yakasimudzwa (white dashed box) inoratidza yakagadziridzwa microvilli pa 3D Caco-2 layer (kurudyi) c, Horizontal kumberi kwekuona kwakasimbiswa Caco-2 3D, claudin (ZO-1, tsvuku) uye inoenderera bhurashi bhodha membranes yakanyorwa F-actin (girinhi) uye nuclei (blue) confectionalization yemasero emumunifluores. machipisi.Miseve inonongedza kuchirongwa chepakati inoratidza nzvimbo yendege yakatarisa kune imwe neimwe yakatarisana.d, Nguva yenguva yekuchinja kwemorphological mu organoid yakasimudzwa pachip yakawanikwa nechikamu chekusiyanisa maikorosikopu pamazuva 3, 7, 9, 11, uye 13.Inoiswa (kumusoro kurudyi) inoratidza kukwidziridzwa kwepamusoro kweiyo yakapihwa mufananidzo we3, guICpi pikicha yeDganoid yakagadzwa muDganoid micrograph kana DICPI yemufananidzo wakapihwa. pachidimbu chakatorwa pazuva rechinomwe LGR5 inoratidzira pasina chiratidzo cheMUC2.Mifananidzo yeFluorescence inoratidza epithelial microstructure (kurudyi) ye3D organoid epithelium yakagadzika muura pane chip nekusvibisa plasma membrane neCellMask dhayi (kurudyi) pazuva rechi13 retsika.Scale bar is 50 μm kunze kwekunge yataurwa neimwe mvumo kubva kuReferensi.b 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 kuti ibudirire ECM coating.Muiyi protocol, tinoshandisa nzira mbiri dzakasiyana dzekugadzirisa hydrophobicity yePDMS membranes.Nekurima masero eCaco-2, kushandiswa kwepamusoro neUV / ozone kurapwa chete kwakanga kwakakwana kuderedza hydrophobicity yeECM uye kuisa pamusoro peCaatco-PDMS uye kuisa pamusoro peCaatco-2 masero eCaatco. PDMS membrane.Zvisinei, 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 uye akaiswa mukati mePDMSA yakagadziriswa uye yakaunzwa mu isosopidft yePDMSA yakatangwa. masero akabatanidzwa, iyo microfluidic cell culture inotanga nekunhuhwirira chete mukati mukati me microchannel yepamusoro kusvikira masero aita monolayer yakakwana, nepo microchannel yakadzika inochengetedza mamiriro ezvinhu.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 nhanho dzakaoma dzepretreatment kuti uvhure pamusoro pekuisa porous.Kukura kweCaco-2 masero paTranswell inserts, ECM coating pane porous inserts inomhanyisa kusungirirwa kweakaparadzaniswa neCaco-2 masero (<1 awa) uye yakasimba junction barrier formation (<1-2 mazuva) . kune membrane pamusoro (<3 h) uye yakachengetedzwa kusvikira organoids ichiita monolayer yakakwana ine chipingamupinyi kutendeseka .Transwell tsika dzinoitwa mu 24-tsime mahwendefa pasina kushandisa 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 introlateral fluid kuyerera kwekombita yemvura inoyerera mu colateral kuyerera kwekombita inoyerera (inotungamira kuyerera kwekomputa) kuyerera kwekombita. secreted morphogen inhibitors.Kupa chikafu chakakwana uye serum kumaseru akasungwa pane porous membranes uye kugadzira luminal shear stress, tinowanzo shandisa kuyerera kwekaviri muura pane chip.Muma hybrid chips, Transwell inserts ine epithelial monolayers yakaiswa muma hybrid chips.Zvino, svikiro yakashandiswa pasi pechikamu chebasolateral chechikamu cheporoussis 3 chenji che Transphonnel 3 yakaitika kuburikidza neporousgene 3 transphonnel. mushure mekutangwa kwebasolateral kuyerera mumapuratifomu ese etsika.
