Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version koj siv tau txwv kev txhawb nqa rau CSS.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis kaw qhov kev sib raug zoo hauv Internet Explorer).
Tib neeg lub plab morphogenesis tsim crypt-villus nta ntawm 3D epithelial microarchitecture thiab spatial lub koom haum.Qhov no tshwj xeeb qauv yog yuav tsum tau tswj lub plab homeostasis los ntawm kev tiv thaiv cov qia cell niche nyob rau hauv lub basal crypt los ntawm exogenous microbial antigens thiab lawv cov metabolites. Tsis tas li ntawd, cov kab mob plab hnyuv muaj peev xwm tiv thaiv cov kab mob hauv lub cev nrog rau cov kab mob hauv lub cev. Kev thaiv ntawm txoj hnyuv mucosal nto.Yog li ntawd, rov tsim cov txheej txheem 3D epithelial yog qhov tseem ceeb rau kev tsim kho hauv vitro plab qauv.Nco ntsoov, cov organic mimetic plab-on-a-chip tuaj yeem ua rau muaj 3D morphogenesis ntawm txoj hnyuv epithelium nrog kev txhim kho thiab kev ua haujlwm ntawm peb lub cev. Txhawm rau ua kom muaj zog hauv plab hnyuv morphogenesis hauv plab rau ntawm microfluidic nti nrog rau hauv Transwell embedded hybrid nti.Peb piav qhia ntxaws txog cov txheej txheem rau kev tsim khoom, kev coj noj coj ua ntawm Caco-2 lossis plab hnyuv organoid epithelial hlwb nyob rau hauv cov chaw ib txwm zoo li ntawm lub microfluisis platform 3D, induction ntawm cov noob caj noob ces, thiab kev tsim cov noob caj noob ces. Ntau yam kev ntsuas kev ntsuas .Qhov kev cai no ua tiav cov kev tsim kho ntawm lub plab microarchitecture los ntawm kev tswj cov kua dej basolateral rau 5 d.Our in vitro morphogenesis txoj kev ntiav cov kws kho mob muaj feem cuam tshuam txog kev ntxhov siab thiab cov tshuab ua haujlwm thiab tsis tas yuav tsum muaj kev ua haujlwm ntawm tes los yog kev tswj xyuas, uas tuaj yeem ua tau zoo dua lwm cov txheej txheem uas twb muaj lawm. biomedical tshawb fawb zej zog, muab ib txoj hauv kev los tsim 3D plab hnyuv epithelial txheej hauv vitro rau biomedical, kho mob, thiab tshuaj siv.
Cov kev sim ua kom pom tias plab hnyuv epithelial Caco-2 hlwb kab lis kev cai hauv plab-on-a-chip1,2,3,4,5 lossis bilayer microfluidic devices6,7 tuaj yeem ua raws li 3D morphogenesis hauv vitro yam tsis muaj kev nkag siab meej ntawm lub hauv paus txheej txheem. tsim nyog thiab txaus los ntxias 3D epithelial morphogenesis hauv vitro, uas tau pom los ntawm Caco-2 thiab cov neeg mob tau txais cov kab mob hauv plab hnyuv. Nyob rau hauv txoj kev tshawb no, peb tau tsom mus rau lub cell ntau lawm thiab concentration tis ntawm ib tug haib Wnt antagonist, Dickkopf-1 (DKK-1), nyob rau hauv lub plab-on-a-chip thiab hloov kho microfluidic li uas muaj Transwell inserts, termed "Hybrid Chip". repressor 1, secreted frizzled-related protein 1, los yog Soggy-1) mus rau hauv-chip plab inhibits morphogenesis los yog cuam tshuam lub prestructured 3D epithelial txheej, tawm tswv yim hais tias antagonistic kev nyuaj siab nyob rau hauv kab lis kev cai yog lub luag hauj lwm rau plab hnyuv morphogenesis nyob rau hauv vitro. Yog li ntawd, ib tug tswv yim mus kom ze rau lub epithelial interfacing los yog genetic mus kom ze. minimally tswj cov theem ntawm Wnt antagonists nyob rau hauv lub basolateral compartment los ntawm active flushing (xws li, nyob rau hauv lub plab-on-a-chip los yog hybrid-on-a-chip platforms) los yog diffusion .Basolateral media (xws li, los ntawm Transwell inserts rau hauv loj basolateral reservoirs nyob rau hauv lub qhov dej).
Hauv cov txheej txheem no, peb muab cov txheej txheem ntxaws ntxaws rau kev tsim cov plab hnyuv microdevices thiab Transwell-insertable hybrid chips (cov kauj ruam 1-5) rau kab mob plab hnyuv epithelial hlwb ntawm polydimethylsiloxane (PDMS)-raws li cov ntaub so ntswg (qib 6A, 7A, 8, 9Bs) lossis polyester 8, 9) thiab induced 3D morphogenesis hauv vitro (qib 10).Peb kuj tau txheeb xyuas cov cellular thiab molecular nta qhia txog cov ntaub so ntswg tshwj xeeb histogenesis thiab lineage-dependent cellular sib txawv los ntawm kev siv ntau yam kev ntsuas (qib 11-24). organoids, nyob rau hauv ob hom kab lis kev cai nrog kev paub meej nrog rau saum npoo ntawm cov porous membranes, creation ntawm 2D monolayers, thiab plab hnyuv biochemical thiab Reproduction ntawm lub biomechanical microenvironment.in vitro.To induce 3D morphogenesis los ntawm 2D epithelial monolayers, peb tshem tawm morphogen antagonists nyob rau hauv nruab nrab ntawm cov kab lis kev cai. kab lis kev cai.Thaum kawg, peb muab ib qho kev sawv cev ntawm cov khoom siv hluav taws xob ntawm cov txheej txheem 3D epithelial uas tuaj yeem siv los ua qauv morphogen-dependent epithelial kev loj hlob, longitudinal host-microbiome co-cultures, kab mob, inflammatory raug mob, epithelial barrier dysfunction, thiab probiotic-based therapies.
