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Tib neeg lub plab morphogenesis tsim kom muaj cov yam ntxwv crypt-villus ntawm 3D epithelial microarchitecture thiab spatial organization. Cov qauv tshwj xeeb no yog qhov yuav tsum tau ua kom lub plab homeostasis los ntawm kev tiv thaiv lub stem cell niche hauv basal crypt los ntawm exogenous microbial antigens thiab lawv cov metabolites. Tsis tas li ntawd, plab hnyuv villi thiab secreting mucus nthuav tawm cov functionally differentiated epithelial cells nrog ib tug tiv thaiv barrier ntawm lub plab hnyuv mucosal nto. Yog li ntawd, rov tsim cov qauv 3D epithelial yog qhov tseem ceeb rau kev tsim cov qauv plab hauv vitro. Qhov tseem ceeb, cov organic mimetic plab-on-a-chip tuaj yeem ua rau spontaneous 3D morphogenesis ntawm plab hnyuv epithelium nrog kev txhim kho physiological functions thiab biomechanics. Ntawm no, peb muab ib tug reproducible protocol los robustly induce plab hnyuv morphogenesis hauv plab hnyuv ntawm ib tug microfluidic chip nrog rau hauv ib tug Transwell embedded hybrid chip. Peb piav qhia txog cov txheej txheem ntxaws rau cov khoom siv fabrication, culturing ntawm Caco-2 lossis plab hnyuv organoid epithelial cells hauv cov chaw ib txwm muaj nrog rau ntawm ib tug microfluidic platform, induction ntawm 3D morphogenesis, thiab kev piav qhia txog cov epithelia 3D uas tau tsim los ntawm kev siv ntau hom kev thaij duab. Cov txheej txheem no ua tiav kev rov ua dua tshiab ntawm cov kab mob plab hnyuv microarchitecture los ntawm kev tswj cov kua dej hauv basolateral rau 5 d. Peb txoj kev morphogenesis hauv vitro siv kev ntxhov siab shear thiab kev txav mus los ntawm lub cev thiab tsis xav tau kev tsim kho cell lossis kev tswj hwm nyuaj, uas yuav ua tau zoo dua lwm cov txheej txheem uas twb muaj lawm. Peb xav tias peb cov txheej txheem tau hais tseg yuav muaj cov txiaj ntsig dav dav rau lub zej zog tshawb fawb biomedical, muab ib txoj hauv kev los tsim cov txheej txheem epithelial plab hnyuv 3D hauv vitro rau kev siv biomedical, kev kho mob, thiab kev siv tshuaj.
Cov kev sim ua pov thawj tias cov hlwb Caco-2 epithelial hauv plab hnyuv uas tau cog rau hauv cov khoom siv gut-on-a-chip1,2,3,4,5 lossis bilayer microfluidic6,7 tuaj yeem dhau los ntawm 3D morphogenesis hauv vitro yam tsis muaj kev nkag siab meej txog lub hauv paus mechanism. Hauv peb txoj kev tshawb fawb tsis ntev los no, peb pom tias kev tshem tawm cov basolaterally secreted morphogen antagonists los ntawm cov khoom siv kab lis kev cai yog qhov tsim nyog thiab txaus los ua rau 3D epithelial morphogenesis hauv vitro, uas tau pom los ntawm Caco-2 thiab cov kab mob plab hnyuv uas tau los ntawm tus neeg mob. Cov hlwb epithelial tau raug lees paub. Hauv kev tshawb fawb no, peb tau tsom mus rau kev tsim cov hlwb thiab kev faib tawm ntawm cov tshuaj Wnt antagonist muaj zog, Dickkopf-1 (DKK-1), hauv cov khoom siv gut-on-a-chip thiab cov khoom siv microfluidic hloov kho uas muaj Transwell inserts, hu ua "Hybrid Chip". Peb qhia tau tias kev ntxiv cov tshuaj Wnt antagonists sab nraud (xws li DKK-1, Wnt repressor 1, secreted frizzled-related protein 1, lossis Soggy-1) rau hauv lub plab on-chip inhibits morphogenesis lossis cuam tshuam cov txheej txheem 3D epithelial uas tau teeb tsa ua ntej, qhia tias kev ntxhov siab antagonistic thaum lub sijhawm kab lis kev cai yog lub luag haujlwm rau kev hloov pauv hauv plab hnyuv hauv vitro. Yog li ntawd, txoj hauv kev ua tau zoo los ua kom tiav cov morphogenesis muaj zog ntawm qhov sib txuas epithelial yog tshem tawm lossis tsawg kawg nkaus tswj cov qib ntawm Wnt antagonists hauv qhov chaw basolateral los ntawm kev ntxuav nquag (piv txwv li, hauv cov platforms gut-on-a-chip lossis hybrid-on-a-chip) lossis diffusion. Basolateral media (piv txwv li, los ntawm Transwell inserts rau hauv cov pas dej loj basolateral hauv qhov dej).
Hauv cov txheej txheem no, peb muab ib txoj kev qhia ntxaws rau kev tsim cov microdevices gut-on-a-chip thiab Transwell-insertable hybrid chips (kauj ruam 1-5) los cog qoob loo cov hlwb epithelial hauv plab hnyuv ntawm polydimethylsiloxane (PDMS)-raws li porous membranes (kauj ruam 6A, 7A, 8, 9) lossis polyester membranes ntawm Transwell inserts (kauj ruam 6B, 7B, 8, 9) thiab ua rau 3D morphogenesis hauv vitro (kauj ruam 10). Peb kuj tau txheeb xyuas cov yam ntxwv ntawm tes thiab molecular qhia txog cov ntaub so ntswg-tshwj xeeb histogenesis thiab lineage-dependent cellular differentiation los ntawm kev siv ntau yam imaging modalities (kauj ruam 11-24). Peb ua rau morphogenesis siv tib neeg cov hlwb epithelial hauv plab hnyuv, xws li Caco-2 lossis cov kab mob hauv plab hnyuv, hauv ob hom kab lis kev cai nrog cov ntsiab lus kev suav nrog kev hloov kho ntawm qhov chaw ntawm cov hlwb porous, kev tsim 2D monolayers, thiab cov kab mob hauv plab hnyuv biochemical thiab Kev tsim dua tshiab ntawm biomechanical microenvironment.in vitro.Txhawm rau ua rau 3D morphogenesis los ntawm 2D epithelial monolayers, peb tau tshem tawm cov morphogen antagonists hauv ob daim ntawv cog qoob loo los ntawm kev ntws cov khoom nruab nrab mus rau hauv qhov chaw basolateral ntawm cov kab lis kev cai. Thaum kawg, peb muab ib qho kev sawv cev ntawm qhov siv tau ntawm cov txheej epithelial 3D uas rov tsim tau uas tuaj yeem siv los ua qauv rau kev loj hlob ntawm morphogen-dependent epithelial, longitudinal host-microbiome co-cultures, pathogen infection, inflammatory injury, epithelial barrier dysfunction, thiab probiotic-based therapies Piv txwv li.influences.
