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.Cov qauv tshwj xeeb no 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, txoj hnyuv sib txawv ntawm cov kab mob hauv lub cev tiv thaiv kab mob thiab cov kab mob hauv lub cev. plab hnyuv mucosal nto.Yog li ntawd, rov tsim 3D cov txheej txheem 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 3D morphogenesis ntawm txoj hnyuv epithelium nrog kev txhim kho physiological kev ua haujlwm rau hauv peb lub cev. Inal morphogenesis nyob rau hauv lub plab ntawm microfluidic nti nrog rau hauv Transwell embedded hybrid nti.Peb piav qhia ntxaws txog cov txheej txheem rau kev tsim cov cuab yeej, culturing ntawm Caco-2 los yog plab hnyuv organoid epithelial hlwb nyob rau hauv cov pa chaw raws li zoo raws li nyob rau hauv ib tug microfluidic platform, induction ntawm 3D morphogenesis ntawm ntau yam kev tsim, thiab kev tsim kho ntawm 3D morphogenesis. Regeneration ntawm lub plab microarchitecture los ntawm kev tswj basolateral fluid flow rau 5 d.Our in vitro morphogenesis txoj kev siv physiologically cuam tshuam shear stress thiab mechanical motion thiab tsis xav tau complex cell engineering los yog manipulation, uas tej zaum yuav outperform lwm yam uas twb muaj lawm cov tswv yim.Peb xav hais tias peb lub tswv yim raws tu qauv yuav muaj broad genetic kev tshawb fawb nyob rau hauv lub zej zog implication. lial khaubncaws sab nraud povtseg nyob rau hauv vitro rau biomedical, kho mob, thiab tshuaj daim ntaub ntawv.
Cov kev sim ua kom pom tias cov kab mob hauv plab hnyuv epithelial Caco-2 cov kab mob hauv plab-on-a-chip1,2,3,4,5 lossis bilayer microfluidic devices6,7 tuaj yeem ua rau 3D morphogenesis hauv vitro yam tsis muaj kev nkag siab meej ntawm lub hauv paus txheej txheem. duce 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 faib ntawm ib tug haib Wnt antagonist, Dickkopf-1 (DKK-1), nyob rau hauv plab-on-a-chip thiab hloov kho microfluidic li uas muaj Transwell inserts, termed "Hybrid Chip". , secreted frizzled-related protein 1, los yog Soggy-1) mus rau hauv lub plab hnyuv inhibits morphogenesis los yog cuam tshuam cov 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 kom tau lub epithelial robust nyob rau hauv lub intermediate ntawm cov genesis ntawm cov genesis. ists 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 (qib 1-5) rau kab mob plab hnyuv epithelial hlwb ntawm polydimethylsiloxane (PDMS)-based porous membranes (qib 6A, 7A, 8, 9B) thiab polyester membrane induced 3D morphogenesis in vitro (qib 10).Peb kuj tau txheeb xyuas cov cellular thiab molecular nta qhia txog cov ntaub so ntswg tshwj xeeb histogenesis thiab kab-dependent cellular sib txawv los ntawm kev siv ntau yam kev yees duab (qib 11-24).Peb induce morphogenesis siv tib neeg txoj hnyuv hom kab mob hauv cov kab mob hauv cov kab mob hauv cov kab mob, cov kab mob hauv cov kab mob hauv cov kab mob hauv cov kab mob hauv cov kab mob 2. Cov ntsiab lus suav nrog kev hloov kho saum npoo ntawm daim nyias nyias, tsim 2D monolayers, thiab plab hnyuv biochemical thiab Reproduction ntawm biomechanical microenvironment.in vitro.To induce 3D morphogenesis los ntawm 2D epithelial monolayers, peb tshem tawm morphogen antagonists nyob rau hauv ob qho tib si kab lis kev cai los ntawm ntws qhov nruab nrab ntawm cov kab lis kev cai ntawm cov kab lis kev cai baso. erable 3D epithelial txheej uas tuaj yeem siv los ua qauv morphogen-dependent epithelial kev loj hlob, longitudinal host-microbiome co-cultures, kab mob kis, inflammatory raug mob, epithelial barrier dysfunction, thiab probiotic-raws li kev kho mob Example.influences.