Peb cov txheej txheem yuav muaj txiaj ntsig zoo rau ntau yam ntawm cov kws tshawb fawb hauv theem pib (piv txwv li, plab hnyuv mucosal biology, qia cell biology, thiab kev loj hlob biology) thiab siv kev tshawb fawb (xws li, kuaj tshuaj preclinical, kab mob qauv, cov ntaub so ntswg engineering, thiab gastroenterology) broad impact.Vim yog cov reproducibility thiab robustness ntawm peb cov proclinical 3. epithelium hauv vitro, peb xav tias peb cov tswv yim kev tshaj lij tuaj yeem tshaj tawm rau cov neeg tuaj saib kev kawm txog kev hloov pauv ntawm lub xov tooj ntawm tes thaum lub sij hawm plab hnyuv loj hlob, rov tsim dua lossis homeostasis . Tsis tas li ntawd, peb cov txheej txheem muaj txiaj ntsig zoo rau kev soj ntsuam cov kab mob nyob rau hauv ntau yam kab mob xws li Norovirus 8, Severe Acute Respiratory Syndrome CoV-2), SARS-Monella. 9 los yog Vibrio cholerae. Cov neeg tuaj saib ntawm cov kab mob pathology thiab cov kab mob pathogenesis kuj tseem muaj txiaj ntsig.Kev siv ntawm on-chip plab microphysiology system tuaj yeem tso cai rau kev sib koom ua ke ntev 10 thiab kev soj ntsuam tom qab ntawm tus tswv tsev tiv thaiv, tiv thaiv kab mob thiab kab mob cuam tshuam kev raug mob hauv plab hnyuv (GI) kab mob 11 .Lwm cov kab mob GI, kab mob hauv lub cev, ulcerative colitis, pouchitis, los yog chim siab plob tsis so tswj syndrome yuav simulated thaum 3D plab hnyuv epithelial txheej yog npaj siv tus neeg mob lub 3D plab hnyuv epithelial txheej, cov kab mob no muaj xws li villous atrophy, crypt shortening, mucosal puas, los yog impaired epithelial barrier.Biopsyal cell. organoids12,13.To ua qauv zoo dua qhov nyuaj ntawm cov kab mob ib puag ncig, cov neeg nyeem yuav xav txog kev ntxiv cov kab mob ntsig txog ntawm tes, xws li cov neeg mob peripheral ntshav mononuclear hlwb (PBMCs), rau cov qauv uas muaj 3D plab hnyuv villus-crypt microarchitectures. cov ntaub so ntswg tshwj xeeb tiv thaiv kab mob, 5.
Txij li thaum 3D epithelial microstructure tuaj yeem kho thiab pom yam tsis muaj cov txheej txheem ntu, cov neeg saib ua haujlwm ntawm spatial transcriptomics thiab kev daws teeb meem siab lossis kev daws teeb meem zoo tshaj plaws yuav txaus siab rau peb daim duab qhia ntawm spatiotemporal dynamics ntawm cov noob thiab cov proteins ntawm epithelial niches. Txaus siab rau kev siv technology.Tshuaj rau cov kab mob microbial lossis tiv thaiv kab mob stimuli. Tsis tas li ntawd, longitudinal host-microbiome crosstalk 10, 14 uas tswj lub plab homeostasis tuaj yeem tsim nyob rau hauv 3D txoj hnyuv mucosal txheej los ntawm kev sib koom ua ke ntau yam kab mob microbial, cov zej zog microbial lossis fecal microbiota-, tshwj xeeb tshaj yog nyob rau hauv cov kab mob microbiota. nyob rau hauv lub platform.Qhov no yog tshwj xeeb tshaj yog txaus nyiam rau cov neeg tuaj saib kawm mucosal immunology, gastroenterology, tib neeg microbiome, culturomics thiab soj ntsuam microbiology nrhiav cultivate yav tas los uncultured plab microbiota nyob rau hauv lub laboratory.Yog hais tias peb nyob rau hauv vitro morphogenesis raws tu qauv yuav tau yoog mus rau scalable kab lis kev cai 32 8 zoo li cov ntaub ntawv, cov phaj uas txuas ntxiv ntxiv basolateral compartments, cov txheej txheem kuj tseem tuaj yeem nthuav tawm rau cov neeg tsim tshuaj, biomedical lossis kev tshuaj ntsuam xyuas siab lossis kev siv tau zoo rau kev lag luam zaub mov.Raws li cov ntaub ntawv pov thawj, peb tsis ntev los no tau pom qhov ua tau ntawm multiplex high-throughput morphogenesis awell formats 2. Cov khoom siv hauv nruab nrog cev-on-a-chip tau ua lag luam16,17,18.Yog li ntawd, kev siv tau ntawm peb txoj hauv kev hauv vitro morphogenesis tuaj yeem nrawm dua thiab muaj peev xwm tau txais los ntawm ntau lub chaw soj ntsuam kev tshawb fawb, kev lag luam lossis tsoomfwv thiab cov koom haum tswj hwm kom nkag siab txog cellular reprogramming ntawm hauv vitro plab morphogenesis ntawm qib transcriptomic therapeutics rau kev kuaj tshuaj los yog cov tshuaj absorption. soj ntsuam siv 3D plab surrogates los yog siv kev cai los yog kev lag luam hauv nruab nrog cev-on-a-chip qauv los ntsuam xyuas lub reproducibility ntawm lub plab morphogenesis txheej txheem.
Ib tug tsawg tus naj npawb ntawm tib neeg cov qauv kev sim tau siv los kawm txoj hnyuv epithelial morphogenesis, feem ntau yog vim tsis muaj cov txheej txheem siv tau los ua rau 3D morphogenesis hauv vitro.Qhov tseeb, ntau qhov kev paub txog lub plab morphogenesis yog raws li kev tshawb fawb tsiaj (xws li, zebrafish20, mice21H) los yog qaib, thiab 2. ethically questionable, thiab tseem ceeb tshaj, tsis txhob precisely txiav txim siab tib neeg txoj kev loj hlob txheej txheem.Cov qauv no kuj muaj tsawg heev nyob rau hauv lawv lub peev xwm los mus soj ntsuam nyob rau hauv ib tug ntau txoj kev scalable yam.Yog li ntawd, peb raws tu qauv rau regenerating 3D cov ntaub so ntswg nyob rau hauv vitro outperforms nyob rau hauv vivo tsiaj qauv raws li zoo raws li lwm yam tsoos zoo li qub 2D cell kab lis kev cai qauv.As yav tas los epilization rau peb tshuaj xyuas lub spatial localization ntawm txawv hlwb nyob rau hauv lub crypt-villus axis teb rau ntau yam mucosal los yog lub cev tiv thaiv stimuli.3D epithelial txheej tuaj yeem muab qhov chaw los kawm seb cov hlwb microbial sib tw li cas rau cov spatial niches thiab ecological evolution nyob rau hauv cov lus teb rau host yam tseem ceeb (piv txwv li, anti sab hauv tiv thaiv cov hnoos qeev sab nraud, peptides). Tsis tas li ntawd, 3D epithelial morphology tuaj yeem ua rau peb nkag siab tias lub plab microbiota txhim kho nws cov zej zog li cas thiab sib koom ua ke tsim cov microbial metabolites (piv txwv li, luv-chain fatty acids) uas zoo li lub koom haum cellular thiab qia cell niches nyob rau hauv basal vicrypts.Cov nta no tsuas yog tsim los ntawm cov txheej txheem 3.
