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morphogenesis nke mmadụ na-eguzobe crypt-villus atụmatụ nke 3D epithelial microarchitecture na spatial organization.Nke a pụrụ iche Ọdịdị chọrọ iji nọgide na-enwe gut homeostasis site na ichebe azuokokoosisi niche na basal crypt si exogenous microbial antigens na ha metabolites.Na mgbakwunye, eriri afọ villi na secreting mucus na epithelial sel na-arụ ọrụ dị iche iche na-echebe ya na sel basal na-arụ ọrụ ugbu a. N'ihi ya, imepụtaghachi ihe owuwu epithelial 3D dị oke mkpa maka iwulite ụdị in vitro gut. N'ụzọ dị ịrịba ama, organic mimetic gut-on-a-chip nwere ike ime ka morphogenesis 3D na-enweghị onwe ya nke epithelium intestinal na-arụ ọrụ physiological na biomechanics. N'ebe a, anyị na-enye ike ịmaliteghachi prost. morphogenesis na eriri afọ na mgbawa microfluidic yana na Transwell agbakwunyere ngwakọ ngwakọ. Anyị na-akọwa ụzọ zuru ezu maka imepụta ngwaọrụ, ịzụlite Caco-2 ma ọ bụ sel organoid epithelial intestinal na ntọala ọdịnala yana n'elu ikpo okwu microfluidic, ntinye nke 3D morphogenesis, na njirimara nke emebere site na iji multiple 3D imaging moto. functional gut microarchitecture site na ịchịkwa basolateral fluid eruba maka 5 d.Our in vitro morphogenesis usoro na-arụ ọrụ physiologically mkpa shiee nrụgide na n'ibu ngagharị na-adịghị achọ mgbagwoju cell engineering ma ọ bụ aghụghọ, nke nwere ike karịa ndị ọzọ dị techniques.We na-eche na anyị tụrụ aro protocol nwere ike inwe sara mbara mmetụta maka biomedical research obodo, inye a oyi akwa usoro regenerate obodo. maka ngwa biomedical, clinical, and pharmaceutical ngwa.
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 ịmalite morphogenesis 3D na-enweghị isi na vitro na-enweghị nghọta doro anya nke usoro dị n'okpuru. 3D epithelial morphogenesis in vitro, nke Caco-2 gosipụtara na organoids intestinal nke ndị ọrịa na-enweta. A kwadoro mkpụrụ ndụ epithelial. N'ọmụmụ ihe a, anyị na-elekwasị 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 gbanwetụrụ microfluidic ngwaọrụ nwere ihe ntinye Transwell, nke a na-akpọ " Chip Hybrid ". frizzled-metụtara protein 1, ma ọ bụ Soggy-1) na on-chip gut inhibit morphogenesis ma ọ bụ na-akpaghasị prestructured 3D epithelial oyi akwa , na-atụ aro na antagonistic nchegbu n'oge omenala bụ n'ihi na intestinal morphogenesis in vitro.Ya mere, a bara uru obibia iji nweta siri ike morphogenesis na epithelial interface nke dị ntakịrị bụ iji wepu ọkwa nke morphogenesis na epithelial nke dị ntakịrị. ngalaba site na ịgbanye mmiri na-arụsi ọrụ ike (dịka ọmụmaatụ, na gut-on-a-chip ma ọ bụ ngwakọ-na-a-chip nyiwe) ma ọ bụ mgbasa .Basolateral media (dịka ọmụmaatụ, site na ntinye Transwell n'ime nnukwu mmiri mmiri basolateral na olulu mmiri).