Peb cov txheej txheem yuav muaj txiaj ntsig zoo rau ntau yam ntawm cov kws tshawb fawb hauv kev yooj yim (piv txwv li, plab hnyuv mucosal biology, qia cell biology, thiab kev loj hlob biology) thiab siv kev tshawb fawb (xws li, kev kuaj tshuaj preclinical, kab mob qauv, cov ntaub so ntswg engineering, thiab gastroenterology) broad impact.Vim hais tias ntawm lub reproducibility thiab robustness ntawm peb cov genetic robustness 3. 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 tshiab lossis homeostasis .Ntxiv rau, peb cov txheej txheem muaj txiaj ntsig zoo rau kev nug cov kab mob nyob rau hauv ntau yam kab mob xws li Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV, Salvium-2) Clofilest. .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 cov kab mob ntsig txog kev raug mob hauv lub plab zom mov (GI) kab mob 11 .Lwm cov kab mob GI, kab mob ntawm cov kab mob hauv lub cev, itis, pouchitis, lossis chim siab plob tsis so tswj tau simulated thaum 3D plab hnyuv epithelial txheej tau 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.Biopsy los yog cov kab mob ntau dua ntawm cov kab mob 1. 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 tuaj yeem hloov mus rau scalable plates 4 cov kab lis kev cai tsis tu ncua 8, xws li ish 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 khoom noj.Raws li cov ntaub ntawv pov thawj, peb tsis ntev los no tau qhia txog qhov ua tau ntawm multiplex high-throughput morphogenesis system scalable to a multi-plate-6. ,17,18.Yog li ntawd, qhov kev siv tau ntawm peb txoj hauv kev vitro morphogenesis tuaj yeem nrawm dua thiab muaj peev xwm tau txais los ntawm ntau lub chaw tshawb fawb, kev lag luam lossis tsoomfwv thiab cov koom haum tswj hwm kom nkag siab txog cellular reprogramming ntawm in vitro gut morphogenesis ntawm qib transcriptomic los kuaj tshuaj lossis biotherapeutics Kev nqus thiab kev thauj mus los ntawm kev siv tshuaj los yog kev thauj mus los ntawm cov neeg sib tw-3 surrogess -chip qauv los ntsuas qhov kev tsim tawm ntawm lub plab morphogenesis txheej txheem.
Ib tug tsawg tus naj npawb ntawm tib neeg cov qauv kev sim tau raug 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 tam sim no txog plab morphogenesis yog raws li kev tshawb fawb tsiaj (xws li zebrafish20, mice21H). Qhov tseem ceeb tshaj plaws, tsis txhob txiav txim siab txog cov txheej txheem kev loj hlob ntawm tib neeg.Cov qauv no kuj txwv tsis pub dhau lawv lub peev xwm los sim ntau txoj hauv kev scalable.Yog li ntawd, peb cov txheej txheem 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.Raws li yav tas los tau piav qhia, qhov sib txawv ntawm cov ntaub so ntswg nyob rau hauv vitro outperforms nyob rau hauv vivo tsiaj qauv raws li zoo raws li lwm yam tsoos static 2D cell kab lis kev cai qauv. hlwb nyob rau hauv crypt-villus axis teb rau ntau yam mucosal los yog kev tiv thaiv kab mob stimuli.3D epithelial txheej tuaj yeem muab qhov chaw los kawm seb cov kab mob microbial sib tw ua spatial niches thiab ecological evolution nyob rau hauv teb rau host yam (piv txwv li, puab tiv thaiv cov mucus txheej, secretion ntawm IgA thiab antimicrobial peptide ntau dua ). Yuav ua li cas lub plab microbiota tsim nws cov zej zog thiab synergistically tsim microbial metabolites (piv txwv li, luv-chain fatty acids) uas zoo li lub koom haum cellular thiab qia cell niches nyob rau hauv lub basal crypts.Cov nta no tsuas yog pom tau thaum 3D epithelial txheej raug tsim nyob rau hauv vitro.