Ntxiv nrog rau peb txoj kev tsim 3D plab hnyuv epithelial cov qauv, muaj ntau txoj hauv kev hauv vitro.Intestinal organoid kab lis kev cai yog lub xeev-of-the-art cov ntaub so ntswg engineering txheej txheem raws li kev cog qoob loo ntawm cov kab mob hauv cov kab mob hauv cov kab mob hauv cov kab mob tshwj xeeb morphogen 23,24,25.Txawm li cas los xij, kev siv 3D organoid-cultures feem ntau yog cov qauv kev thauj mus los los yog chassis. lub plab hnyuv lumen yog nyob rau hauv lub organoid thiab, yog li ntawd, kev taw qhia ntawm luminal Cheebtsam xws li microbial hlwb los yog exogenous antigens yog txwv. Kev nkag mus rau organoid lumens tuaj yeem txhim kho tau siv microinjector, 26,27 tab sis txoj kev no yog qhov cuam tshuam thiab ua haujlwm hnyav thiab yuav tsum tau muaj kev paub tshwj xeeb los ua. Tsis tas li ntawd, cov kab lis kev cai organoid tsoos khaws cia hauv hydrogel scaffolds nyob rau hauv cov xwm txheej zoo li qub tsis muaj kev cuam tshuam rau hauv vivo biomechanics.
Lwm txoj hauv kev ua haujlwm los ntawm ntau pab pawg tshawb fawb siv cov txheej txheem ua ntej 3D hydrogel scaffolds los ua cov qauv ntawm lub plab epithelial los ntawm kev tsim cov kab mob hauv tib neeg cov hnyuv ntawm cov gel nto.Fabricate hydrogel scaffolds siv 3D-luam, micro-milled, los yog lithographically fabricated pwm. physiologically cuam tshuam morphogen gradients, tsim kom muaj ib tug high nam piv epithelial qauv thiab stroma-epithelial crosstalk los ntawm xws li stromal hlwb nyob rau hauv lub scaffold.Txawm li cas los, qhov xwm ntawm prestructured scaffolds yuav tiv thaiv cov zaub ntawm cov spontaneous morphogenetic txheej txheem ntawm nws tus kheej. Cov kab mob hauv plab yuav tsum tau mus rau morphogenesis thiab nce physiological muaj nuj nqi.Lwm txoj kev tshawb fawb tsis ntev los no tau siv hydrogel scaffolds hauv microfluidic platform thiab cov qauv plab hnyuv epithelial siv cov txheej txheem laser-etching.Mouse plab hnyuv organoids ua raws li cov qauv etched los ua cov kab mob hauv plab hnyuv, thiab cov kua dej intraluminal. qauv tsis muaj cov txheej txheem morphogenetic tshwm sim los yog tsis suav nrog lub plab mechanobiological txav.3D tshuab luam ntawv los ntawm tib pab pawg tuaj yeem tsim cov kab me me nrog cov txheej txheem morphogenetic.Txawm hais tias txoj kev tsim ntawm cov plab hnyuv sib txawv hauv lub raj, cov qauv no kuj tsis muaj cov qauv ua haujlwm tsis zoo. txwv tsis pub, tshwj xeeb tshaj yog tom qab cov txheej txheem bioprinting tiav, ua rau muaj kev cuam tshuam kev sim lossis kev sib cuam tshuam ntawm cell-to-cell.Xwb, peb cov txheej txheem npaj tau muab cov plab hnyuv morphogenesis, physiologically cuam tshuam shear stress, biomechanics uas mimic plab motility, kev nkag tau ntawm ywj siab apical thiab basolateral compartments ntawm kev ua haujlwm ntawm peb lub cev. nyob rau hauv vitro 3D morphogenesis raws tu qauv yuav muab ib tug ntxiv mus kom ze rau kov yeej cov kev cov nyom ntawm cov uas twb muaj lawm txoj kev.
Peb cov txheej txheem yog tag nrho tsom rau 3D epithelial morphogenesis, tsuas yog cov hlwb epithelial hauv kab lis kev cai thiab tsis muaj lwm yam ntawm cov hlwb nyob ib puag ncig xws li mesenchymal cells, endothelial cells, thiab immune cells.Raws li tau piav qhia dhau los, lub hauv paus ntawm peb cov txheej txheem yog induction ntawm epithelial morphogenesis los ntawm kev tshem tawm qhov nruab nrab ntawm cov morphogen. lub zog modularity ntawm peb lub plab-on-a-chip thiab hybrid-on-a-chip tso cai rau peb rov tsim cov undulating 3D epithelial txheej, ntxiv lom complexities xws li epithelial-mesenchymal kev sib cuam tshuam 33,34, extracellular Matrix (ECM) deposition 35 thiab, nyob rau hauv peb cov qauv ntawm cov ntaub ntawv, crypts bass lus. tseem yuav tsum tau txiav txim siab ntxiv.Stromal hlwb (xws li, fibroblasts) hauv mesenchyme ua lub luag haujlwm tseem ceeb hauv kev tsim cov ECM cov proteins thiab kev tswj cov plab hnyuv morphogenesis hauv vivo35,37,38.Qhov sib ntxiv ntawm cov hlwb mesenchymal rau peb cov qauv txhim kho cov txheej txheem morphogenetic thiab cell txuas efficiency.Cov txheej txheem endothelial molecular (piv txwv li, lub luag haujlwm tseem ceeb hauv cov kab mob qog noj ntshav). transport39 thiab tiv thaiv cell recruitment40 nyob rau hauv lub plab microenvironment. Tsis tas li ntawd, vasculature Cheebtsam uas yuav tsum tau kev cob cog rua ntawm cov ntaub so ntswg qauv yog ib tug prerequisite thaum cov ntaub so ntswg qauv yog tsim los ua kom pom ntau yam hauv nruab nrog cev.Yog li ntawd, endothelial hlwb yuav tsum tau muab tso rau hauv cov qauv ntau yog physiological yam ntxwv nrog organ-theem kev daws teeb meem.Patient-derived hlwb nyob rau hauv immune cov lus teb. Kev nthuav qhia antigen, innate adaptive immune crosstalk, thiab cov ntaub so ntswg tshwj xeeb tiv thaiv kab mob hauv cov ntsiab lus ntawm kev ua cov kab mob plab hnyuv.
Kev siv cov chips hybrid yog yooj yim dua li lub plab-on-a-chip vim tias cov cuab yeej teeb tsa tau yooj yim dua thiab kev siv Transwell inserts tso cai rau cov kab lis kev cai ntawm lub plab epithelium.Txawm li cas los xij, cov khoom lag luam muaj Transwell inserts nrog polyester membranes tsis elastic thiab tsis tuaj yeem simulate peristaltic zoo li txav mus. Tsis tas li ntawd, cov nti ntawm qhov chaw nres tsheb apicalis muab tso rau hauv apical hybrid compartment. Tsis muaj kev ntxhov siab nyob rau sab apical.Qhov tseeb, cov khoom zoo li qub nyob rau hauv lub apical compartment tsis tshua muaj peev xwm ua rau cov kab mob sib kis tau ntev hauv hybrid chips.Thaum peb tuaj yeem ua kom muaj zog 3D morphogenesis hauv Transwell inserts thaum siv hybrid chips, qhov tsis txaus ntawm physiologically cuam tshuam rau biomechanical chips hybrids thiab apical hybrid platform hybrids. muaj peev xwm siv tau.
