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A na-achọkwa ihe gabigara Gut na Cryphogenitititate nke 3D Epithelittalitis na Spatial Counter.ini na-eji ihe mgbochi na-enweghị ihe mgbochi Uzo nke oma. luidic mgbawa yana na transwell agbakwunyere ngwakọ ngwakọ, na njikọta nke mkpụrụ ego nke microfluidididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididididi. ure site na ịchịkwa mmiri na-agagharị maka 5 d.our na-eme ka usoro nyocha nke ọma na-eme ihe na-adịghị mma. Ngwa ọgwụ.
Nnwale na-egosi na intestinal epithelial Caco-2 sel ndị a na-emepụta na gut-on-a-chip1,2,3,4,5 ma ọ bụ bilayer microfluidic devices6,7 nwere ike ịnweta morphogenesis 3D na-enweghị isi na vitro na-enweghị nghọta doro anya nke usoro dị n'okpuru. morphogenesis in vitro, nke Caco-2 gosipụtara na organoids intestinal nke ndị ọrịa na-enweta.A kwadoro mkpụrụ ndụ epithelial. N'ime ọmụmụ ihe a, anyị lekwasịrị anya kpọmkwem na mmepụta cell na nkesa nkesa nke onye na-emegide Wnt dị ike, Dickkopf-1 (DKK-1), na gut-on-a-chip na ngwaọrụ microfluidic gbanwetụrụ nwere ntinye Transwell, nke a na-akpọ " Chip Hybrid ". , ma ọ bụ Soggy-1) na na-chip gut na-egbochi morphogenesis ma ọ bụ na-akpaghasị 3D epithelial Layer prestructured, na-atụ aro na nrụgide na-emegide onwe ya n'oge omenala na-ahụ maka morphogenesis intestinal in vitro. Ya mere, ụzọ dị irè iji nweta morphogenesis siri ike na interface epithelial bụ iji wepụ ma ọ bụ na-arụsi ọrụ ike na-eme ka ọkpụkpụ na-arụ ọrụ na-arụ ọrụ. -on-a-chip ma ọ bụ ngwakọ-na-a-chip nyiwe) ma ọ bụ mgbasa .Basolateral media (dịka ọmụmaatụ, site na Transwell ntinye n'ime nnukwu basolateral reservoirs na olulu mmiri).
N'ime usoro a, anyị na-enye usoro zuru ezu maka ịmepụta microdevices gut-on-a-chip na transwell-insertable hybrid chips (nzọụkwụ 1-5) na mkpụrụ ndụ epithelial intestinal omenala na polydimethylsiloxane (PDMS) -based porous membranes (nzọụkwụ 6A, 7A, 8, 9) ma ọ bụ 6A, 7A, 8, 9) ma ọ bụ transwell 6 polyester. kpalitere 3D morphogenesis in vitro (nzọụkwụ 10) .Anyị chọpụtakwara cellular na molecular features egosi nke anụ ahụ-kpọmkwem histogenesis na lineage-adabere cellular dị iche iche site n'itinye multiple imaging modalities (nzọụkwụ 11-24) .Anyị na-eme ka morphogenesis na-eji mmadụ eriri afọ epithelial cell sel, dị ka Cacoal-2 nkọwa nke ọdịdị ọdịdị ọdịdị ọdịdị elu, dị ka ọdịdị ọdịdị ọdịdị ọdịdị nke Cacorous, ma ọ bụ ọdịdị ihu abụọ nke Cacorous. e kere eke 2D monolayers, na intestinal biochemical na Mmeputakwa nke biomechanical microenvironment.in vitro.To induce 3D morphogenesis si 2D epithelial monolayers, anyị wepụrụ morphogen antagonists na ma omenala ụdị site eruba na-ajụ n'ime basolateral compartment nke omenala. N'ikpeazụ, anyị nwere ike na-eji ihe nlereanya nke epitheliD na-anọchi anya ihe nlereanya. morphogen na-adabere epithelial uto, longitudinal host-microbiome co-cultures, pathogen infection, inflammatory unfunction, epithelial barrier dysfunction, and probiotics based probiotics Example.influences.
Usoro anyị nwere ike ịba uru nye ndị ọkà mmụta sayensị sara mbara (dịka ọmụmaatụ, biology mucosal intestinal, biology stem cell, na bayoloji mmepe) yana nyocha etinyere (dịka ọmụmaatụ, nnwale ọgwụ preclinical, nhazi ọrịa, injin anụ ahụ, na gastroenterology) mmetụta sara mbara. Enwere ike ịgbasa atụmatụ maka ndị na-ege ntị na-amụ ike nke mgbaàmà cell n'oge mmepe intestinal, ịmaliteghachi ma ọ bụ homeostasis .Na mgbakwunye, usoro anyị bara uru maka ịgba ajụjụ gbasara ọrịa n'okpuru ọrịa dị iche iche na-efe efe dị ka Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridiummumon difficile.Ndị na-ege ntị nke ọrịa pathology na pathogenesis na-abakwa uru.Ojiji nke usoro microphysiology nke on-chip gut nwere ike ikwe ka ogologo oge co-omenala 10 na nyocha na-esote nke nchebe ndị ọbịa, nzaghachi ndị na-alụso ọrịa ọgụ na mmerụ ahụ metụtara ọrịa na-edozi ahụ na tract gastrointestinal (GI) tract 11 . Nsogbu ndị ọzọ GI jikọtara ya na leaky gutwel syndrome, ọrịa na-egbu egbu, ọrịa Crohn, ọrịa Crohn, ọrịa crohn, ma ọ bụ ọrịa Crohn. 3D eriri afọ epithelial layers na-akwado ya site na iji onye ọrịa 3D eriri afọ epithelial layers , ọrịa ndị a na-agụnye villous atrophy, crypt shortening, mucosal mmebi, ma ọ bụ mebie epithelial barrier.Biopsy ma ọ bụ stem cell-ewepụtara intestinal organoids12,13. Iji mma nlereanya na-eme ka ndị ọrịa na-agụ ụdị ọrịa ndị dị otú ahụ dị mgbagwoju anya. mkpụrụ ndụ nuklia (PBMCs), ka ụdị nwere 3D intestinal villus-crypt microarchitectures.mkpụrụ ndụ na-alụso ọrịa ọgụ kpọmkwem, 5.
