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Inda ya morfogenezi yumuntu ishyiraho ibintu byitwa crypt-villus biranga 3D epithelial microarchitecture hamwe nu muteguro wahantu.Iyi miterere idasanzwe irasabwa kubungabunga amara homeostasis ikingira ingirangingo ngengabuzima ya selile yo mu bwoko bwa mikorobe ya antogen na mikorobe zabo. Byongeye kandi, villi yo mu nda hamwe no gusohora ururenda rwinshi rwihishwa rwimyanya myanya myanya myanya ndangagitsina. ya moderi ya vitro gut.Ntibisanzwe, imiterere ya mimetic gut-on-a-chip irashobora gutera ubwonko bwa 3D morfogenezi ya epitelium yo munda hamwe nibikorwa byimikorere ya physiologique hamwe na biomehanike.Dore hano, turatanga protocole yimyororokere kugirango itere imbaraga morfogenezi zo munda mu nda kuri chip ya microfluidic kimwe na chip ya Hyde ya Hyde. epithelial selile mumiterere isanzwe kimwe no kuri microfluidic platform, kwinjiza morfogenezi ya 3D, no kuranga epithelia yashizweho hakoreshejwe uburyo bwinshi bwo gufata amashusho .Iyi protocole igera ku kuvugurura imikorere yimitsi mikorobe ikora igenzura imigendekere yimyunyu ngugu ya 5 d.Uburyo bwacu bwa tekinoroji ya tekinoroji ya tekinoroji ya tekinoroji ya tekinoroji ya tekinoroji ya tekinike kandi idasaba ko hakoreshwa ubundi buryo bwa tekinike ya tekinike. Ingaruka kumuryango wubushakashatsi bwibinyabuzima, utanga uburyo bwo kuvugurura ibice bitatu byo mu nda epithelia muri vitro kubuzima bwa biomedical, clinique, na farumasi.
Ubushakashatsi bwerekana ko epiteliyale yo mu nda ya Caco-2 ikura mu nda-kuri-chip-1,2,3,4,5 cyangwa mikorobe ya microfluidic ya bilayeri6,7 ishobora kwandura 3D morfogenezi muri vitro idasobanutse neza uburyo bwashingiweho.Mu bushakashatsi duherutse gukora, twasanze kuvanaho indwara ya morfogene yatewe na virusi itera indwara ya virusi. 2 na organoide yo munda ikomoka ku barwayi.Epithelia selile yemejwe. Muri ubu bushakashatsi, twibanze cyane cyane ku musemburo wa selile no gukwirakwiza imbaraga za Wnt antagonist ikomeye, Dickkopf-1 (DKK-1), mu nda-kuri-chip ndetse n’ibikoresho bya microfluidic byahinduwe birimo insimburangingo ya Transwell, byitwa "Hybrid Chip". -1) kuri on-chip amara abuza morfogenezi cyangwa guhungabanya urwego rwa 3D epiteliyale yubatswe, byerekana ko guhangayikishwa na antagonistique mugihe cyumuco ari nyirabayazana ya morfogenezi yo munda muri vitro. Kubwibyo rero, uburyo bufatika bwo kugera kuri morfogenezi ikomeye kuri epiteleial interfeque ni ugukuraho cyangwa kugabanya urwego rwa Wnt antagonistes mu gice cya fonctionnement-yibice-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-by-ibice-byuzuye-by-ibice-byuzuye-by-ibice-by-ibice-byuzuye-by-ibice-byuzuye. gukwirakwiza .Itangazamakuru ryibanze (urugero, kuva Transwell yinjiza mubigega binini byibanze mu mariba).
Muri iyi protocole, turatanga uburyo burambuye bwo guhimba amara-kuri-chip-microdevices hamwe na Transwell-insertable hybrid chips (intambwe 1-5) kumico ya epithelial selile yo mu mara kuri polydimethylsiloxane (PDMS) ishingiye kumyanya ndangagitsina (intambwe 6A, 7A, 8, 9) cyangwa polyester membrane ya Transwell yinjizwamo (intambwe 6B, intambwe ya 6B, intambwe ya 6B, intambwe ya 9B). selile na molekuline biranga ibimenyetso byerekana amateka yihariye ya histogenezi no gutandukanya umurongo utandukanijwe na selile dukoresheje uburyo bwinshi bwo gufata amashusho (intambwe 11-24) .Twinjiza morfogenezi dukoresheje selile zo mu nda zo mu nda, nka Caco-2 cyangwa organoide zo mu nda, muburyo bubiri bwumuco hamwe nibisobanuro bya tekiniki harimo guhinduranya ibinyabuzima bya mikorobe hamwe na mikorobe ya mikorobe. 3D morphogenez muri 2D epithelial monolayers, twakuyeho antagonistes ya morfogene muburyo bwombi bwumuco twinjiza imiyoboro mugice cyibanze cyumuco.Mu kurangiza, turatanga ishusho yerekana akamaro ka epiteliyale ya 3D ishobora kuvugururwa ishobora gukoreshwa muburyo bwo gukura kwa morfogene, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara, kwanduza indwara. luences.