Tag nrho cov kev tsim kho tshiab ntawm tib neeg crypt-villus axis nyob rau hauv plab-on-a-chip thiab hybrid-on-a-chip kab lis kev cai tsis tau tsim kom tiav.Vim li cas morphogenesis pib los ntawm ib tug epithelial monolayer, 3D microarchitectures tsis tas muab morphological zoo sib xws rau crypts cov neeg nyob hauv vivo. domain nyob rau hauv microengineered 3D epithelium, cov cheeb tsam crypt thiab villous tsis tau meej meej demarcated.Txawm hais tias siab dua cov kab ntawm cov nti ua rau kom qhov siab ntawm microengineered epithelium, qhov siab tshaj plaws yog tseem txwv rau ~ 300-400 µm.Qhov tseeb qhov tob ntawm tib neeg txoj hnyuv crypts nyob rau hauv lub me me thiab loj 0 ~ 4µm 3 µmtest, qhov tseeb qhov tob ntawm tib neeg plab hnyuv crypts nyob rau hauv lub me thiab loj 0 ~ 1 µm Feem ntau, thiab qhov siab ntawm lub plab hnyuv me yog ~ 600 µm41.
Los ntawm kev pom kev pom, nyob rau hauv qhov kev daws teeb meem zoo tshaj plaws ntawm 3D microarchitectures yuav raug txwv rau lub plab ntawm lub nti, txij li qhov yuav tsum tau ua haujlwm deb ntawm lub hom phiaj lo ntsiab muag mus rau txheej txheej epithelial yog nyob rau ntawm qhov kev txiav txim ntawm ob peb millimeters.Yuav kom kov yeej qhov teeb meem no, lub hom phiaj nyob deb yuav tsum tau. Tsis tas li ntawd, ua nyias ntu ntawm challing speciming rau kev npaj siab. PDMS.Tsis tas li ntawd, txij li txheej txheej microfabrication ntawm lub plab ntawm lub nti cuam tshuam nrog kev ua kom ruaj khov ntawm txhua txheej, nws yog qhov nyuaj heev los qhib lossis tshem tawm cov txheej sab saud los tshuaj xyuas cov qauv saum npoo ntawm cov txheej txheej epithelial.Piv txwv li, los ntawm kev siv lub tshuab ntsuas hluav taws xob (SEM).
Lub hydrophobicity ntawm PDMS tau yog ib qho kev txwv nyob rau hauv microfluidic-raws li kev tshawb fawb soj ntsuam nrog hydrophobic me me molecules, txij li thaum PDMS tuaj yeem tsis tshwj xeeb adsorb xws li hydrophobic molecules. Lwm txoj hauv kev rau PDMS yuav raug txiav txim siab nrog lwm cov ntaub ntawv polymeric.Xwb, kev hloov kho deg ntawm PDMS (xws li, txheej txheej nrog polyethylene 423 g). ) tuaj yeem txiav txim siab kom txo qis adsorption ntawm hydrophobic molecules.
Thaum kawg, peb txoj kev tsis tau ua tau zoo nyob rau hauv cov nqe lus ntawm kev muab cov kev ntsuam xyuas siab los yog "ib-loj-fits-tag nrho" cov neeg siv khoom siv hauv kev sim platform.Cov txheej txheem tam sim no yuav tsum muaj lub twj tso kua mis rau ib lub microdevice, uas siv qhov chaw hauv CO2 incubator thiab tiv thaiv cov kev sim loj loj.Qhov kev txwv no tuaj yeem txhim kho tau zoo los ntawm cov qauv scalability ntawm 2, piv txwv li, nov. 96-zoo, los yog 384-zoo porous inserts uas tso cai rau txuas ntxiv ntxiv thiab tshem tawm ntawm basolateral media).
Txhawm rau ntxias 3D morphogenesis ntawm tib neeg txoj hnyuv epithelium hauv vitro, peb siv lub microfluidic nti plab hnyuv cov cuab yeej uas muaj ob qho tib si microchannels thiab elastic porous daim nyias nyias nyob nruab nrab ntawm los tsim cov lumen-capillary interface. Transwell inserts.Nyob rau hauv ob qho tib si platforms, morphogenesis ntawm ntau yam tib neeg txoj hnyuv epithelial hlwb tuaj yeem pom tau los ntawm kev siv cov kev taw qhia ntawm kev khiav kom tshem tawm cov morphogen antagonists los ntawm basolateral compartment.Cov txheej txheem kev sim tag nrho (Daim duab 1) muaj tsib ntu: (i) microfabrication ntawm lub plab chipb 5 (ntxig 1-step), transwell. (ii) kev npaj ntawm plab hnyuv epithelial hlwb (Caco-2 hlwb) los yog tib neeg plab hnyuv organoids; thawv 2-5), (iii) kab lis kev cai ntawm plab hnyuv epithelial hlwb ntawm plab hnyuv chips los yog hybrid chips (qib 6-9), (iv) induction ntawm 3D morphogenesis nyob rau hauv vitro (qib 10) thiab (v) ) kom characterize lub 3D epithelial microstructure (cov kauj ruam 11-24) tsim nyog pab pawg neeg hauv qab no. validate cov hauj lwm zoo ntawm nyob rau hauv vitro morphogenesis los ntawm kev sib piv epithelial morphogenesis rau spatial, temporal, conditional, los yog txheej txheem tswj.
Peb siv ob hom kab lis kev cai sib txawv: lub plab-on-a-chip nrog cov kab ncaj nraim lossis cov kab tsis sib txuas, lossis cov chips hybrid uas muaj Transwell (TW) ntxig rau hauv cov khoom siv microfluidic, tsim raws li tau piav qhia hauv Box 1, thiab qib 1 -5. "Device Fabrication" qhia cov kauj ruam tseem ceeb hauv kev ua ib qho ntawm tes los yog ib qho hybrid ntawm tes piav qhia. qhov chaw (Caco-2 los yog tib neeg plab hnyuv organoids) thiab kab lis kev cai siv nyob rau hauv no raws tu qauv. tso tawm kom pom hauv qab txhua tus xub.Cov ntawv thov muab cov piv txwv ntawm kev tsim cov kab mob hauv plab hnyuv li cas tuaj yeem siv tau, piv txwv li, hauv kev sib txawv ntawm tes tus cwj pwm, kev tshawb fawb lub plab, kev tsim cov tswv tsev-microbiome ecosystems, thiab cov qauv kab mob.Immunofluorescence dluab hauv "Cell Differentiation" qhia nuclei, F-actin thiab MUC3dli txheej hauv epico2. gut chip.MUC2 signaling yog tam sim no nyob rau hauv goblet hlwb thiab mucos secreted los ntawm mucosal nto.Fluorescent dluab nyob rau hauv lub plab Physiology qhia cov mucus uas ua los ntawm staining rau sialic acid thiab N-acetylglucosamine residues siv fluorescent nplej kab mob agglutinin.Cov ob overlapping dluab nyob rau hauv "Host-Cult-Microbis" tus neeg sawv cev ntawm co-co. lub plab ntawm lub nti.Lub vaj huam sib luag sab laug qhia txog kev sib koom ua ke ntawm E. coli nthuav qhia ntsuab fluorescent protein (GFP) nrog microengineered 3D Caco-2 epithelial cells.Lub vaj huam sib luag sab xis qhia tau hais tias qhov chaw ntawm GFP E. coli co-cultured nrog 3D Caco-2 epithelial hlwb, ua raws li cov immunofluorescence (F thiab nucleus) stainiscleared (F. Kev ua qauv qhia txog kev noj qab haus huv piv rau cov plab hnyuv hauv plab o nyob rau hauv kev sib tw physiological nrog cov kab mob antigens (piv txwv li, lipopolysaccharide, LPS) thiab lub cev tiv thaiv kab mob (piv txwv li, PBMC; ntsuab).Caco-2 hlwb tau coj los tsim kom muaj 3D epithelial txheej.Scale bar, 50 µm kev tso cai ntawm cov hlwb hauv qab: kev hloov pauv hauv qab. los ntawm reference.2. Oxford University Xovxwm; Luam tawm nrog kev tso cai los ntawm Ref.5. NAS; "Host-Microbe Co-Culture" yoog nrog kev tso cai los ntawm ref.3. NAS; "Tus Qauv Kab Mob" yoog nrog kev tso cai los ntawm kev siv.5. NAS.