N'ime usoro a, anyị na-enye usoro zuru ezu maka ịmepụta microdevices gut-on-a-chip na ngwakọ ngwakọ transwell-insertable (nzọụkwụ 1-5) na mkpụrụ ndụ epithelial intestinal omenala na polydimethylsiloxane (PDMS) -based porous membranes (nzọụkwụ 6A, 7A, 8, 9) ma ọ bụ transwells 6 polyester membranes. 8, 9) na-emetụta 3D morphogenenesis na vitro (nzọụkwụ 10) site na itinye mkpụrụ ndụ ihe omimi site na itinye mkpụrụ osisi clonoid, ma ọ bụ eriri afọ abụọ teknuzu nkọwa gụnyere n'elu mgbanwe nke porous membranes, e kere 2D monolayers, na intestinal biochemical na Mmeputakwa nke biomechanical microenvironment.in vitro. Iji mee ka 3D morphogenesis si 2D epithelial monolayers, anyị wepụrụ morphogen antagonists na ma omenala ụdị site na-eru na-ajụ n'ime basolateral compartment nke na-anọchi anya nke a na-anọchi anya basolateral. 3D epithelial oyi akwa na-emezigharị nke enwere ike iji mee ka ụdị uto epithelial na-adabere na morphogen, ogologo oge host-microbiome co-cultures, ọrịa nje, mmerụ ahụ ọkụ, nkwụsị nke ihe mgbochi epithelial, na ọgwụgwọ ndị dabeere na probiotic 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) na nyocha etinyere (dịka ọmụmaatụ, nyocha ọgwụ preclinical, nhazi ọrịa, injin anụ ahụ, na gastroenterology) mmetụta sara mbara. chee echiche na enwere ike ịgbasa atụmatụ teknụzụ anyị nye 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-Costridium ma ọ bụ 2), Cloffiimumrium , Salphilo-Costridium , Cloffiimumrium . cholerae. 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 ọnyá afọ, ọrịa Crohn, ọrịa Crohn, ọrịa crohn, ma ọ bụ ọrịa celiac. simulated mgbe 3D intestinal epithelial layers na-akwado ya site na iji onye ọrịa 3D intestinal epithelial layers , ọrịa ndị a na-agụnye villous atrophy, crypt shortening, mucosal mmebi, ma ọ bụ na-adịghị mma epithelial mgbochi.Biopsy ma ọ bụ stem cell-ewepụtara eriri afọ organoids , na-atụle ka mma nlereanya nke ọrịa organoids12,13. ụdị, dị ka mkpụrụ ndụ mononuclear ọbara ndị ọrịa (PBMCs), na 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. n'elu ikpo okwu. Nke a na obibia kacha mma 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 anyị in vitro morphogenesis protocol nwere ike imeghari ka scalable omenala formats, dị ka multiwell, na-aga n'ihu na-etinye n'ime 624 platenish. Basolateral compartments, protocol nwekwara ike gbasaa na ndị na-emepe emepe ọgwụ, biomedical Ma ọ bụ elu-throughput screening ma ọ bụ nkwado nyiwe maka nri ụlọ ọrụ.Dịka ihe akaebe-nke-ụkpụrụ, anyị na-adịbeghị anya gosiri na a pụrụ ime nke a multiplex elu-throughput morphogenesis usoro scalable na a 24-nke ọma platechi usoro. commercialized16,17,18.Ya mere, nkwado nke usoro in vitro morphogenesis anyị nwere ike mee ngwa ngwa ma nwee ike ịnabata ya site na ọtụtụ ụlọ nyocha nyocha, ụlọ ọrụ ma ọ bụ gọọmentị na ụlọ ọrụ na-achịkwa iji ghọta cellular reprogramming nke in vitro gut morphogenesis na ọkwa transcriptomic iji nwalee ọgwụ ma ọ bụ biotherapeutics Ndị na-eme ntuli aka na-eji gut ma ọ bụ ebufe nke ọgwụ 3. ụdị akụkụ-on-a-chip iji chọpụta mmụgharị nke usoro morphogenesis gut.