Ebe ọ bụ na 3D epithelial microstructure nwere ike idozi ma na-ahụ anya na-enweghị usoro nkebi, ndị na-ekiri na-arụ ọrụ na transcriptomics spatial transcriptomics na nnukwu mkpebi ma ọ bụ nnukwu mkpebi nwere ike ịmasị na map anyị nke spatiotemporal dynamics of genes and proteins on epithelial niches.Enwere mmasị na teknụzụ. Nzaghachi maka microbial ma ọ bụ ihe mkpali na-alụso ọrịa ọgụ. Ọzọkwa, longitudinal host-microbiome crosstalk 10, 14 that coordinate gut homeostasis nwere ike guzobe na 3D eriri afọ mucosal oyi akwa site co-culturing dị iche iche microbial ụdị, microbial obodo ma ọ bụ fecal microbiota- karịsịa na fecal microbiota.na n'elu ikpo okwu.Nke a obibia na-adọrọ mmasị karịsịa na-ege ntị na-amụ mucosal immunology, gastroenterology, ụmụ mmadụ microbiome, culturomics na clinical microbiology na-achọ ịzụlite na mbụ uncultured gut microbiota na laabu.If our in vitro morphogenesis protocol nwere ike na-emegharị ka scalable omenala formats, dị ka multiwell, na-aga n'ihu na ntinye nke ọma na 624 platenish. , A pụkwara ịgbasa usoro ahụ na ndị na-emepe emepe ọgwụ, biomedical Ma ọ bụ elu-throughput screening ma ọ bụ nkwado nyiwe maka ụlọ ọrụ nri. Dị ka ihe akaebe-nke-ụkpụrụ, anyị na-adịbeghị anya gosiri na a ga-ekwe omume nke multiplex elu-throughput morphogenesis usoro scalable ka a 24-ọma plate.Na mgbakwunye, 1-1 nke a na-emepụta ihe na-emepụta ihe. nkwado nke usoro morphogenesis nke in vitro anyị nwere ike ịgbatị ma nwee ike ịnabata ya site na ọtụtụ ụlọ nyocha nyocha, ụlọ ọrụ ma ọ bụ gọọmentị na ụlọ ọrụ na-ahụ maka njikwa iji ghọta cellular reprogramming nke in vitro gut morphogenesis na ọkwa transcriptomic iji nwalee ọgwụ ma ọ bụ biotherapeutics A na-enyocha nnabata na mbufe nke ndị na-achọ ọgwụ ike site na iji 3D gut surrogates ma ọ bụ iji usoro nhazi nke usoro azụmaahịa ma ọ bụ usoro nhazi nke nhazi usoro. usoro.
A na-eji ọnụ ọgụgụ dị nta nke ụdị nnwale ndị dị mkpa nke mmadụ mee ihe iji mụọ morphogenesis intestinal epithelial morphogenesis, tumadi n'ihi enweghị usoro iwu nke nwere ike ime ka 3D morphogenesis in vitro. cisely chọpụta usoro mmepe mmadụ. Ụdị ndị a dịkwa oke oke n'ikike ha na-anwale n'ọtụtụ ụzọ scalable. Ya mere, usoro anyị maka ịmaliteghachi ọkpụkpụ anụ ahụ 3D na vitro pụtara ìhè na ụdị anụ ọhịa vivo na ụdị omenala 2D cell static ndị ọzọ. Dị ka akọwara mbụ, tinye n'ọrụ 3D epithelial sel nke dị iche iche na-enye ohere ka anyị nweta nzaghachi n'ime sel dị iche iche. dị iche iche mucosal ma ọ bụ dịghịzi stimuli.3D epithelial n'ígwé nwere ike inye ohere na-amụ otú microbial mkpụrụ ndụ mpi etolite spatial niches na ecological evolushọn na nzaghachi na-anabata ihe (eg, n'ime versus mpụta imi n'ígwé, secretion nke IgA na antimicrobial peptides) .Ọzọkwa, 3D epithelial morpholoji na-ekwe ka anyị microbial morphology na-ekwe ka anyị na-eme ka microbial morphology na-eme ka anyị nwee ike ime ka microbial morphology na-emepụta ihe. metabolites (dịka ọmụmaatụ, fatty acids nke dị mkpụmkpụ) nke na-akpụzi nzukọ cellular na niches cell niches na basal crypts. A pụrụ igosipụta njirimara ndị a naanị mgbe 3D epithelial layers na-eguzobe na vitro.
Na mgbakwunye na usoro anyị nke ịmepụta 3D intestinal epithelial structures, e nwere ọtụtụ ụzọ in vitro. Intestinal organoid culture is a state-of-the-art-art tissue engineering technique based on cultivation of intestinal stem cell n'okpuru kpọmkwem morphogen ọnọdụ23,24,25. Otú ọ dị, iji 3D organoid organoid host-models na-emekarị na-eme njem nlegharị anya n'ihi na ọ bụ ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe. a na-agbanye n'ime organoid na, ya mere, iwebata ihe ndị na-egbuke egbuke dị ka mkpụrụ ndụ microbial ma ọ bụ antigens exogenous dị oke.Enwere ike imeziwanye ohere ịnweta lumen organoid site na iji microinjector,26,27 ma usoro a na-akpa ike ma na-arụsi ọrụ ike ma na-achọ ihe ọmụma pụrụ iche iji rụọ ọrụ. Ọzọkwa, omenala organoid omenala na-echekwa na hydrogel scaffolds n'okpuru ọnọdụ static adịghị egosipụta n'ụzọ ziri ezi na-arụ ọrụ na vivo biomechanics.