Ob lub plab-on-chip thiab hybrid chips tau tsim los ntawm kev siv PDMS replicas uas tau demolded los ntawm silicon pwm los ntawm mos lithography1,44 thiab qauv nrog SU-8.Qhov tsim ntawm microchannels nyob rau hauv txhua nti yog txiav txim siab los ntawm kev xav txog hydrodynamics xws li shear stress thiab hydrodynamic pressure1,4,12.The thawj plab-on-achi design. juxtaposed parallel ncaj microchannels, tau hloov zuj zus mus rau hauv lub plab-on-a-chip (Ntxiv cov ntaub ntawv Fig. 1b) uas muaj xws li ib khub ntawm nkhaus microchannels los ua kom cov kua dej nyob hauv lub sijhawm, cov qauv tsis sib luag, thiab ntau qhov deformation ntawm cov kab lis kev cai (Fig. 2a-f) complex recessed gut 12. Gut-on-a-chips tuaj yeem xaiv tau.Peb tau pom tias lub convoluted Gut-Chip kuj tseem ua rau 3D morphogenesis nyob rau lub sijhawm zoo sib xws nrog cov qib zoo sib xws ntawm kev loj hlob ntawm epithelial piv rau tus thawj Gut-Chip, tsis hais cov kab lis kev cai ntawm tes.Yog li ntawd, kom induces complex 3D morphogenesis on PD-chip, replicas thiab reconstruction. kho ntawm silicon pwm nrog SU-8 cov qauv muab cov yam ntxwv tsis zoo tom qab demolding (Daim duab 2a).Txhim kho lub plab ntawm ib nti, cov txheej txheej PDMS tau npaj ua ntu zus mus rau PDMS zaj duab xis ntxeem tau thiab tom qab ntawd ua raws cov txheej txheem PDMS qis los ntawm kev sib koom ua ke tsis siv neeg siv cov tshuaj kho corona (Daim duab 2). tau txuas rau iav slides los tsim ib leeg-channel microfluidic pab kiag li lawm uas yuav haum Transwell inserts (Daim duab. 2h thiab Extended Data Fig. 2).Cov txheej txheem kev sib txuas yog ua los ntawm kev kho qhov chaw ntawm PDMS replica thiab iav nrog oxygen plasma los yog corona kev kho mob.Tom qab sterilization ntawm microfabricated 3D tube gene phoov tau npaj cov khoom siv rau kev sib txuas rau cov khoom siv silicone. ntawm txoj hnyuv epithelium (Daim duab 2g).
a, Schematic illustration ntawm kev npaj ntawm PDMS qhov chaw los ntawm SU-8 patterned silicon moulds.Cov uncured PDMS tov yog poured mus rau ib tug silicon pwm (sab laug), kho ntawm 60 ° C (nruab nrab) thiab demolded (txoj cai).Lub demolded PDMS raug txiav mus rau hauv daim thiab huv si rau sab sauv use.b, Photograph ntawm lub silicon txheej. silicon pwm siv los fabricate lub PDMS ntxeem tau membrane.d, ib tug series ntawm cov duab ntawm lub sab sauv thiab sab PDMS Cheebtsam thiab lub assembled on-chip plab hnyuv device.e, Schematic ntawm kev sib raug zoo ntawm lub sab sauv, daim nyias nyias, thiab qis PDMS Cheebtsam.Txhua txheej yog irreversibly bonded los ntawm cov ntshav los yog cov ntaub so ntswg coronautated-s.f, superimposed convoluted microchannels thiab lub tshuab nqus tsev chambers.g, Teeb lub plab-on-a-chip rau microfluidic cell kab lis kev cai.Lub plab hnyuv ntawm ib nti sib sau ua ke nrog silicone tube thiab syringe tau muab tso rau ntawm ib lub hau npog.Cov cuab yeej nti tau muab tso rau ntawm lub hau ntawm 150 mm Petri phaj rau kev ua.Cov khoom siv silicone yog kaw. hybrid nti fabrication thiab 3D morphogenesis siv hybrid chips.Transwell inserts npaj ntawm nws tus kheej mus rau kab lis kev cai 2D monolayers ntawm plab hnyuv epithelial hlwb raug muab tso rau hauv lub hybrid nti kom induce plab hnyuv 3D morphogenesis.Qhov nruab nrab yog perfused los ntawm microchannels hauv qab lub cell txheej tsim los ntawm barale. 1 cm tso cai. Elsevier.
Nyob rau hauv cov txheej txheem no, Caco-2 cell kab thiab plab hnyuv organoids tau siv los ua epithelial qhov chaw (Fig. 3a).Ob hom hlwb tau kab lis kev cai ntawm nws tus kheej (Box 2 thiab Box 5) thiab siv los noob ECM-coated microchannels ntawm on-chip plab los yog Transwell inserts.Thaum cov cell muaj confluent coverr (> 9) Caco-2 hlwb (nruab nrab ntawm nqe lus 10 thiab 50) nyob rau hauv T-flasks yog sau los npaj dissociated cell suspensions los ntawm trypsinization kua (lub thawv 2). Tib neeg plab hnyuv organoids los ntawm txoj hnyuv biopsies los yog phais resections tau cultured nyob rau hauv Matrigel scaffold domes nyob rau hauv 24-zoo cov txheej txheem. morphogens (xws li Wnt, R-spondin, thiab Noggin) thiab cov khoom loj hlob tau npaj raws li tau piav qhia hauv Box 3 tau ntxiv txhua hnub kom txog rau thaum cov organoids loj hlob mus txog ~ 500 µm inch. Tag nrho cov organoids tau sau thiab dissociated rau hauv ib lub hlwb rau cov noob rau hauv plab lossis Transwell inserts ntawm 5 nti (Box) tau tshaj tawm yav dhau los. hom 12,13 (piv txwv li ulcerative colitis, Crohn's disease, mob qog noj ntshav, lossis ib txwm pub), qhov chaw qhov txhab (piv txwv li, qhov txhab tiv thaiv qhov chaw tsis muaj qhov txhab) thiab qhov chaw nyob hauv plab hnyuv (xws li, duodenum, jejunum, ileum, cecum, colon, lossis lub qhov quav 5). (coloids) uas feem ntau xav tau ntau dua ntawm morphogens dua li cov hnyuv me.
