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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 models.Ha e le hantle, organic mimetic gut-on-a-chip e ka etsa hore ho be le 3D morphogenesis ea intestinal epithelium ka mesebetsi e ntlafetseng ea physiological le biomechanics e fana ka protocol in reprotebustcible. morphogenesis ka maleng ka har'a chip ea microfluidic hammoho le ho Transwell embedded hybrid chip.Re hlalosa mekhoa e qaqileng ea ho etsa lisebelisoa, ho hlaolela Caco-2 kapa lisele tsa intestinal organoid epithelial ka maemo a tloaelehileng hammoho le sethaleng sa microfluidic, induction of 3D morphogenesis, and characterization of the established 3D protocols achievement. ea microarchitecture e sebetsang ea mala ka ho laola phallo ea mokelikeli oa basolateral bakeng sa 5 d. Mokhoa oa rona oa in vitro morphogenesis o sebelisa khatello ea kelello ea ho kuta le ho sisinyeha ha mochine 'me ha o hloke boenjiniere bo rarahaneng ba lisele kapa ho qhekella, tse ka' nang tsa feta mekhoa e meng e teng. bakeng sa lits'ebetso tsa bongaka, tsa bongaka le tsa meriana.
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. epithelial morphogenesis in vitro, e bontšitsoeng ke Caco-2 le li-organoids tsa mala a 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 li-insert tsa Transwell, tse bitsoang "Hybrid Chip". protheine e amanang le frizzled 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. ka phaposing ya basolateral ka ho phuphusela ka mafolofolo (mohlala, ka hara mala-on-a-chip kapa hybrid-on-a-chip platforms) kapa diffusion .Basolateral media (mohlala, ho tloha Transwell e kenya matamong 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 epithelial tsa mala ka polydimethylsiloxane (PDMS) -e thehiloeng ho li-membrane tse nang le porous (mehato 6A, 7A, 8, 9) kapa li-membrane tsa TranswellB, 8 9) le ho kenya 3D morphogenesis in vitro (mohato oa 10) .Re boetse re tseba 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) . ka lintlha tsa tekheniki ho kenyelletsa ho fetoloa ha li-membrane tsa porous, ho bōptjoa ha 2D monolayers, le intestinal biochemical and Reproduction of the biomechanical microenvironment.in vitro.Ho etsa hore 3D morphogenesis ho tloha 2D epithelial monolayers, re tlositse bahanyetsi ba morphogen ka mefuta e 'meli ea setso ka ho phalla bohareng ho ea karolong ea basolateral e fana ka sebaka sa ho fana ka sebaka sa basolateral. regeneable 3D epithelial layer e ka sebelisoang ho etsa mohlala oa kholo ea epithelial e itšetlehileng ka morphogen, 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.litšusumetso.
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, le tsoelo-pele ea baeloji) le lipatlisiso tse sebelisoang (mohlala, tlhahlobo ea meriana ea pele, mohlala oa mafu, boenjiniere ba lisele, le gastroenterology) tšusumetso e pharaletseng. leano la tekheniki le ka fetisetsoa ho bamameli ba ithutang matla a mats'oao a lisele nakong ea nts'etsopele ea mala, ho nchafatsoa kapa homeostasis .Ho feta moo, protocol ea rona e na le thuso bakeng sa ho hlahloba ts'oaetso tlas'a mahlahana a fapaneng a tšoaetsanoang a kang Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium Salmonelera Salmonephilla , Vifila 9. Bamameli ba lefu la mafu le likokoana-hloko 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 GIceky a amanang le lefu la lefu la Croace, lefu la Croace le lefu la lets ulcerative colitis, pouchitis, kapa irritable bowel syndrome e ka etsisoa ha 3D intestinal epithelial layers e lokisoa ho sebelisoa 3D intestinal epithelial layers , mafu ana a kenyelletsa villous atrophy, crypt shortening, mucosal damage, kapa impaired epithelial barrier. ho rarahana ho hoholo ha tikoloho ea mafu, babali ba ka nahana ho eketsa mefuta ea lisele tse amanang le mafu, joalo ka lisele tsa mokuli tsa peripheral blood