Ụzọ ndị ọzọ na-arụ ọrụ dị iche iche na-eme nnyocha na-eji 3D hydrogel scaffolds prestructured iji ṅomie eriri afọ epithelial Ọdịdị site n'ịzụlite mkpụrụ ndụ eriri afọ mmadụ dịpụrụ adịpụ na gel surface. gradients, guzosie ike a akụkụ dị elu ratio epithelial Ọdịdị na stroma-epithelial crosstalk site gụnyere stromal cell na scaffold. Otú ọ dị, ọdịdị nke prestructured scaffolds nwere ike igbochi ngosi nke spontaneous morphogenetic usoro n'onwe ya. Ndị a ụdị na-adịghị enye dynamic dynamic ma ọ bụ interstitial shemorpha sel na-arụ ọrụ nke mmiri na-eruba n'okpuru mmiri na-eruba na-arụ ọrụ na-arụ ọrụ nke na-arụ ọrụ nke ọma. Ọ dịghị ọzọ na-adịbeghị anya ọmụmụ na-eji hydrogel scaffolds na a microfluidic ikpo okwu na ụkpụrụ intestinal epithelial Ọdịdị na-eji laser-etching techniques.Mouse intestinal organoids na-eso etched ụkpụrụ na-etolite eriri afọ tubular Ọdịdị, na intraluminal mmiri eruba ike recapitulated site na iji a microfluidics modul. Otú ọ dị, nke a nlereanya ọ bụghị exhibits gunyere usoro nke gut. Site n'otu ìgwè ahụ nwere ike ịmepụta obere tubes gut tubes na usoro morphogenetic na-enweghị ihe ọ bụla. N'agbanyeghị mgbagwoju anya nke akụkụ dị iche iche nke eriri afọ n'ime tube ahụ, ihe nlereanya a enweghịkwa luminal fluid flow and mechanical deformation. Tụkwasị na nke ahụ, arụmọrụ ihe nlereanya nwere ike ịdị oke, karịsịa mgbe usoro bioprinting zuru ezu, na-emebi ọnọdụ nnwale ma ọ bụ ihe na-eme ka anyị nwee ike ime ihe na-eme ka morphogene . , physiologically mkpa shear stress, biomechanics nke na-eṅomi gut motility, nnweta nke onwe apical na basolateral compartments, na re-mepụta nke mgbagwoju biological microenvironments nke modularity.Ya mere, anyị in vitro 3D morphogenesis protocol nwere ike inye a mmekota uzo iji merie ihe ịma aka nke ụzọ ndị dị adị.
Usoro anyị na-elekwasị anya kpamkpam na 3D epithelial morphogenesis, na-enwe nanị mkpụrụ ndụ epithelial na omenala na ọ dịghị ụdị ndị ọzọ dị gburugburu sel dị ka mesenchymal cell, endothelial cell, na dịghịzi sel. Dị ka akọwara na mbụ, isi nke anyị protocol bụ ntinye nke epithelial morphogenesis site na-ewepụ morphogen inhibitors zoro ezo na akụkụ nke robost-mkpụrụ ndụ anyị. -chip na ngwakọ-on-a-chip na-enye anyị ohere ịmegharị akwa epithelial 3D na-adịghị agwụ agwụ, ihe mgbagwoju anya ndị ọzọ dị ka epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 na, n'ime ihe nlereanya anyị, crypt-villus atụmatụ na-ebuga stem cell niches na basal crypts, .S. na mmepụta nke protein ECM na nhazi nke intestinal morphogenesis na vivo35,37,38.Mgbakwunye nke mkpụrụ ndụ mesenchymal na ihe nlereanya anyị kwalitere usoro morphogenetic na ntinye cell nke ọma.The endothelial Layer (ie, capillaries or lymphatics) na-arụ ọrụ dị mkpa n'ịchịkwa molecular transport3440 microvacult cell . nke nwere ike ijikọ n'etiti ụdị anụ ahụ bụ ihe dị mkpa mgbe e mere ụdị anụ ahụ iji gosipụta mmekọrịta dị iche iche nke akụkụ ahụ. Ya mere, mkpụrụ ndụ endothelial nwere ike ịdị mkpa ka etinyere ya iji mee ka njirimara physiological ziri ezi na mkpebi nke akụkụ ahụ.
Ojiji nke ngwakọ ibe n'ụzọ kwụ ọtọ karịa gut-on-a-chip n'ihi na ngwaọrụ nhazi dị mfe na iji Transwell ntinye na-enye ohere maka scalable omenala nke gut epithelium. Otú ọ dị, azụmahịa dị Transwell inserts na polyester membranes na-adịghị na-agbanwe na-enweghị ike simulate peristaltic-dị ka mmegharị. Ọzọkwa, na shei na-etinye na apical n'ọdụ ụgbọ mmiri na-etinye na apical na-etinye na apical n'ọdụ ụgbọ mmiri na-etinye na apical na-etinye na apical n'ọdụ ụgbọ mmiri. Apical side.N'ụzọ doro anya, na static Njirimara na apical ngalaba adịkarịghị aka ogologo oge nje co-omenala na ngwakọ chips.While anyị nwere ike robustly induce 3D morphogenesis na Transwell inserts mgbe eji ngwakọ ibe, ụkọ physiologically mkpa biomechanics na apical mmiri eruba nwere ike na-amachi ngwa ngwa chipsibility nke hybrid.
Nrụgharị zuru oke nke mmadụ crypt-villus axis na gut-on-a-chip na ngwakọ-na-a-chip omenala eguzobebeghị nke ọma. Ebe ọ bụ na morphogenesis na-amalite site na monolayer epithelial, 3D microarchitectures adịghị enye morphological myirịta na crypts na vivo. Ọ bụ ezie na 3D na-eme ka ọnụ ọgụgụ nke microcrypter na-eme ka ọnụ ọgụgụ nke microcrypter dị nso na-eme ka ọnụ ọgụgụ ndị na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-eme ka ọnụ ọgụgụ nke microcrypter dị nso na-eme ka ọnụ ọgụgụ nke microcrypter. lium, crypt na villous region e akọwapụtaghị nke ọma. Ọ bụ ezie na ọwa elu dị elu na mgbawa na-eduga n'ịdị elu nke microengineered epithelium, elu kachasị elu ka na-ejedebe na ~ 300-400 µm. N'ezie omimi nke eriri afọ mmadụ crypts na obere na nnukwu eriri afọ bụ ~ 135 µm, ịdị elu nke villi na ~ 45 µm. 600 µm41.