Porotokole yacu irashobora kuba ingirakamaro kubahanga benshi mubumenyi bwibanze (urugero, ibinyabuzima byo mu mara byo mu mara, ibinyabuzima byo mu bwoko bwa stem selile, na biologiya yiterambere) hamwe nubushakashatsi bwashyizwe mu bikorwa (urugero, gupima imiti y’ibanze, kwerekana indwara, gukora ingirabuzimafatizo, hamwe na gastroenterology) ingaruka nini.Kubera imyororokere n’imbaraga za protocole yacu kugira ngo itere imbaraga za enterineti zifata intungamubiri za enterinete muri vitamine. .Abumva indwara y’indwara na patogenezi na bo ni ingirakamaro. Gukoresha sisitemu yo mu bwoko bwa microphysiologiya ya chip irashobora kwemerera umuco-wo-kure-10-hamwe no gusuzuma nyuma yo kwirwanaho, ibisubizo by’ubudahangarwa no gukomeretsa indwara ziterwa na virusi mu gice cya gastrointestinal (GI) 11 .Indi ndwara ya GI ifitanye isano na syndrome de munda, indwara ya celiac, indwara ya kanseri yanduye, indwara ya kanseri yanduye. umurwayi wa 3D epiteliyale yumurwayi wa 3D, izi ndwara zirimo atrophyi mbi, kugabanya amarira, kwangirika kwa mucosal, cyangwa inzitizi ya epiteliyale.ingirabuzimafatizo yihariye yumubiri, 5.
Kubera ko microstructure ya 3D epiteliyale ishobora gukosorwa no kugaragara neza nta buryo bwo gutandukanya ibice, abayireba bakora kuri transcriptomique yumwanya hamwe n’ibisubizo bihanitse cyane cyangwa amashusho yerekana amashusho arashobora gushimishwa no gushushanya imbaraga za spatiotemporal dinamike ya gen na proteyine kuri niche epithelia.Ushishikajwe n'ikoranabuhanga. Igisubizo ku bitera mikorobe cyangwa ubudahangarwa bw'umubiri.Ikindi kandi, igihe kirekire cyakira-microbiome crosstalk 10, 14 gihuza amara homeostasis yo mu nda irashobora gushyirwaho mugice cya 3D cyo mu mara cyo mu nda hamwe no guhuza ubwoko butandukanye bwa mikorobe, mikorobe cyangwa mikorobe ya fecal, cyane cyane mu nda-kuri-chip.muri platifomu. Ubu buryo burashimishije cyane cyane kubantu bumva immunologiya ya mucosal, gastroenterology, microbiome yabantu, culturomics na microbiologiya ivura bashaka guhinga microbiota yo mu nda mbere idafite umuco muri laboratoire. Niba porotokole ya vitro morphogenez ishobora guhuzwa nimiterere yimico minini, nka progaramu ya farumasi ikomatanyirizwamo ibice byombi. omedical Cyangwa ibicuruzwa byinshi byapimwe cyangwa kwemeza inganda zibiribwa.Nkibihamya-ngenderwaho, duherutse kwerekana uburyo bushoboka bwa sisitemu ya Multlex nini-yinjiza cyane ya morfogenezi igereranywa nimiterere ya plaque 24. Byongeye kandi, ibicuruzwa byinshi-kuri-chip byashyizwe mubucuruzi byinganda zikora ubushakashatsi hamwe na laboratoire zishobora gukoreshwa muburyo bwa laboratwari hamwe na laboratoire zishobora gukoreshwa muburyo bwa laboratoire hamwe na laboratoire zishobora gukoreshwa na laboratoire ya leta hamwe na laboratoire ishobora gukoreshwa na laboratoire ya leta. ya muri vitro gut morfogenezi kurwego rwa transcriptomic kugirango bapime ibiyobyabwenge cyangwa biotherapeutics Kwinjira no gutwara abakandida b'ibiyobyabwenge byasuzumwe hifashishijwe amara ya 3D surrogate cyangwa hakoreshejwe imiterere gakondo cyangwa ubucuruzi-kuri-chip kugirango hamenyekane imyororokere yimikorere ya mara.
Umubare ntarengwa wubushakashatsi bwerekeranye nubumuntu bwakoreshejwe mukwiga epithelial morfogenezi yo munda, cyane cyane bitewe no kubura protocole zishyirwa mubikorwa zo gutera morfogenezi ya 3D muri vitro.Mu byukuri, ubumenyi bwinshi buriho bujyanye na morfogenezi yo mu nda bushingiye ku bushakashatsi bw’inyamaswa (urugero, zebrafish20, imbeba21 cyangwa inkoko22) .Nyamara, ni ngombwa, kandi ni ngombwa cyane, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa cyane, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa, kandi ni ngombwa cyane. icyitegererezo nacyo kigarukira cyane mubushobozi bwabo bwo gupimwa muburyo butandukanye bwagutse.Niyo mpamvu, protocole yacu yo kuvugurura imiterere ya tissue tissue ya 3D mumikorere ya vitro yinyamanswa mubyitegererezo byinyamanswa za vivo kimwe nubundi buryo bwa gakondo bwa 2D 2D umuco w’umuco. Nkuko byavuzwe haruguru, gukoresha imiterere ya epiteliyale ya 3D byadushoboje gusuzuma aho utandukanyirizo twinshi twinshi twerekana uburyo butandukanye bw’imyanya ndangagitsina. Ubwihindurize bwa ologiya mugusubiza kubintu byakiriwe (urugero, imbere imbere na mucus yo hanze, gusohora kwa IgA na peptide ya anticicrobial) .Ikindi kandi, morphologie ya epithelale ya 3D irashobora kudufasha gusobanukirwa nuburyo mikorobe yo munda itunganya imiterere yabaturage bayo kandi igahuza mikorobe ya mikorobe (urugero, amavuta ya acide aciriritse) yerekana imitunganyirize ya selile.