Peb cov txheej txheem yuav pab tau rau ntau tus kws tshawb fawb hauv kev tshawb fawb yooj yim (piv txwv li, kev tshawb fawb txog cov qog nqaij hlav hauv plab, kev tshawb fawb txog cov noob qes hauv lub plab, thiab kev tshawb fawb txog kev loj hlob) thiab kev tshawb fawb siv (piv txwv li, kev kuaj tshuaj ua ntej, kev tsim qauv kab mob, kev tsim cov ntaub so ntswg, thiab kev kho mob plab) qhov cuam tshuam dav. Vim yog qhov rov ua dua thiab kev ruaj khov ntawm peb cov txheej txheem los ua kom muaj 3D morphogenesis ntawm cov epithelium hauv plab hauv vitro, peb xav tias peb txoj kev npaj txuj ci tuaj yeem tshaj tawm rau cov neeg tuaj saib kawm txog kev ua haujlwm ntawm cov cell signaling thaum lub sijhawm loj hlob ntawm plab hnyuv, kev rov tsim dua lossis homeostasis. Tsis tas li ntawd, peb cov txheej txheem muaj txiaj ntsig zoo rau kev nug txog kev kis kab mob hauv ntau yam kab mob sib kis xws li Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 lossis Vibrio cholerae. Cov neeg tuaj saib txog kab mob thiab pathogenesis kuj muaj txiaj ntsig zoo. Kev siv lub cev microphysiology hauv plab hauv lub chip yuav tso cai rau kev sib koom ua ke 10 thiab kev ntsuam xyuas tom qab ntawm kev tiv thaiv tus tswv tsev, kev tiv thaiv kab mob thiab kev kho mob ntsig txog kab mob hauv txoj hnyuv (GI) 11. Lwm yam kab mob GI cuam tshuam nrog kev mob plab hnyuv, kab mob celiac, kab mob Crohn, mob ulcerative colitis, pouchitis, lossis mob plab hnyuv tuaj yeem ua piv txwv thaum cov khaubncaws sab nraud povtseg 3D plab hnyuv tau npaj siv tus neeg mob cov khaubncaws sab nraud povtseg 3D plab hnyuv, cov kab mob no suav nrog villous atrophy, crypt shortening, mucosal puas tsuaj, lossis impaired epithelial barrier. Biopsy lossis stem cell-derived intestinal organoids12,13. Txhawm rau kom zoo dua qhov nyuaj ntawm qhov chaw mob, cov neeg nyeem yuav xav txog kev ntxiv cov hom cell cuam tshuam nrog kab mob, xws li cov neeg mob peripheral ntshav mononuclear cells (PBMCs), rau cov qauv uas muaj 3D plab hnyuv villus-crypt microarchitectures. cov cell tiv thaiv kab mob tshwj xeeb, 5.
Vim tias 3D epithelial microstructure tuaj yeem kho thiab pom tau yam tsis muaj cov txheej txheem sectioning, cov neeg saib ua haujlwm ntawm spatial transcriptomics thiab high-resolution lossis super-resolution imaging yuav xav paub txog peb daim ntawv qhia ntawm spatiotemporal dynamics ntawm cov noob thiab cov protein ntawm epithelial niches. Xav paub txog thev naus laus zis. Teb rau microbial lossis immunity stimuli. Ntxiv mus, longitudinal host-microbiome crosstalk 10, 14 uas sib koom tes hauv plab homeostasis tuaj yeem tsim tau hauv 3D plab hnyuv mucosal txheej los ntawm kev sib koom ua ke ntau hom microbial, microbial zej zog lossis fecal microbiota, tshwj xeeb tshaj yog hauv plab-rau-ib-chip. nyob rau hauv lub platform. Txoj hauv kev no yog qhov tshwj xeeb tshaj yog rau cov neeg tuaj saib uas kawm txog mucosal immunology, gastroenterology, human microbiome, culturomics thiab clinical microbiology uas tab tom nrhiav kev cog qoob loo cov kab mob plab uas tsis tau cog qoob loo hauv chav kuaj mob. Yog tias peb cov txheej txheem morphogenesis hauv vitro tuaj yeem hloov kho rau cov qauv kab lis kev cai scalable, xws li multiwell inserts hauv 24, 96 lossis 384 lub phaj uas txuas ntxiv rov ua dua cov chaw basolateral, cov txheej txheem kuj tseem tuaj yeem faib rau cov neeg tsim cov tshuaj, biomedical lossis cov chaw tshuaj ntsuam xyuas lossis kev lees paub rau kev lag luam zaub mov. Raws li pov thawj ntawm lub hauv paus ntsiab lus, peb nyuam qhuav ua pov thawj qhov ua tau ntawm lub kaw lus multiplex high-throughput morphogenesis scalable rau 24-well phaj hom ntawv. Tsis tas li ntawd, ntau yam khoom organ-on-a-chip tau raug lag luam16,17,18. Yog li ntawd, kev lees paub ntawm peb txoj kev morphogenesis hauv vitro tuaj yeem ua kom nrawm dua thiab muaj peev xwm txais yuav los ntawm ntau lub chaw soj ntsuam, kev lag luam lossis tsoomfwv thiab cov koom haum tswj hwm kom nkag siab txog kev tsim kho dua tshiab ntawm cov kab mob hauv vitro ntawm qib transcriptomic los sim cov tshuaj lossis kev kho mob biotherapy Kev nqus thiab kev thauj mus los ntawm cov neeg sib tw tshuaj tau raug soj ntsuam siv 3D plab hloov pauv lossis siv cov qauv kev cai lossis kev lag luam organ-on-a-chip los ntsuas qhov rov ua dua ntawm cov txheej txheem morphogenesis plab.
Muaj tsawg tus qauv sim uas cuam tshuam rau tib neeg tau siv los kawm txog kev hloov pauv ntawm txoj hnyuv epithelial, feem ntau yog vim tsis muaj cov txheej txheem siv tau los ua kom muaj 3D morphogenesis hauv vitro. Qhov tseeb, ntau yam kev paub tam sim no txog kev hloov pauv ntawm txoj hnyuv yog raws li kev tshawb fawb tsiaj (piv txwv li, zebrafish20, nas21 lossis qaib22). Txawm li cas los xij, lawv yog cov neeg ua haujlwm thiab tus nqi ntau, tuaj yeem ua rau muaj kev tsis ntseeg txog kev ncaj ncees, thiab qhov tseem ceeb tshaj plaws, tsis txiav txim siab meej txog cov txheej txheem kev loj hlob ntawm tib neeg. Cov qauv no kuj tseem txwv heev hauv lawv lub peev xwm los sim ntau txoj hauv kev scalable. Yog li ntawd, peb cov txheej txheem rau kev tsim cov qauv ntaub so ntswg 3D hauv vitro ua tau zoo dua hauv cov qauv tsiaj hauv vivo nrog rau lwm cov qauv kab lis kev cai 2D ib txwm muaj. Raws li tau piav qhia ua ntej, kev siv cov qauv epithelial 3D tau tso cai rau peb los tshuaj xyuas qhov chaw nyob ntawm cov hlwb sib txawv hauv crypt-villus axis teb rau ntau yam mucosal lossis kev tiv thaiv kab mob. 3D epithelial txheej tuaj yeem muab qhov chaw los kawm txog seb cov hlwb microbial sib tw li cas los tsim cov niches spatial thiab kev hloov pauv ecological teb rau cov yam ntxwv ntawm tus tswv tsev (piv txwv li, cov txheej sab hauv piv rau cov txheej sab nraud, kev tso tawm ntawm IgA thiab cov tshuaj tua kab mob peptides). Ntxiv mus, 3D epithelial morphology tuaj yeem tso cai rau peb nkag siab tias cov kab mob plab hnyuv tsim nws cov zej zog li cas thiab ua ke tsim cov tshuaj lom neeg microbial metabolites (piv txwv li, cov roj fatty acids luv luv) uas ua rau lub koom haum cellular thiab cov hlwb cell niches hauv cov basal crypts. Cov yam ntxwv no tsuas yog tuaj yeem pom thaum cov txheej epithelial 3D tau tsim hauv vitro.