Ntxiv nrog rau peb txoj kev tsim cov txheej txheem 3D plab hnyuv epithelial, 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-cov qauv kev kuaj mob feem ntau yog charobis rau kev thauj mus los. lumen yog nyob rau hauv lub organoid thiab, yog li ntawd, cov 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 siv zog ua haujlwm thiab xav tau 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 prestructured 3D hydrogel scaffolds los ua kom lub plab epithelial qauv los ntawm culturing cais tib neeg cov hnyuv hlwb ntawm lub gel nto.Fabricate hydrogel scaffolds siv 3D-luam, micro-milled, los yog lithographically fabricated pwm. morphogen gradients, tsim kom muaj qhov sib piv siab ntawm epithelial qauv thiab stroma-epithelial crosstalk los ntawm kev suav nrog stromal hlwb nyob rau hauv scaffold.Txawm li cas los xij, qhov xwm txheej ntawm prestructured scaffolds yuav tiv thaiv cov zaub ntawm cov txheej txheem morphogenetic spontaneous nws tus kheej.Cov qauv no kuj tsis muab cov dynamic flumorphobic ndlwg nyob rau hauv lub cev tsis muaj zog, interst genetic tester nws tus kheej. sis thiab nce physiological muaj nuj nqi.Lwm txoj kev tshawb fawb tsis ntev los no tau siv hydrogel scaffolds nyob rau hauv lub microfluidic platform thiab qauv plab hnyuv epithelial qauv siv laser-etching cov tswv yim.Mouse plab hnyuv organoids ua raws li cov qauv etched los ua cov kab mob plab hnyuv, thiab cov kua dej intraluminal tuaj yeem rov ua dua siv cov qauv microfluither, exfoliate modules. Cov txheej txheem tic tsis suav nrog lub plab mechanobiological txav.3D tshuab luam ntawv los ntawm tib pab pawg tuaj yeem tsim cov kab me me hauv plab nrog cov txheej txheem morphogenetic.Txawm hais tias cov txheej txheem nyuaj ntawm cov plab hnyuv sib txawv hauv cov raj, cov qauv no tseem tsis muaj luminal fluid flow thiab mechanical deformation. Tsis tas li ntawd, kev ua tiav ntawm cov txheej txheem ntawm tes los yog kev ua tiav ntawm cov txheej txheem bioprinting. Kev sib cuam tshuam ntawm tes.Xwb, peb cov txheej txheem npaj tau muab spontaneous plab morphogenesis, physiologically cuam tshuam shear kev nyuaj siab, biomechanics uas mimic plab motility, kev nkag tau ntawm ywj siab apical thiab basolateral compartments, thiab rov tsim cov complex biological microenvironments ntawm modularity.Yog li ntawd, peb cov kev sib tw hauv vitrogenes tuaj yeem ua rau muaj kev sib tw ntau dua. txoj kev uas twb muaj lawm.
Peb cov txheej txheem yog tag nrho tsom mus 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. 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 tshuam33,34, extracellular Matrix (ECM) deposition 35 thiab, nyob rau hauv peb cov qauv, crypt conveys trophys nyob rau hauv trophy. (piv txwv li, fibroblasts) hauv mesenchyme ua lub luag haujlwm tseem ceeb hauv kev tsim cov ECM cov proteins thiab kev tswj cov 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.The endothelial txheej (piv txwv li, capillaries lossis lymphatics) ua lub luag haujlwm tseem ceeb hauv kev thauj mus los ntawm lymphatics. microenvironment.Tsis tas li ntawd, vasculature Cheebtsam uas tuaj yeem txuas ntawm cov qauv ntaub so ntswg yog qhov yuav tsum tau ua ua ntej thaum cov qauv ntaub so ntswg tsim los ua kom pom kev sib cuam tshuam hauv nruab nrog cev.Yog li ntawd, cov kab mob endothelial yuav tsum tau muab tso rau hauv cov qauv ntawm lub cev muaj zog ntau dua nrog kev daws teeb meem hauv nruab nrog cev. Cov neeg mob tau txais cov kab mob tiv thaiv kab mob kuj tseem ceeb rau kev nthuav tawm cov kab mob hauv nruab nrog cev, tiv thaiv kab mob sib kis. fic tiv thaiv nyob rau hauv cov ntsiab lus ntawm mimicking plab hnyuv kab mob.