mononuclear (PBMCs), ho mehlala 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 holimo-limo 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 khahla haholo-holo ho bamameli ba ithutang mucosal immunology, gastroenterology, human microbiome, culturomics and clinical microbiology ba batlang ho lema microbiota ea pele e neng e sa tloaeleha ka laboratoring.Haeba in vitro morphogenesis protocol ea rona e ka ikamahanya le lifomate tsa setso tse scalable, joalo ka lipoleiti tse 43 hantle tse kenyang 46 hantle kapa tse 46 ka ho fetisisa. replenish basolateral compartments, protocol e ka boela ea fetisetsoa ho ba ntseng ba ntlafatsa meriana, biomedical Kapa high-throughput screening or validation platforms for the food industry.E le bopaki ba molao-motheo, re sa tsoa bonts'a ho khoneha ha multiplex high-throughput morphogenesis system scalable to a 24-multiple format. Ka hona, netefatso ea mokhoa oa rona oa in vitro morphogenesis e ka potlakisoa mme ea ka amoheloa ke lilaboratori tse ngata tsa lipatlisiso, indasteri kapa mmuso le mekhatlo ea taolo ho utloisisa reprogramming ea cellular ea in vitro gut morphogenesis maemong a transcriptomic ho lekola lithethefatsi kapa biotherapeutics. mekhoa ea khoebo ea organ-on-a-chip ho hlahloba ho ikatisa ha 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 likhoho-ho sa tsotellehe hore na li ka ba labor2 joang). e belaetsang, 'me ea bohlokoa ka ho fetisisa, ha e tsebe hantle mekhoa ea tsoelo-pele ea batho.Mehlala ena e boetse e fokotsehile haholo ka bokhoni ba bona ba ho lekoa ka mekhoa e mengata e ka senyehang.Ka lebaka leo, protocol ea rona bakeng sa ho tsosolosa mehaho ea 3D ea li-tissue ka har'a vitro e ntle ka ho fetisisa ka mehlala ea liphoofolo tsa vivo hammoho le mefuta e meng ea setso e tsitsitseng ea 2D ea lisele. lisele tse ka har'a crypt-villus axis ka lebaka la 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 sebaka le ho iphetola ha lintho tsa tikoloho ho arabela lintho tse amohelang batho (mohlala, ka hare le ka ntle ho li-mucus layers, secretion ea IgA le antimicrobial pepithelies e ka lumellang li-pepide tsa antimicrobial ho utloisisa hore na u ka utloisisa joang hore na u ka utloisisa joang hore na u ka etsa joang hore li-ecological pepimore3D). gut microbiota e theha metse ea eona 'me ka synergistically e hlahisa metabolites ea microbial (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 'maloa ea in vitro.Meetlo ea organoid ea intestinal ke mokhoa oa morao-rao oa boenjiniere ba lisele tse thehiloeng holim'a ho lengoa ha lisele tsa intestinal stem 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 cosmetology kapa li-microculture tsa cosmetology. lumen e koaletsoe ka har'a organoid 'me, 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. physiologically relevant morphogen gradients, ho theha karolo e phahameng ea sebopeho sa epithelial le crosstalk ea stroma-epithelial ka ho kenyelletsa lisele tsa stromal ka har'a scaffold. morphogenesis and gain physiological function.Phuputso e 'ngoe ea morao tjena e sebelisitsoeng hydrogel scaffolds in a microfluidic platform and patterned intestinal epithelial structures using laser-etching techniques.Mouse intestinal organoids e latela mekhoa e tsitsitsoeng ho etsa intestinal tubular structures, le intraluminal fluid flow can be recapitulated this microfluidics process nor spolufonogen. ebile ha e kenyeletse metsamao ea gut mechanobiological. Mekhoa ea khatiso ea 3D 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 ho senyeha ha mechine. proposed protocol e fana ka spontaneous gut morphogenesis, physiologically relevant shear stress, biomechanics e etsisang motility ea mala, ho fumaneha ha likarolo tse ikemetseng tsa apical le basolateral, le ho bopa bocha ha likokoana-hloko tse rarahaneng tsa likokoana-hloko tsa modularity.