Usibye uburyo bwacu bwo gukora epithelale ya 3D yo mu nda, hariho uburyo bwinshi bwa vitro.Umuco wa organoide wo mu nda nubuhanga bugezweho bwa tekinike yubuhanga bushingiye ku guhinga ingirangingo zo mu mara mu bihe byihariye bya morfogene 23,24,25.Nyamara, gukoresha imiterere ya 3D organoide mu gusesengura ubwikorezi cyangwa mikorobe ikomatanya akenshi ni ingorabahizi kubera ko intangangore ziba mu mitsi hamwe na mikorobe ikomatanyirijwe hamwe. ial selile cyangwa antigen antigens igarukira.Kugera kuri lisansi ya organoide birashobora kunozwa hifashishijwe microinjector, 26.27 ariko ubu buryo buratera kandi busaba akazi kandi busaba ubumenyi bwihariye bwo gukora.Ikindi kandi, imico gakondo ya organoide ikomezwa muri hydrogel scafolds mubihe bihamye ntigaragaza neza mubikorwa bya vivo biomechanics.
Ubundi buryo bukoreshwa nitsinda ryinshi ryubushakashatsi bukoresha hydrogel scafolds yubatswe kugirango bigane imiterere ya epiteliyale yo mu nda hifashishijwe ingirabuzimafatizo zo mu nda zitaruye abantu ku buso bwa gel. Gukora hydrogel scafolds ukoresheje 3D yacapishijwe 3D, yasya mikorobe, cyangwa ibihimbano byakozwe na lithographie. igishishwa ushizemo utugingo ngengabuzima muri scafold.Nyamara, imiterere ya scafolds yubatswe irashobora kubuza kwerekana uburyo bwimikorere ya morfogeneetike ubwayo. Izi ngero nazo ntizitanga imbaraga za luminal cyangwa interstinterial flow, zidafite impungenge zo gutembera kwamazi yo mu nda ikenera mikorobe ya etoritifike ikorwa na hydrogène etesteque yubushakashatsi bwakoreshejwe na hydrogène scrofed. tekinike ya ching.Imbeba zo mu nda zo mu nda zikurikiza uburyo bwashizweho kugirango zibe imitsi yigitereko cyo munda, kandi itembera ryamazi yo munda irashobora gusubirwamo hifashishijwe module ya microfluidics.Nyamara, iyi moderi ntago igaragaza imikorere ya morfogeneque cyangwa ngo ikore imitsi ya mikorobiologiya.3D tekiniki yo gucapura ibice bitobito hamwe na mato mato mato mato mato mato mato mato mato mato. Amazi atemba no guhindura imashini. Byongeye kandi, imikorere yicyitegererezo irashobora kuba mike, cyane cyane nyuma yuburyo bwa bioprinting irangiye, bikabangamira imiterere yubushakashatsi cyangwa imikoranire ya selile-selile. Ahubwo, protocole yacu yatanzwe itanga ubwonko bwo mu nda bwihuse, ihindagurika ryimiterere yimitsi, ibinyabuzima bigizwe na mikorobe yibinyabuzima bigizwe na mikorobe ya mikorobe yibinyabuzima, hamwe na mikorobe yimbaraga za mikorobe. protokole irashobora gutanga inzira yuzuzanya kugirango ikemure ibibazo byuburyo buriho.
Porotokole yacu yibanze cyane kuri 3D epithelial morphogenez, hamwe na selile epithelale gusa mumuco kandi ntayindi moko yingirabuzimafatizo nka selile mesenchymal, selile endothelial selile, na selile selile.Nkuko byasobanuwe mbere, intandaro ya protocole yacu ni iyinjizwa rya morfogene yo mu bwoko bwa chip-ya-ya-ya-ya-ya-ya-ya-ya-ya-ya-ya-ya- yariye igipimo cya 3D epithelial layer, inyongera yibinyabuzima nka epithelial-mesenchymal imikoranire 33,34, Matrix idasanzwe ya Matrix (ECM) yoherejwe 35 kandi, murugero rwacu, ibintu byitwa crypt-villus byerekana utugingo ngengabuzima twibanze mu bice byibanze bikomeje gusuzumwa. enchymal selile kuri moderi yacu yazamuye imikorere ya morfogeneque hamwe no gukora neza kwingirangingo.Icyiciro cya endoteliyale (ni ukuvuga capillaries cyangwa lymphatics) kigira uruhare runini mugutwara ubwikorezi bwa molekile 39 hamwe no kwinjiza ingirabuzimafatizo 40 muri mikorobe yangiza ibidukikije. gukemura.Ingirabuzimafatizo zikomoka ku barwayi nazo ni ngombwa mu kwerekana ibisubizo by’ubudahangarwa bw'umubiri, kwerekana antigen, ubudahangarwa bw'umubiri wavutse, hamwe n'ubudahangarwa bwihariye bw'umubiri mu rwego rwo kwigana indwara zo mu nda.