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. Tsis tas li ntawd, cov nti ntawm qhov chaw nres tsheb tsis muaj kev ntxhov siab. Lub sab apical.Ntawm qhov tseeb, cov khoom zoo li qub nyob rau hauv lub apical compartment tsis tshua muaj peev xwm ua rau cov kab mob ntev 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 lub cev muaj feem cuam tshuam txog biomechanics thiab apical chip hybrid 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. engineered 3D epithelium, lub crypt thiab villous cheeb tsam twb tsis meej demarcated.Txawm hais tias siab dua nyob rau hauv lub 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 cov hnyuv me thiab loj yog ~ 0 µm, thiab qhov siab ntawm 13 μm, 0 μm thiab 13 μm. testinal villi yog ~ 600 µm41.
Los ntawm kev pom kev pom, nyob rau hauv qhov kev daws teeb meem ntawm 3D microarchitectures yuav raug txwv rau lub plab ntawm ib nti, txij li qhov yuav tsum tau ua hauj lwm nyob deb ntawm lub hom phiaj lens mus rau epithelial txheej yog nyob rau hauv qhov kev txiav txim ntawm ob peb millimeters.Kom kov yeej qhov teeb meem no, ib tug deb lub hom phiaj tej zaum yuav tsum tau. Tsis tas li ntawd, ua nyias ntu ntawm kev npaj rau PDur ntxiv rau cov speciming. txij li txheej txheej-los ntawm-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 txheej txheem saum npoo ntawm epithelial txheej.Piv txwv li, los ntawm kev siv lub tshuab ntsuas hluav taws xob scanning (SEM).
Lub hydrophobicity ntawm PDMS yog qhov txwv tsis pub muaj 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 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 lipophilic eth43 g) tuaj yeem suav tias yog cov khoom siv polyethylene 43 g. minimize adsorption ntawm hydrophobic molecules.
Thaum kawg, peb cov txheej txheem 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-phooj ywg kev sim platform.Cov txheej txheem tam sim no yuav tsum muaj lub twj tso kua mis ib lub microdevice, uas siv qhov chaw nyob hauv CO2 incubator thiab tiv thaiv cov kev sim loj.Qhov kev txwv no tuaj yeem txhim kho tau zoo los ntawm cov kab lis kev cai 2, 8, scalability ntawm 2, 8, 2. 4-zoo ntxeem tau ntxig uas tso cai 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 membrane nyob nruab nrab los tsim kom muaj lumen-capillary interface.Peb kuj ua kom pom kev siv ib leeg-channel microfluidic ntaus ntawv (ib txheej hybrid nti). ob lub 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 morphogen antagonists los ntawm basolateral compartment.Cov txheej txheem kev sim tag nrho (Daim duab 1) muaj tsib ntu: (i) microfabrication ntawm lub plab nti los yog Transwell5 insertable 1 Box hybrid (1. hlwb (Caco-2 hlwb) los yog tib neeg txoj 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 rau kev tswj pab pawg F (qib 11-24). 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 xws, lossis cov chips hybrid uas muaj Transwell (TW) ntxig rau hauv cov khoom siv microfluidic, fabricated raws li tau piav qhia hauv Box 1, thiab kauj ruam 1 -5 "Device Fabrication" qhia cov kauj ruam tseem ceeb hauv kev ua ib lub nti los yog ib qho hybrid ntawm tib neeg cov chips. 