Site na onyonyo onyonyo, n'ọnọdụ dị oke oke onyonyo nke 3D microarchitectures nwere ike ịbụ naanị eriri afọ na mgbawa, ebe ọ bụ na ebe a na-arụ ọrụ dị anya site na lens lens na epithelial oyi akwa dị n'usoro nke millimeters ole na ole. Iji merie nsogbu a, enwere ike ịchọ ebumnuche dị anya. , Ebe ọ bụ na microfabrication nke oyi akwa nke eriri afọ na mgbawa na-agụnye adhesion na-adịgide adịgide n'etiti oyi akwa ọ bụla, ọ na-esiri ike imeghe ma ọ bụ wepụ elu elu iji nyochaa ọdịdị elu nke epithelial Layer. Dịka ọmụmaatụ, site na iji nyocha eletrọnịkị eletrọnịkị (SEM).
The hydrophobicity nke PDMS bụ ihe na-amachi ihe na microfluidic dabeere ọmụmụ na-emeso hydrophobic obere ụmụ irighiri ihe, ebe PDMS nwere ike nonspecifically adsorb ndị dị otú ahụ hydrophobic ụmụ irighiri. Nhọrọ na PDMS nwere ike na-atụle na ihe ndị ọzọ polymeric. N'aka nke ọzọ, n'elu mgbanwe mgbanwe nke PDMS (dịka ọmụmaatụ, mkpuchi na lip lip 4 polym4 3 lipophilic ihe) ize adsorption nke hydrophobic molekul.
N'ikpeazụ, usoro anyị akọwabeghị nke ọma n'ihe gbasara ịnye nyocha dị elu ma ọ bụ "otu-size-dabara-niile" onye ọrụ-enyi na enyi nnwale platform. Usoro nke ugbu a chọrọ mgbapụta sirinji kwa microdevice, nke na-eweghara ohere na CO2 incubator ma na-egbochi nnukwu nnwale. Nke a nwere ike ime ka mmachi dịkwuo mma site na scalability, scalability, 24-9, dịka ọmụmaatụ, 6, scalability, ma ọ bụ 4-nke ọma-4. -nke ọma porous inserts na-ekwe ka na-aga n'ihu replenishment na mwepụ nke basolateral media).
Iji mee ka 3D morphogenesis nke epithelium eriri afọ mmadụ na vitro, anyị na-eji ngwaọrụ eriri afọ microfluidic nke nwere microchannels abụọ yiri ya na akpụkpọ ahụ na-agbanwe agbanwe n'etiti iji mepụta lumen-capillary interface. morphogenesis nke mkpụrụ ndụ epithelial intestinal dị iche iche nke mmadụ nwere ike igosi site n'itinye ntụziaka ntụziaka nke na-asọpụta iji wepụ morphogen antagonists site na akụkụ basolateral. Usoro nyocha dum (Njirimara 1) nwere akụkụ ise: (i) microfabrication nke mgbawa gut ma ọ bụ Transwell ntinye ngwakọ ngwakọ (nzọụkwụ 1-5); (nkwụsị 1-5 ma ọ bụ sel epite) sel anụ ahụ n'ime sel 1), (nkwadebe nke sel n'ime sel anụ ahụ). stinal organoids;igbe 2-5), (iii) omenala nke eriri afọ epithelial sel na eriri afọ ibe ma ọ bụ ngwakọ ibe (nzọụkwụ 6-9), (iv) induction nke 3D morphogenesis in vitro (nzọụkwụ 10) na (v) )) iji mara 3D epithelial microstructure (nzọụkwụ 11-24 kwesịrị ekwesị na-arụ ọrụ n'okpuru) e mere n'ihu vitro. morphogenesis site na iji epithelial morphogenesis atụnyere ohere, nwa oge, ọnọdụ, ma ọ bụ njikwa usoro.
Anyị na-eji usoro ọdịnala abụọ dị iche iche: gut-on-a-chip nwere ọwa kwụ ọtọ ma ọ bụ ọwa na-enweghị njikọ, ma ọ bụ ngwakọ ngwakọ nwere ihe ntinye Transwell (TW) na ngwaọrụ microfluidic, nke arụpụtara dị ka akọwara na Igbe 1, yana nzọụkwụ 1 -5. In vitro morphogenesis" na-egosi usoro nke usoro nke Caco-2 ma ọ bụ organoid sitere na epithelial sel na-etolite na mgbawa eriri afọ ma ọ bụ na ntinye nke Transwell nke mgbawa ngwakọ, sochiri ntinye nke 3D morphogenesis na nguzobe nke usoro ihe ngosi nke usoro ihe atụ n'okpuru usoro ihe atụ nke usoro ihe omume ọ bụla. Enwere ike iji akwa epithelial stinal, dịka ọmụmaatụ, na njirimara cell differentiation characterization, gut physiology studies, nguzobe nke host-microbiome ecosystems, and disease modeling.Immunofluorescence oyiyi na "Cell Differentiation" na-egosi nuclei, F-actin na MUC2 nke egosipụtara na 3D Caco-2 epithelial epithelial mucose cell chipd ugbu a na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe. N'elu elu. Ihe oyiyi fluorescent na Gut Physiology na-egosi imi na-emepụta site na nsị maka sialic acid na N-acetylglucosamine residues iji fluorescent wheat germ agglutinin.The abụọ overlapping on "Host-Microbe Co-Cultures" na-egosi onye nnọchiteanya host-microbiome co-cultures in the gut on a chip. gineered 3D Caco-2 epithelial cell. The ziri ezi panel na-egosi localization nke GFP E. coli co-cultured na 3D Caco-2 epithelial sel, sochiri immunofluorescence staining na F-actin (red) na nuclei (acha anụnụ anụnụ) .Mwepụta ọrịa na-egosi ahụike na-agba agba na gut na-egbu egbu n'okpuru lipological chips, L. na sel ndị na-alụso ọrịa ọgụ (dịka ọmụmaatụ, PBMC;green) .Caco-2 mkpụrụ ndụ ka e mere ka e guzobe 3D epithelial Layer.Scale bar, 50 µm.Images na ala n'ahịrị: "iche nke mkpụrụ ndụ" na-emegharị na ikike site ntụaka.2.Mahadum Oxford;Emegharịrị site na ikike sitere na Ref.5.NAS;“Co-Culture Ọbịa-Microbe” emebere site na ikike sitere na ref.3.NAS;“Ndele ọrịa” emebere site na ikike sitere na ntụaka.5.NAS.
