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Human gut morphogenesis e theha likarolo tsa crypt-villus tsa 3D epithelial microarchitecture le spatial organization.Sebopeho sena se ikhethang se hlokeha ho boloka mala a homeostasis ka ho sireletsa stem cell niche ho basal crypt ho tloha exogenous microbial antigens le metabolites ea bona. Ka hona, ho tsosolosa 3D epithelial structures ke habohlokoa bakeng sa kaho ea in vitro gut model. fluidic chip hammoho le ka Transwell embedded hybrid chip.Re hlalosa mekhoa e qaqileng ea ho etsa lisebelisoa, ho hlaolela Caco-2 kapa lisele tsa intestinal organoid epithelial ka litlhophiso tse tloaelehileng hammoho le sethaleng sa microfluidic, induction of 3D morphogenesis, and characterization of established 3D epithelia bakeng sa 5 d. Mokhoa oa rona oa in vitro morphogenesis o sebelisa khatello ea kelello ea ho kuta le ho sisinyeha ha mechine 'me ha e hloke boenjiniere bo rarahaneng ba lisele kapa ho qhekella, tse ka' nang tsa feta mekhoa e meng e teng.
Liteko li bonts'a hore lisele tsa intestinal epithelial Caco-2 tse hlahisitsoeng ka har'a gut-on-a-chip1,2,3,4,5 kapa bilayer microfluidic device6,7 li ka etsoa ka mokhoa o ikemetseng oa 3D morphogenesis in vitro ntle le kutloisiso e hlakileng ea mochine oa motheo. sis in vitro, e bontšitsoeng ke Caco-2 le li-organoids tsa mala tse tsoang ho mokuli.Lisele tsa Epithelial li ne li tiisitsoe. Thutong ena, re tsepamisitse maikutlo ka ho khetheha tlhahiso ea lisele le kabo ea mahloriso ea mohanyetsi ea matla oa Wnt, Dickkopf-1 (DKK-1), ka li-gut-on-a-chip le lisebelisoa tse fetotsoeng tsa microfluidic tse nang le lisebelisoa tsa Transwell, tse bitsoang "Hybrid Chip". d-e amanang le protheine 1, kapa Soggy-1) ho on-chip gut e thibela morphogenesis kapa e senya 3D epithelial layer e hlophisitsoeng esale pele, e fana ka maikutlo a hore khatello ea maikutlo e hanyetsanang nakong ea setso ke eona e ikarabellang bakeng sa intestinal morphogenesis in vitro. ho phuphusela (mohlala, ka har'a gut-on-a-chip kapa hybrid-on-a-chip platforms) kapa ho hasana .Basolateral media (mohlala, ho tloha Transwell ho kenya matamo a maholo a basolateral ka lilibeng).
Protocol ena, re fana ka mokhoa o qaqileng oa ho etsa li-microdevices tsa "gut-on-a-chip" le "Transwell-insertable hybrid chips" (mehato ea 1-5) ho lisele tsa intestinal epithelial ka polydimethylsiloxane (PDMS) -e thehiloeng ho li-membrane tse nang le porous (mehato 6A, 7A, 8, 9) kapa li-membrane tsa TranswellB le li-membrane tsa TranswellB, 8 3D morphogenesis in vitro (mohato oa 10) .Re boetse re khethile likarolo tsa cellular le limolek'hule tse bontšang histogenesis e khethehileng ea lisele le phapang ea lisele tse itšetlehileng ka moloko ka ho sebelisa mekhoa e mengata ea litšoantšo (mehato ea 11-24) . , pōpo ea 2D monolayers, le intestinal biochemical and Reproduction of the biomechanical microenvironment.in vitro.Ho induce 3D morphogenesis from 2D epithelial monolayers, re tlositse bahanyetsi ba morphogen ka mefuta e 'meli ea setso ka ho phalla bohareng ho kena ka har'a setsi sa basolateral sa setso.Qetellong, regeneation ea regene e ka sebelisoang ho etsa mohlala oa D epithelial. kholo ea epithelial e itšetlehileng ka phogen, longitudinal host-microbiome co-cultures, tšoaetso ea pathogen, kotsi ea ho ruruha, ho se sebetse ha epithelial barrier, le liphekolo tse thehiloeng ho probiotic Mohlala.tšusumetso.
Protocol ea rona e ka 'na ea e-ba molemo ho mefuta e mengata ea bo-rasaense litabeng tsa motheo (mohlala, intestinal mucosal biology, stem cell biology, and developmental biology) le lipatlisiso tse sebelisitsoeng (mohlala, tlhahlobo ea lithethefatsi ea preclinical, mohlala oa mafu, boenjiniere ba lisele, le gastroenterology) tšusumetso e pharaletseng. e rometsoe ho bamameli ba ithutang matla a pontšo ea lisele nakong ea tsoelo-pele ea mala, tsosoloso kapa homeostasis .Ho feta moo, protocol ea rona e na le thuso bakeng sa ho hlahloba tšoaetso tlas'a likokoana-hloko tse fapaneng tse tšoaetsanoang tse kang Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonellae Salmonellae 9.Bamameli ba lefu la mafu le pathogenesis le tsona li na le thuso.Tšebeliso ea on-chip gut microphysiology system e ka lumella longitudinal co-culture 10 le tlhahlobo e latelang ea tšireletso ea moeti, likarabo tsa 'mele oa ho itšireletsa mafung le ho lokisoa ha kotsi e amanang le pathogen ka har'a pampitšana ea 11 .Mafu a mang a GI acolitis, lefu la ho ruruha ha lisosa tse ling tse amanang le lefu la Croceky, lefu la ho ruruha ha lesapo la mokokotlo, lefu la lisosa le lefuba pouchitis, kapa irritable bowel syndrome e ka etsisoa ha 3D intestinal epithelial layers li lokisoa ho sebelisoa 3D intestinal epithelial layers , mafu ana a kenyelletsa villous atrophy, crypt shortening, mucosal damage, kapa impaired epithelial barrier.Biopsy or stem cell-derived the 2000 ka ho ntlafala ha lisele tsa 3D, ho feta moo, ho ka etsahala hore motho a be le bothata ba ho ba le lefu la 3D. nahana ka ho eketsa mefuta ea lisele tse amanang le lefu, tse kang mokuli peripheral blood mononuclear cells (PBMCs), ho mefuta e nang le 3D intestinal villus-crypt microarchitectures.lisele tsa 'mele tsa ho itšireletsa mafung, 5.