Ntxiv rau peb txoj kev tsim cov qauv epithelial plab hnyuv 3D, muaj ntau txoj hauv kev hauv vitro. Kev cog qoob loo organoid plab hnyuv yog cov txheej txheem tsim cov ntaub so ntswg zoo tshaj plaws raws li kev cog qoob loo ntawm cov hlwb plab hnyuv hauv qab cov xwm txheej morphogen tshwj xeeb23,24,25. Txawm li cas los xij, kev siv cov qauv 3D organoid rau kev tshuaj xyuas kev thauj mus los lossis cov kab lis kev cai ntawm tus tswv tsev-microbiome feem ntau nyuaj vim tias lub plab hnyuv lumen raug kaw hauv organoid thiab, yog li ntawd, kev qhia txog cov khoom siv luminal xws li cov hlwb microbial lossis cov antigens exogenous yog txwv. Kev nkag mus rau organoid lumens tuaj yeem txhim kho siv microinjector,26,27 tab sis txoj kev no yog kev nkag mus thiab kev siv zog ntau thiab xav tau kev paub tshwj xeeb los ua. Ntxiv mus, cov kab lis kev cai organoid ib txwm muaj nyob rau hauv hydrogel scaffolds nyob rau hauv cov xwm txheej zoo li qub tsis qhia meej txog kev ua haujlwm hauv vivo biomechanics.
Lwm txoj kev siv los ntawm ntau pawg tshawb fawb siv cov qauv 3D hydrogel scaffolds uas tau teeb tsa ua ntej los ua raws li cov qauv epithelial ntawm lub plab los ntawm kev cog cov hlwb plab tib neeg cais tawm ntawm qhov chaw gel. Tsim cov qauv hydrogel siv cov pwm 3D-luam tawm, micro-milled, lossis lithographically fabricated. Txoj kev no qhia txog kev teeb tsa tus kheej ntawm cov hlwb epithelial cais tawm hauv vitro nrog cov qauv morphogen gradients, tsim kom muaj cov qauv epithelial siab thiab stroma-epithelial crosstalk los ntawm kev suav nrog cov hlwb stromal hauv scaffold. Txawm li cas los xij, qhov xwm txheej ntawm cov qauv scaffolds uas tau teeb tsa ua ntej yuav tiv thaiv kev tso saib ntawm cov txheej txheem morphogenetic spontaneous nws tus kheej. Cov qauv no kuj tsis muab cov dej ntws dynamic luminal lossis interstitial, tsis muaj kev ntxhov siab ntawm cov kua dej uas cov hlwb plab yuav tsum tau dhau los ntawm morphogenesis thiab tau txais kev ua haujlwm ntawm lub cev. Lwm txoj kev tshawb fawb tsis ntev los no siv cov qauv hydrogel hauv lub platform microfluidic thiab cov qauv epithelial plab hnyuv patterned siv cov txheej txheem laser-etching. Cov nas plab hnyuv organoids ua raws li cov qauv etched los tsim cov qauv tubular plab hnyuv, thiab cov kua dej ntws intraluminal tuaj yeem rov ua dua siv a microfluidics module. Txawm li cas los xij, tus qauv no tsis qhia txog cov txheej txheem morphogenetic spontaneous thiab tsis suav nrog cov kev txav mus los ntawm lub plab. Cov txheej txheem luam ntawv 3D los ntawm tib pawg tau tsim cov raj plab me me nrog cov txheej txheem morphogenetic spontaneous. Txawm hais tias muaj kev tsim cov ntu plab sib txawv hauv lub raj, tus qauv no kuj tsis muaj cov kua dej ntws thiab kev hloov pauv tshuab. Tsis tas li ntawd, kev ua haujlwm ntawm tus qauv yuav raug txwv, tshwj xeeb tshaj yog tom qab cov txheej txheem bioprinting tiav lawm, cuam tshuam rau cov xwm txheej kev sim lossis kev sib cuam tshuam ntawm tes. Hloov chaw, peb cov txheej txheem tau muab cov morphogenesis plab spontaneous, kev ntxhov siab shear cuam tshuam, biomechanics uas ua raws li kev txav mus los ntawm lub plab, kev nkag mus rau cov chaw apical thiab basolateral ywj pheej, thiab rov tsim cov chaw ua haujlwm microenvironments ntawm modularity. Yog li ntawd, peb cov txheej txheem morphogenesis hauv vitro 3D yuav muab txoj hauv kev sib koom ua ke los kov yeej cov teeb meem ntawm cov txheej txheem uas twb muaj lawm.
Peb cov txheej txheem yog tsom mus rau 3D epithelial morphogenesis, tsuas yog cov hlwb epithelial hauv kev cog qoob loo thiab tsis muaj lwm hom hlwb nyob ib puag ncig xws li cov hlwb mesenchymal, cov hlwb endothelial, thiab cov hlwb tiv thaiv kab mob. Raws li tau piav qhia ua ntej, lub hauv paus ntawm peb cov txheej txheem yog qhov kev tsim cov epithelial morphogenesis los ntawm kev tshem tawm cov tshuaj morphogen inhibitors secreted ntawm sab basolateral ntawm cov khoom nruab nrab. Thaum lub zog modularity ntawm peb lub plab-rau-ib-chip thiab hybrid-rau-ib-chip tso cai rau peb rov ua dua cov txheej undulating 3D epithelial, ntxiv cov teeb meem biological xws li epithelial-mesenchymal kev sib cuam tshuam33,34, extracellular Matrix (ECM) deposition35 thiab, hauv peb tus qauv, crypt-villus nta uas xa cov qia hlwb niches hauv basal crypts tseem yuav tsum tau txiav txim siab ntxiv. Stromal hlwb (piv txwv li, fibroblasts) hauv mesenchyme ua lub luag haujlwm tseem ceeb hauv kev tsim cov protein ECM thiab kev tswj hwm ntawm txoj hnyuv morphogenesis hauv vivo35,37,38. Qhov ntxiv ntawm mesenchymal cov hlwb rau peb tus qauv txhim kho cov txheej txheem morphogenetic thiab kev ua haujlwm ntawm kev txuas ntawm cov hlwb. Lub txheej endothelial (piv txwv li, capillaries lossis lymphatics) ua lub luag haujlwm tseem ceeb hauv kev tswj hwm kev thauj mus los molecular39 thiab kev nrhiav cov hlwb tiv thaiv kab mob40 hauv qhov chaw ib puag ncig ntawm lub plab. Ntxiv mus, cov khoom siv vasculature uas tuaj yeem txuas nrog cov qauv ntaub so ntswg yog qhov yuav tsum tau ua thaum cov qauv ntaub so ntswg tau tsim los qhia txog kev sib cuam tshuam ntawm ntau lub cev. Yog li ntawd, cov hlwb endothelial yuav tsum tau suav nrog los ua qauv ntau yam ntxwv ntawm lub cev nrog kev daws teeb meem ntawm lub cev. Cov hlwb tiv thaiv kab mob los ntawm tus neeg mob kuj tseem ceeb rau kev ua kom pom cov lus teb tiv thaiv kab mob, kev nthuav qhia antigen, kev sib txuas lus tiv thaiv kab mob, thiab kev tiv thaiv kab mob tshwj xeeb hauv cov ntsiab lus ntawm kev ua raws li kab mob plab.
Kev siv cov hybrid chips yooj yim dua li gut-on-a-chip vim tias qhov kev teeb tsa ntawm lub cuab yeej yooj yim dua thiab kev siv Transwell inserts tso cai rau kev loj hlob ntawm cov epithelium plab. Txawm li cas los xij, cov inserts Transwell muaj nyob rau hauv kev lag luam nrog polyester membranes tsis elastic thiab tsis tuaj yeem ua rau muaj kev txav mus los zoo li peristaltic. Ntxiv mus, qhov chaw apical ntawm Transwell insert tso rau hauv hybrid chip tseem nyob ruaj khov tsis muaj kev ntxhov siab ntawm sab apical. Nws yog qhov tseeb, cov khoom zoo li qub hauv qhov chaw apical tsis tshua muaj peev xwm ua rau muaj kev sib koom ua ke ntev hauv cov hybrid chips. Txawm hais tias peb tuaj yeem ua rau muaj 3D morphogenesis hauv Transwell inserts thaum siv cov hybrid chips, qhov tsis txaus ntawm cov biomechanics thiab apical fluid flow yuav txwv tsis pub muaj hybrid chip platforms rau cov ntawv thov.