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.Joalokaha ho hlalositsoe pele, motheo oa protocol ea rona ke ho kenngoa ha epithelial morphogenesis ka ho tlosa morphogen inhibitors e hlahisoang ka lehlakoreng la robust modular ea rona. gut-on-a-chip le hybrid-on-a-chip e re lumella ho tsosolosa lesela le sa tsitsang la 3D epithelial, lisebelisoa tse eketsehileng 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 li-cell stem cell niches, li ntse li nkoa e le lisele tse ling tsa li-cell. fibroblasts) ka mesenchyme e phetha karolo ea bohlokoa tlhahisong ea liprotheine tsa ECM le taolo ea intestinal morphogenesis ka vivo35,37,38. Ho eketsoa ha lisele tsa mesenchymal ho mohlala oa rona ho ntlafalitse mokhoa oa morphogenetic le katleho ea ho kopanya lisele. microenvironment.Ho feta moo, likaroloana tsa vasculature tse ka kopanngoa pakeng tsa mehlala ea lisele ke ntho e hlokahalang ha mehlala 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 o nepahetseng haholoanyane oa likarolo tsa' mele ka qeto ea boemo ba setho. ho itšireletsa boemong ba ho etsisa lefu la mala.
Tšebeliso ea li-chips tse nyalisitsoeng li otlolohile 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 se kotsi sa mala a epithelium. Apical side.Ho hlakile hore thepa e sa fetoheng ka har'a komporo ea apical ha e khonehe nako e telele ea baktheria ea nako e telele ea li-chips tse nyalisitsoeng.Ha re ntse re ka khona ho kenya 3D morphogenesis ho li-chips tsa Transwell ha u sebelisa li-chips tse nyalisitsoeng, khaello ea li-biomechanics tse amanang le physiologically le phallo ea mokelikeli oa apical e ka fokotsa monyetla oa ho sebelisa sethala sa hybrid.
Full-scale 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. 3D epithelium, crypt le libaka tse mpe li ne li sa khetholloa ka ho hlaka. Le hoja likanale tse holimo tsa chip li lebisa bophahamong bo eketsehileng ba microengineered epithelium, bophahamo bo sa ntse bo lekantsoe ho ~300–400 µm. Botebo ba 'nete ba maleng a motho ka har'a mala a manyane le a maholo, ~5mµmµmµmµm50,0 mme bophahamo ba mala a manyane a mala ke ~600 µm41.
Ho ea ka pono ea ho nahana, in situ super-resolution imaging of 3D microarchitectures e ka lekanyetsoa ho mala a 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. PDMS.Ho feta moo, kaha microfabrication ea lera-by-layer ea mala holim'a chip e kenyelletsa ho khomarela ka ho sa feleng pakeng tsa lera ka 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 e bile ntho e fokotsang liphuputsong tse thehiloeng ho microfluidic tse sebetsanang le limolek'hule tse nyenyane tsa hydrophobic, kaha PDMS e ka khona ho hlahisa limolek'hule tse joalo tsa hydrophobic. Mekhoa e meng ea PDMS e ka nkoa ka lisebelisoa tse ling tsa polymeric. glycol) 43 ) e ka nkoa e fokotsa ho ata ha limolek'hule tsa hydrophobic.
Qetellong, mokhoa oa rona ha o e-s'o ka o tsejoa hantle mabapi le ho fana ka tlhahlobo e phahameng ea lisebelisoa kapa "mosebetsi o le mong-o lekana" sethaleng sa liteko tsa basebelisi. Protocol ea hona joale e hloka pompo ea syringe ka microdevice, e nkang sebaka ka CO2 incubator le ho thibela liteko tse kholo.Moeli ona o ka ntlafatsoa haholo ka mokhoa oa scalability oa 94well, setso sa 96well, kapa mekhoa e mecha ea 9 384-ho kenya li-porous hantle tse lumellang ho tlatsa le ho tlosoa ha mecha ea litaba ea basolateral).