Kaha 3D epithelial microstructure e ka ts'oaroa le ho bonoa ntle le ts'ebetso ea ho arola, bashebelli ba sebetsanang le li-transcriptomics tsa sebaka le litšoantšo tse phahameng kapa tse phahameng haholo ba ka 'na ba thahasella 'mapa oa rona oa matla a sebaka sa liphatsa tsa lefutso le liprotheine ho li-epithelial niches.Ho thahasella thekenoloji.Response to microbial or immune stimuli.Ho feta moo, longitudinal host-microbiome crosstalk 10, 14 e hokahanyang gut homeostasis e ka thehoa ka har'a 3D intestinal mucosal layer ka ho kopanya mefuta e sa tšoaneng ea likokoana-hloko, metse ea likokoana-hloko kapa fecal microbiota-gut-a-gut-a-gut-a-gut-a-gut-a-gut-a.sethaleng.Mokhoa ona o khahleha ka ho khetheha ho bamameli ba ithutang mucosal immunology, gastroenterology, human microbiome, culturomics and clinical microbiology batla ho hlaolela pele e neng e sa tloaeleha mala microbiota laboratoring.Haeba in vitro morphogenesis protocol ea rona e ka ikamahanya le lifomate tsa setso sa scalable, joalo ka poleiti ea 43 hantle, e kenyang 46 hantle kapa 46 e tsoela pele hantle. li-compartments, protocol e ka boela ea ajoa ho ba ntseng ba tsoela pele ho hlahloba meriana, biomedical Kapa lisebelisoa tse phahameng tsa ho hlahloba kapa ho netefatsa lisebelisoa bakeng sa indasteri ea lijo.E le bopaki ba molao-motheo, re sa tsoa bonts'a ho khoneha ha multiplex high-throughput morphogenesis system scalable to a 24-well-plate format.Ho phaella moo, ho na le lihlahisoa tse ngata tsa pele, tse 16, pele ho moo, ho na le lihlahisoa tse ngata tsa pele tse 16. Mokhoa oa rona oa in vitro morphogenesis o ka potlakisoa mme oa amoheloa ke lilaboratori tse ngata tsa lipatlisiso, indasteri kapa mmuso le mekhatlo e laolang ho utloisisa reprogramming ea cellular ea in vitro gut morphogenesis maemong a transcriptomic ho lekola lithethefatsi kapa biotherapeutics. mokhoa oa gut morphogenesis.
Palo e lekanyelitsoeng ea mekhoa ea liteko e amanang le batho e 'nile ea sebelisoa ho ithuta intestinal epithelial morphogenesis, haholo-holo ka lebaka la ho haelloa ke mekhoa e ka sebelisoang ho etsa hore 3D morphogenesis in vitro.Ha e le hantle, boholo ba tsebo ea morao-rao mabapi le gut morphogenesis e thehiloe lithutong tsa liphoofolo (mohlala, zebrafish20, mice21 kapa li-mice21 kapa likhoho, li ka ba bohlokoa ka ho fetisisa, li ka ba le li-labor2 tsa bohlokoa ka ho fetisisa). ly, u se ke ua tseba hantle mekhoa ea tsoelo-pele ea batho.Mehlala ena e boetse e fokotsehile haholo ka bokhoni ba eona ba ho lekoa ka mokhoa o fapaneng oa mekhoa e mengata.Ka hona, protocol ea rona ea ho tsosolosa mekhoa ea 3D ea li-tissue ka har'a vitro ho feta mefuta e meng ea liphoofolo tse phelang hammoho le mefuta e meng ea setso e tsitsitseng ea 2D ea lisele. ka karabelo ea li-mucosal tse fapa-fapaneng kapa li-immune stimuli.3D epithelial layers e ka fana ka sebaka sa ho ithuta hore na lisele tsa likokoana-hloko li hlōlisana joang ho theha libaka tsa libaka le ho iphetola ha lintho tsa tikoloho ho arabela lintho tse amohelang batho (mohlala, likarolo tse ka hare le tsa ka ntle tsa mucus, secretion ea IgA le li-peptide tse thibelang likokoana-hloko) . s microbial metabolites (mohlala, short-chain fatty acids) e bōpang mokhatlo oa cellular le stem cell niches in the basal crypts.Likarolo tsena li ka bontšoa feela ha 3D epithelial layers e thehoa in vitro.
Ntle le mokhoa oa rona oa ho theha 3D intestinal epithelial structures, ho na le mekhoa e mengata ea in vitro. Culture organoid ea intestinal ke mokhoa oa morao-rao oa boenjiniere ba lisele tse thehiloeng ho lengoa ha lisele tsa mala a mala tlas'a maemo a khethehileng a morphogen23,24,25. Leha ho le joalo, tšebeliso ea 3D organoid models for transportation ea li-module tsa organoid hangata ke ho hlahloba likokoana-hloko tsa "hostestinal" kapa "hosting" ea "hostestinal" ea "hostestinal" kapa "model" d ka hare ho organoid mme, ka hona, ho kenngoa ha likarolo tsa luminal tse kang lisele tsa likokoana-hloko kapa li-antigens tse sa tloaelehang li lekanyelitsoe.Ho fihlella ho lumens ea organoid ho ka ntlafatsoa ho sebelisoa microinjector,26,27 empa mokhoa ona oa hlasela ebile o sebetsa ka matla 'me o hloka tsebo e khethehileng ea ho etsa.Ho feta moo, meetlo ea setso ea organoid e bolokiloeng ka scaffolds ea hydrogel tlas'a maemo a tsitsitseng ha e bontše ka nepo e sebetsang ka har'a vivo biomechanics.