Kev tsim kho dua tshiab ntawm tib neeg lub crypt-villus axis hauv cov kab lis kev cai gut-on-a-chip thiab hybrid-on-a-chip tseem tsis tau tsim tag nrho. Txij li thaum morphogenesis pib los ntawm epithelial monolayer, 3D microarchitectures tsis tas yuav muab cov morphological zoo sib xws rau crypts hauv vivo. Txawm hais tias peb tau piav qhia txog cov pej xeem proliferating cell nyob ze ntawm lub basal crypt domain hauv microengineered 3D epithelium, cov crypt thiab villous cheeb tsam tsis tau demarcated meej. Txawm hais tias cov channel siab dua ntawm lub chip ua rau qhov siab ntawm microengineered epithelium, qhov siab tshaj plaws tseem txwv rau ~ 300–400 µm. Qhov tob tiag tiag ntawm tib neeg lub plab hnyuv crypts hauv cov hnyuv me thiab loj yog ~ 135 µm thiab ~ 400 µm, feem, thiab qhov siab ntawm cov hnyuv me villi yog ~ 600 µm41.
Los ntawm qhov pom ntawm kev thaij duab, kev thaij duab zoo heev ntawm 3D microarchitectures tej zaum yuav txwv rau lub plab ntawm lub nti, vim tias qhov deb ua haujlwm xav tau los ntawm lub lens mus rau txheej epithelial yog nyob rau ntawm qhov kev txiav txim ntawm ob peb millimeters. Txhawm rau kov yeej qhov teeb meem no, lub hom phiaj deb yuav tsum tau ua. Ntxiv mus, ua cov ntu nyias rau kev npaj cov qauv thaij duab yog qhov nyuaj vim yog qhov elasticity siab ntawm PDMS. Ntxiv mus, txij li thaum txheej-los-txheej microfabrication ntawm lub plab ntawm lub nti cuam tshuam nrog kev sib txuas tas mus li ntawm txhua txheej, nws yog qhov nyuaj heev rau qhib lossis tshem tawm txheej sab saud los tshuaj xyuas cov qauv ntawm cov txheej epithelial. Piv txwv li, los ntawm kev siv lub tshuab scanning electron microscope (SEM).
Qhov hydrophobicity ntawm PDMS tau yog qhov txwv tsis pub ua hauv kev tshawb fawb microfluidic uas cuam tshuam nrog cov molecules me me hydrophobic, vim PDMS tuaj yeem nqus cov hydrophobic molecules tsis tshwj xeeb. Lwm txoj hauv kev rau PDMS yuav raug txiav txim siab nrog lwm cov ntaub ntawv polymeric. Xwb, kev hloov kho qhov chaw ntawm PDMS (piv txwv li, txheej nrog cov ntaub ntawv lipophilic 42 lossis poly (ethylene glycol) 43) tuaj yeem raug txiav txim siab kom txo qis kev nqus ntawm cov hydrophobic molecules.
Thaum kawg, peb txoj kev tsis tau piav qhia zoo txog kev muab kev tshuaj ntsuam xyuas siab lossis "ib qho loj-haum-txhua tus" lub platform sim uas yooj yim rau tus neeg siv. Cov txheej txheem tam sim no xav tau lub twj tso kua mis rau txhua lub microdevice, uas siv qhov chaw hauv lub tshuab CO2 thiab tiv thaiv kev sim loj. Qhov kev txwv no tuaj yeem txhim kho tau zoo los ntawm kev nthuav dav ntawm cov qauv kab lis kev cai tshiab (piv txwv li, 24-qhov, 96-qhov, lossis 384-qhov porous inserts uas tso cai rau kev ua kom tiav thiab tshem tawm cov xov xwm basolateral tas mus li).
Txhawm rau kom muaj 3D morphogenesis ntawm tib neeg txoj hnyuv epithelium hauv vitro, peb tau siv lub microfluidic chip intestinal device uas muaj ob lub parallel microchannels thiab ib lub elastic porous membrane nyob nruab nrab los tsim ib lub lumen-capillary interface. Peb kuj qhia txog kev siv ib lub single-channel microfluidic device (ib lub hybrid chip) uas muab cov dej ntws basolateral tas mus li hauv qab cov polarized epithelial txheej uas loj hlob ntawm Transwell inserts. Hauv ob lub platform, morphogenesis ntawm ntau yam tib neeg txoj hnyuv epithelial hlwb tuaj yeem pom los ntawm kev siv kev coj ua ntawm kev ntws kom tshem tawm morphogen antagonists los ntawm lub basolateral compartment. Tag nrho cov txheej txheem sim (Daim Duab 1) muaj tsib ntu: (i) microfabrication ntawm lub plab chip lossis Transwell insertable hybrid chip (kauj ruam 1-5; Box 1), (ii) kev npaj ntawm cov plab hnyuv epithelial hlwb (Caco-2 hlwb) lossis tib neeg txoj hnyuv organoids; thawv 2-5), (iii) kev cog qoob loo ntawm cov hlwb epithelial hauv plab hnyuv ntawm cov chips plab hnyuv lossis cov chips sib xyaw (kauj ruam 6-9), (iv) kev tsim kom muaj 3D morphogenesis hauv vitro (kauj ruam 10) thiab (v)) los piav qhia txog 3D epithelial microstructure (kauj ruam 11-24). Thaum kawg, ib pawg tswj hwm tsim nyog (tham ntxiv hauv qab no) tau tsim los lees paub qhov ua tau zoo ntawm in vitro morphogenesis los ntawm kev sib piv epithelial morphogenesis rau spatial, temporal, conditional, lossis procedural controls.
Peb siv ob lub platform kab lis kev cai sib txawv: gut-on-a-chip nrog cov channel ncaj lossis nonlinear convoluted channels, lossis hybrid chips uas muaj Transwell (TW) inserts hauv microfluidic device, 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 chip lossis hybrid chip. "Kev Kab Lis Kev Cai ntawm Tib Neeg Intestinal Epithelial Cells" piav qhia txog qhov chaw ntawm lub hlwb (Caco-2 lossis tib neeg intestinal organoids) thiab cov txheej txheem kab lis kev cai siv hauv cov txheej txheem no. "In vitro morphogenesis" qhia cov kauj ruam tag nrho uas Caco-2 lossis organoid-derived epithelial cells raug cog rau ntawm lub plab hnyuv chip lossis ntawm Transwell inserts ntawm lub hybrid chip, ua raws li kev tsim ntawm 3D morphogenesis thiab kev tsim ntawm cov qauv epithelial. Tus lej kauj ruam ntawm qhov kev pab cuam lossis tus lej thawv tau tso tawm hauv qab txhua tus xub. Daim ntawv thov muab cov piv txwv ntawm yuav ua li cas cov txheej txheem epithelial plab hnyuv tau tsim, piv txwv li, hauv kev sib txawv ntawm tes, kev kawm txog lub cev plab, kev tsim cov ecosystems-microbiome, thiab kab mob. qauv. Cov duab Immunofluorescence hauv "Cell Differentiation" qhia txog nuclei, F-actin thiab MUC2 qhia tawm hauv 3D Caco-2 epithelial txheej tsim tawm ntawm lub plab chip. MUC2 signaling muaj nyob rau hauv goblet hlwb thiab mucus secreted los ntawm mucosal surfaces. Cov duab fluorescent hauv Gut Physiology qhia mucus tsim los ntawm kev staining rau sialic acid thiab N-acetylglucosamine residues siv fluorescent wheat germ agglutinin. Ob daim duab overlapping hauv "Host-Microbe Co-Cultures" qhia cov sawv cev ntawm tus tswv tsev-microbiome co-cultures hauv lub plab ntawm lub chip. Lub vaj huam sib luag sab laug qhia txog kev sib koom ua ke ntawm E. coli qhia txog ntsuab fluorescent protein (GFP) nrog microengineered 3D Caco-2 epithelial hlwb. Lub vaj huam sib luag sab xis qhia txog qhov chaw ntawm GFP E. coli co-cultured nrog 3D Caco-2 epithelial hlwb, ua raws li immunofluorescence staining nrog F-actin (liab) thiab nuclei (xiav). Kev ua qauv kab mob qhia txog kev noj qab haus huv piv rau leaky plab hauv Cov kab mob plab hnyuv nyob rau hauv kev sib tw physiological nrog cov kab mob bacterial antigens (piv txwv li, lipopolysaccharide, LPS) thiab cov hlwb tiv thaiv kab mob (piv txwv li, PBMC; ntsuab). Caco-2 hlwb tau cog qoob loo los tsim kom muaj 3D epithelial txheej. Scale bar, 50 µm. Cov duab hauv kab hauv qab: "Kev sib txawv ntawm cov hlwb" hloov kho nrog kev tso cai los ntawm kev siv.2. Oxford University Press; Rov tsim dua nrog kev tso cai los ntawm Ref.5. NAS; "Host-Microbe Co-Culture" hloov kho nrog kev tso cai los ntawm ref.3. NAS; "Disease Modeling" hloov kho nrog kev tso cai los ntawm kev siv.5. NAS.