a, Workflow rau induction ntawm plab morphogenesis nyob rau hauv lub plab chip.Caco-2 tib neeg plab hnyuv epithelium thiab plab hnyuv organoids yog siv nyob rau hauv no raws tu qauv los qhia 3D morphogenesis.Lub cais epithelial hlwb tau seeded nyob rau hauv lub npaj plab-rau-ib-chip ntaus ntawv (chip npaj) (nyob rau hauv uas txuas nrog MS hlwb). porous membrane nyob rau hnub 0 (D0), apical (AP) ntws yog pib thiab tswj rau thawj 2 hnub (ntws, AP, D0-D2).Basolateral (BL) ntws kuj pib nrog rau cyclic stretching motions (stretch, flow, AP thiab BL) thaum ib tug ua tiav 2D monolayer yog tsim.Intestinal 3D kab lis kev cai ntawm morphoflocyte hnub tom qab morphoflocytes. (morphogenesis, D5).Phase contrast dluab qhia tus neeg sawv cev morphology ntawm Caco-2 hlwb ntawm txhua theem kev sim los yog lub sij hawm taw tes (bar graph, 100 µm). Plaub schematic daim duab qhia qhov sib thooj cascades ntawm plab morphogenesis (sab saum toj sab xis).Dashed xub nyob rau hauv lub fluidity ntawm schematic flowb sawv cev. 3D Caco-2 epithelium (sab laug).Lub inset tseem ceeb thaj chaw magnified (dawb dashed lub thawv) qhia cov regenerated microvilli ntawm 3D Caco-2 txheej (txoj cai).c, Kab rov tav pem hauv ntej saib ntawm tsim Caco-2 3D, claudin (ZO-1, liab) thiab nruam txhuam ciam teb daim ntawv lo (Nucluenence) thiab fluoricles daim nyias nyias confocal visualization ntawm epithelial hlwb ntawm plab hnyuv chips.Xub taw tes rau nruab nrab schematic qhia qhov chaw ntawm lub dav hlau focal rau txhua confocal view.d, Lub sij hawm kawm ntawm morphological hloov nyob rau hauv organoids kab lis kev cai ntawm ib nti tau los ntawm theem contrast microscopy nyob rau hnub 3, 7, 9, 11, thiab 13.Qhov zoo ntawm cov duab IC. photomicrograph ntawm organoid 3D epithelium tsim nyob rau hauv lub plab ntawm daim hlais noj nyob rau hnub 7.f, Overlaid immunofluorescence dluab qhia cov cim rau cov qia hlwb (LGR5; magenta), goblet hlwb (MUC2; ntsuab), F-actin (grey) thiab nuclei (cyan) loj hlob ntawm plab chips rau 3 hnub (Left) thiab cov kab mob hauv nruab nrab. ntawm cov txheej txheem epithelial.Saib Ntxiv Cov Ntaub Ntawv Daim Duab 3, uas qhia txog LGR5 teeb liab yam tsis muaj MUC2 signaling.Fluorescence cov duab uas qhia cov epithelial microstructure (txoj cai) ntawm 3D organoid epithelium tsim nyob rau hauv lub plab ntawm ib nti los ntawm staining lub plasma membrane nrog CellMask dye (txoj cai 3 hnub) tsis muaj kab lis kev cai. stated.b Reprinted nrog kev tso cai los ntawm reference.2. Oxford University Xovxwm; c Tau txais kev tso cai los ntawm Reference.2. Oxford University Xovxwm; e thiab f hloov nrog kev tso cai los ntawm kev siv.12 Raws li Creative Commons License CC BY 4.0.
Nyob rau hauv lub plab ntawm ib nti, nws yog ib qho tsim nyog los hloov qhov hydrophobic nto ntawm PDMS ntxeem tau daim nyias nyias rau kev ua tiav ECM txheej.Nyob rau hauv cov txheej txheem no, peb siv ob txoj kev sib txawv los hloov cov hydrophobicity ntawm PDMS membranes.Rau culturing Caco-2 hlwb, nto activation los ntawm UV / ozone kev kho mob ib leeg yog txaus kom txo tau cov hydrophobicity ntawm PDMS cov ntaub so ntswg. PDMS membrane.Txawm li cas los xij, microfluidic kab lis kev cai ntawm organoid epithelium yuav tsum tau siv tshuaj raws li qhov chaw ua haujlwm kom ua tiav cov txiaj ntsig zoo ntawm ECM cov proteins los ntawm kev siv cov polyethyleneimine (PEI) thiab glutaraldehyde rau PDMS microchannels.Tom qab kev hloov kho deg, ECM cov proteins tau muab tso rau hauv cov txheej txheem ua haujlwm ntawm PDMS thiab ua kom pom tseeb. Tom qab cov hlwb txuas nrog, cov kab lis kev cai microfluidic cell pib los ntawm perfusing tsuas yog nruab nrab mus rau sab sauv microchannel kom txog rau thaum lub hlwb tsim ib tug ua tiav monolayer, thaum lub qis microchannel tswj cov xwm txheej zoo li qub.Qhov no optimized txoj kev ua kom nto thiab ECM txheej enables lub Symptoms ntawm organoid epithelium induce 3D morphogenesis ntawm lub PD.
Transwell kab lis kev cai kuj yuav tsum tau ECM txheej ua ntej cell noob; Txawm li cas los xij, Transwell kab lis kev cai tsis tas yuav ua kom cov txheej txheem ua ntej ua kom lub ntsej muag ntxeem tau.Rau qhov loj hlob Caco-2 hlwb ntawm Transwell inserts, ECM txheej ntawm cov ntxeem tau nrawm nrawm qhov txuas ntawm cov kab mob Caco-2 dissociated (<1 teev) thiab nruj hlws ris txheej txheem tsim (<1-2 hnub). inserts, txuas rau daim nyias nyias (<3 h) thiab khaws cia kom txog rau thaum cov organoids tsim ib qho kev ua tiav monolayer nrog kev ruaj ntseg .Transwell kab lis kev cai yog ua nyob rau hauv 24-zoo daim hlau yam tsis tas siv hybrid chips.