Gukoresha imashini ya Hybrid iroroshye cyane kuruta gut-on-a-chip kubera ko igikoresho cyoroshye cyoroshye kandi gukoresha insimburangingo ya Transwell ituma umuco wagutse wa epitelium yo mu nda.Nyamara, ibicuruzwa byinjira mu bucuruzi byinjira mu bucuruzi hamwe na polyester ntabwo byoroshye kandi ntibishobora kwigana ibintu bisa na perisitique byashyizwemo na sitasiyo ya Hybrid yashyizwe kuri sitasiyo ya Hybrid. Ibice bidasanzwe ntibishobora gutuma bagiteri zigihe kirekire zifatanya mumico ya Hybride.Mu gihe dushobora gutera imbaraga za morfogenezi ya 3D muri inseri ya Transwell mugihe dukoresheje imashini ya Hybrid, ibura rya biomehanike ifitanye isano na physiologique hamwe nibisukari byamazi bishobora kugabanya uburyo bwo gukoresha imiyoboro ya Hybrid ishobora gukoreshwa.
Iyubakwa ryuzuye ryimikorere ya crypt-villus axis mumara-kuri-a-chip na hybrid-on-a-chip imico ntirashyirwaho neza.Kubera ko morfogenezi itangirira kuri epiteliyale monolayeri, microarchitecture ya 3D ntabwo byanze bikunze itanga morfologiya ihuye na kode muri vivo.Nubwo twaranze umubare w'abaturage b'utugari twinshi twinshi muri virusi ya virusi. imiyoboro yo hejuru kuri chip iganisha ku kongera uburebure bwa epitelium ya microengineered, uburebure ntarengwa buracyagarukira kuri ~ 300-400 µm. Ubujyakuzimu nyabwo bw’imitsi yo mu nda y’abantu mu mara mato manini kandi manini ni ~ 135 µm na ~ 400 µm, kandi uburebure bwa villi ntoya yo mu nda ni ~ 600 µm41.
Urebye kumashusho, muburyo bwa super-reaction yerekana amashusho ya microarchitecture ya 3D irashobora kugarukira kumara kuri chip, kubera ko intera isabwa kuva kumurongo wintego kugeza kuri epiteliyale iri kuri gahunda ya milimetero nkeya.Kugira ngo ukemure iki kibazo, intego ya kure irashobora gusabwa.Ikindi kandi, gukora ibice bito byerekana amashusho byateguwe bitoroshye bitewe nubushakashatsi bukabije bwa PDMS.Ikindi. layer, biragoye cyane gufungura cyangwa gukuraho urwego rwo hejuru kugirango dusuzume imiterere yubuso bwa epiteliyale.Urugero, ukoresheje scanning electron microscope (SEM).
Hydrophobicity ya PDMS yabaye imbogamizi mubushakashatsi bushingiye kuri microfluidic bujyanye na molekile ntoya ya hydrophobique, kubera ko PDMS ishobora kumenyekanisha mu buryo budasanzwe molekile ya hydrophobique.Ibisobanuro kuri PDMS bishobora gutekerezwa hamwe nibindi bikoresho bya polymeriki. Ubusanzwe, guhindura isura ya PDMS (urugero, gutwikira ibikoresho bya lipofilique 42) cyangwa poli.
Hanyuma, uburyo bwacu ntabwo bwaranzwe neza mubijyanye no gutanga ibicuruzwa byinshi byinjira cyangwa "imwe-imwe-ihuza-byose" urubuga rwikigereranyo rwifashisha igeragezwa. Porotokole iriho ubu isaba pompe ya syringe kuri microdevice, ifata umwanya muri incubator ya CO2 kandi ikabuza igeragezwa rinini.Iyi mbogamizi irashobora kunozwa cyane nubunini bwimiterere yumuco udushya -4, urugero-24 itangazamakuru).
Kugirango dushyireho morfogenezi ya 3D ya epitelium yo munda yumuntu muri vitro, twakoresheje microfluidic chip intestinal igikoresho kirimo microchannel ebyiri zibangikanye hamwe na elastique porous membrane hagati kugirango dukore interineti ya lumen-capillary.Twerekana kandi ko dukoresha umuyoboro umwe wa microfluidic (chip ya Hybrid) itanga urujya n'uruza rwinshi munsi ya polarisiyumu ya enterineti. Kugaragazwa no gukoresha manipulation yicyerekezo kugirango ukureho antagonistes ya morfogene mubice byibanze.Uburyo bwose bwo kugerageza (Igishusho 1) bugizwe nibice bitanu: (i) microfabrication ya chip yo munda cyangwa Transwell yinjizwamo chip (intambwe 1-5; Agasanduku 1), (ii) gutegura ingirabuzimafatizo zo mu nda (Caco-2 selile) cyangwa intangangore zo mu nda;agasanduku 2-5), (iii) umuco w'uturemangingo twa epiteliyale yo mu mara ku mitsi yo mu nda cyangwa imvange ya Hybrid (intambwe 6-9), (iv) kwinjiza 3D morfogenezi muri vitro (intambwe ya 10) na (v)) kuranga microstructure ya 3D epiteliyale (intambwe 11-24) .Mu kurangiza, itsinda rishinzwe kugenzura ibintu (byaganiriweho hepfo) ryakozwe kugirango hamenyekane neza imikorere ya vitro morogene. kugenzura.