Ob qho tib si gut-on-chip thiab hybrid chips tau tsim los ntawm kev siv PDMS replicas uas tau demolded los ntawm silicon pwm los ntawm cov lithography mos 1,44 thiab patterned nrog SU-8. Kev tsim cov microchannels hauv txhua lub chip yog txiav txim siab los ntawm kev xav txog hydrodynamics xws li shear stress thiab hydrodynamic pressure 1,4,12. Tus thawj gut-on-a-chip tsim (Extended Data Fig. 1a), uas muaj ob lub juxtaposed parallel ncaj microchannels, tau hloov zuj zus mus rau hauv ib qho nyuaj gut-on-a-chip (Extended Data Fig. 1b) uas suav nrog ib khub ntawm curved microchannels los ua kom muaj zog ntxiv cov kua dej nyob, cov qauv ntws tsis linear, thiab multiaxial deformation ntawm cov hlwb cog qoob loo (Fig. 2a-f) 12. Thaum cov biomechanics ntawm plab hnyuv ntau dua yuav tsum tau rov tsim dua, cov complex gut-on-a-chips tuaj yeem xaiv tau. Peb tau ua pov thawj tias convoluted Gut-Chip kuj tseem ua rau muaj 3D morphogenesis hauv lub sijhawm zoo sib xws nrog qib zoo sib xws ntawm epithelial kev loj hlob piv rau thawj Gut-Chip, tsis hais hom cell kab lis kev cai. Yog li ntawd, txhawm rau ua kom muaj 3D morphogenesis, cov qauv tsim linear thiab complex on-chip gut yog sib pauv tau. PDMS replicas kho rau ntawm silicon pwm nrog SU-8 qauv muab cov yam ntxwv tsis zoo tom qab demolding (Daim duab 2a). Txhawm rau tsim lub plab ntawm lub chip, cov txheej PDMS sab saud uas tau npaj tseg tau sib txuas ua ke rau zaj duab xis PDMS porous thiab tom qab ntawd sib phim nrog cov txheej PDMS qis dua los ntawm kev sib txuas tsis rov qab siv lub tshuab kho corona (Daim duab 2b-f). Txhawm rau tsim cov chips hybrid, cov PDMS replicas kho tau sib txuas rau cov iav slides los tsim cov khoom siv microfluidic ib-channel uas tuaj yeem haum rau Transwell inserts (Daim duab 2h thiab Extended Data Fig. 2). Cov txheej txheem sib txuas yog ua los ntawm kev kho cov nto ntawm PDMS replica thiab iav nrog oxygen plasma lossis corona kho. Tom qab sterilization ntawm lub cuab yeej microfabricated txuas rau lub raj silicone, lub cuab yeej teeb tsa tau npaj txhij los ua 3D morphogenesis ntawm cov epithelium plab hnyuv (Daim duab 2g).
a, Daim duab kos duab ntawm kev npaj cov khoom PDMS los ntawm cov pwm silicon patterned SU-8. Cov tshuaj PDMS tsis tau kho tau nchuav rau ntawm cov pwm silicon (sab laug), kho ntawm 60 ° C (nruab nrab) thiab demolded (sab xis). Cov PDMS demolded tau txiav ua tej daim thiab ntxuav rau kev siv ntxiv.b, Duab ntawm cov pwm silicon siv los npaj cov txheej sab saud PDMS.c, Duab ntawm cov pwm silicon siv los tsim cov PDMS porous membrane.d, Ib qho ntawm cov duab ntawm cov khoom PDMS sab saud thiab sab qis thiab cov khoom siv plab hnyuv sib dhos.e, Daim duab kos duab ntawm kev sib dhos ntawm cov khoom PDMS sab saud, daim nyias nyias, thiab sab qis. Txhua txheej yog irreversibly bonded los ntawm plasma lossis corona kho.f, Daim duab kos duab ntawm cov khoom siv plab hnyuv-rau-ib-chip nrog cov microchannels convoluted superimposed thiab vacuum chambers.g, Teeb tsa ntawm plab hnyuv-rau-ib-chip rau microfluidic cell kab lis kev cai. Cov plab hnyuv tsim ntawm ib lub chip sib dhos nrog lub raj silicone thiab syringe tau muab tso rau ntawm lub npog. Lub cuab yeej chip tau muab tso rau ntawm Lub hau ntawm lub tais Petri 150 hli rau kev ua tiav. Lub binder yog siv los kaw lub raj silicone.h, Cov duab pom ntawm kev tsim cov chip hybrid thiab 3D morphogenesis siv cov chips hybrid.Transwell inserts npaj ywj pheej rau kev cog qoob loo 2D monolayers ntawm cov hlwb epithelial plab hnyuv tau ntxig rau hauv cov chip hybrid los ua kom cov hnyuv 3D morphogenesis. Lub nruab nrab yog perfused los ntawm microchannels hauv qab lub cell txheej tsim rau ntawm Transwell insert.Scale bar, 1 cm.h Rov luam tawm nrog kev tso cai los ntawm kev siv.4. Elsevier.
Hauv cov txheej txheem no, cov kab Caco-2 cell thiab cov kab mob hauv plab hnyuv tau siv ua cov chaw epithelial (Daim Duab 3a). Ob hom cell tau cog qoob loo ywj pheej (Lub thawv 2 thiab Lub thawv 5) thiab siv los cog cov ECM-coated microchannels ntawm on-chip gut lossis Transwell inserts. Thaum cov cell sib xyaw (> 95% kev them nyiaj hauv flasks), cov kab mob Caco-2 uas tau cog qoob loo tas li (ntawm cov kab lus 10 thiab 50) hauv T-flasks raug sau los npaj cov cell suspensions dissociated los ntawm trypsinization kua (lub thawv 2). Cov kab mob hauv plab hnyuv tib neeg los ntawm cov biopsies hauv plab hnyuv lossis kev phais mob tau cog qoob loo hauv Matrigel scaffold domes hauv 24-qhov phaj los txhawb nqa qhov chaw ib puag ncig. Cov khoom nruab nrab uas muaj cov morphogens tseem ceeb (xws li Wnt, R-spondin, thiab Noggin) thiab cov yam ntxwv loj hlob npaj raws li tau piav qhia hauv Lub thawv 3 tau ntxiv txhua hnub kom txog thaum cov organoids loj hlob mus txog ~ 500 µm hauv txoj kab uas hla. Cov organoids loj hlob tag nrho raug sau thiab dissociated rau hauv ib qho cov hlwb rau kev cog rau ntawm txoj hnyuv lossis Transwell ntxig rau ntawm lub chip (Lub thawv 5). Raws li peb tau tshaj tawm ua ntej, nws tuaj yeem sib txawv raws li hom kab mob 12,13 (piv txwv li ulcerative colitis, Crohn's kab mob, colorectal cancer, lossis tus neeg pub dawb ib txwm), qhov chaw raug mob (piv txwv li, qhov raug mob piv rau thaj chaw tsis raug mob) thiab qhov chaw hauv txoj hnyuv (piv txwv li, duodenum, jejunum, ileum, cecum, colon, lossis rectum). Peb muab cov txheej txheem zoo tshaj plaws hauv Lub thawv 5 rau kev cog qoob loo colonic organoids (coloids) uas feem ntau xav tau cov morphogens ntau dua li cov organoids me me hauv plab hnyuv.