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. Ihe dị mkpa, adịghị ekpebi kpọmkwem usoro mmepe mmadụ. Ụdị ndị a dịkwa oke oke na ikike ha nwere ike ịnwale n'ọtụtụ ụzọ scalable. Ya mere, usoro anyị maka ịmaliteghachi ihe ọkpụkpụ 3D anụ ahụ na vitro na-apụta na vivo anụmanụ ụdị nakwa dị ka ndị ọzọ omenala static 2D cell omenala ụdị. Dị ka akọwara na mbụ, tinye n'ọrụ na spa 3D Ọdịdị dị iche iche na-eme ka e nwee sel dị iche iche na-eme ka e nwee usoro nhazi nke ime obodo. na crypt-villus axis na nzaghachi dị iche iche mucosal ma ọ bụ dịghịzi stimuli.3D epithelial layers nwere ike inye ohere iji mụọ otú microbial sel na-asọ mpi iji mepụta spatial niches na ecological evolution na nzaghachi nye ihe ndị ọbịa (dịka, n'ime versus mpụta imi n'ígwé, secretion nke IgA na antimicrobial peptides) nwere ike ikwe ka guzuozu 3 ọzọ. microbiota na-ahazi obodo ya na synergistically na-emepụta microbial metabolites (dịka ọmụmaatụ, abụba abụba dị mkpụmkpụ) nke na-akpụzi nzukọ cellular na stem 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 omenala bụ usoro nkà na ụzụ nke anụ ahụ na-emepụta ihe na-adabere n'ịzụlite mkpụrụ ndụ intestinal n'okpuru ọnọdụ morphogen kpọmkwem23,24,25. Otú ọ dị, iji 3D organoid organoid host-models-emekarị na-eme njem na-eme 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-emechi lumen intestinal 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 nke ọtụtụ ndị nchọpụta na-eji 3D hydrogel scaffolds prestructured iji na-eṅomi usoro epithelial gut site n'ịmepụta mkpụrụ ndụ eriri afọ mmadụ dịpụrụ adịpụ na gel elu. mkpa morphogen gradients, guzobe a elu akụkụ 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 morphogenetic usoro onwe ya. Ndị a ụdị na-adịghị enye dynamic luminal ma ọ bụ lacking na-erugharị n'ime mkpụrụ ndụ interstitite nke na-eme ka ọ bụrụ ihe na-egbuke egbuke nke na-eme ka ọ bụrụ ihe na-eme ka ọkpụkpụ na-egbuke egbuke na-eme ka ọkpụkpụ na-egbuke egbuke na-eme ka ọkpụkpụ na-egbuke egbuke na-eme ka ọkpụkpụ na-egbuke egbuke na-eme ka ọkpụkpụ na-eme ka ọ ghara ịdị na-eme ka ọ bụrụ ihe na-eme ka ọkpụkpụ na-egbuke egbuke na-eme ka ọ dị mkpa n'ime ahụ. morphogenesis na nweta ọrụ physiological. Nnyocha ọzọ na-adịbeghị anya na-eji hydrogel scaffolds na microfluidic ikpo okwu na nhazi nke eriri afọ epithelial na-eji usoro laser-etching usoro. gụnyere gut mechanobiological movements.3D printing techniques from the same group were ike ịmepụta miniature gut tubes with spontaneous morphogenetic processes. N'agbanyeghị na mgbagwoju anya nke dị iche iche gut segments n'ime tube, ihe nlereanya a nwekwara enweghị luminal mmiri eruba na n'ibu deformation. Ọzọkwa, nlereanya operability nwere ike na-ejedebeghị, karịsịa mgbe usoro nke bioprinting ma ọ bụ cell na-emecha usoro a na-emecha. interactions.Kama, anyị na-atụ aro protocol na-enye spontaneous gut morphogenesis, physiologically mkpa shiee stress, biomechanics nke na-eṅomi gut motility, accessibility nke onwe apical na basolateral compartments, na re-mepụta nke mgbagwoju biological microenvironments nke modularity.Ya mere, anyị in vitro 3D morphto nwere ike imeri usoro mgbagwoju anya nke usoro mgbagwoju anya.
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 a kọwara na mbụ, isi nke protocol anyị bụ ntinye nke epithelial morphogenesis site n'iwepụ morphogen inhibitors zoro ezo na akụkụ nke robust anyị. gut-on-a-chip na ngwakọ-on-a-chip na-enye anyị ohere ịmegharị oyi akwa epithelial 3D na-adịghị agwụ agwụ, ihe mgbagwoju anya ndị ọzọ dị ka epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 na, na ihe nlereanya anyị, crypt-villus atụmatụ na-ebuga stem cell niches na basal . fibroblasts) na mesenchyme na-ekere òkè dị mkpa na mmepụta nke protein ECM na nhazi nke morphogenesis intestinal na vivo35,37,38. Mgbakwunye nke mkpụrụ ndụ mesenchymal na ihe nlereanya anyị kwalitere usoro morphogenetic na ntinye sel nke ọma. na gut microenvironment. Ọzọkwa, vasculature components nke nwere ike jikọọ n'etiti ụdị anụ ahụ bụ ihe dị mkpa mgbe a na-emepụta ụdị anụ ahụ iji gosipụta mmekọrịta dị iche iche nke akụkụ ahụ. Ya mere, mkpụrụ ndụ endothelial nwere ike ịdị mkpa ka a na-etinye ya ka ọ bụrụ ihe dị mkpa iji gosipụta njirimara physiological nke ziri ezi na mkpebi nke akụkụ ahụ. mgbochi anụ ahụ kpọmkwem n'ọnọdụ nke iṅomi ọrịa eriri afọ.