Mekhoa e meng e sebelisoang ke lihlopha tse 'maloa tsa lipatlisiso e sebelisa scaffolds ea 3D hydrogel e hlophisitsoeng esale pele ho etsisa sebopeho sa mala ka ho hlahisa lisele tsa mala a motho a le mong holim'a gel. malebela a morphogen gradients, ho theha karolo e phahameng ea sebopeho sa epithelial le stroma-epithelial crosstalk ka ho kenyelletsa lisele tsa stromal ka har'a scaffold. mosebetsi.Phuputso e 'ngoe ea morao-rao e sebelisitseng hydrogel scaffolds sethaleng sa microfluidic le mekhoa e metle ea mala a epithelial e sebelisang mekhoa ea laser-etching.Mouse intestinal organoids e latela mekhoa e tsitsitseng ea ho etsa li-tubular tsa mala, le phallo ea intraluminal fluid e ka khutlisetsoa ho sebelisa mochine oa microfluidics. mekhoa ea ing e tsoang sehlopheng se le seng e ile ea khona ho etsa li-tubes tse nyenyane tse nang le mekhoa e sa tloaelehang ea morphogenetic.Ho sa tsotellehe ho etsoa ka mokhoa o rarahaneng oa likarolo tse fapaneng tsa mala ka har'a tube, mohlala ona o boetse o haelloa ke phallo ea mokelikeli oa luminal le deformation ea mechine.Ho phaella moo, ts'ebetso ea mohlala e ka fokotsoa, ​​haholo-holo ka mor'a hore ts'ebetso ea bioprinting e phethoe, e senya maemo a liteko kapa cell-to-cell interpositions proposed gut-cell, cell-to-cell. khatello ea kelello e amehang ea ho kuta, li-biomechanics tse etsisang motility ea mala, ho fumaneha ha likarolo tse ikemetseng tsa apical le basolateral, le ho tsosolosoa ha likokoana-hloko tse rarahaneng tsa likokoana-hloko tsa modularity.Ka hona, in vitro 3D morphogenesis protocol ea rona e ka fana ka mokhoa o tlatsetso oa ho hlōla mathata a mekhoa e teng.
Protocol ea rona e tsepamisitsoe ka ho feletseng ho 3D epithelial morphogenesis, e nang le lisele tsa epithelial feela setso 'me ha ho na mefuta e meng ea lisele tse potolohileng tse kang lisele tsa mesenchymal, lisele tsa endothelial, le lisele tsa' mele tsa ho itšireletsa mafung. chip le hybrid-on-a-chip e re lumella ho tsosolosa sekhahla sa 3D epithelial e sa tsitsang, lintho tse ling tse rarahaneng tsa likokoana-hloko tse kang li-epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 le, ka mohlala oa rona, likarolo tsa crypt-villus tse fetisang stem cell niches in basal cryptsmes in the basal cryptsmes in the basal cryptsmes in the basal crypts. tlhahisong ea liprotheine tsa ECM le taolo ea morphogenesis ea mala ka vivo35,37,38. Ho eketsoa ha lisele tsa mesenchymal ho mohlala oa rona ho ile ha ntlafatsa mokhoa oa morphogenetic le katleho ea ho kopanya lisele.The endothelial layer (ke hore, capillaries kapa lymphatics) e phetha karolo ea bohlokoa ho laola lipalangoang tsa limolek'hule39 le lisele tsa 'mele tsa ho itšireletsa mafung li ka ba karolo ea lisele tsa 'mele tsa ho itšireletsa mafung. ho kopanngoa pakeng tsa mefuta ea lisele ke ntho e hlokahalang ha mefuta ea lisele e etselitsoe ho bontša ho sebelisana ha litho tse ngata.Ka hona, lisele tsa endothelial li ka 'na tsa hloka ho kenyelletsoa ho etsa mohlala oa likarolo tse nepahetseng tsa fisioloji ka qeto ea boemo ba setho.Lisele tsa 'mele tsa ho itšireletsa mafung tse tsoang ho mokuli le tsona li bohlokoa bakeng sa ho bontša likarabo tsa tlhaho tsa ho itšireletsa mafung, tlhahiso ea antigen, innate adaptive immune crosstalk, le tissue-immunity immunity intestinfic disease.
Tšebeliso ea li-chips tse nyalisitsoeng li bonolo ho feta li-gut-on-a-chip hobane sesebelisoa sa ho seta se bonolo 'me tšebeliso ea Transwell inserts e lumella hore ho be le setso sa scalable sa gut epithelium. Leha ho le joalo, re ka khona ho kenya 3D morphogenesis ho Transwell ha re sebelisa li-chips tse nyalisitsoeng, khaello ea li-biomechanics tse amanang le fisioloji le phallo ea mokelikeli oa apical e ka fokotsa bokhoni ba lisebelisoa tsa hybrid chip platforms.
Ka ho feletseng reconstructions of the human crypt-villus axis in gut-on-a-chip le hybrid-on-a-chip cultures ha e so thehoe ka botlalo.Kaha morphogenesis e qala ho tloha ho epithelial monolayer, 3D microarchitectures do not necessarily provide morphological similarity to crypts in vivo. Leha ho le joalo likanale tse holimo ho chip li lebisa bophahamong bo eketsehileng ba microengineered epithelium, bophahamo bo boholo bo ntse bo lekanyelitsoe ho ~300–400 µm. Botebo ba 'nete ba maleng a motho ka maleng a manyane le a maholo ke ~µm, le 135m, le bolelele ba lisenthimithara tse 135,000,000 le 135 µm, le 135 µm, le 135 µm. lli ke ~ 600 µm41.
Ho ea ka pono ea ho nahana, in situ super-resolution imaging of 3D microarchitectures e ka lekanyetsoa feela ho mala ho chip, kaha sebaka se hlokahalang sa ho sebetsa ho tloha lense ea sepheo ho ea ho epithelial layer ke ka tatellano ea limilimithara tse seng kae. Ho hlōla bothata bona, ho ka hlokahala sepheo se hōle. by-layer microfabrication ea mala holim'a chip e kenyelletsa ho khomarela ka ho sa feleng pakeng tsa lera le leng le le leng, ho thata haholo ho bula kapa ho tlosa lera le ka holimo ho hlahloba sebopeho sa bokaholimo ba epithelial layer.Ka mohlala, ka ho sebelisa microscope ea elektronike ea ho hlahloba (SEM).
The hydrophobicity of PDMS has been limiting factor in microfluidic-based based to hydrophobic small molecules, since PDMS can unspecifically adsorb such hydrophobic molecules 3) e ka nkoa e fokotsa ho ata ha limolek'hule tsa hydrophobic.