Ho susumetsa 3D morphogenesis ea epithelium ea mala a motho, re sebelisitse sesebelisoa sa microfluidic chip intestinal se nang le li-microchannel tse peli tse bapisang le lesela le rekere la porous lipakeng tsa ho theha sebopeho sa lumen-capillary. kenya.Ho li-platform ka bobeli, morphogenesis ea lisele tse fapaneng tsa mala a motho a ka bontšoa ka ho sebelisa tataiso ea tataiso ea phallo ho tlosa bahanyetsi ba morphogen ho tloha karolong ea basolateral.Tsela eohle ea liteko (Setšoantšo sa 1) e na le likarolo tse hlano: (i) microfabrication ea gut chip kapa Transwellable chip (5step chip) chipwellable chip (5step Box) lisele tsa intestinal epithelial (lisele tsa Caco-2) kapa li-organoids tsa mala a motho; mabokose a 2-5), (iii) setso sa lisele tsa intestinal epithelial ka li-chips tsa mala kapa li-chips tse nyalisitsoeng (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 ho latela sehlopha se nepahetseng. in vitro morphogenesis ka ho bapisa epithelial morphogenesis le taolo ea sebaka, ea nakoana, ea maemo, kapa ea tsamaiso.
Re sebelisitse lipolanete tse peli tse fapaneng tsa setso: gut-on-a-chip e nang le likanale tse otlolohileng kapa likanale tse sa koaeloang ke letho, 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. (Caco-2 kapa human intestinal organoids) le mokhoa oa setso o sebelisitsoeng ho protocol ena." In vitro morphogenesis "e bonts'a mehato e akaretsang eo lisele tsa epithelial tse tsoang ho organoid li hlahisoang ka har'a chip ea mala kapa ka ho kenya Transwell ea chip e nyalisitsoeng, e lateloe ke ho kenngoa ha 3D morphogenesis le sebopeho sa epithelial se seng le se seng ka tlase. motsu.Kopo e fana ka mehlala ea hore na likarolo tsa epithelial tsa intestinal tse thehiloeng li ka sebelisoa joang, mohlala, ho khethollo ea lisele, lithuto tsa fisioloji ea mala, ho thehoa ha li-host-microbiome ecosystems, le ho etsa mohlala oa mafu.Litšoantšo tsa Immunofluorescence ka "Cell Differentiation" e bontšang nuclei, F-actin le MUC2D Capilicoal e hlahisoang ka har'a 3D Capilicoal layer2. chip.MUC2 pontšo e teng ka liseleng tsa goblet le mucus e patiloeng ho tloha holim'a mucosal mala holim'a chip.Panele e ka ho le letšehali e bonts'a co-culture ea E. coli e hlalosang protheine e tala ea fluorescent (GFP) e nang le microengineered 3D Caco-2 lisele tsa epithelial. Letlapa le letona le bontša sebaka sa GFP E. coli co-cultured le 3D Caco-2 epithelial cells, e lateloa ke immunofluored with F-Diinclease nuclease nuclear mohlala o bonts'a bophelo bo botle khahlanong le leaky mala ka har'a phephetso ea 'mele e nang le li-antigens tsa baktheria (mohlala, lipopolysaccharide, LPS) le lisele tsa' mele tsa 'mele (mohlala, PBMC; tala) Lisele tsa Caco-2 li ne li bōpiloe ho theha 3D epithelial layer.Scale bar, 50 µsmage in the bottomed in . tshupiso.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 ne li entsoe ka ho sebelisa likopi 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. li-microchannel tse peli tse tšoanang tse tšoanang tse otlolohileng, 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 ho be le nako e eketsehileng ea ho lula mokelikeli, mekhoa e sa tšoaneng ea phallo, le ho senyeha ha lisele tse ngata tse nang le mekhoa e metle (setšoantšo sa 2a-f) ho hlokahala hore ho be le li-biochannel tse rarahaneng ho feta 12. li-gut-on-a-chips li ka khethoa.Re bontšitse hore convoluted Gut-Chip e boetse e kenyelletsa 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 ea setso. ka mekhoa ea SU-8 e fane ka likarolo tse mpe ka mor'a ho senya (setšoantšo sa 2a) . Ho etsa mala holim'a chip, lera le lokisitsoeng le ka holimo la PDMS le ne le kopantsoe ka sequentially ho filimi ea PDMS e nang le porous ebe e lumellana le karolo e ka tlaase ea PDMS ka tlamahano e ke keng ea qojoa ho sebelisa mochine oa corona (Fig. 2b-f) . lisebelisoa tsa microfluidic tse nang le mochine o le mong o ka khonang ho kenya lisebelisoa tsa Transwell (setšoan. (Setšoantšo sa 2g).