Twakoresheje uburyo bubiri butandukanye bwumuco: gut-on-a-chip hamwe numuyoboro ugororotse cyangwa imiyoboro itagabanije, cyangwa imashini ya Hybrid irimo Transwell (TW) yinjizamo ibikoresho bya microfluidic, yahimbwe nkuko byasobanuwe mu gasanduku ka 1, n'intambwe ya 1 -5. protocole. "Muri vitro morphogenez" yerekana intambwe rusange aho selile ya Caco-2 cyangwa organoide ikomoka kuri organoide iterwa na chip yo munda cyangwa kuri Transwell yinjizamo chip ya Hybrid, hanyuma igakurikirwa no kwinjiza morfogenezi ya 3D hamwe no gushiraho imiterere ya epiteliyale.Intambwe yintambwe ya nomero cyangwa agasanduku kagaragazwa munsi ya buri mwambi. ya host-microbiome ecosystems, hamwe no kwerekana imiterere yindwara.Imashusho ya Imunofluorescence muri "Cell Differentiation" yerekana nuclei, F-actin na MUC2 yerekanwe muri 3D Caco-2 epithelial layer yakozwe kuri chip yo munda.Ikimenyetso cya MUC2 kiboneka muri selile ya goblet na mucus ziva mumisemburo ya acide ya acide ya globe ya acide. agglutinin. Amashusho abiri yuzuzanya muri "Host-Microbe Co-Cultures" yerekana abahagarariye host-microbiome bafatanije imico munda kuri chip.Ikibaho cyibumoso cyerekana ubufatanye bwa E. coli bwerekana poroteyine yicyatsi kibisi (GFP) hamwe na microcineine 3D Caco-2 epithelial selile hamwe na selile ya GFP E. coli. actin (umutuku) na nuclei (ubururu) .Icyitegererezo cyindwara cyerekana ubuzima bwiza nigifu kiva mumyanya yo gutwika amara mugihe cyibibazo bya physiologique hamwe na antigene za bagiteri (urugero, lipopolysaccharide, LPS) hamwe ningirabuzimafatizo (urugero, PBMC;icyatsi) .Caco-2 selile zari zifite umuco wo gushiraho epiteliyale ya 3D. Akabari kangana, 50 µm. Amashusho kumurongo wo hasi: "Itandukaniro rya selile" yahujwe nimpushya zitangwa.2.Itangazamakuru rya kaminuza ya Oxford;Yabyaye uruhushya rutangwa na Ref.5.NAS;"Umucumbitsi-Microbe Co-Umuco" yahujwe nimpushya zitangwa na ref.3.NAS;"Kwerekana Indwara" byahujwe nimpushya zitangwa.5.NAS.
Chip-on-chip hamwe na chip ya Hybride byahimbwe hakoreshejwe kopi ya PDMS yamanuwe kuva mubibumbano bya silicon na lithographie yoroshye1,44 ikanashushanywa na SU-8.Igishushanyo mbonera cya microchannel muri buri chip igenwa no gusuzuma hydrodynamic nka stress ya shear hamwe na hydrodynamic igitutu cyashushanyijeho ibice bibiri bigizwe na 1. gut-on-a-chip (Ikwirakwizwa ryamakuru Igicapo 1b) gikubiyemo imiyoboro ya mikorobe igoramye kugirango yongere igihe cyo gutura cyamazi yo guturamo, uburyo bwo gutembera kumurongo, hamwe no guhindura ibintu byinshi mumyanya ndangagitsina (Igicapo 2a - f) 12.Iyo mikorobe igoye cyane yo mu nda ikeneye guhindurwa, igifu gikaze kandi kigahita gishyirwa mu majwi. yo gukura kwa epiteliyale ugereranije na Gut-Chip yumwimerere, hatitawe ku bwoko bwimiterere yimikorere yimico.Niyo mpamvu, kugirango itere morfogenezi ya 3D, umurongo kandi utoroshye kuri chip yo mu nda irashobora guhinduranya. Kopi ya PPMS yakize kubibumbano bya silicon hamwe na SU-8 byatanze ibintu bibi nyuma yo kumeneka (Ishusho.. bikozwe no kuvura ubuso bwa kopi ya PDMS hamwe nikirahure hamwe na plasma ya ogisijeni cyangwa kuvura corona.Nyuma yo guhagarika ibikoresho bya microfabrica bifatanye na silicone, ibikoresho byashizweho byari biteguye gukora morfogenezi ya 3D ya epitelium yo munda (Ishusho 2g).
a. by'ibice byo hejuru na hepfo ya PDMS hamwe nibikoresho byateranijwe kuri chip-inteste.e, Igishushanyo cyo guhuza ibice byo hejuru, membrane, hamwe na PDMS yo hepfo. Buri cyiciro gihujwe kuburyo budasubirwaho nubuvuzi bwa plasma cyangwa corona.f, Igishushanyo cyibikoresho byahimbwe-kuri-chip hamwe na mikorobe yuzuye ya chim-hamwe na vacuum hamwe na selile ya selile. umuyoboro umwe na syringe byashyizwe ku gipfukisho. Igikoresho cya chip cyashyizwe ku gipfundikizo cya mm 150 ya Petri yo gutunganya.Binder ikoreshwa mu gufunga umuyoboro wa silicone.h, Amashusho agaragara yerekana ibihimbano bya Hybrid hamwe na morfogenezi ya 3D ukoresheje chip ya Hybride. munsi ya selile ya selile yashizwe kumurongo wa Transwell. Umwanya muto, cm 1.h Yongeye gusubirwamo uruhushya rutangwa.4.Elsevier.