a, Kev Ua Haujlwm rau kev ua kom muaj kev hloov pauv ntawm lub plab hauv cov chip plab. Caco-2 tib neeg lub plab epithelium thiab cov kab mob plab yog siv rau hauv cov txheej txheem no los qhia txog 3D morphogenesis. Cov hlwb epithelial cais tau cog rau hauv lub cuab yeej npaj plab-rau-ib-chip (chip npaj). Thaum cov hlwb tau cog (seeded) thiab txuas (txuas) rau PDMS porous membrane rau hnub 0 (D0), apical (AP) ntws pib thiab tswj hwm rau thawj 2 hnub (ntws, AP, D0-D2). Basolateral (BL) ntws kuj pib nrog rau kev txav mus los ntawm kev ncab (ncab, ntws, AP thiab BL) thaum ua tiav 2D monolayer. Intestinal 3D morphogenesis tshwm sim spontaneous tom qab 5 hnub ntawm microfluidic kab lis kev cai (morphogenesis, D5). Cov duab sib piv theem qhia txog morphology sawv cev ntawm Caco-2 hlwb ntawm txhua kauj ruam sim lossis lub sijhawm (bar graph, 100 µm). Plaub daim duab schematic piav qhia txog cov cascades sib xws ntawm plab. morphogenesis (sab xis saum toj). Cov xub dashed hauv daim duab qhia txog kev coj ntawm cov kua dej ntws.b, daim duab SEM qhia txog qhov chaw saum npoo ntawm 3D Caco-2 epithelium uas tau tsim (sab laug). Daim duab inset qhia txog thaj chaw loj (lub thawv dawb dashed) qhia txog cov microvilli uas tau tsim dua tshiab ntawm 3D Caco-2 txheej (sab xis).c, Saib kab rov tav ntawm Caco-2 3D uas tau tsim, claudin (ZO-1, liab) thiab cov txhuam txuas ntxiv uas muaj daim ntawv lo F-actin (ntsuab) thiab nuclei (xiav) Immunofluorescence confocal visualization ntawm cov hlwb epithelial ntawm cov chips plab hnyuv. Cov xub taw rau daim duab nruab nrab qhia txog qhov chaw ntawm lub dav hlau focal rau txhua qhov kev pom confocal.d, Lub sijhawm ntawm kev hloov pauv morphological hauv organoids cog rau ntawm lub chip tau los ntawm kev siv lub tshuab microscopy sib piv rau hnub 3, 7, 9, 11, thiab 13. Daim duab inset (sab xis saum toj) qhia txog kev loj hlob siab ntawm daim duab muab.e, DIC photomicrograph ntawm organoid 3D epithelium uas tau tsim. hauv plab hnyuv ntawm daim uas tau thaij rau hnub 7.f, Cov duab immunofluorescence uas tau muab tso rau saum toj qhia cov cim rau cov hlwb qia (LGR5; magenta), cov hlwb goblet (MUC2; ntsuab), F-actin (grey) thiab nuclei (cyan) uas loj hlob rau ntawm cov chips plab hnyuv rau 3 hnub, raws li (Sab laug) thiab 13-hnub (nruab nrab) organoids ntawm txheej epithelial.Saib ntxiv Cov Ntaub Ntawv Ntxiv Daim Duab 3, uas qhia txog LGR5 signaling yam tsis muaj MUC2 signaling. Cov duab fluorescence uas qhia txog epithelial microstructure (sab xis) ntawm 3D organoid epithelium uas tau tsim nyob rau hauv plab hnyuv ntawm ib lub chip los ntawm kev pleev xim rau daim nyias nyias plasma nrog CellMask zas (sab xis) rau hnub 13 ntawm kev cog qoob loo. Qhov ntsuas bar yog 50 μm tshwj tsis yog hais lwm yam.b Luam tawm dua nrog kev tso cai los ntawm kev siv.2. Oxford University Press; c Hloov kho nrog kev tso cai los ntawm Kev Siv.2. Oxford University Press; e thiab f hloov kho nrog kev tso cai los ntawm kev siv.12 Nyob rau hauv Creative Commons License CC BY 4.0.
Hauv lub plab hnyuv ntawm ib lub chip, nws yog qhov tsim nyog los hloov kho qhov chaw hydrophobic ntawm PDMS porous membrane rau kev ua tiav ECM txheej. Hauv cov txheej txheem no, peb siv ob txoj hauv kev sib txawv los hloov kho qhov hydrophobicity ntawm PDMS daim nyias nyias. Rau kev cog qoob loo Caco-2 hlwb, kev ua haujlwm ntawm qhov chaw los ntawm UV / ozone kev kho mob ib leeg txaus los txo qhov hydrophobicity ntawm PDMS nto, txheej ECM thiab txuas Caco-2 hlwb rau PDMS daim nyias nyias. Txawm li cas los xij, microfluidic kab lis kev cai ntawm organoid epithelium xav tau kev ua haujlwm ntawm qhov chaw tshuaj lom neeg kom ua tiav kev tso cov protein ECM los ntawm kev siv cov polyethyleneimine (PEI) thiab glutaraldehyde rau PDMS microchannels. Tom qab kev hloov kho ntawm qhov chaw, cov protein ECM tau tso rau kom npog qhov chaw PDMS ua haujlwm thiab tom qab ntawd qhia rau hauv cov organoid epithelium cais. Tom qab cov hlwb raug txuas, cov kab lis kev cai ntawm microfluidic pib los ntawm kev nkag mus rau hauv microchannel sab saud kom txog thaum cov hlwb tsim ib qho monolayer tiav, thaum cov microchannel qis dua tswj cov xwm txheej zoo li qub. Txoj kev zoo tshaj plaws no rau kev ua haujlwm ntawm qhov chaw thiab ECM txheej ua rau kev txuas ntawm organoid epithelium. kom ua rau 3D morphogenesis ntawm PDMS nto.
Cov kab lis kev cai Transwell kuj xav tau ECM txheej ua ntej cog noob; txawm li cas los xij, Transwell kab lis kev cai tsis xav tau cov kauj ruam ua ntej ua kom lub ntsej muag ntawm cov ntxig porous. Rau kev loj hlob Caco-2 hlwb ntawm Transwell inserts, ECM txheej ntawm cov ntxig porous ua kom nrawm dua qhov txuas ntawm cov hlwb Caco-2 dissociated (<1 teev) thiab kev sib txuas nruj nreem tsim (<1-2 hnub). Txhawm rau cog cov organoids ntawm Transwell inserts, cov organoids cais tawm raug cog rau ntawm ECM-coated inserts, txuas rau ntawm daim nyias nyias (<3 teev) thiab khaws cia kom txog thaum cov organoids tsim ib qho monolayer tiav nrog kev ruaj ntseg thaiv. Transwell kab lis kev cai raug ua tiav hauv 24-qhov phaj yam tsis siv cov chips hybrid.