Qetellong, mokhoa oa rona ha o e-s'o hlahe hantle mabapi le ho fana ka tlhahlobo e phahameng ea ho hlahloba kapa "mosebetsi o le mong" oa tekolo ea basebelisi.Protocol ea hona joale e hloka pompo ea syringe ka microdevice, e nkang sebaka ho CO2 incubator le ho thibela liteko tse kholo.Moeli ona o ka ntlafatsoa haholo ka mokhoa oa scalability oa 94well, setso sa 94well, 34well, setso sa 94well, 94well, 94well, setso sa 94well, 34-well ho kenya li-porous hantle tse lumellang ho tlatsa le ho tlosoa ha mecha ea litaba ea basolateral).
Ho etsa hore 3D morphogenesis ea intestinal epithelium in vitro, re sebelisitse microfluidic chip intestinal device e nang le li-microchannel tse peli tse bapileng le lera le reketla la porous lipakeng tsa ho theha sebopeho sa lumen-capillary. s, morphogenesis ea lisele tse sa tšoaneng tsa mala a mala a motho a ka bontšoa ka ho sebelisa tataiso ea tataiso ea phallo ho tlosa bahanyetsi ba morphogen ho tloha karolong ea basolateral.Tsamaiso eohle ea liteko (setšoantšo sa 1) e na le likarolo tse hlano: (i) microfabrication ea gut chip kapa Transwell hybrid chip 1-5; co-2 lisele) kapa li-organoid tsa mala a motho;mabokose a 2-5), (iii) setso sa lisele tsa intestinal epithelial ka li-chips tsa mala kapa li-chips tsa lebasetere (mehato ea 6-9), (iv) ho kenngoa ha 3D morphogenesis in vitro (mohato oa 10) le (v)) ho khetholla 3D epithelial microstructure (mehato ea 11-24) e ile ea etsoa ka mokhoa o nepahetseng ka tlas'a taolo ea morphogenesis ea morphogenesis (mohato oa 10) le (v)) genesis ka ho bapisa epithelial morphogenesis le taolo ea sebaka, ea nakoana, ea maemo, kapa ea tsamaiso.
Re sebelisitse li-platform tse peli tse fapaneng tsa setso: gut-on-a-chip e nang le likanale tse otlolohileng kapa likanale tse sa koahelehang, kapa li-chips tse nyalisitsoeng tse kentsoeng Transwell (TW) ka sesebelisoa sa microfluidic, se entsoeng joalo ka ha se hlalositsoe ho Lebokose la 1, le mohato oa 1 -5. "In vitro morphogenesis" e bontša mehato e akaretsang eo lisele tsa epithelial tsa Caco-2 kapa organoid-derived-derived epithelial li hlahisoa ka har'a chip ea mala kapa ho kenya Transwell ea chip ea lebasetere, e lateloa ke ho kenngoa ha 3D morphogenesis le ho thehoa ha palo ea epithelial e bontšitsoeng ka tlase. li-epithelial layering li ka sebelisoa, ka mohlala, ho khethollo ea lisele, lithuto tsa physiology ea mala, ho thehoa ha li-host-microbiome ecosystems, le mohlala oa mafu.Litšoantšo tsa Immunofluorescence ka "Cell Differentiation" e bontšang nuclei, F-actin le MUC2 e hlalositsoeng ka 3D Caco-2 epithelial epithelial e hlahisoang ka har'a li-cell mucus e hlahisoang ka har'a li-mucus tse hlahisoang ka har'a li-cells tsa sekhukhu. mucosal surfaces.Lits'oants'o tsa fluorescent in Gut Physiology show mucus produced by staining for sialic acid and N-acetylglucosamine residues using fluorescent wheat germ agglutinin. The two overlapping images in "Host-Microbe Co-Cultures" show representing host-microbiome gut left on the co-culture of fluolichip co-culture. protheine ea rescent (GFP) e nang le microengineered 3D Caco-2 epithelial cells.Panele e nepahetseng e bonts'a sebaka sa GFP E. coli e kopantsoeng le lisele tsa 3D Caco-2 epithelial, e lateloa ke immunofluorescence staining e nang le F-actin (e khubelu) le nuclei (blue)) . s (mohlala, lipopolysaccharide, LPS) le lisele tsa 'mele tsa ho itšireletsa mafung (mohlala, PBMC;tala).Lisele tsa Caco-2 li ne li entsoe ka mokhoa oa ho theha 3D epithelial layer.Scale bar, 50 µm.Lits'oants'o tse moleng o ka tlase: "Phapang ea lisele" e fetotsoe ka tumello ho tsoa ho litšupiso.2.Oxford University Press;E hlahisitsoe hape ka tumello ho tsoa ho Ref.5.NAS;"Host-Microbe Co-Culture" e hlophisitsoe ka tumello ho tsoa ho ref.3.NAS;“Moetso oa Mafu” o ikamahanyang le tumello ho tsoa ho litšupiso.5.NAS.
Ka bobeli li-gut-on-chip le li-hybrid chips li entsoe ka ho sebelisa li-replicas tsa PDMS tse ileng tsa theoleloa ho tloha hlobo ea silicon ka lithography e bonolo1,44 'me e entsoe ka SU-8.Moqapi oa li-microchannels ka har'a chip e' ngoe le e 'ngoe e khethoa ka ho nahana ka hydrodynamics e kang ho shear stress le khatello ea hydrodynamic1,4,12.The original chip chip-on-a 12. li-microchannels tse otlolohileng tse tšoanang, li fetohile ka har'a li-gut-on-a-chip tse rarahaneng (Extended Data Fig. 1b) e kenyelletsang li-microchannel tse kobehileng ho etsa hore nako e eketsehileng ea ho lula mokelikeli, mekhoa ea ho phalla e se nang moeli, le deformation ea multiaxial ea lisele tse tsoetseng pele (Setšoantšo sa 2a-f) 12. Re bontšitse hore convoluted Gut-Chip e boetse e susumetsa ka matla 3D morphogenesis ka nako e ts'oanang le tekanyo e ts'oanang ea kholo ea epithelial ha e bapisoa le ea pele ea Gut-Chip, ho sa tsotellehe mofuta oa sele e hlahisitsoeng.2a) .Ho etsa mala holim'a chip, lera le lokiselitsoeng la PDMS le ka holimo le ne le tlameletsoe ka tatellano ho filimi ea PDMS e nang le porous ebe e tsamaisana le karolo e ka tlaase ea PDMS ka tlamahano e ke keng ea khutlisetsoa ka ho sebelisa setlolo sa corona (setšoantšo sa 2b-f) . Setšoantšo sa 2h le Boitsebiso bo Atolositsoeng Fig. 2) Ts'ebetso ea ho tlamahane e etsoa ka ho tšoara libaka tsa PDMS replica le khalase e nang le plasma ea oksijene kapa phekolo ea corona. Ka mor'a hore ho be le sterilization ea sesebelisoa sa microfabricated se kopantsoeng le tube ea silicone, sesebelisoa sa sesebelisoa se ne se itokiselitse ho etsa 3D morphogenesis ea epithelium ea mala 2g (Figure 2g).