Nyob rau hauv vitro 3D morphogenesis tuaj yeem pib los ntawm kev siv cov kua dej ntws mus rau basolateral nam ntawm ib txheej txheej epithelial.Nyob rau hauv lub plab ntawm ib nti, epithelial morphogenesis pib thaum nruab nrab perfused mus rau sab sauv thiab qis microchannels (Fig. 3a).Raws li yav tas los tau piav, nws yog ib qho tseem ceeb rau kev qhia cov kua dej txuas ntxiv ntawm cov bass nyob rau hauv cov ntaub so ntswg. Cov kev taw qhia secreted morphogen inhibitors.Rau muab cov as-ham txaus thiab cov ntshav rau cov hlwb khi rau ntawm daim nyias nyias thiab tsim cov luminal shear kev nyuaj siab, peb feem ntau siv dual ntws hauv plab rau ntawm lub nti.In hybrid chips, Transwell inserts uas muaj epithelial monolayers tau muab tso rau hauv hybrid chips.Tom qab ntawd, qhov nruab nrab tau siv rau hauv qhov chaw nruab nrab ntawm lub baso. morphogenesis tshwm sim 3-5 hnub tom qab pib ntawm basolateral ntws nyob rau hauv ob qho tib si kab lis kev cai platforms.
Cov yam ntxwv morphological ntawm microengineered 3D epithelial txheej tuaj yeem txheeb xyuas los ntawm kev siv ntau yam kev ntsuas, suav nrog theem sib piv microscopy, qhov sib txawv ntawm qhov sib txawv (DIC) microscopy, SEM, lossis immunofluorescence confocal microscopy (Daim duab 3 thiab 4). protrusion ntawm 3D epithelial khaubncaws sab nraud povtseg.Vim lub optical transparency ntawm PDMS thiab polyester zaj duab xis, ob lub plab-on-a-chip thiab hybrid nti platforms muaj peev xwm muab real-time nyob rau hauv situ imaging yam tsis tas yuav tsum tau rau sectioning los yog disassembly ntawm lub ntaus ntawv.Thaum ua yeeb yam immunofluorescence imaging (4 3 lub hlwb, figures, figures 1. nrog 4% (wt/vol) paraformaldehyde (PFA), ua raws li Triton X-100 thiab 2% (wt/vol) ) bovine serum albumin (BSA), nyob rau hauv kev txiav txim.Raws li hom cell, txawv fixatives, permeabilizers, thiab blocking agents yuav siv tau.Primary antibodies tsom kab mob los yog cov kab mob tseem ceeb hauv cell nyob rau ntawm nti, ua raws li cov tshuaj tiv thaiv kab mob thib ob nrog rau cov tshuaj tua kab mob uas tsom rau cov nucleus (piv txwv li, 4′,6-diamidino-2-phenylene) indole, DAPI) lossis F-actin (piv txwv li, fluorescently sau npe phalloidin).Fluorescence-raws li nyob imaging kuj tau ua nyob rau hauv qhov chaw "FiC. physiology"), random colonization ntawm microbial hlwb (Fig. 1, "Host-microbe co-culture"), kev recruitment ntawm lub cev tiv thaiv kab mob (Fig. 1, 'Disease Modeling') los yog lub contours ntawm 3D epithelial morphology (Fig. 3c, f thiab 4b nyob rau hauv lub sab sauv txheej ntawm lub microbial morphology). txheej, raws li tau piav qhia hauv daim duab 2, 3D epithelial morphology nrog rau microvilli ntawm apical txhuam ciam teb tuaj yeem pom los ntawm SEM (Daim duab 3b).Kev qhia ntawm cov cim sib txawv tuaj yeem ntsuas los ntawm kev ua kom muaj nuj nqis PCR5 lossis ib leeg-cell RNA txheej txheem ntawm cov txheej txheem no. chips los yog hybrid chips yog sau los ntawm trypsinization thiab ces siv rau molecular los yog genetic tsom xam.
a, Workflow rau induction ntawm plab hnyuv morphogenesis nyob rau hauv ib tug hybrid nti.Caco-2 thiab plab hnyuv organoids yog siv nyob rau hauv no raws tu qauv los qhia 3D morphogenesis nyob rau hauv ib tug hybrid nti platform.Dissociated epithelial hlwb tau seeded nyob rau hauv npaj Transwell inserts (TW prepnce; saib cov hlwb hauv qab no). inserts, tag nrho cov hlwb raug coj los nyob rau hauv cov xwm txheej zoo li qub (TW kab lis kev cai).Tom qab 7 hnub, ib qho Transwell ntxig uas muaj 2D monolayer ntawm cov hlwb epithelial tau muab tso rau hauv ib qho hybrid nti los qhia txog qhov dej ntws ntawm basolateral (Flow, BL), uas thaum kawg coj mus rau tiam 3D epithelial txheej (morphogenesis micrographs). muab tau los ntawm ib txwm pub (C103 kab) ascending nyuv nyob rau hauv txhua theem sim los yog lub sij hawm taw tes.Cov schematics nyob rau hauv lub sab sauv txheej qhia txog kev sim configuration rau txhua kauj ruam.b, Hybrid chips (sab laug schematic) tuaj yeem ua rau 3D morphogenesis ntawm organoid epithelial hlwb nrog rau sab saum toj-down confocal sib txawv txoj hauj lwm; schematic thiab coj cov kab dotted). pom pom tseeb morphological yam ntxwv.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled saib) ntawm organoid-derived epithelial hlwb cultured nyob rau hauv static Transwell (TW; inset nyob rau hauv dawb dashed box) piv rau hybrid nti (loj tshaj tag nrho kev txhaj tshuaj) piv ntawm 2. 2D ntsug cross-cut views (inset nyob rau sab xis sab xis; "XZ") kuj qhia 2D thiab 3D nta.Scale bar, 100 µm.c Reprinted nrog kev tso cai los ntawm reference.4. Elsevier.
Kev tswj hwm tuaj yeem npaj tau los ntawm kev coj ua tib lub hlwb (Caco-2 los yog plab hnyuv organoid epithelial hlwb) mus rau hauv ob-dimensional monolayers nyob rau hauv cov kab lis kev cai zoo li qub.Notably, nutrient depletion tej zaum yuav tshwm sim vim lub ntim ntim ntawm microchannels (piv txwv li ~ 4 µL nyob rau sab saum toj channel ntawm thawj lub plab-chip epithelium tsim). kuj muab piv.
Cov txheej txheem mos lithography yuav tsum tau ua nyob rau hauv ib chav huv si.Rau txhua txheej ntawm cov nti (sab sauv thiab sab qis thiab daim nyias nyias) thiab hybrid chips, photomasks sib txawv tau siv thiab fabricated rau nyias silicon wafers vim hais tias qhov siab ntawm microchannels txawv.Lub hom phiaj siab ntawm lub siab thiab qis microchannels ntawm lub plab ntawm lub nti yog 500 µm thiab qhov siab ntawm lub hom phiaj. hybrid nti yog 200 µm.