2 los yog tib neeg plab hnyuv organoids) thiab kab lis kev cai siv nyob rau hauv no raws tu qauv "In vitro morphogenesis" qhia tag nrho cov kauj ruam nyob rau hauv uas Caco-2 los yog organoid-derived epithelial hlwb yog kab lis kev cai ntawm lub plab hnyuv nti los yog nyob rau hauv Transwell inserts ntawm ib tug hybrid nti, ua raws li induction ntawm 3D morphogenesis. muab cov piv txwv ntawm yuav ua li cas tsim cov hnyuv epithelial txheej yuav siv tau, piv txwv li, nyob rau hauv lub cell sib txawv characterization, plab physiology kev tshawb fawb, tsim host-microbiome ecosystems, thiab kab mob modeling.Immunofluorescence dluab nyob rau hauv "Cell Differentiation" qhia nuclei, F-actin thiab MUC2 qhia nyob rau hauv lub epithelial txheej Caco-2 MUC2. thiab hnoos qeev secreted los ntawm mucosal nto.Fluorescent duab nyob rau hauv lub plab Physiology qhia cov hnoos qeev uas tsim los ntawm staining rau sialic acid thiab N-acetylglucosamine residues siv fluorescent nplej kab mob agglutinin.Ob tug overlapping duab nyob rau hauv "Host-Microbe Co-Cultures" qhia tus neeg sawv cev party-microbiome nyob rau hauv lub co-culture ntawm lub vaj huam sib luag. ing ntsuab fluorescent protein (GFP) nrog microengineered 3D Caco-2 epithelial hlwb.Lub vaj huam sib luag txoj cai qhia lub localization ntawm GFP E. coli co-cultured nrog 3D Caco-2 epithelial hlwb, ua raws li immunofluorescence staining nrog F-actin (liab) thiab nuclei (xiav chips). 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 raug coj los tsim kom muaj 3D epithelial txheej.Scale bar, 50 µm.Images nyob rau hauv qab kab: "Differentiation ntawm hlwb" yoog nrog kev tso cai 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 hauv txhua nti yog txiav txim siab los ntawm kev txiav txim siab hydrodynamics xws li shear stress thiab hydrodynamic pressure1,4,12.The thawj plab-on-achi design.Extend 1. ed parallel ncaj microchannels, tau hloov zuj zus mus rau hauv lub plab-ntawm-ib-chip (Ntxiv Cov Ntaub Ntawv Fig. 1b) uas suav nrog ib khub ntawm cov kab nkhaus microchannels txhawm rau 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) 12.When ntau cov txheej txheem yuav tsum tau ua kom muaj kev sib xyaw ua ke. .Peb tau pom tias lub convoluted Gut-Chip kuj muaj zog induces 3D morphogenesis nyob rau hauv ib tug zoo sib xws lub sij hawm ncej nrog ib tug zoo xws li degree ntawm epithelial loj hlob piv rau tus thawj Gut-Chip, tsis hais hom kab lis kev cai ntawm tes.Yog li ntawd, induce 3D morphogenesis, linear thiab complex on-chip gutable pwm curplicas MS yog cov qauv kev sib tshuam. tom qab demolding (Fig.2a).Txhim kho lub plab rau ntawm ib nti, cov txheej txheej PDMS tau npaj ua ntu zus mus rau cov yeeb yaj kiab PDMS ntxeem tau thiab tom qab ntawd ua raws cov txheej txheem PDMS qis los ntawm kev sib txuas tsis tau siv cov tshuaj kho corona (Daim duab 2b–f).To fabricate hybrid chips, kho PDMS replicas tau muab tso rau hauv ib qho kev sib txuas ntawm cov iav fiber ntau. g. 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 kev kho mob corona.Tom qab ua kom tsis muaj menyuam ntawm cov khoom siv microfabricated txuas rau lub raj silicone, cov cuab yeej teeb tsa tau npaj ua 3D morphogenesis ntawm txoj hnyuv (Fig2urelium).