a, Schematic papiso ea ho lokisoa ha likarolo tsa PDMS ho tloha SU-8 patterned silicon molds.Tharollo e sa phekolehang ea PDMS e ile ea tšeloa holim'a hlobo ea silicon (ka ho le letšehali), e phekotsoeng ka 60 °C (bohareng) le ho theoleloa (ka ho le letona).The demolded PDMS e ile ea khaoloa likotoana le ho hloekisoa bakeng sa tšebeliso e tsoelang pele.b, Photograph of the silicon mold mold of the silicon Photograph. etsa PDMS porous membrane.d, Letoto la linepe tsa likarolo tse ka holimo le tse tlase tsa PDMS le sesebelisoa se kopaneng sa on-chip intestinal.e, Schematic of the alignment of the top, membrane, and lower PDMS components.E mong le e mong lera o irreversibly bonded by plasma kapa corona treatment.f, Schematic- vamkom-contact-microchips-contact s.g, Setup of gut-on-a-chip for microfluidic cell culture.Marete a entsoeng ka chip a kopantsoeng le tšupu ea silicone le sering e behiloe holim'a sekoahelo.Sesebelisoa sa chip se ile sa behoa holim'a sekoahelo sa sejana sa Petri sa 150 mm bakeng sa ho sebetsa.Setlamo se sebelisetsoa ho koala silicone tube.h, Visual hybrids chipssd genesis. e lokiselitsoeng ka mokhoa o ikemetseng ho setso sa 2D monolayers ea lisele tsa epithelial tsa mala li ile tsa kenngoa ka har'a chip ea lebasetere ho susumetsa mala a 3D morphogenesis. The medium is perfused through microchannels under the cell layer thehed on the Transwell insert.Scale bar, 1 cm.h E hatisitsoe hape ka tumello e tsoang ho referense.4.Elsevier.
Protocol ena, Caco-2 cell line le intestinal organoids li ne li sebelisoa e le mehloli ea epithelial (Fig. 3a) . Mefuta e 'meli ea lisele e ne e hlahisoa ka mokhoa o ikemetseng (Lebokose la 2 le Lebokose la 5) 'me e sebelisetsoa ho hlahisa li-microchannels tse entsoeng ka ECM tsa on-chip gut kapa Transwell inserts. 0 le 50) ka T-flasks kotuloa ho lokisetsa dissociated cell suspensions ka trypsinization mokelikeli (lebokose la 2) . Motho intestinal organoids ho tswa ho biopsies intestinal kapa opereishene resections ba ne ba cultured Matrigel scaffold domes ka 24-liliba lipoleiti ho tšehetsa structural tlhophiso ea lintho tse bonahalang Nophogennmedium, Angpogennt, Espogennt microenvironment, Espogennt, Espogennt, Espogennt, Espogennt, Esnophogennt, Espogennt, Espogennt, Espogennt, Espogennt, Esspogennt, Esspogennt, Esspogennt, Esspogennt, Esspogennt, Esspogennt, Esspogennt, Espogenment, Espogenment. lintlha tsa khōlo tse lokiselitsoeng joalokaha li hlalositsoe ho Lebokose la 3 li ile tsa tlatsetsoa letsatsi le leng le le leng ho fihlela li-organoids li hōla ho ~ 500 µm ka bophara. Li-organoid tse hōlileng ka ho feletseng li kotuloa 'me li aroloa ho ba lisele tse le 'ngoe bakeng sa ho jala maleng kapa li kenngoa ka Transwell holim'a chip (Lebokose la 5) .Joalokaha re se re tlalehile pele, e ka khetholloa ho ea ka lefu la mofuta oa 12, 13, mohlala, lefu la ulcerative, lesapo la mokokotlo, lefu la ho ruruha, kapa lesapo la mokokotlo (lefu la lisosa, lesapo la mokokotlo, lesapo la mokokotlo, lesapo la mokokotlo, lets'ollo, kankere ea matsoele) sebaka (mohlala, leqeba khahlano le sebaka se se nang maqeba) le sebaka sa mala ka har'a pampitšana (mohlala, duodenum, jejunum, ileum, cecum, colon, kapa rectum). Re fana ka protocol e ntlafalitsoeng ka har'a Lebokose la 5 bakeng sa ho lema li-colonic organoids (coloids) tseo ka tloaelo li hlokang hore ho be le likarolo tse phahameng tsa morphogens kapa tse nyane.