Muri iyi protocole, umurongo wa selile Caco-2 hamwe na organoide yo munda byakoreshejwe nkisoko ya epiteliyale (Igishusho 3a) .Ubwoko bubiri bwingirabuzimafatizo bwaranzwe nubwigenge (agasanduku 2 na agasanduku 5) kandi bwakoreshwaga mu gutera mikorobe ya ECM ikozwe mu nda ya chip cyangwa insimburangingo ya Transwell. ated selile ihagarikwa na trypsinisation fluid (agasanduku 2) .Ibinyabuzima byo munda byumuntu biva muri biopies yo munda cyangwa kubaga kubaga byatewe mumatongo ya Matrigel scafold mumasahani 24 yuzuye kugirango ashyigikire ibidukikije byangiza ibidukikije. Medium irimo morfogene zingenzi (nka Wnt, R-spondin, na Noggin) hamwe nibintu bikura byateguwe nkuko byasobanuwe mumasanduku ya 3. gusarurwa no gutandukanywa mu ngirabuzimafatizo imwe yo gutera imbuto mu nda cyangwa Transwell yinjizamo chip (Agasanduku 5) .Nkuko twigeze kubivuga, irashobora gutandukana ukurikije ubwoko bwindwara12,13 (urugero: colitis ulcerative colitis, indwara ya Crohn, kanseri yibara, cyangwa abaterankunga basanzwe), urubuga rwakomeretse (urugero, lesion na non-lesion, ducum, ducum, ducum, ducum, ) .Dutanga protocole nziza muri Box 5 yo guhuza ibinyabuzima bya coloide (coloide) mubisanzwe bisaba kwibanda cyane kuri morfogene kuruta organoide zo munda.
a. iminsi 2 yambere (gutemba, AP, D0-D2) .Isoko ryibanze (BL) naryo ritangizwa hamwe no kurambura cycle (kurambura, gutemba, AP na BL) mugihe hashyizweho monolayeri ya 2D yuzuye. Igishushanyo mbonera cyacu cyerekana caskade ihuye na morfogenezi yo munda (hejuru iburyo) .Imyambi yacagaguritse mubishushanyo yerekana icyerekezo cyamazi yatemba.b, ishusho ya SEM yerekana topologiya yubuso bwa epitelium ya 3D Caco-2 (ibumoso) .Icyerekezo cyerekana agace gakomeye (agasanduku gashushanyijeho) kuri microcilli ya 3D ya Caco. muri (ZO-1, umutuku) hamwe no guhanagura umupaka wibibabi byanditseho F-actin (icyatsi) na nuclei (ubururu) Immunofluorescence confocal visualisation ya selitile epithelia kumyanya yo munda.Imyambi yerekana igishushanyo mbonera cyerekana aho indege yibanze kuri buri cyerekezo giteye ubwoba.d, Igihe cyimpinduka zijyanye na morphologi muri organoide zifite umuco kuri microset ya 9 ification yishusho yatanzwe.magenta), selile ya goblet (MUC2; icyatsi), F-actin (imvi) na nuclei (cyan) ikura kumitsi yamara iminsi 3, kimwe (Ibumoso) hamwe niminsi 13 (hagati) organoide kurwego rwa epiteliyale. Reba kandi Data Data Yaguye Ishusho ya 3, yerekana ibimenyetso bya LGR5 byerekana ibimenyetso bya epitelium ya selile. baza irangi (iburyo) kumunsi wa 13 wumuco. Akabari kangana ni 50 mm keretse bivuzwe ukundi.b Byasubiwemo uruhushya rutangwa.2.Itangazamakuru rya kaminuza ya Oxford;c Yahinduwe nu ruhushya rutangwa na.2.Itangazamakuru rya kaminuza ya Oxford;e na f byahujwe nu ruhushya kubisobanuro.12 Munsi ya Creative Commons Uruhushya CC BY 4.0.
Mu mara kuri chip, birakenewe guhindura hydrophobique yubuso bwa PDMS porous membrane kugirango igere neza kuri ECM.Muri iyi protocole, dukoresha uburyo bubiri butandukanye bwo guhindura hydrophobicity ya membrane ya PDMS. Kubijyanye no gutsimbataza selile Caco-2, gukora hejuru yubutaka bwa UV / ozone byonyine byari bihagije kugirango ugabanye hydrophobic ya selile ya PDMSH hamwe na CMS ya PDMS. umuco wa epitelium ya organoide isaba imikorere yubutaka bushingiye kumiti kugirango igere neza kuri proteine ​​za ECM ukoresheje uburyo bukurikiranye bwa polyethyleneimine (PEI) na glutaraldehyde kuri microchannel ya PDMS.Nyuma yo guhindura isura, proteyine za ECM zashyizwe kugirango zipfundikire hejuru ya PDMS hanyuma zinjizwe mumyanya mikorobe yonyine itangizwa na selile microfluidic selile. , mugihe microchannel yo hepfo ikomeza ibintu bihagaze neza.Ubu buryo bwiza bwogukora hejuru yubutaka hamwe na ECM bifasha guhuza epitelium ya organoide kugirango itere morfogenezi ya 3D hejuru ya PDMS.
Imico ya Transwell isaba kandi ECM gutwikira mbere yo gutera imbuto;icyakora, imico ya Transwell ntisaba intambwe igoye yo kwitegura kugirango ikore hejuru yubuso bwuzuye.Ku gukura ingirabuzimafatizo za Caco-2 ku iyinjizwa rya Transwell, ECM itwikiriye insina zihuta byihutisha guhuza ingirabuzimafatizo za Caco-2 (Muri vitro 3D morfogenezi irashobora gutangizwa mugukoresha amazi atembera mubice byibanze byurwego rwashizweho.Mu nda yo kuri chip, epithelial morphogenez yatangiriye mugihe uburyo bwakoreshwaga muri mikorobe yo hejuru no hepfo (Igicapo 3a) .Nkuko byasobanuwe mbere, ni ngombwa kwinjiza ibintu bitembera mungingo zuzuye za poroteyine. ranes no kubyara luminal shear stress, mubisanzwe dushyira imigozi ibiri mumara kuri chip.Mu chipi ya Hybrid, insimburangingo ya Transwell irimo epithelial monolayers yinjijwe muri chip ya Hybrid.