Ma gut-on-chip and hybrid chips e kere site na iji PDMS replicas nke e mebiri si silicon ebu site soft lithography1,44 na patterned na SU-8.The imewe nke microchannels na nke ọ bụla mgbawa na-ekpebisi ike site n'ịtụle hydrodynamics dị ka shiee nrụgide na hydrodynamic pressure1,4,12.The mbụ gut-on-a-chip data mejupụtara ogologo Fig. microchannels, etolitela n'ime gut-on-a-chip dị mgbagwoju anya (Extended Data Fig. 1b) nke na-agụnye otu ụzọ microchannels gbagọrọ agbagọ iji mee ka oge obibi mmiri na-abawanye ụba, usoro na-asọ oyi na-adịghị na ya, na nrụrụ multiaxial nke mkpụrụ ndụ omenala (Fig 2a-f) 12. Mgbe mgbagwoju anya biomechanted dị mgbagwoju anya, ọ dị mkpa ka e nwee mgbagwoju anya. ted Gut-Chip na-akpalikwa morphogenesis nke 3D ike n'otu oge ahụ na ogo nke epithelial na-eto eto ma e jiri ya tụnyere Gut-Chip mbụ, n'agbanyeghị ụdị cell omenala.Ya mere, iji mee ka 3D morphogenesis, linear na mgbagwoju anya na-chip gut designs na-agbanwe.2a) . Iji mepụta eriri afọ na mgbawa, PDMS oyi akwa a kwadebere n'usoro jikọtara ya na ihe nkiri PDMS porous wee kwekọọ na oyi akwa PDMS dị ala site na njikọ na-enweghị ike ịgbanwe site na iji onye na-agwọ ọrịa corona (Fig 2b-f) . Iji mepụta ngwakọ ngwakọ, PDMS replicas gwọrọ na-ejikọta ya na slides glazed na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-emepụta ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe eji eme ihe. Extended Data Fig. 2) A na-eme usoro njikọ ahụ site n'ịgwọ ihe ndị dị n'elu nke PDMS oyiri na iko na plasma oxygen ma ọ bụ ọgwụgwọ corona. Mgbe sterilization nke microfabricated ngwaọrụ jikọtara na silicone tube, ngwaọrụ ntọala dị njikere ịrụ 3D morphogenesis nke intestinal epithelium (Figure 2g).
a, Schematic ihe atụ nke nkwadebe nke PDMS akụkụ si SU-8 patterned silicon Molds.The uncured PDMS ngwọta wụsara n'elu a silicon ebu (n'aka ekpe), gwọrọ na 60 Celsius C (n'etiti) na demolded (nri) .The demolded PDMS e bee n'ime iberibe na kpochara maka n'ihu ojiji.b, Foto nke silicon PDMS eji akwa oyi akwa na-akwadebe silicon molds. rous membrane.d, Usoro foto nke akụkụ PDMS nke elu na nke dị ala na ihe ejikọtara on-chip intestinal device.e, Usoro nhazi nke elu, akpụkpọ ahụ, na PDMS dị ala. oyi akwa ọ bụla bụ nke na-adịghị agbanwe agbanwe site na plasma ma ọ bụ ọgwụgwọ corona.f, Schematic nke a kpụrụ akpụ nke gut-on-a-chip ngwaọrụ na supervacuum champion nwere nnukwu ihe mgbagwoju anya. on-a-chip maka microfluidic cell culture. A na-etinye eriri a na-emepụta na mgbawa na-ejikọta ya na tube silicone na sirinji na mkpuchi mkpuchi. A na-etinye ngwaọrụ mgbawa na mkpuchi nke efere 150 mm Petri maka nhazi. A na-eji ihe nkedo na-emechi silicone tube.h, Visual snapshots nke ngwakọ chipsmorphation omenala na 3D hybrid onwe ya kwadebere omenala chips2D. ers of intestinal cell epithelial ka etinyere n'ime ngwakọ ngwakọ iji mee ka eriri afọ 3D morphogenesis. A na-agbaze ihe na-esi na ya site na microchannels n'okpuru oyi akwa cell nke etinyere na ntinye Transwell.Scale bar, 1 cm.h Edegharịrị ya na ikike site na ntụaka.4.Elsevier.
N'ime usoro iwu a, a na-eji eriri Caco-2 cell na intestinal organoids mee ihe dị ka isi mmalite epithelial (Fig 3a) . A na-emepụta ụdị abụọ nke mkpụrụ ndụ n'adabereghị (Igbe 2 na Igbe 5) ma jiri mkpụrụ ECM kpuchie microchannels nke on-chip gut ma ọ bụ Transwell inserts. Mgbe sel ndị confluent (> sk95% na-ekpuchi sel na-eme ihe na-eme ka ọkpụkpụ na-eme ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-emepụta ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-emepụta ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-eme ) 50) na T-flasks ka a na-egbute iji kwadebe nkwụsịtụ cell nkewapụrụ site na mmiri mmiri trypsinization (igbe 2) . Ụmụ mmadụ intestinal organoids sitere na intestinal biopsies ma ọ bụ ịwa ahụ resections bụ omenala na Matrigel scaffold domes na 24-well plates iji kwado microenvironment structural .Medium na ihe ndị dị mkpa nke morphogent, ihe ndị dị mkpa na-emepụta ihe. dị ka akọwara na Igbe 3 na-agbakwunye kwa ụbọchị ruo mgbe organoids na-eto eto ruo ~ 500 µm na dayameta. A na-egbute organoids zuru oke ma kesaa ya n'ime otu mkpụrụ ndụ maka ịgha mkpụrụ n'ime eriri afọ ma ọ bụ ihe ntinye Transwell na mgbawa (Igbe 5) .Dịka anyị kwuburu na mbụ, ọ nwere ike ịdị iche dị ka ọrịa type12,13, ọrịa anụ ahụ nkịtị, ma ọ bụ ọrịa cancer nke anụ ahụ (dịka ọmụmaatụ, ọrịa anụ ahụ na-egbuke egbuke). vesos non-lesioned area) na eriri afọ na ọnọdụ na tract (eg, duodenum, jejunum, ileum, cecum, colon, ma ọ bụ ikensi ike) .Anyị na-enye ihe kacha protocol na Igbe 5 maka culturing colonic organoids (coloids) nke a na-achọkarị elu mkpokọta nke morphogens karịa obere eriri afọ organoids.