a, Workflow for induction of gut morphogenesis in the gut chip.Caco-2 epithelium ea mala a motho le intestinal organoids e sebelisoa ho protocol ena ho bontša 3D morphogenesis. Lisele tse ka thōko tsa epithelial li ne li behiloe ka har'a sesebelisoa se lokiselitsoeng sa gut-on-a-chip (chipboard) . D0), phallo ea apical (AP) e qalisoa le ho bolokoa matsatsing a pele a 2 (phallo, AP, D0-D2) Phallo ea Basolateral (BL) e boetse e qalisoa hammoho le mekhoa ea ho otlolla cyclic (ho otlolla, ho phalla, AP le BL) ha ho thehoa monolayer e feletseng ea 2D. Intestinal 3D morphogenesis e etsahetse matsatsi a tloaelehileng a 3D morphogenesis, moemeli oa micromorphose ka mor'a matsatsi a 5 ka mor'a hore ho be le liphetoho tsa micromorphogenesis. ea lisele tsa Caco-2 mohatong o mong le o mong oa liteko kapa nako ea nako (bar graph, 100 µm). Litšoantšo tse 'nè tsa schematic tse bontšang li-cascade tse tsamaisanang tsa gut morphogenesis (ka holimo ka ho le letona) .Metsu e phunyeletsoeng ka har'a schematic e emela tataiso ea phallo ea mokelikeli.b, setšoantšo sa SEM se bontšang topology ea holim'a sebaka se thehiloeng sa 3D Cacolisheding (lebokose le lesoeu) e bonts'a microvilli e nchafalitsoeng holim'a 3D Caco-2 layer (ka ho le letona).c, Horizontal frontal view of the established Caco-2 3D, claudin (ZO-1, red) le continuous brush border membranes e ngotsweng F-actin (green) le nuclei (blue) Immunofluorescence confocal visualization of the epistinal pointing chips to the middle of the middle of the forrow of the middle of the middle of the forrow confocal view.d, Nako ea nako ea liphetoho tsa morphological organoids tse hlahisitsoeng ka chip e fumanoeng ke phasepase microscopy matsatsing a 3, 7, 9, 11, le 13. The inset (holimo ka ho le letona) e bonts'a kholo e phahameng ea setšoantšo se fanoeng.e, DIC photomicrograph ea organoid 3D epithelium e thehiloeng ka har'a mala a letsatsi le li-slice tsa mahlaseli a letsatsi, tse nkiloeng ka har'a lisele tsa mots'eare. GR5;magenta), li-goblet cell (MUC2; tala), F-actin (grey) le nuclei (cyan) tse hōlileng ka li-gut chips bakeng sa matsatsi a 3, ka ho latellana (Ka ho le letšehali) le matsatsi a 13 (bohareng) organoids holim'a epithelial layer.Sheba le Extended Data Figure 3, e totobatsang LGR5 pontso ea litšoantšo ntle le pontšo ea microstructure3 ea MUCF2. organoid epithelium e thehilweng ka maleng ho chip ka ho silafatsa lera la plasma ka dae ya CellMask (ka ho le letona) ka letsatsi la 13 la setso.Scale bar ke 50 μm ntle le ha ho boletswe ka tsela e nngwe.b E hatisitswe hape ka tumello ho tswa ho referense.2.Oxford University Press;c E hlophisitsoe ka tumello ho tsoa ho Reference.2.Oxford University Press;e le f e fetotsoe ka tumello ka litšupiso.12 Under Creative Commons License CC BY 4.0.
Ka maleng ka chip, hoa hlokahala ho fetola holim'a hydrophobic holim'a PDMS porous membrane bakeng sa ho roala ECM e atlehileng.Ho protocol ena, re sebelisa mekhoa e 'meli e fapaneng ea ho fetola hydrophobicity ea li-membrane tsa PDMS.Ho hlaolela lisele tsa Caco-2, ts'ebetso ea holim'a metsi ka phekolo ea UV / ozone feela e ne e lekane ho fokotsa hydrophobicity ea hydrophobicity ea CaCMMS le ho kenya lisele tsa hydrophobicity tsa Caco-2 ho Caco-2. Leha ho le joalo, setso sa microfluidic sa organoid epithelium se hloka hore ho sebelisoe lik'hemik'hale tse thehiloeng holim'a lik'hemik'hale ho finyella ts'ebetso e nepahetseng ea liprotheine tsa ECM ka ho sebelisa polyethyleneimine (PEI) le glutaraldehyde ho li-microchannels tsa PDMS. setso e qala ka perfusing feela seaplane ho kena ka holimo microchannel ho fihlela lisele tse theha monolayer e feletseng, ha microchannel e ka tlaase e boloka maemo a tsitsitseng.Mokhoa ona o ntlafalitsoeng bakeng sa ts'ebetso ea holim'a metsi le ho roala ha ECM ho nolofalletsa ho khomarela epithelium ea organoid ho kenya 3D morphogenesis holim'a PDMS holim'a metsi.
Litloaelo tsa Transwell li boetse li hloka ho roala ha ECM pele ho peo ea sele;leha ho le joalo, litloaelo tsa Transwell ha li hloke mehato e rarahaneng ea pele ho ts'ebetso ho kenya tšebetsong bokaholimo ba li-porous inserts.Ho holisa lisele tsa Caco-2 ka li-insert tsa Transwell, ho roala ha ECM holim'a li-porous inserts ho potlakisa ho khomarela lisele tsa Caco-2 tse arohaneng (<1 hora) le sebopeho se thata sa li-junction barriers (<1-2 matsatsi) . <3 h) le ho hlokomeloa ho fihlela li-organoids li theha monolayer e feletseng e nang le barrier integrity .Meetlo ea Transwell e etsoa ka lipoleiti tsa liliba tse 24 ntle le tšebeliso ea li-chips tse nyalisitsoeng.
In vitro 3D morphogenesis e ka qalisoa ka ho sebelisa phallo ea mokelikeli ho karolo ea basolateral ea epithelial layer e thehiloeng. Ka maleng a chip, epithelial morphogenesis e qalile ha sehare se ne se kenngoa ka har'a li-microchannels tse ka holimo le tse ka tlaase (Setšoantšo sa 3a) . Joalokaha ho hlalositsoe pele, ke habohlokoa ho hlahisa metsi a sekhukhu a tsamaiso ea motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako oa motsoako o tsoelang pele. Ho fana ka limatlafatsi tse lekaneng le serum ho lisele tse tlanngoeng ka lera la porous le ho hlahisa khatello ea kelello ea luminal shear, re atisa ho sebelisa phallo e 'meli ka mpeng holim'a chip.Ka har'a li-chips tse nyalisitsoeng, li-transwell tse nang le li-monolayers tsa epithelial li ile tsa kenngoa ka har'a li-chips tse nyalisitsoeng.Ebe, sehare se ne se sebelisoa tlas'a lehlakore la basolateral la porous Transwell flowing3 matsatsi a ho phalla ha transwell5 ka mor'a matsatsi a ho phalla ha transwell5. sethala ka bobeli setso.