Imiterere ya morfologiya yibice bya epithelale ya microengineered irashobora gusesengurwa ukoresheje uburyo butandukanye bwo gufata amashusho, harimo microscopi yo gutandukanya icyiciro, itandukaniro rya interineti itandukanye (DIC) microscopi, SEM, cyangwa microscopi ya immunofluorescence (Ishusho ya 3 n'iya 4) .Ibice bitandukanye cyangwa amashusho ya DIC birashobora gukorwa byoroshye mugihe icyo aricyo cyose mugihe cyumuco kugirango harebwe imiterere na primaire ya 3D ya epiteliyale. chip platform irashobora gutanga igihe-nyacyo mumashusho idakenewe gukenera kugabanywa cyangwa gusenya igikoresho.Iyo ukora amashusho ya immunofluorescence (Ishusho 1, 3c, f na 4b, c), selile zisanzwe zishyirwaho na 4% (wt / vol) paraformaldehyde (PFA), ikurikirwa na Triton X-100 na 2% (wt / vol)) ers, hamwe nuguhagarika ibintu birashobora gukoreshwa. Antibodiyumu yibanze yibasira ingirabuzimafatizo zishingiye kumurongo cyangwa ibimenyetso byakarere bikoreshwa mukugaragaza ingirabuzimafatizo zidafite aho zihurira na chip, hanyuma zigakurikirwa na antibodiyumu ya kabiri hamwe n irangi ryo kurwanya irangi ryibasira nucleus (urugero, 4 ′, 6-diamidino-2-fenylene) ryerekana indo, DAPI) cyangwa F-actin (Flucine). (Ishusho.1. epithelial morphologie kimwe na microvilli kumupaka wa apical brush irashobora kugaragazwa na SEM (Igishusho 3b) .Imvugo yerekana ibimenyetso bitandukanya irashobora gusuzumwa mugukora PCR5 cyangwa selile selile RNA ikurikirana.Muri iki gihe, ibice bya 3D byingirabuzimafatizo zikura mumyanya ndangagitsina cyangwa chip ya Hybride bisarurwa na trypsinisation hanyuma bigakoreshwa mubisesengura bya molekile cyangwa genetique.
a. iminsi, insimburangingo imwe ya Transwell irimo 2D monolayeri yingirabuzimafatizo ya epiteliyale yinjijwe muri chip ya Hybrid kugirango itangire urujya n'uruza rw'ibanze (Flow, BL), amaherezo yatumye havuka urwego rwa epiteliyale ya 3D (morfogenezi) .Icyiciro cya mikoro itandukanye yerekana imiterere ya morfologiya yibice bigize intangangore ya buri munsi. chip (ibishushanyo byibumoso) birashobora kuganisha kuri 3D morfogenezi ya selile ya epiteliyale ya organoid hamwe na microscopi yo hejuru-hepfo ya conocal yafashwe ku myanya itandukanye ya Z (hejuru, hagati, na hepfo;reba ibishushanyo biboneye kandi bihuye n'imirongo yerekana utudomo).yerekanye ibimenyetso biranga morphologie. c Byasubiwemo uruhushya rutangwa.4.Elsevier.
Igenzura rirashobora gutegurwa muguhuza ingirabuzimafatizo zimwe (Caco-2 cyangwa intestinal organoid epithelial selile) mo monolayers ebyiri-zimiterere yimiterere yumuco usanzwe.Ntabwo bivuze ko kugabanuka kwintungamubiri bishobora kuvamo bitewe nubushobozi buke bwimikorere ya mikorobe (ni ukuvuga ~ 4 µL mumuyoboro wo hejuru kumurongo wambere wa chip-chip) .Nuko rero, epithelia morphologie mbere na nyuma yo gukoreshwa).
Uburyo bworoshye bwa lithographie bugomba gukorerwa mucyumba gisukuye.Kuri buri cyiciro kuri chip (hejuru no hepfo yo hepfo na membrane) hamwe na chip ya Hybrid, fotomasi zitandukanye zarakoreshejwe kandi zihimbwa kuri wafer ya silicon itandukanye kuko uburebure bwa microchannel bwari butandukanye. Uburebure bwintego za microchannel yo hejuru no hepfo ya chip ni 500 µm na 200 µm.
Shira wafer ya silicon ya santimetero 3 mu isahani hamwe na acetone. Kuzenguruka witonze isahani amasegonda 30, hanyuma umuyaga wumishe wafer. Hindura wafer ku isahani hamwe na IPA, hanyuma uzenguruke isahani kuri 30 s kugirango usukure.
Umuti wa piranha (uruvange rwa hydrogène peroxide na acide sulfurike yibanze, 1: 3 (vol / vol)) urashobora gukoreshwa muburyo bwo gukuraho ibisigazwa kama kama hejuru ya silicon wafer.