Muab 3-nti silicon wafer rau hauv ib lub tais nrog acetone. Maj mam tig lub phaj rau 30 vib nas this, ces cua qhuav lub wafer.Hloov lub wafer mus rau ib lub phaj nrog IPA, ces tig lub phaj rau 30 s los ntxuav.
Ib qho tshuaj piranha (sib tov ntawm hydrogen peroxide thiab concentrated sulfuric acid, 1: 3 (vol / vol)) tuaj yeem xaiv tau los ua kom tshem tawm cov organic residues los ntawm silicon wafer nto.
Piranha kua yog corrosive heev thiab generates tshav kub.Kev ceev faj txog kev nyab xeeb ntxiv yog tsim nyog.Rau pov tseg, cia cov tshuaj kom txias thiab hloov mus rau ib tug huv, qhuav ntim ntim.Siv ob lub thawv thiab muab cov ntawv ntim cov khib nyiab kom zoo.Thov ua raws li qhov chaw cov txheej txheem kev nyab xeeb rau cov txheej txheem kom ntxaws ntxiv.
Dehydrate lub wafers los ntawm muab tso rau ntawm 200 ° C kub phaj rau 10 feeb.
Ncuav ~ 10 g ntawm photoresist SU-8 2100 mus rau hauv nruab nrab ntawm cov silicon wafer huv si.Siv tweezers kom kis cov photoresist tusyees ntawm lub wafer. Qee zaus tso lub wafer rau ntawm 65 ° C kub phaj kom cov photoresist tsis nplaum thiab yooj yim dua kis tau.Tsis txhob tso lub phaj ncaj qha.
SU-8 tau sib npaug ntawm cov wafer los ntawm kev khiav cov txheej txheej.Txhim kho qhov kev sib hloov ntawm SU-8 rau 5-10 s kom nthuav tawm ntawm 500 rpm ntawm qhov nrawm ntawm 100 rpm / s.Set lub ntsiab spin rau 200 µm thickness patterning ntawm 1,500 aµ rpm 500 µm qhov siab rau txheej sab sauv ntawm lub plab ntawm lub nti;
Lub ntsiab spin ceev tuaj yeem hloov kho raws li lub hom phiaj tuab ntawm SU-8 qauv ntawm silicon wafer.
Txhawm rau tsim cov qauv SU-8 ntawm 500 µm qhov siab rau txheej sab sauv ntawm lub plab ntawm lub nti, cov txheej txheem spinning thiab mos ci cov kauj ruam ntawm lub thawv no (qib 7 thiab 8) tau rov ua dua (saib kauj ruam 9) los tsim ob txheej txheej ntawm 250 µm Ib txheej tuab ntawm SU-8, uas tuaj yeem muab txheej txheej thiab koom nrog hauv 0 2 txheej txheej ntawm 0 ex. µm siab.
Mos ci lub SU-8 coated wafers los ntawm ua tib zoo muab cov wafers rau ntawm lub phaj kub ntawm 65 ° C rau 5 min, ces hloov qhov chaw rau 95 ° C thiab incubate rau ib tug ntxiv 40 min.
Txhawm rau ua tiav 500 μm qhov siab ntawm SU-8 tus qauv nyob rau sab sauv microchannel, rov ua cov kauj ruam 7 thiab 8 kom tsim tau ob 250 μm tuab SU-8 txheej.
Siv lub UV Mask Aligner, ua lub teeb ntsuas raws li cov chaw tsim khoom cov lus qhia los xam lub sij hawm raug ntawm lub wafer.(lub sij hawm raug, ms) = ( kis tau, mJ / cm2) / (teeb lub zog, mW / cm2).
Tom qab txiav txim siab lub sij hawm raug, muab daim npog ntsej muag rau ntawm daim npog ntsej muag ntawm UV daim npog ntsej muag thiab muab daim npog ntsej muag rau ntawm SU-8 coated wafer.
Muab cov ntawv luam tawm ntawm daim npog ntsej muag ncaj qha rau ntawm SU-8 coated sab ntawm silicon wafer kom txo tau UV dispersion.
Tshem tawm SU-8 coated wafer thiab photomask vertically mus rau 260 mJ / cm2 ntawm UV teeb rau lub sij hawm raug predetermined (saib kauj ruam 10 ntawm lub thawv no).
Tom qab UV raug, SU-8-coated silicon wafers tau ci ntawm 65 ° C rau 5 min thiab 95 ° C rau 15 min ntawm txhua lub phaj kub los tsim cov qauv nrog qhov siab ntawm 200 μm.Ntev lub sijhawm tom qab ci ntawm 95 ° C rau 30 min los tsim cov qauv nrog qhov siab ntawm 50 µm.
Tus tsim tawm yog nchuav rau hauv ib lub khob tais, thiab cov ci wafer muab tso rau hauv lub tais.Qhov ntim ntawm SU-8 tus tsim tawm yuav txawv nyob ntawm seb qhov loj ntawm lub khob phaj.Ua kom siv cov SU-8 tsim kom txaus kom tshem tawm tsis tau SU-8.Piv txwv li, thaum siv 150 hli txoj kab uas hla iav tais nrog 1 L muaj peev xwm, siv ~ 300 mL ntawm cov pwm tsim. qee zaus maj mam tig.
Yaug cov pwm tsim nrog ~ 10 mL ntawm tus tsim tawm tshiab ua raws li IPA los ntawm kev txau cov tshuaj siv lub pipette.
Muab cov wafer tso rau hauv lub tshuab nqus tsev plasma thiab nthuav tawm cov pa plasma (cov pa hauv huab cua, lub hom phiaj siab 1 × 10−5 Torr, lub zog 125 W) rau 1.5 min.
Muab cov wafer tso rau hauv lub tshuab nqus tsev vacuum nrog ib lub iav swb sab hauv.Wafers thiab slides tuaj yeem muab tso rau ntawm ib sab.Yog tias lub tshuab nqus tsev desiccator muab faib ua ob peb txheej los ntawm ib lub phaj, muab cov slides tso rau hauv qab chamber thiab cov wafers nyob rau hauv lub sab sauv chamber.Drop 100 μL ntawm trichloro (1H, 1H, 1H, 1H, 1H) tshuaj swb thiab siv lub tshuab nqus tsev rau silanization.
Thaw ib lub vial ntawm khov Caco-2 hlwb nyob rau hauv ib da dej 37 ° C, ces hloov lub hlwb thawed mus rau ib tug T75 hwj uas muaj 15 mL ntawm 37 ° C prewarmed Caco-2 nruab nrab.
Kom dhau Caco-2 hlwb ntawm ~ 90% confluency, thawj sov Caco-2 nruab nrab, PBS, thiab 0.25% trypsin / 1 mM EDTA nyob rau hauv ib da dej 37 ° C.
Aspirate nruab nrab los ntawm lub tshuab nqus tsev aspiration. Ntxuav cov hlwb ob zaug nrog 5 mL ntawm sov PBS los ntawm kev rov ua lub tshuab nqus tsev aspiration thiab ntxiv PBS tshiab.