a, A na-eji usoro a na-arụ ọrụ maka ntinye nke morphogenesis n'ime eriri afọ.Caco-2 mmadụ intestinal epithelium na intestinal organoids na-eji na usoro a iji gosipụta morphogenesis 3D. A na-akụ mkpụrụ ndụ epithelial dịpụrụ adịpụ na ngwaọrụ gut-on-a-chip a kwadebere (njikwa PD) . Otu oge mkpụrụ ndụ na-amịpụta (seeded) na akpụkpọ anụ (seeded). A na-ebute mmiri apical (AP) ma na-echekwa maka ụbọchị 2 mbụ (flow, AP, D0-D2) .Basolateral (BL) eruba na-ebutekwa ya na mgbatị cyclic na-agbatị (stretch, flow, AP na BL) mgbe a na-emepụta monolayer 2D zuru ezu. Intestinal 3D morphogenesis mere n'otu ntabi anya nke ọdịdị ọdịdị ọdịdị ọdịdị nke microgenesis (5 ụbọchị 5). co-2 mkpụrụ ndụ na nke ọ bụla nnwale nzọụkwụ ma ọ bụ oge ebe ( mmanya graph, 100 µm) anọ schematic diagrams na-egosi kwekọrọ ekwekọ cascades nke gut morphogenesis (n'elu aka nri) .The dashed akụ na schematic na-anọchi anya ntụziaka nke fluid flow.b, SEM image na-egosi elu topology nke guzosie ike 3D Caco-2 epithelift na-egosi na igbe dị ebube (nke na-egbuke egbuke na mpaghara). villi na 3D Caco-2 oyi akwa (n'aka nri).c, kehoraizin n'ihu echiche nke guzosie ike Caco-2 3D, claudin (ZO-1, red) na-aga n'ihu ahịhịa ókèala membranes labeled F-actin (green) na nuclei (acha anụnụ anụnụ) Immunofluorescence confocal visualization nke epithelial cell na-egosi n'ihi na nke ọ bụla n'ime ha na-atụ uche na mbara igwe echiche. .d, Oge usoro mgbanwe mgbanwe nke morphological na organoids omenala na mgbawa nwetara site na microscopy frasiconspy na ụbọchị 3, 7, 9, 11, na 13. Inset (n'elu aka nri) na-egosi mmụba dị elu nke ihe oyiyi ahụ nyere.magenta), goblet cell (MUC2; green), F-actin (isi awọ) na nuclei (cyan) toro na gut ibe maka 3 ụbọchị, n'otu n'otu (ekpe) na 13-ụbọchị (n'etiti) organoids na epithelial oyi akwa. Leekwa Extended Data Figure 3, nke na-egosi LGR5 n'ókè na-egosi na LGR5 na-egosi enweghị ihe ngosi MUC2. organoid epithelium guzosie ike n'ime eriri afọ na mgbawa site n'itinye akpụkpọ ahụ plasma na cellMask dye (n'aka nri) n'ụbọchị 13 nke omenala. Ogwe ọnụ ahịa bụ 50 μm ma ọ bụrụ na ekwughị ya.Mahadum Oxford;c Ewere ikike sitere na ntụaka.2.Mahadum Oxford;e na f emegharịrị site na ikike site na ntụaka.12 N'okpuru ikikere Creative Commons CC BY 4.0.
N'ime eriri afọ na mgbawa, ọ dị mkpa iji gbanwee elu hydrophobic nke PDMS porous membrane maka ihe ịga nke ọma ECM mkpuchi. N'ime usoro a, anyị na-etinye ụzọ abụọ dị iche iche iji gbanwee hydrophobicity nke PDMS membranes.N'ihi na culturing Caco-2 mkpụrụ ndụ, elu activation site UV / ozone ọgwụgwọ naanị bụ zuru ezu iji belata hydrophobicity nke ECM n'elu sel na coPDC. , Microfluidic omenala nke organoid epithelium chọrọ chemical dabeere n'elu functionalization iji nweta ịrụ ọrụ nke ọma deposition nke ECM protein site sequentially itinye polyethyleneimine (PEI) na glutaraldehyde na PDMS microchannels. Mgbe elu mgbanwe, ECM proteins e debere na-ekpuchi functionalized PDMS elu na mgbe ahụ, a na-etinye usoro microfludic n'ime sel ahụ. s site na-esi ísì naanị ọkara n'ime microchannel elu ruo mgbe mkpụrụ ndụ na-etolite monolayer zuru oke, ebe microchannel dị ala na-edobe ọnọdụ static. Usoro a kachasị mma maka ịgbalite elu na mkpuchi ECM na-enyere aka ntinye nke epithelium organoid na-eme ka 3D morphogenesis na elu PDMS.
Omenala Transwell chọkwara mkpuchi ECM tupu mkpụrụ mkpụrụ ndụ;Otú ọ dị, Transwell omenala adịghị achọ mgbagwoju pretreatment nzọụkwụ iji rụọ ọrụ elu nke porous inserts. Maka na-eto eto Caco-2 mkpụrụ ndụ na Transwell inserts, ECM mkpuchi on porous inserts accelerates na mgbakwunye nke dissociated Caco-2 cell (<1 hour) na tight junction mgbochi formation (<1-2 ụbọchị) .To omenala organoids on Transwell n'elu ihe ntinye nke ECM na-etinye aka na ntinye nke ECM. 3 h) ma na-echekwa ruo mgbe organoids na-etolite monolayer zuru oke na ihe mgbochi iguzosi ike n'ezi ihe .A na-eme omenala Transwell na efere 24-ọma na-ejighi ngwakọ ibe.
In vitro 3D morphogenesis nwere ike ịmalite site n'itinye mmiri mmiri na-asọba na akụkụ basolateral nke epithelial Layer guzosie ike.N'ime eriri afọ na mgbawa, epithelial morphogenesis malitere mgbe a na-agbanye ihe n'ime elu na elu microchannels (Fig 3a) .Dị ka akọwara na mbụ, ọ dị oké mkpa ịmebata mmiri mmiri na-asọba na nke dị ala (basolateral) na-enye ntụziaka na-aga n'ihu nke morphoge. serum ka mkpụrụ ndụ ejikọta on porous membranes na n'ịwa luminal shiee stress, anyị na-emekarị etinye dual eruba na eriri afọ na a chip.Na ngwakọ ibe, Transwell inserts nwere epithelial monolayers e etinyere n'ime ngwakọ ibe. Mgbe ahụ, na-ajụ e etinyere n'okpuru basolateral n'akụkụ nke porous Transwell ntinye site na microchannelogenesis mgbe morphso omenala n'elu ikpo okwu 3 ụbọchị.