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 kwemaitiro uye tsika yeDIC inogona kuitwa nyore nyore panguva ipi zvayo yemaitiro e3D epithelial layers.Nekuda kwekujeka kwemaziso ePDMS nemafirimu epolyester, zvose gut-on-a-chip uye hybrid chip platforms zvinogona kupa nguva chaiyo in situ imaging pasina kudiwa kwechikamu kana kupatsanurwa kwechigadzirwa.Paunenge uchiita immunofluorescence imaging (Figure 1, 3, 4b, 4, c, c, c, c, f, c, f, masero). (wt / vol) paraformaldehyde (PFA), inoteverwa neTriton X-100 uye 2% (wt / vol) ) bovine serum albumin (BSA), mukurongeka.Zvichienderana nemhando yesero, zvakasiyana-siyana zvinogadzirisa, permeabilizers, uye blocking agents zvinogona kushandiswa.Masoja ekutanga anotarisa mutsara-inotsamira sero kana masero emudunhu anotevedzwa nemasero emagetsi anoshandiswa nemasero emagetsi anoshandiswa nemasero emagetsi anoshandiswa. pamwe chete nedhayi yecounterstain inotarisa chero nucleus (eg, 4′, 6-diamidino-2-phenylene) indole, DAPI) kana F-actin (eg, fluorescently labeled phalloidin) .Fluorescence-based live imaging inogonawo kuitwa in situ kuti ione kugadzirwa kwemakasi (Fig. Cell siyana uye "Cell" yerandomology, "Guru 1", "Cell" isina kurongwa microbial cells (Fig. 1, "Host-microbe co-culture") , kuunganidza masero ekudzivirira zvirwere (Fig. 1, 'Disease Modeling') kana kuti contours ye 3D epithelial morphology (Fig. 3c, f uye 4b, c) . Paunenge uchigadzirisa gut pane chip kuti uparadzanise chidimbu chepamusoro. 2, iyo 3D epithelial morphology pamwe chete ne microvilli pamuganhu weapical brush inogona kuonekwa neSEM (Fig. 3b) .Kutaura kwekusiyanisa zviratidzo kunogona kuongororwa nekuita uwandu PCR5 kana single-cell RNA sequencing.Muchiitiko ichi, 3D layers epithelicular epithelicular anoshandiswa negoho remachips epithelicular uye edza machips epithelial anoshandiswa negoho remachips epithelial masero uye edza machips epithelial masero anoedza. genetic analysis.
a, Workflow for induction of intestinal morphogenesis in a hybrid chip.Caco-2 uye intestinal organoids inoshandiswa mune iyi protocol kuratidza 3D morphogenesis mu hybrid chip platform.Dissociated epithelial cells dzakasimwa mukugadzirwa kweTranswell inserts (TW prep; ona mufananidzo pazasi) yakasimudzwa pasi pemamiriro ekugadzirisa (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 yakazoita kuti kuve nekugadzirwa kwe 3D epithelial layer (morphogenesis) . (C103 mutsara) inokwira colon pane imwe neimwe yekuedza nhanho kana nguva yenguva.Zvirongwa 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-siyana, nzvimbo dzepakati uye dzakadzika dzeZ (yepamusoro-soro uye maonero epazasi). 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 3D morphology 2D 2D vertical cross-cut views (yakaiswa mukona yepamusoro yekurudyi; “XZ”) inoratidzawo 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 tsika. zvakare kuenzaniswa.
Iyo yakapfava lithography process inofanira kuitwa mukamuri rakachena.Pane imwe neimwe layer pane chip (yepamusoro uye yakaderera layers uye membranes) uye mahybrid chips, akasiyana mafotomasks akashandiswa uye akagadzirwa pane akasiyana silicon wafers nekuti hurefu hwema microchannels hwakanga hwakasiyana.Iyo inotariswa kureba kwepamusoro uye yakaderera microchannels yemudumbu pa chip ndeye 500 µmµ kuremerera kwetarem hybrid chip ndeye 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 kwe5–10 s kuti ipararire pa500 rpm nekukasira kwe100 rpm/s.Seta main spin ye200 µm ukobvu patterning pa1,500 rpm kana kuwana 500 rpm 500 µm kureba kwechikamu chepamusoro chehura pane chip; ona "Matanho Akakosha" pazasi) akaiswa pakumhanyisa kwe300 rpm/s 30 masekonzi pa1,200 rpm.
Iyo huru spin yekumhanyisa inogona kugadziridzwa zvinoenderana nekwakavavarira ukobvu hweSU-8 patani pane silicon wafer.
Kugadzira SU-8 mapatani e500 µm kureba kwekumusoro kwehumbu pane chip, iyo spin yekubikira uye yakapfava yekubheka nhanho dzeBhokisi iri (nhanho 7 uye 8) dzakadzokororwa sequentially (ona nhanho 9) kuburitsa maviri akaturikidzana e250 µm Iyo gobvu layer yeSU-8, iyo inogona kuvharidzirwa ye2 nhanho uye kubatanidzwa 0 bhokisi ne0. µm yakakwirira.
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.Huwandu hweSU-8 mugadziri hunogona kusiyana zvichienderana nekukura kweplate yegirazi.Iva nechokwadi chekushandisa zvakakwana SU-8 developer kubvisa zvachose SU-8.Somuenzaniso, paunenge uchishandisa 150 mm dhayamita yegirazi ndiro ine 1 L kukwanisa, shandisa ~ 300 dhizaini ye 300 mL . apo neapo kutenderera kunyoro.
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 padivi nedivi.Kana vacuum desiccator yakakamurwa muzvikamu zvakawanda nendiro, isa masiraidhi mukamuri rezasi uye zvitete mukamuri yepamusoro.Drop 100 μL ye trichloro,1H,2H 2H-perfluorooctyl) silane mhinduro pane siraidhi yegirazi uye isa vacuum yekuisa 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.