a, Papiso ea moralo oa ho lokisoa ha likarolo tsa PDMS ho tloha ho 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 phekotsoe 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 eketsehileng.b, Photograph of the silicon mold of the silicon graph. silicon mold e sebelisetsoang ho etsa PDMS porous membrane.d, Letoto la linepe tsa likarolo tse ka holimo le tse tlaase tsa PDMS le sesebelisoa sa mala se kopaneng sa on-chip. convoluted microchannels and vacuum chambers.g, Setup of gut-on-a-chip for microfluidic cell culture.Mare a entsoeng holim’a chip a kopantsoeng le tšupu ea silicone le sering e ile ea behoa holim’a sekoahelo.Sesebelisoa sa chip se ile sa behoa holim’a sekoahelo sa sejana sa Petri sa 150 mm bakeng sa ho sebetsa.The binder hybrid silicone e sebelisoa ho koala li-silicone tsa chip. tlhahiso le 3D morphogenesis ka ho sebelisa li-chips tse nyalisitsoeng 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 koahetsoeng ke ECM tsa on-chip gut kapa Transwell inserts. (pakeng tsa litemana tsa 10 le 50) ho li-flasks tsa T li kotuloa ho lokisetsa ho emisoa ha lisele tse arohaneng ka trypsinization fluid (lebokose la 2) . Li-intestinal organoids tsa motho tse tsoang ho intestinal biopsies kapa li-resections tsa ho buuoa li ile tsa hlahisoa matlong a Matrigel scaffold ka lipoleiti tsa 24-well ho tšehetsa structural W. R-spondin, le Noggin) le lintlha tsa kholo tse lokiselitsoeng joalokaha li hlalositsoe ho Lebokose la 3 li ile tsa tlatsetsoa letsatsi le leng le le leng ho fihlela li-organoids li ntse li hōla ho fihlela ho ~ 500 µm ka bophara. Crohn's disease, colorectal cancer, or normal donor), sebaka sa leqeba (mohlala, leqeba khahlano le sebaka se sa jeoang) le sebaka sa mala ka har'a pampitšana (mohlala, duodenum, jejunum, ileum, cecum, colon, kapa rectum). li-organoids tsa mala.