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 tau txiav mus rau hauv daim thiab ntxuav rau ntxiv use.b, Diam duab ntawm silicon pwm siv rau txheej txheej silic. noj cov PDMS ntxeem tau membrane.d, ib tug series ntawm cov duab ntawm lub sab sauv thiab sab qis PDMS Cheebtsam thiab cov assembled on-chip plab hnyuv device.e, Schematic ntawm cov kev sib raug zoo ntawm lub Upper, membrane, thiab qis PDMS Cheebtsam.Txhua txheej yog irreversibly bonded los ntawm cov ntshav los yog corona treatment.f, Schematicon-consultant-vascular ntaus ntawv thiab cov khoom siv super-vasco-vaj huam sib luag. 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 ib lub raj silicone thiab syringe tau muab tso rau ntawm ib lub hau npog.Cov khoom siv nti tau muab tso rau ntawm lub hau ntawm 150 mm Petri phaj rau kev ua.Cov binder yog siv los kaw lub silicone tube.h, ntaub chiphybridshot 3. .Transwell inserts npaj ntawm nws tus kheej mus rau kab lis kev cai 2D monolayers ntawm plab hnyuv epithelial hlwb tau muab tso rau hauv hybrid nti kom induce plab hnyuv 3D morphogenesis.Qhov nruab nrab yog perfused los ntawm microchannels hauv qab cell txheej tsim nyob rau hauv Transwell insert.Scale bar, 1 cm.h Reprinted nrog kev tso cai los ntawm reference.4.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 cell tau cultured ntawm nws tus kheej (Box 2 thiab Box 5) thiab siv los noob cov ECM-coated microchannels ntawm on-chip plab los yog Transwell inserts.Thaum cov cell muaj confluent coverr (> 9) cov kab lis kev cai ntawm Caco-2. nyob rau hauv nqe 10 thiab 50) hauv T-flasks raug 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 yog cultured nyob rau hauv Matrigel scaffold domes nyob rau hauv 24-zoo daim hlau los txhawb lub structural . din, thiab Noggin) thiab cov khoom loj hlob tau npaj raws li tau piav qhia hauv lub thawv 3 tau ntxiv txhua hnub kom txog rau thaum cov organoids loj hlob mus txog ~ 500 µm inch.Fully grown organoids are harvested and dissociated into single cells for seeding on gut or Transwell inserts on a chip (Box 5).Raws li peb tau tshaj tawm yav dhau los, kab mob sib txawv, raws li hom kab mob 2. mob qog noj ntshav, lossis ib txwm pub dawb), qhov chaw mob (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, txoj hnyuv, lossis lub qhov quav). s.
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 noob nyob rau hauv lub npaj plab-rau-ib-chip ntaus ntawv (chip npaj) (nyob rau hauv lub cell sedation ntawm PD thiab sedation). 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 ua tiav 2D monolayer yog tsim.Intestinal 3D morponphotaneous kab lis kev cai tshwm sim tom qab 5 hnub. ase contrast dluab qhia tus neeg sawv cev morphology ntawm Caco-2 hlwb ntawm txhua kauj ruam kev sim los yog lub sij hawm taw tes (bar graph, 100 µm). Plaub schematic daim duab qhia txog tus coj cascades ntawm lub plab morphogenesis (sab saum toj sab xis).Dashed xub nyob rau hauv lub schematic sawv cev rau cov kev taw qhia ntawm cov kua dej ntws.b, 3. inset tseem ceeb thaj chaw loj (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 sau npe F-actin (ntsuab) thiab nuclei (xiav) cov kab mob ntawm cov kab mob hauv lub cev. mus 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.The inset (sab saum toj sab xis) qhia tau hais tias lub siab magnification ntawm lub muab duab.e, DIC photomicrograph hnub ntawm daim duab 3. tso cov duab immunofluorescence uas 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, raws li (Sab laug) thiab 13-hnub (nruab nrab) organoids ntawm lub epithelial txheej.Saib Ntxiv Cov Ntaub Ntawv Daim duab 3, uas qhia txog LGR5 teeb liab. D organoid epithelium tsim nyob rau hauv lub plab ntawm ib nti los ntawm staining lub plasma membrane nrog CellMask dye (txoj cai) nyob rau hnub 13 ntawm kab lis kev cai.Scale bar yog 50 μm tshwj tsis yog hais tias lwm yam.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 cov 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 ua kom los ntawm UV / ozone kev kho mob ib leeg yog txaus los txo cov hydrophobicity ntawm PDMS daim nyias nyias, thiab txuas rau Caco-2 hlwb. .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 mus rau PDMS microchannels.Tom qab kev hloov kho deg, ECM cov proteins tau muab tso rau los npog cov kev ua haujlwm ntawm cov kab mob hauv cov kab mob PDMA. fluidic cell kab lis kev cai 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 zoo li qub.Qhov no optimized txoj kev ua kom nto thiab ECM txheej enables qhov txuas ntawm organoid epithelium induce 3D morphogenesis ntawm PDMS nto.