Hauv vitro 3D morphogenesis tuaj yeem pib los ntawm kev siv cov kua dej ntws mus rau sab basolateral ntawm ib txheej epithelial uas tau tsim tsa. Hauv plab hnyuv ntawm ib lub chip, epithelial morphogenesis pib thaum cov khoom nruab nrab tau perfused rau hauv cov microchannels sab saud thiab sab qis (Daim duab 3a). Raws li tau piav qhia ua ntej, nws yog qhov tseem ceeb kom qhia cov kua dej ntws hauv qhov qis dua (basolateral) compartment rau kev tshem tawm tas mus li ntawm cov tshuaj morphogen inhibitors. Txhawm rau muab cov as-ham thiab serum txaus rau cov hlwb khi rau ntawm cov ntaub so ntswg porous thiab tsim kom muaj kev ntxhov siab luminal shear, peb feem ntau siv ob npaug ntws hauv plab hnyuv ntawm ib lub chip. Hauv cov chips hybrid, Transwell inserts uas muaj epithelial monolayers tau ntxig rau hauv cov chips hybrid. Tom qab ntawd, cov khoom nruab nrab tau siv nyob rau hauv qab sab basolateral ntawm porous Transwell insert los ntawm microchannel. Intestinal morphogenesis tshwm sim 3-5 hnub tom qab pib ntawm basolateral ntws hauv ob lub platform kab lis kev cai.
Cov yam ntxwv morphological ntawm microengineered 3D epithelial txheej tuaj yeem tshuaj xyuas los ntawm kev siv ntau yam imaging modalities, suav nrog theem sib piv microscopy, differential interference contrast (DIC) microscopy, SEM, lossis immunofluorescence confocal microscopy (Daim Duab 3 thiab 4). Theem sib piv lossis DIC imaging tuaj yeem ua tau yooj yim txhua lub sijhawm thaum lub sijhawm kab lis kev cai los saib xyuas cov duab thiab protrusion ntawm 3D epithelial txheej. Vim yog qhov pom tseeb ntawm PDMS thiab polyester zaj duab xis, ob qho tib si gut-on-a-chip thiab hybrid chip platforms tuaj yeem muab cov duab tiag tiag hauv situ yam tsis tas yuav txiav lossis disassembly ntawm lub cuab yeej. Thaum ua immunofluorescence imaging (Daim Duab 1, 3c, f thiab 4b, c), cov hlwb feem ntau raug kho nrog 4% (wt / vol) paraformaldehyde (PFA), ua raws li Triton X-100 thiab 2% (wt / vol)) bovine serum albumin (BSA), raws li kev txiav txim. Nyob ntawm hom hlwb, cov khoom kho sib txawv, Cov tshuaj permeabilizers, thiab cov neeg sawv cev thaiv kab mob siv tau. Cov tshuaj tiv thaiv kab mob thawj zaug uas tsom mus rau cov cim ntawm cov hlwb lossis thaj chaw uas nyob ntawm cov noob caj noob ces siv los qhia cov hlwb uas tsis txav mus los hauv qhov chaw ntawm lub nti, ua raws li cov tshuaj tiv thaiv kab mob thib ob nrog rau cov xim pleev xim uas tsom mus rau cov nucleus (piv txwv li, 4′,6-diamidino-2-phenylene) indole, DAPI) lossis F-actin (piv txwv li, fluorescently labeled phalloidin). Kev thaij duab raws li fluorescence kuj tseem tuaj yeem ua tau hauv qhov chaw los ntes cov hnoos qeev (Daim Duab 1, "Cell differentiation" thiab "Gut physiology"), kev tsim cov hlwb microbial random (Daim Duab 1, "Host-microbe co-culture"), kev nrhiav cov hlwb tiv thaiv kab mob (Daim Duab 1, 'Disease Modeling') lossis cov duab ntawm 3D epithelial morphology (Daim Duab 3c, f thiab 4b, c). Thaum hloov kho lub plab ntawm lub nti kom cais cov txheej sab saud los ntawm cov txheej microchannel qis dua, raws li tau piav qhia hauv ref.Raws li qhia hauv Daim Duab 2, 3D Cov qauv ntawm epithelial nrog rau cov microvilli ntawm ntug txhuam apical tuaj yeem pom tau los ntawm SEM (Daim Duab 3b). Kev qhia tawm ntawm cov cim sib txawv tuaj yeem soj ntsuam los ntawm kev ua PCR5 lossis ib lub cell RNA sequencing. Hauv qhov no, cov txheej 3D ntawm cov hlwb epithelial uas loj hlob hauv cov chips plab lossis cov chips hybrid raug sau los ntawm trypsinization thiab tom qab ntawd siv rau kev tshuaj xyuas molecular lossis genetic.
a, Kev Ua Haujlwm rau kev ua kom cov morphogenesis ntawm txoj hnyuv hauv ib lub hybrid chip. Caco-2 thiab cov organoids hauv txoj hnyuv siv rau hauv cov txheej txheem no los qhia txog 3D morphogenesis hauv ib lub platform hybrid chip. Cov hlwb epithelial uas tau tawg lawm tau cog rau hauv cov ntawv sau Transwell uas tau npaj tseg (TW prep; saib daim duab hauv qab no). Thaum cov hlwb tau cog (seeded) thiab txuas rau polyester membranes hauv Transwell inserts, txhua lub hlwb tau cog qoob loo hauv qab cov xwm txheej zoo li qub (TW culture). Tom qab 7 hnub, ib qho Transwell ntxig uas muaj 2D monolayer ntawm cov hlwb epithelial tau koom ua ke rau hauv ib lub hybrid chip los qhia txog cov dej ntws basolateral (Flow, BL), uas thaum kawg ua rau muaj cov txheej epithelial 3D (morphogenesis). Cov duab sib piv ntawm theem qhia txog cov yam ntxwv ntawm cov hlwb epithelial tib neeg uas tau los ntawm cov neeg pub dawb ib txwm (C103 kab) nce mus rau hauv plab hnyuv ntawm txhua kauj ruam sim lossis lub sijhawm. Cov duab kos hauv cov khaubncaws sab saud qhia txog kev teeb tsa sim rau txhua kauj ruam.b, Cov chips Hybrid (schematic sab laug) tuaj yeem ua rau 3D morphogenesis ntawm cov hlwb epithelial organoid. nrog rau sab saum toj-down confocal microscopy saib ntawm ntau qhov chaw Z sib txawv (sab saud, nruab nrab, thiab qis dua; saib sab xis schematic thiab cov kab dotted sib xws). qhia pom tseeb morphological yam ntxwv.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled view) ntawm organoid-derived epithelial cells cultured hauv static Transwell (TW; inset hauv lub thawv dawb dashed) piv rau hybrid chip (qhov loj tshaj plaws tag nrho txhaj tshuaj) piv rau 2D piv rau 3D morphology, feem.Ib khub ntawm 2D ntsug cross-cut views (inset hauv kaum sab xis saum toj; "XZ") kuj qhia 2D thiab 3D nta. Scale bar, 100 µm.c Rov luam tawm nrog kev tso cai los ntawm kev siv.4. Elsevier.
Cov kev tswj hwm tuaj yeem npaj tau los ntawm kev cog cov hlwb zoo sib xws (Caco-2 lossis cov hlwb epithelial hauv plab hnyuv) rau hauv ob-seem monolayers nyob rau hauv cov xwm txheej kab lis kev cai zoo li qub. Qhov tseem ceeb, kev noj zaub mov tsis txaus yuav tshwm sim vim yog lub peev xwm ntim tsawg ntawm cov microchannels (piv txwv li ~ 4 µL hauv cov channel sab saum toj ntawm tus qauv tsim plab-chip thawj). Yog li ntawd, epithelial morphology ua ntej thiab tom qab daim ntawv thov ntawm basolateral ntws kuj tuaj yeem piv.
Cov txheej txheem lithography mos mos yuav tsum tau ua nyob rau hauv chav huv. Rau txhua txheej ntawm lub chip (cov txheej sab saud thiab qis dua thiab cov membranes) thiab cov chips hybrid, cov photomasks sib txawv tau siv thiab tsim rau ntawm cov silicon wafers sib cais vim tias qhov siab ntawm cov microchannels sib txawv. Qhov siab ntawm cov microchannels sab saud thiab qis dua ntawm lub plab ntawm lub chip yog 500 µm thiab 200 µm, raws li. Qhov siab ntawm cov channel ntawm lub hybrid chip yog 200 µm.