The morphological features of microengineered 3D epithelial layers ka sekasekwa ka ho sebedisa mekgwa e fapaneng ya ho etsa imaging, ho kenyeletswa phase difference microscopy, differential interference difference (DIC) microscopy, SEM, or immunofluorescence confocal microscopy (Figures 3 le 4). Phase phapano kapa DIC imaging nakong ea protrupision DIC nako e ka etsoa habonolo ka nako efe kapa efe ea DIC. e ho ponaletso ea optical ea PDMS le lifilimi tsa polyester, li-platform tsa gut-on-a-chip le hybrid chip li ka fana ka litšoantšo tsa nako ea sebele ntle le tlhokahalo ea ho arola kapa ho qhaqhoa ha sesebelisoa. ton X-100 le 2% (wt/vol) ) bovine serum albumin (BSA), ka tatellano.Ho ea ka mofuta oa sele, li-fixatives tse fapaneng, li-permeabilizers, le li-blocking agents li ka sebelisoa.Li-antibodies tsa mantlha tse shebileng lisele tse itšetlehileng ka mela kapa matšoao a sebaka li sebelisoa ho totobatsa lisele tse sa tsamaeeng ka har'a situ holim'a chip, dae ea bobeli e lateloa ke dae ea 6, le li-antibodies diamidino-2-phenylene) indole, DAPI) kapa F-actin (mohlala, fluorescently labeled phalloidin) . Imaging e phelang e thehiloeng ho Fluorescence e ka boela ea etsoa ka situ ho lemoha tlhahiso ea mucus (Feie.1, "Pharohano ea lisele" le "Gut physiology"), kolone e sa reroang ea lisele tsa likokoana-hloko (setšoantšo sa 1, "Host-microbe co-culture"), ho hira lisele tsa 'mele tsa ho itšireletsa mafung (setšoan. karolo e ka tlaase ea microchannel, joalokaha e hlalositsoe ho ref.Joalokaha ho bontšitsoe setšoantšong sa 2, 3D epithelial morphology hammoho le microvilli moeling oa brush ea apical e ka bonoa ka SEM (Fig. 3b) li kotuloa ka trypsinization ebe li sebelisetsoa tlhahlobo ea limolek'hule kapa liphatsa tsa lefutso.
a, Workflow for induction of intestinal morphogenesis in a hybrid chip.Caco-2 le intestinal organoids li sebelisoa ho protocol ena ho bontša 3D morphogenesis ka sethaleng sa hybrid chip.Lisele tse arohaneng tsa epithelial li ne li kenngoa ka li-transwell tse lokiselitsoeng (TW prep; bona setšoantšo se ka tlase) .Hang ha lisele li ne li kenngoa ka tlas'a maemo a polystatic (lisele tse kenngoeng) li ne li kenngoa ka har'a lisele tse peō li kenngoa ka tlas'a lisele tse peō. (T setso sa TW) .Ka mor'a matsatsi a 7, ho kenngoa ha Transwell e le 'ngoe e nang le 2D monolayer ea lisele tsa epithelial e ile ea kenngoa ka har'a chip ea lebasetere ho hlahisa phallo ea basolateral (Flow, BL), e ileng ea qetella e lebisitse ho moloko oa 3D epithelial layer (morphogenesis) . Phase phapang micrographs e bontšang likarolo tsa morphological tsa morphological organor C e 'ngoe le e 'ngoe ea lisele tsa setho sa motho C depithelical e 'ngoe le e 'ngoe ea lisele tsa motho. mohato kapa nako ea nako.Merero ea mekhahlelo e ka holimo e bontša tlhophiso ea liteko bakeng sa mohato o mong le o mong.b, Hybrid chips (left schematic) e ka lebisa ho 3D morphogenesis ea organoid epithelial cell with top-down confocal microscopy views tse nkiloeng libakeng tse fapaneng tsa Z (holimo, bohareng le tlaase;bona kemiso e nepahetseng le mela e matheba e tsamaellanang).e bontšitse litšobotsi tse hlakileng tsa morphological.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled view) ea organoid-derived epithelial celld cultured in static Transwell (TW; inset within white dashed box) khahlanong le hybrid chip (kholo ka ho fetisisa e feletseng e thunngoa) bapisa pairivevertical morphus2D 2D morphology. maikutlo (e kentsoe hukung e kaholimo ho le letona; “XZ”) e boetse e bontša likarolo tsa 2D le 3D.Scale bar, 100 µm.c E hatisitsoe hape ka tumello ho tsoa ho referense.4.Elsevier.
Litaolo li ka lokisoa ka ho hlaolela lisele tse tšoanang (Caco-2 kapa intestinal organoid epithelial cell) hore e be li-monolayers tse peli tlas'a maemo a tloaelehileng a static culture.Ho hlokomelehang, ho fokotseha ha limatlafatsi ho ka 'na ha fella ka lebaka la matla a fokolang a molumo oa li-microchannels (ke hore ~ ​​4 µL ka kanaleng e ka holimo holim'a phallo ea pele ea gut-chippiso le ho feta ha ho bapisoa le moralo oa pele oa "chippiso".
Ts'ebetso e bonolo ea lithography e lokela ho etsoa ka phapusing e hloekileng. Bakeng sa lera le leng le le leng holim'a chip (likarolo tse ka holimo le tse tlase le li-membrane) le li-chips tse nyalisitsoeng, li-photomasks tse fapaneng li ile tsa sebelisoa 'me tsa etsoa ka li-wafers tsa silicon tse arohaneng hobane bophahamo ba li-microchannel li ne li fapane. 200µm.
Beha sephaphatha sa silicon se lisenthimithara tse 3 ka sejana se nang le acetone. Fetola poleiti ka bonolo ka metsotsoana e 30, ebe u omisa sephaphatha ka moea. Fetisetsa sephaphatha poleiting e nang le IPA, ebe u ohla poleiti bakeng sa 30 s ho hloekisa.
Tharollo ea piranha (motsoako oa hydrogen peroxide le concentrated sulfuric acid, 1:3 (vol/vol)) ka boikhethelo e ka sebelisoa ho eketsa ho tlosoa ha masalla a lintho tse phelang ho tloha holim'a silicon oafer.
Tharollo ea Piranha e senya ka ho feteletseng 'me e hlahisa mocheso.Ho hlokahala mehato e meng ea tšireletso.Bakeng sa ho lahla litšila, lumella tharollo hore e pholile ebe e fetisetsoa setšelong se hloekileng, se omeletseng sa lithōle.Sebelisa lijana tsa bobeli le ho ngola ka nepo lijana tsa litšila.Ka kopo latela litataiso tsa polokeho ea setsi bakeng sa lits'ebetso tse felletseng.
Hlakola li-wafers ka ho li beha holim'a sejana se chesang sa 200 ° C bakeng sa metsotso e 10. Ka mor'a ho felloa ke metsi, sephaphatha se ile sa sisinyeha ka makhetlo a mahlano moeeng hore se pholile.