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 tsum tau ua cov txheej txheem ua ntej kom ua kom lub ntsej muag ntxeem tau.Rau cov Caco-2 hlwb loj hlob ntawm Transwell inserts, ECM txheej ntawm cov ntxig ntxig nrawm nrawm rau qhov txuas ntawm cov kab mob Caco-2 dissociated (<1 teev) thiab nruj junction barrier formation (<1-2 hnub). daim nyias nyias (<3 h) thiab khaws cia kom txog rau thaum cov organoids tsim ib tug ua tiav monolayer nrog barrier kev ncaj ncees .Transwell kab lis kev cai yog ua nyob rau hauv 24-zoo daim hlau yam tsis muaj kev 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 ua kom paub cov kua dej tsis tu ncua ntawm cov kev taw qhia ntawm cov bass nyob rau hauv cov ntaub so ntswg. Txhawm 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 ntawm ib 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 nyob rau hauv lub basolateral sab ntawm cov porous 3 hnub tom qab qhov kev ntsuam xyuas. 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). D epithelial khaubncaws sab nraud povtseg.Vim lub optical transparency ntawm PDMS thiab polyester films, 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 hauj lwm immunofluorescence imaging (Daim duab 1, 4bd), cov kab mob feem ntau yog 4, 3c, formaldehyde (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 thaiv cov neeg sawv cev yuav siv tau.Primary antibodies targeting lineage-dependent cell los yog cheeb tsam chipsimmo ntawm cov tshuaj tiv thaiv kab mob. tain dye targeting ob lub 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 tuaj yeem ua nyob rau hauv qhov chaw txhawm rau kuaj cov hnoos qeev (Fig.1, “Cell differentiation” thiab “Gut physiology”), random colonization of microbial cells (Fig. 1, “Host-microbe co-culture”), kev recruitment of immune cells (Fig. 1, 'Disease Modeling') or the contours of 3D epithelial morphology. g. cais cov txheej sab saud los ntawm cov txheej txheem microchannel qis, raws li tau piav qhia hauv daim duab 2, 3D epithelial morphology nrog rau microvilli ntawm apical txhuam ciam teb tuaj yeem pom tau los ntawm SEM (Daim duab 3b).Qhov kev qhia ntawm cov cim sib txawv tuaj yeem soj ntsuam los ntawm kev ua kom muaj nuj nqis ntawm cov txheej txheem PCR5 los yog ib leeg-txheej txheej. nyob rau hauv plab 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 prep; saib cov hlwb txuas hauv qab no). tau kab lis kev cai 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 ua kom paub cov basolateral flow (Flow, BL), uas thaum kawg coj mus rau tiam 3D epithelial txheej (morphogenesis). 03 kab) ascending nyuv ntawm txhua theem kev sim lossis lub sijhawm taw qhia.Cov schematics nyob rau hauv cov khaubncaws sab nraud povtseg sab sauv 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 microscopy views coj ntawm qhov sib txawv Z, nruab nrab thiab qis dua.saib txoj cai schematic thiab coj cov kab dotted).qhia 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 D) piv ntawm 2. 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 lossis plab hnyuv organoid epithelial hlwb) rau hauv ob sab hauv monolayers raws li cov kab lis kev cai zoo li qub.Notably, nutrient depletion tej zaum yuav tshwm sim vim qhov txwv ntim ntawm microchannels (piv txwv li ~ 4 µL nyob rau sab saum toj channel ntawm thawj lub plab-chip epithelium tsim).
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 qhov siab ntawm cov microchannels txawv.Lub hom phiaj siab ntawm sab sauv thiab qis microchannels ntawm lub plab ntawm lub nti yog 500 μm hybrid channel. 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 lwm cov 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 μm thickness 1,500 µm / min. rau txheej sab sauv ntawm lub plab ntawm lub nti; saib "Cov kauj ruam tseem ceeb" hauv qab no) teeb tsa ntawm qhov nrawm ntawm 300 rpm / s 30 vib nas this ntawm 1,200 rpm.
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 txheej sab sauv ntawm lub plab ntawm lub nti, cov txheej txheem spin thiab mos ci cov kauj ruam ntawm lub thawv no (cov kauj ruam 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 sib koom ua ke ntawm 0 μm txheej txheej no, 0 μm txheej txheej 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 qhov loj ntawm lub khob phaj.Ua kom siv cov SU-8 txaus kom tshem tawm cov khoom tsis raug 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 tawm. kev ua.
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 lub iav swb sab hauv.Wafers thiab slides tuaj yeem muab tso rau ntawm ib sab.Yog hais 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, 2H, 1H) cov tshuaj fluoro, 1H. um 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.