Muab ib daim wafer silicon 3-nti tso rau hauv ib lub tais uas muaj acetone. Maj mam tig lub phaj rau 30 vib nas this, tom qab ntawd ziab lub wafer. Muab lub wafer tso rau hauv ib lub phaj uas muaj IPA, tom qab ntawd tig lub phaj rau 30 vib nas this kom huv.
Ib qho tshuaj piranha (sib tov ntawm hydrogen peroxide thiab concentrated sulfuric acid, 1: 3 (vol/vol)) tuaj yeem siv tau los ua kom tshem tawm cov organic residues ntawm qhov chaw silicon wafer.
Cov kua Piranha muaj zog heev thiab tsim cua sov. Yuav tsum muaj kev ceev faj ntxiv. Yog xav pov tseg cov khib nyiab, cia cov kua txias thiab muab tso rau hauv lub thawv khib nyiab huv thiab qhuav. Siv cov thawv thib ob thiab sau cov ntawv pov tseg kom raug. Thov ua raws li cov lus qhia kev nyab xeeb ntawm lub chaw kom paub ntau ntxiv.
Ua kom cov wafers qhuav los ntawm kev muab tso rau ntawm lub phaj kub 200 ° C rau 10 feeb. Tom qab ua kom qhuav, cov wafer raug co tsib zaug hauv huab cua kom txias.
Ncuav li 10 g ntawm photoresist SU-8 2100 rau hauv nruab nrab ntawm lub silicon wafer uas tau ntxuav lawm. Siv cov tweezers los nthuav cov photoresist kom sib npaug rau ntawm lub wafer. Qee zaum muab lub wafer tso rau ntawm lub phaj kub 65°C kom cov photoresist tsis nplaum thiab yooj yim nthuav. Tsis txhob muab lub wafer tso ncaj qha rau ntawm lub phaj kub.
SU-8 tau muab faib sib npaug zos rau ntawm lub wafer los ntawm kev khiav lub txheej txheej tig. Tsim kom muaj kev tig ntawm SU-8 rau 5–10 vib nas this kom nthuav dav ntawm 500 rpm ntawm qhov nrawm ntawm 100 rpm / s. Teem lub ntsiab tig rau 200 µm tuab patterning ntawm 1,500 rpm, lossis ua tiav 250 µm tuab (ua kom muaj 500 µm siab rau txheej sab saud ntawm lub plab ntawm lub chip; 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.
Qhov ceev ntawm lub tshuab tig tseem ceeb tuaj yeem hloov kho raws li lub hom phiaj tuab ntawm tus qauv SU-8 ntawm lub silicon wafer.
Yuav kom tsim tau cov qauv SU-8 ntawm 500 µm qhov siab rau txheej sab saud ntawm lub plab ntawm lub nti, cov txheej txheej tig thiab cov kauj ruam ci mos ntawm lub thawv no (cov kauj ruam 7 thiab 8) tau rov ua dua ib zaug ntxiv (saib cov kauj ruam 9) los tsim ob txheej ntawm 250 µm Ib txheej tuab ntawm SU-8, uas tuaj yeem muab txheej thiab koom nrog los ntawm kev raug UV hauv kauj ruam 12 ntawm lub thawv no, ua ib txheej 500 µm siab.
Ci cov wafers uas tau coated SU-8 kom mos mos los ntawm kev muab cov wafers tso rau ntawm lub phaj kub ntawm 65 ° C rau 5 feeb, tom qab ntawd hloov qhov chaw mus rau 95 ° C thiab incubate rau ntxiv 40 feeb.
Yuav kom ua tiav qhov siab 500 μm ntawm tus qauv SU-8 hauv lub microchannel sab saud, rov ua cov kauj ruam 7 thiab 8 los tsim ob txheej SU-8 tuab 250 μm.
Siv lub UV Mask Aligner, ua qhov kev sim teeb raws li cov lus qhia ntawm cov chaw tsim khoom kom xam lub sijhawm raug ntawm lub wafer. (lub sijhawm raug, ms) = (koob tshuaj raug, mJ / cm2) / (lub zog teeb, mW / cm2).
Tom qab txiav txim siab lub sijhawm raug, muab lub photomask tso rau ntawm lub qhov tuav lub ntsej muag ntawm lub tshuab sib dhos UV thiab muab lub photomask tso rau ntawm lub wafer uas tau coated SU-8.
Muab qhov chaw luam tawm ntawm lub photomask ncaj qha rau ntawm SU-8 coated sab ntawm lub silicon wafer kom txo qis UV dispersion.
Tso cov wafer thiab photomask uas tau coated SU-8 rau ntawm 260 mJ/cm2 ntawm lub teeb UV rau lub sijhawm raug teeb tsa ua ntej (saib kauj ruam 10 ntawm lub thawv no).
Tom qab raug UV, SU-8-coated silicon wafers tau ci ntawm 65 ° C rau 5 feeb thiab 95 ° C rau 15 feeb ntawm txhua lub phaj kub kom tsim cov qauv nrog qhov siab ntawm 200 μm. Ncua lub sijhawm tom qab ci ntawm 95 ° C mus rau 30 feeb kom tsim cov qauv nrog qhov siab ntawm 500 µm.
Cov tshuaj developer raug nchuav rau hauv lub tais iav, thiab cov wafer ci raug muab tso rau hauv lub tais. Qhov ntim ntawm SU-8 developer yuav txawv nyob ntawm qhov loj ntawm lub phaj iav. Nco ntsoov siv cov tshuaj developer SU-8 txaus kom tshem tawm tag nrho cov SU-8 uas tsis tau pom. Piv txwv li, thaum siv lub tais iav 150 hli txoj kab uas hla nrog lub peev xwm 1 L, siv ~ 300 mL ntawm SU-8 developer. Tsim cov pwm rau 25 feeb nrog kev tig maj mam qee zaum.
Yaug cov pwm uas tau tsim lawm nrog ~ 10 mL ntawm cov tshuaj developer tshiab ua raws li IPA los ntawm kev txau cov tshuaj siv lub pipette.
Muab lub wafer tso rau hauv lub tshuab ntxuav plasma thiab tso rau hauv cov pa oxygen (cov pa roj atmospheric, lub hom phiaj siab 1 × 10−5 Torr, lub zog 125 W) rau 1.5 feeb.
Muab cov wafer tso rau hauv lub tshuab nqus tsev uas muaj lub iav swb sab hauv. Cov wafer thiab cov slides tuaj yeem tso ua ib sab. Yog tias lub tshuab nqus tsev raug faib ua ntau txheej los ntawm ib lub phaj, muab cov slides tso rau hauv chav qis dua thiab cov wafer tso rau hauv chav sab saud. Tso 100 μL ntawm trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane kua rau ntawm lub iav swb thiab siv lub tshuab nqus tsev rau silanization.
Yaj ib lub raj mis ntawm cov hlwb Caco-2 khov rau hauv lub dab dej 37 ° C, tom qab ntawd hloov cov hlwb uas yaj mus rau hauv lub raj mis T75 uas muaj 15 mL ntawm 37 ° C prewarmed Caco-2 nruab nrab.
Yuav kom dhau Caco-2 hlwb ntawm ~ 90% confluency, ua ntej sov Caco-2 nruab nrab, PBS, thiab 0.25% trypsin/1 mM EDTA hauv 37°C dej da dej.
Siv lub tshuab nqus tsev nqus cov khoom nruab nrab. Ntxuav cov hlwb ob zaug nrog 5 mL ntawm PBS sov los ntawm kev rov ua dua lub tshuab nqus tsev nqus thiab ntxiv PBS tshiab.