Enwere ike nyochaa njirimara morphological nke microengineered 3D epithelial layers site n'itinye usoro ihe ngosi dị iche iche, gụnyere microscopy nke dị iche iche, ọdịiche nnyonye anya dị iche iche (DIC) microscopy, SEM, ma ọ bụ immunofluorescence confocal microscopy (Figures 3 na 4) .Phase iche ma ọ bụ DIC imaging nwere ike ịrụ ọrụ nke ọma n'oge ọ bụla n'oge protrue oyi akwa. ical transparency nke PDMS na polyester films, ma gut-on-a-chip na ngwakọ mgbawa nyiwe nwere ike inye ezigbo oge na situ imaging na-enweghị mkpa nke ngalaba ma ọ bụ disassembly nke ngwaọrụ. Mgbe na-eme immunofluorescence imaging (Figures 1, 3c, f na 4b, c), mkpụrụ ndụ na-emekarị edozi na paradaịs 1% (wt) 1. 2% (wt / vol) ) bovine serum albumin (BSA), n'usoro. Dabere n'ụdị cell, dị iche iche fixatives, permeabilizers, na blocking agents nwere ike iji. indole, DAPI) ma ọ bụ F-actin (dịka ọmụmaatụ, fluorescently labeled phalloidin) . Enwere ike ịme ihe onyonyo ndụ dabere na fluorescence na ọnọdụ iji chọpụta mmepụta imi (Fig.1, "Cell di iche iche" na "Gut physiology"), random colonization nke microbial cell (Fig. 1, "Ọbịa-microbe co-omenala"), na mbanye nke dịghịzi mkpụrụ ndụ (Fig. 1, 'Disease Modeling') ma ọ bụ contours nke 3D epithelial morphology (Fig. 3c modifying na elu oyi akwa, na microchannel). nel oyi akwa, dị ka akọwara na ref. Dị ka e gosiri na fig. 2, na 3D epithelial morphology nakwa dị ka microvilli na apical brush ókè nwere ike visualized site SEM (Fig. 3b) Okwu nke di iche iche nrịbama nwere ike na-enyocha site n'ịrụ quantitative PCR5 ma ọ bụ otu-cell RNA sequencing.Na nke a ikpe, chips 3D oyi akwa na-eto eto na chips sel 3. trypsinization wee jiri ya mee nyocha nke molecular ma ọ bụ mkpụrụ ndụ ihe nketa.
a, Workflow maka ntinye nke morphogenesis intestinal na ngwakọ ngwakọ. A na-eji Caco-2 na intestinal organoids mee ihe na usoro a iji gosipụta morphogenesis nke 3D na ngwakọ ngwakọ ngwakọ. (TW Culture) .Mgbe ụbọchị 7 gasịrị, otu ntinye Transwell nke nwere 2D monolayer nke mkpụrụ ndụ epithelial agbakwunyere n'ime mgbawa ngwakọ iji webata mmiri nke basolateral (Flow, BL), nke mechara mee ka ọgbọ nke 3D epithelial oyi akwa (morphogenesis) .Phase iche ntinye micrographs na-egosi njirimara morphological nke akụkụ ahụ mmadụ epithelial donor sel oge ọ bụla deri. schematics na elu n'ígwé na-egosi na nnwale nhazi maka nzọụkwụ ọ bụla.b, Ngwakọta ibe (ekpe schematic) nwere ike iduga 3D morphogenesis nke organoid epithelial cell na n'elu-ala confocal microscopy echiche weere na dị iche iche Z ọnọdụ (elu, n'etiti, na ala;hụ schematic ziri ezi na ahịrị ntụpọ kwekọrọ).gosiri n'ụzọ doro anya àgwà morphological.F-actin (cyan), nucleus (isi awọ).c, Fluorescence confocal micrographs (3D angled view) nke organoid-ewepụtara epithelial cell cultured in static Transwell (TW; inset within white dashed box) versus ngwakọ mgbawa (kasị zuru ezu ogbugba) atụnyere 3D morphology versus versus 2D paired. etinyere na akuku aka nri elu;“XZ”) gosikwara ihe 2D na 3D. Ogwe ọnụ ọgụgụ, 100 µm.c edegharịrị site na ikike sitere na ntụaka.4.Elsevier.
Enwere ike ịkwadebe njikwa site na ịzụlite otu mkpụrụ ndụ (Caco-2 ma ọ bụ intestinal organoid epithelial cell) n'ime monolayers abụọ n'okpuru ọnọdụ omenala static omenala. N'ụzọ dị ịrịba ama, nkwụsịtụ nri nwere ike ịkpata n'ihi oke ụda olu nke microchannels (ie ~ 4 µL na n'elu ọwa na mbụ gut-chip morphology nwere ike iji ya tụnyere). .
A ghaghị ịrụ usoro lithography dị nro n'ime ụlọ dị ọcha. N'ihi na oyi akwa ọ bụla na mgbawa (n'elu na ala n'ígwé na membranes) na ngwakọ ibe, dị iche iche photomasks e ji mee ihe dị iche iche photomasks na iche iche silicon wafers n'ihi na elu nke microchannels dị iche iche. The lekwasịrị elu nke elu na ala microchannels nke eriri afọ na mgbawa bụ 500 µm nke elu, n'ebe ịdị elu nke ọwa. 200 µm.
Tinye 3-inch silicon wafer na efere na acetone. Jiri nwayọọ gbanye efere ahụ maka 30 sekọnd, wee kpochaa wafer. Nyefee wafer na efere na IPA, wee tụgharịa efere ahụ maka 30 s ka ọ dị ọcha.
Enwere ike iji ngwọta piranha (ngwakọta nke hydrogen peroxide na sulfuric acid gbanyere mkpọrọgwụ, 1: 3 (vol/vol)) iji mee ka mwepụ nke ihe ndị dị ndụ na-esi na silicon wafer elu.