Ojiji nke ngwakọ ngwakọ dị mfe karịa gut-on-a-chip n'ihi na nhazi ngwaọrụ dị mfe na iji ihe ntinye Transwell na-enye ohere maka omenala scalable nke gut epithelium. Otú ọ dị, azụmahịa dị na Transwell ntinye na polyester membranes na-adịghị na-agbanwe na-enweghị ike simulate peristaltic-dị ka mmegharị. Ọzọkwa, na apical etinyere na apical ọdụ na-etinye n'ime apical ọdụ na-enweghị ike itinye n'ime apical ọdụ. shiee stress on the apical side.N'ụzọ doro anya, na static Njirimara na apical ngalaba adịkarịghị enyere 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 mmiri n'elu ikpo okwu eruba nwere ike na-amachi ngwa ngwa ngwa ngwa.
Nrụgharị zuru oke nke mmadụ crypt-villus axis na gut-on-a-chip na ngwakọ-na-a-chip omenala eguzobebeghị n'ụzọ zuru ezu. 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 anyị na-akọwa ngalaba nke microcrypter na nso nso a. 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 n'ime obere eriri afọ na obere eriri afọ bụ ~ 135 µmµ na 45 µ. obere eriri afọ bụ ~ 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 oghere ebumnuche na oyi akwa epithelial dị n'usoro nke milimita ole na ole. Iji merie nsogbu a, enwere ike ịchọ ebumnuche dị anya. Ọzọkwa, ịme akụkụ dị mkpa maka imepụta eserese bụ ihe ịma aka siri ike. PDMS.Ọzọkwa, ebe ọ bụ na oyi akwa-site-Layer microfabrication nke eriri afọ na mgbawa na-agụnye adhesion na-adịgide adịgide n'etiti onye ọ bụla oyi akwa, ọ bụ oké ihe ịma aka imeghe ma ọ bụ wepụ elu oyi akwa iji nyochaa elu Ọdịdị nke epithelial oyi akwa. Dị ka ihe atụ, site na iji a scanning eletrọn microscope (SEM).
The hydrophobicity nke PDMS bụ ihe na-egbochi ihe na microfluidic dabeere na ọmụmụ ihe 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 nke PDMS (dịka ọmụmaatụ, mkpuchi na 4 lipophilic lipophilic ihe) a ga-atụle iji belata mgbasa nke mkpụrụ ndụ hydrophobic.
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 na-egbochi nnukwu nnwale. Nke a mmachi nwere ike budata mma site na scalability nke ọma-dị ka omenala 24, dịka ọmụmaatụ, 6. ma ọ bụ 384-nke ọma porous ntinye na-enye ohere 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 porous n'etiti iji mepụta lumen-capillary interface. Anyị na-egosipụtakwa iji ngwaọrụ microfluidic nke otu-ọwa (a ngwakọ mgbawa) nke na-enye na-aga n'ihu na-aga n'ihu na-aga n'ihu. ntinye.Na n'elu ikpo okwu abụọ, 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 eriri eriri afọ ma ọ bụ Transwell insertable hybrid), (nkwụsị 1-5) igbe igbe 1-5. mkpụrụ ndụ epithelial (sel Caco-2) ma ọ bụ organoids eriri afọ mmadụ; 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). nke in vitro morphogenesis site n'ịtụle epithelial morphogenesis na njikwa oghere, nwa oge, ọnọdụ, ma ọ bụ usoro.