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 tsa epithelial tse ka thōko li ne li behiloe ka har'a sesebelisoa se lokiselitsoeng sa gut-on-a-chip (chipboard) .Hang ha lisele li kenngoa ka har'a li-membrane le li-membrane tse khomaretsoeng. ka letsatsi la 0 (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 ha cyclic (ho otlolla, ho phalla, AP le BL) ha ho thehoa monolayer e feletseng ea 2D. Intestinal 3D culture morphogenesis morphogenesis matsatsi a 5 a morphogenesis e etsahetse ka mor'a matsatsi a 5 a morphogenesis. D5) .Litšoantšo tse fapaneng tsa Phase li bonts'a morphology e emelang 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-cascades tse lumellanang tsa gut morphogenesis (ka holimo ka ho le letona). sets'oants'o se totobatsang sebaka se holisitsoeng (lebokose le lesoeu) se bonts'a microvilli e nchafalitsoeng holim'a 3D Caco-2 layer (ka ho le letona).c, Ho sheba ka pele ka holimo ho Caco-2 3D e thehiloeng, claudin (ZO-1, e khubelu) le li-membrane tsa moeli oa borashe tse ngotsoeng F-actin (e tala) le nuclei (bluence) ea li-immunocalization tsa lisele tse bonoang. chips.Metsu e supang schematic e bohareng e bonts'a sebaka sa sefofane se tsepameng bakeng sa pono e 'ngoe le e 'ngoe ea confocal.d, Nako ea nako ea liphetoho tsa morphological ho organoids e hlahisitsoeng ka chip e fumanoeng ka phapano ea microscopy matsatsing a 3, 7, 9, 11, le 13. Sets'oants'o (ka holimo ka ho le letona) se bontša kholo e phahameng ea setšoantšo se fanoeng sa 3D, GUIC, Dganoid, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ka selae se nkiloeng ka letsatsi la 7.f, litšoantšo tse koahetsoeng tsa immunofluorescence tse bontšang matšoao bakeng sa lisele tsa stem (LGR5; magenta), lisele tsa goblet (MUC2; tala), F-actin (grey) le nuclei (cyan) e hōlileng ka li-chips tsa mala ka matsatsi a 3, ka ho latellana (Ka ho le letšehali) le matsatsi a 13 (bohareng) organolitended le li-organolitended tse lerōleng tsa Epigure. LGR5 e bontšang ntle le pontšo ea MUC2 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 a chip, hoa hlokahala ho fetola holim'a hydrophobic holim'a PDMS porous membrane bakeng sa ho roala ka katleho ECM.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 le ho kopanya lisele tsa hydrophobicity tsa Caatco le ho kopanya lisele tsa hydrophobicity tsa Caatco-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. lisele li kenyelelitsoe, setso sa lisele tsa microfluidic se qala ka ho sebelisa li-perfusing feela ka hare ho microchannel e ka holimo ho fihlela lisele li etsa monolayer e feletseng, ha microchannel e tlaase e boloka maemo a tsitsitseng.Mokhoa ona o ntlafalitsoeng bakeng sa ts'ebetso ea holim'a metsi le ho roala ha ECM ho etsa hore ho kenyelelitsoe epithelium ea organoid ho etsa hore 3D morphogenesis e be holim'a PDMS.
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 ea ka holim'a lera (<3 h) le ho hlokomeloa ho fihlela li-organoids li theha monolayer e feletseng e nang le mokoallo oa botšepehi .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 kenngoa ka har'a li-microchannels tse ka holimo le tse tlaase (Setšoantšo sa 3a) . Ho fana ka limatlafatsi tse lekaneng le serum ho lisele tse tlanngoeng ka li-membrane tsa porous le ho hlahisa khatello ea kelello ea luminal shear, re atisa ho sebelisa phallo e 'meli ka maleng holim'a chip.Ka har'a li-chips tse nyalisitsoeng, li-transwell tse nang le li-epithelial monolayers li ile tsa kenngoa ka har'a li-chips tse nyalisitsoeng. Joale, sehare se ne se sebelisoa tlas'a lehlakore la basolateral la poroussis-3 matsatsi a Transphonnel 3. ka mor'a ho qalisoa ha phallo ea basolateral sethaleng ka bobeli sa setso.