Igisubizo cya Piranha kirashobora kwangirika cyane kandi gitanga ubushyuhe. Harakenewe ingamba zo kwirinda umutekano wongeyeho.Kujugunya imyanda, emera igisubizo gikonje kandi cyimurwe mu kintu cyanduye, cyumye. Koresha ibikoresho bya kabiri kandi wandike neza imyanda. Nyamuneka ukurikize amabwiriza yumutekano yikigo kugirango ubone uburyo burambuye.
Kuvomerera waferi ubishyira ku isahani ishyushye ya 200 ° C mu minota 10. Nyuma yo kubura umwuma, wafer yazunguye inshuro eshanu mu kirere kugira ngo akonje.
Suka ~ 10 g yumufotozi SU-8 2100 hagati ya wafer ya silicon isukuye. Koresha tewers kugirango ukwirakwize fotorezeri kuri wafer. Rimwe na rimwe shyira wafer ku isahani ishyushye ya 65 ° C kugirango fotorester idafatika kandi byoroshye gukwirakwizwa.Ntugashyire wafer mu isahani ishyushye.
SU-8 yagabanijwe neza kuri wafer ukoresheje coating spin.Programme izunguruka yinjira ya SU-8 kuri 5-10 s kugirango ikwirakwize saa kumi nimwe zumugoroba ku muvuduko wa 100 rpm / s. Shyira umuzenguruko nyamukuru kuri 200 µm umubyimba ushushanya kuri 1.500 rpm, cyangwa ugere kuri 250 µm uburebure bwa santimetero 30 kuri rpm nimugoroba.
Umuvuduko wingenzi wa spin urashobora guhindurwa ukurikije uburebure bwintego ya SU-8 kuri wafer ya silicon.
Kugirango uhimbe ishusho ya SU-8 yuburebure bwa 500 µm kurwego rwo hejuru rwigifu kuri chip, kuzunguruka kuzunguruka hamwe nintambwe zoroshye zo guteka ziyi Boxe (intambwe ya 7 na 8) byagarutsweho bikurikiranye (reba intambwe ya 9) kugirango bitange ibice bibiri bya 250 µm Igice kinini cya SU-8, gishobora gushyirwaho kandi kigahuzwa na UV yerekanwe kumurongo wa 12 wiyi sanduku, ugakora urwego 500 µm hejuru.
Byoroshye guteka waferi ya SU-8 ushyira witonze ushyira waferi ku isahani ishyushye kuri 65 ° C muminota 5, hanyuma uhindure igenamiterere kuri 95 ° C hanyuma ushireho iminota 40.
Kugirango ugere kuri 500 mkm z'uburebure bwa SU-8 muri microchannel yo hejuru, subiramo intambwe 7 na 8 kugirango ubyare ibice 250 250 mm bya SU-8.
Ukoresheje UV Mask Aligner, kora ikizamini cyamatara ukurikije amabwiriza yabakozwe kugirango ubare igihe cyo kwerekana wafer.
Nyuma yo kumenya igihe cyo kwerekana, shyira fotomask kuri mask ufashe mask ya UV mask hanyuma ushire fotomasi kuri wafer ya SU-8.
Shyira hejuru yifoto ya fotomask kuruhande rwa SU-8 rwometse kuri wafer ya silicon kugirango ugabanye UV ikwirakwizwa.
Erekana SU-8 isize wafer na fotomask uhagaritse kuri 260 mJ / cm2 yumucyo wa UV mugihe cyagenwe cyagenwe (reba intambwe ya 10 yiyi sanduku).
Nyuma yo kwerekana UV, wafer ya SU-8 yometse kuri silicon yatetse kuri 65 ° C kuminota 5 na 95 ° C kuminota 15 kuri buri sahani ishyushye kugirango ihimbe ibishushanyo bifite uburebure bwa 200 mk.
Iterambere risukwa mu isahani yikirahure, hanyuma wafer yatetse igashyirwa mu isahani. Ingano yuwatezimbere SU-8 irashobora gutandukana bitewe nubunini bwikirahure. Wiyemeze gukoresha SU-8 ihagije kugirango ukureho burundu SU-8.
Kwoza ifu yateye imbere hamwe na ~ 10 mL yiterambere rishya rikurikirwa na IPA utera igisubizo ukoresheje pipette.
Shira wafer mu isuku ya plasma hanyuma uhure na plasma ya ogisijeni (gaze yo mu kirere, umuvuduko w'intego 1 × 10−5 Torr, ingufu 125 W) muminota 1.5.
Shira wafer muri desiccator ya vacuum hamwe nigitereko cyikirahure imbere.Inyanja na slide birashobora gushyirwa muruhande rumwe.Niba desiccator ya vacuum igabanijwemo ibice byinshi hamwe nisahani, shyira amashusho mucyumba cyo hasi hamwe na wafer mucyumba cyo hejuru. Kureka 100 μL ya trichloro (1H, 1H, 2H, 2H-perfluorooct).
Kuramo isahani ya selile Caco-2 yakonje mu bwogero bw’amazi ya 37 ° C, hanyuma wimure ingirabuzimafatizo zashizwe mumashanyarazi ya T75 irimo mL 15 ya 37 ° C yabanjirije Caco-2.
Gutambutsa selile Caco-2 kuri ~ 90% ihurira, ubanza gushyushya Caco-2 giciriritse, PBS, na 0.25% trypsin / 1 mM EDTA mubwogero bwamazi 37 ° C.
Wifuze uburyo ukoresheje icyifuzo cya vacuum. Karaba selile kabiri hamwe na 5 mL yubushyuhe bwa PBS usubiramo ibyifuzo bya vacuum hanyuma wongereho PBS nshya.