Ngwọta Piranha na-emebi emebi nke ukwuu ma na-ebute okpomọkụ. Ihe nchekwa nchekwa ọzọ dị mkpa. Maka ikpofu ihe mkpofu, kwe ka ngwọta ahụ dị jụụ ma nyefee ya na ihe mkpofu dị ọcha, nke akọrọ. Jiri ihe nkedo nke abụọ na-edebanye aha nke ọma nke ọma. Biko soro ntuziaka nchekwa nke ụlọ ọrụ maka usoro zuru ezu karị.
Kpochapu wafers site n'itinye ha na efere ọkụ 200 Celsius C maka nkeji 10. Mgbe mmiri gwụchara, a na-amagharị wafer ugboro ise na ikuku ka ọ dị jụụ.
Wunye ~ 10 g nke photoresist SU-8 2100 n'etiti etiti silicon wafer dị ọcha. Jiri tweezers gbasaa fotoresis na-ekpo ọkụ na wafer. Mgbe ụfọdụ na-etinye wafer na efere ọkụ 65 Celsius C iji mee ka fotoresist ghara ịdị nro ma dị mfe ịgbasa. Edola wafer ozugbo na efere ọkụ.
A na-ekesa SU-8 n'otu n'otu na wafer site n'iji mkpuchi mkpuchi na-agba ọsọ. oyi akwa elu nke eriri afọ na mgbawa; lee “Nzọụkwụ dị oke egwu” n'okpuru) ka edobere na osooso nke 300 rpm / s 30 sekọnd na 1,200 rpm.
Enwere ike gbanwee ọsọ ọsọ nke isi dị ka ọkpụrụkpụ ebumnuche nke ụkpụrụ SU-8 na wafer silicon.
Iji mepụta SU-8 ụkpụrụ nke 500 µm ịdị elu maka elu oyi akwa nke eriri afọ na mgbawa, na spin mkpuchi na nro ime nzọụkwụ nke Igbe a (nzọụkwụ 7 na 8) na-sequentially ugboro ugboro (lee nzọụkwụ 9) na-emepụta abụọ n'ígwé nke 250 µm A oké oyi akwa nke SU-8, nke nwere ike Layered na sonye na 1 a µ oyi akwa nke igbe 5 µ.
Richaa wafers mkpuchi SU-8 dị nro site na iji nlezianya na-etinye wafers na efere ọkụ na 65 Celsius C maka 5 min, wee gbanwee ntọala ahụ na 95 Celsius C ma tinye ya maka nkeji 40 ọzọ.
Iji nweta ogo 500 μm nke ụkpụrụ SU-8 na microchannel dị elu, megharịa usoro 7 na 8 iji mepụta akwa SU-8 gbara ọkpụrụkpụ 250 μm abụọ.
Iji UV Mask Aligner, mee ule oriọna dị ka ntuziaka onye nrụpụta si gbakọọ oge ikpughe nke wafer (oge ikpughe, ms) = ( dose exposure, mJ/cm2) / (ike oriọna, mW/cm2).
Mgbe ị chọpụtachara oge ikpughe, tinye fotomask na ihe nkpuchi mkpuchi nke UV ma tinye fotomask na wafer mkpuchi SU-8.
Debe elu e biri ebi nke fotomask ozugbo n'akụkụ SU-8 mkpuchi nke silicon wafer iji belata mgbasa nke UV.
Wepu SU-8 wafer na fotomask kwụ ọtọ na 260mJ/cm2 nke ọkụ UV maka oge mkpughe a kara aka (lee nzọụkwụ 10 nke igbe a).
Mgbe ikpughe UV, SU-8-coated silicon wafers na-esi na 65 ° C maka 5 min na 95 ° C maka 15 min na efere ọkụ ọ bụla iji mepụta ụkpụrụ na elu nke 200 μm. Gbatịa oge a na-esi nri na 95 ° C ruo 30 min iji mepụta ụkpụrụ na elu 500 µm.
A na-awụsa onye mmepụta ahụ n'ime efere iko, a na-etinyekwa wafer a na-esi nri na efere. Olu nke SU-8 onye mmepụta nwere ike ịdị iche iche dabere na nha nke efere iko ahụ. Jide n'aka na ị ga-eji SU-8 zuru ezu wepụ ihe SU-8 na-enweghị ihe ọ bụla. Dị ka ihe atụ, mgbe ị na-eji 150 mm dayameta iko efere na 1 L ikike, jiri ~ 300 ml nke SU-8 mL nke onye na-emepụta ihe na-eji nwayọọ mee ihe maka oge roro.
Jiri ~ 10 ml nke onye nrụpụta ọhụrụ na-asachapụ ihe ebupụtapụtara ọhụrụ nke IPA na-esochi ya site na ịgbanye ihe ngwọta site na iji pipette.
Debe wafer na ihe nchacha plasma ma kpughee na plasma oxygen (gasị ikuku, nrụgide ebumnuche 1 × 10-5 Torr, ike 125 W) maka 1.5 min.
Debe wafer na onye na-ekpo ọkụ na-ekpo ọkụ na-eji ihe nkiri iko dị n'ime. Enwere ike idowe wafers na slides n'akụkụ n'akụkụ. Ọ bụrụ na a na-ekewa ihe na-ekpo ọkụ na-ekpo ọkụ n'ọtụtụ ọkwa site na efere, tinye ihe nkiri ahụ n'ime ụlọ dị ala na wafers n'ime ụlọ elu. Drop 100 μL nke trichloro (1H, 1Hroo) na slideHrooctane ngwọta maka silil flu. ization.
Kpoo vial nke mkpụrụ ndụ Caco-2 oyi kpọnwụrụ n'ime mmiri ịsa mmiri 37°C, wee bufee mkpụrụ ndụ gbazere na flask T75 nwere 15 ml nke 37°C prewarmed Caco-2 ọkara.
Iji gafere mkpụrụ ndụ Caco-2 na ~ 90% confluency, nke mbụ na-ekpo ọkụ Caco-2 ọkara, PBS, na 0.25% trypsin/1 mM EDTA n'ime mmiri ịsa ahụ 37°C.
Jiri 5 ml nke PBS na-ekpo ọkụ saa mkpụrụ ndụ ugboro abụọ site na ịmegharị agụụ agụụ na ịgbakwunye PBS ọhụrụ.