Tšela ~10 g ea photoresist SU-8 2100 bohareng ba sephaphatha sa silicon se hloekisitsoeng.Sebelisa li-tweezers ho jala photoresist ka ho lekana holim'a sephaphatha.Ka linako tse ling beha sephaphatha holim'a poleiti e chesang ea 65 ° C ho etsa hore photoresist e se ke ea khomarela le ho e hasa habonolo.U se ke ua beha sephaphatha ka ho toba holim'a poleiti e chesang.
SU-8 e ile ea ajoa ka ho lekana holim'a lefiri ka ho tsamaisa spin coating. Lenaneo la ho potoloha ha SU-8 bakeng sa 5-10 s ho phatlalla ka 500 rpm ka lebelo la 100 rpm/s. Beha spin e kholo bakeng sa 200 µm botenya ba patterning ka 1,50king a 5mµm2pm bophahamo ba limithara bakeng sa lera le ka holimo la mala holim'a chip; bona "Mehato ea Bohlokoa" ka tlase) e behiloeng ka lebelo la 300 rpm / s metsotsoana e 30 ka 1,200 rpm.
Lebelo le ka sehloohong la spin le ka fetoloa ho latela botenya ba sepheo sa sebopeho sa SU-8 sepacheng sa silicon.
Ho etsa lipaterone tsa SU-8 tsa bolelele ba 500 µm bakeng sa lera le kaholimo la mala holim'a chip, ho roala le ho baka litepisi tse bonolo tsa Lebokose lena (mehato ea 7 le ea 8) li ile tsa phetoa ka tatellano (sheba mohato oa 9) ho hlahisa likarolo tse peli tsa 250 µm Lera le letenya la SU-8, le ka beoang µm 2 ka lebokoseng le phahameng la 0, le ka beoang µm 2 ka lebokoseng le phahameng. .
Bake li-wafers tse koahetsoeng ka SU-8 ka hloko ka ho beha liphaphatha holim'a poleiti e chesang ka 65 ° C bakeng sa metsotso e 5, ebe u fetola boemo ho 95 ° C 'me u e kenye metsotso e meng e 40.
Ho fihlela bolelele ba 500 μm ea mohlala oa SU-8 ho microchannel e ka holimo, pheta mehato ea 7 le ea 8 ho hlahisa likarolo tse peli tsa 250 μm tse teteaneng tsa SU-8.
U sebelisa UV Mask Aligner, etsa tlhahlobo ea lebone ho latela litaelo tsa moetsi ho bala nako ea ho pepeseha ea sephaphatha.(nako ea ho pepeseha, ms) = (tekanyetso ea ho pepeseha, mJ/cm2)/(matla a lebone, mW/cm2).
Kamora ho tseba nako ea ho pepeseha, beha sets'oants'o sa foto holim'a sets'oants'o sa maske sa UV mask 'me u behe fotomask holim'a sephaphatha se koahetsoeng sa SU-8.
Beha bokaholimo bo hatisitsoeng ba fotomask ka kotloloho lehlakoreng le koahetsoeng la SU-8 la sephaphatha sa silicon ho fokotsa ho hasana ha UV.
Hlahisa sephaephe se koahetsoeng sa SU-8 le maske oa foto ho ea holimo ho 260 mJ/cm2 ea lebone la UV bakeng sa nako e reriloeng esale pele (sheba mohato oa 10 oa lebokose lena).
Ka mor'a ho pepeseha ha UV, li-wafers tsa silicon tse koahetsoeng ka SU-8 li ne li behoa ka 65 ° C bakeng sa 5 min le 95 ° C bakeng sa metsotso e 15 holim'a poleiti e 'ngoe le e' ngoe e chesang ho etsa lipaterone tse nang le bolelele ba 200 μm. Eketsa nako ea ho baka ka mor'a 95 ° C ho 30 min ho etsa mekhoa e nang le bolelele ba 500 µm.
Moqapi o tšeloa ka sejana sa khalase, 'me sephaphatha se besitsoeng se behiloe ka har'a sejana.Bophahamo ba moqapi oa SU-8 bo ka fapana ho itšetlehile ka boholo ba poleiti ea khalase.Etsa bonnete ba hore u sebelisa moqapi oa SU-8 o lekaneng ho tlosa ka ho feletseng SU-8 e sa pepesehang.Ka mohlala, ha u sebelisa sejana sa khalase sa bophara ba 150 mm se nang le bokhoni ba 1 L, sebelisa nako e bonolo ea ~ 300 rovelop2 mold . .
Hlatsoa hlobo e entsoeng ka ~ 10 mL ea moqapi o mocha o lateloa ke IPA ka ho fafatsa tharollo ka pipette.
Beha sephaphatha ka har'a sesebelisoa sa plasma 'me u pepese plasma ea oksijene (khase ea sepakapaka, khatello ea sepheo 1 × 10-5 Torr, matla a 125 W) bakeng sa 1.5 min.
Beha sephaphatha ka har'a vacuum desiccator e nang le slide ea khalase ka hare. Li-wafers le li-slide li ka behoa ka mahlakoreng.Haeba "vacuum desiccator" e arotsoe ka likarolo tse 'maloa ka poleiti, beha li-slide ka kamoreng e ka tlaase le li-wafers ka kamoreng e ka holimo.Drop 100 μL ea trichloro,Hsiper,H2H,H2H ea trichloro,H2H,H,H,H,H,H,H. thella le ho kenya vacuum bakeng sa silanization.
Qhibiliha pial ea lisele tsa Caco-2 tse leqhoa ka bateng ea metsi ea 37 ° C, ebe u fetisetsa lisele tse qhibilihisitsoeng ka har'a botlolo ea T75 e nang le 15 mL ea 37 ° C e futhumetseng ea Caco-2 medium.
Ho fetisa lisele tsa Caco-2 ho ~ 90% confluency, ea pele e futhumetseng ea Caco-2 medium, PBS, le 0.25% trypsin / 1 mM EDTA ka bateng ea metsi ea 37 ° C.
Aspirate medium by vacuum aspiration.Hlatsoa lisele tse habeli ka 5 mL ea PBS e futhumetseng ka ho pheta-pheta vacuum aspiration le ho eketsa PBS e ncha.