The morphological features of microengineered 3D epithelial layers ka sekasekwa ka ho sebedisa mekgwa e fapaneng ya ho nka ditshwantsho, ho kenyeletswa phapano ya microscopy, differential interference difference (DIC) microscopy, SEM, kapa immunofluorescence confocal microscopy (Figure 3 le 4). Phapang ea Phase kapa DIC imaging nakong ea protrusion ea protrusion ho ka etsoa habonolo nakong ea sebopeho leha e le sefe3D. epithelial layers.Ka lebaka la ho pepeseha ha optical ea PDMS le lifilimi tsa polyester, li-platform tsa gut-on-a-chip le hybrid chip platforms li ka fana ka litšoantšo tsa nako ea sebele ka ntle ho tlhokahalo ea ho arola kapa ho qhaqhoa ha sesebelisoa. (wt / vol) paraformaldehyde (PFA), e lateloa ke Triton X-100 le 2% (wt / vol) ) bovine serum albumin (BSA), ka tatellano.Ho itšetlehile ka mofuta oa sele, li-fixatives tse fapaneng, li-permeabilizers, le li-blocking agents li ka sebelisoa.Li-antibodies tse ka sehloohong tse lebisang molokong o itšetlehileng ka moloko oa lisele ho li-antibodies ho li-cell marker tse sebelisoang ka har'a lisele tsa chip li sebelisoa. hammoho le dae ea counterstain e shebileng nucleus (mohlala, 4′, 6-diamidino-2-phenylene) indole, DAPI) kapa F-actin (mohlala, fluorescently labeled phalloidin) .Fluorescence-based live imaging e ka boela ea etsoa in situ ho lemoha tlhahiso ea mucus (Fig. Cell 1, Fig. lisele tsa likokoana-hloko (setšoantšo sa 1, "Host-microbe co-culture"), ho hira lisele tsa 'mele tsa ho itšireletsa mafung (setšoantšo sa 1, 'Mohlala oa lefu lena') kapa li-contours tsa 3D epithelial morphology (setšoantšo sa 3c, f le 4b, c) . 2, 3D epithelial morphology hammoho le microvilli moeling oa brush ea apical e ka bonoa ka SEM (Fig. 3b) tlhahlobo ea liphatsa tsa lefutso.
a, Workflow for induction of intestinal morphogenesis in a hybrid chip.Caco-2 le intestinal organoids li sebelisoa protocol ena ho bontša 3D morphogenesis ka sethaleng sa hybrid chip.Lisele tse arohaneng tsa epithelial li ile tsa jaloa ka li-Transwell inserts tse lokiselitsoeng (TW prep; bona setšoantšo se ka tlase) .Hang ha lisele li ne li kenngoa ka har'a lisele tse peō li kenngoa ka har'a li-polyse cultured tlas'a maemo a tsitsitseng (TW culture) .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 hybrid ho hlahisa phallo ea basolateral (Flow, BL), e ileng ea qetella e lebisitse ho moloko oa 3D epithelial layer (morphogenesis) . (C103 line) e nyolohang colon ka mohato o mong le o mong oa liteko kapa nako ea nako. The schematics ka 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 lisele tsa organoid epithelial tse nang le top-down confocal microscopy maikutlo a nkiloeng ka mahlakoreng a fapaneng a li-microscopy, tse lumellanang le maemo a tlaase a Z, le lipono tse ka tlaase tsa schematic tsa Z. 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 (e kholo ka ho fetisisa e feletseng e thunngoa) ho bapisa 3D morphology 2D morphology. lipono tse tšekaletseng tse tšekaletseng (kentsoe hukung e kaholimo ho le letona; "XZ") e boetse e bonts'a 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 mahlakore a mabeli tlas'a maemo a tloaelehileng a static culture. Ka ho hlakileng, 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-chippisoli ea pele, e ka morao ea kopo ea "chippiso" ea pele le ho feta moo. hape ho bapisoa.
Ts'ebetso e bonolo ea lithography e lokela ho etsoa ka kamoreng e hloekileng. Bakeng sa lera le leng le le leng holim'a chip (ka holimo le ka tlaase 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. hybrid chip ke 200 µm.
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 silicon oafer e 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 ne e ajoa ka ho lekana holim'a lephaka ka ho tsamaisa spin coating. Lenaneo la ho potoloha ha SU-8 bakeng sa 5-10 s ho ata ka 500 rpm ka lebelo la 100 rpm/s. 500 µm bophahamo 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 ho 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, "spin coating" le mehato e bonolo ea ho baka ea 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 ka har'a lebokose le le leng le le leng le le leng le le leng le le leng le le leng, le ka behoang ka har'a lebokose la 1 le ho kopanngoa le 0. µm holimo.
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 ea 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 150 mm bophara se nang le matla a 1 L, sebelisa ~ 300 mold 300 mL . ho potoloha ha bonolo ka nako e itseng.
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. 2H-perfluorooctyl) tharollo ea silane holim'a selaete sa khalase ebe o 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.