Anyị na-eji usoro ọdịnala abụọ dị iche iche: gut-on-a-chip nwere ọwa kwụ ọtọ ma ọ bụ ọwa convoluted na-abụghị, ma ọ bụ ngwakọ ngwakọ nwere ihe ntinye Transwell (TW) na ngwaọrụ microfluidic, nke emere dị ka akọwara na igbe 1, yana nzọụkwụ 1 -5. (Caco-2 ma ọ bụ mmadụ eriri afọ organoids) na omenala usoro eji na nke a protocol. "In vitro morphogenesis" na-egosi n'ozuzu nzọụkwụ nke Caco-2 ma ọ bụ organoid-ewepụtara mkpụrụ ndụ epithelial na-emebe na mgbawa eriri afọ ma ọ bụ na Transwell inserts nke ngwakọ mgbawa, sochiri induction nke 3D morphogenesis na usoro ihe omume nke ọ bụla e ji mara usoro ihe omume. Arrow.The ngwa na-enye ihe atụ nke otú e si guzosie ike eriri afọ epithelial layers nwere ike iji ihe atụ, na cell differentiation characterization, gut physiology studies, nguzobe nke host-microbiome ecosystems, and disease modeling.Immunofluorescence oyiyi na "Cell iche iche" na-egosi nuclei, F-actin na MUC2 nke e gosipụtara na chip-2D Layer epicotlid. ihe nrịbama dị na sel goblet na imi nke zoro ezo site na elu mucosal. Ihe oyiyi na-egbuke egbuke na Gut Physiology na-egosi mmịpụta nke na-emepụta site na nsị maka sialic acid na N-acetylglucosamine residues site na iji fluorescent wheat germ agglutinin. Ihe oyiyi abụọ na-ekpuchi anya na "Ọbịa-Microbe Co-Cultures" na-egosi na mgbawa panel-cultures na-egosi onye na-elekọta ụlọ-microbi. co-omenala nke E. coli ekwupụta green fluorescent protein (GFP) na microengineered 3D Caco-2 epithelial cell. The ziri ezi panel na-egosi na localization nke GFP E. coli co-cultured na 3D Caco-2 epithelial cell, sochiri immunofluorescence staining na F-actin (red) na nustrates na-egbuke egbuke na-acha anụnụ anụnụ na-acha anụnụ anụnụ. ibe n'okpuru physiological ịma aka na nje antigens (eg, lipopolysaccharide, LPS) na dịghịzi sel (dịka, PBMC; green) .Caco-2 mkpụrụ ndụ e omenala guzobe a 3D epithelial Layer.Scale mmanya, 50 µm.Images na ala n'usoro: "iche nke mkpụrụ ndụ" na-emegharị na ikike site na ntụaka. 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 shear stress na hydrodynamic pressure1,4,12.The mbụ gut-on-a-chip data mejupụtara Fig. juxtaposed parallel straight microchannels, evolved into a complex gut-on-a- chip (Extended Data Fig. 1b) nke na-agụnye a ụzọ mkpirisi microchannels na-eme ka mmụba mmiri oge obibi, na-adịghị na-erugharị usoro, na multiaxial deformation nke cultured mkpụrụ ndụ (Fig. 2a-f) 12. Mgbe ihe mgbagwoju anya na-eme ka a na-agbagwoju anya gut-gut. nwere ike họrọ.Anyị egosila na convoluted Gut-Chip na-ike na-akpali 3D morphogenesis na a yiri oge etiti na a yiri ogo nke epithelial ibu tụnyere mbụ Gut-Chip, n'agbanyeghị nke omenala cell ụdị.Ya mere, ime ka 3D morphogenesis, linear na mgbagwoju on-chip gut designs na-rechangeable na-agbanwe agbanwe na-mmetụta silicon atụmatụ na-mgbanwe na-agbanwe agbanwe na PD8. demolding (Fig. 2a) . Iji mepụta eriri afọ na mgbawa, a na-ejikọta oyi akwa PDMS dị elu nke a kwadebere na fim PDMS porous wee kwekọọ na oyi akwa PDMS nke dị ala site na njikọ na-apụghị ịgbagha agbagha site na iji corona treater (Fig 2b-f) . Iji mepụta ngwakọ ngwakọ, PDMS oyiri PDMS nwere ike kee ya na ngwaọrụ mmịfe nke nwere ike jikọta ya na ngwaọrụ micro-flute. Ihe ntinye nke transwell (Fig 2h na Extended Data Fig. 2) A na-eme usoro njikọ ahụ site n'ịgwọ ihu nke PDMS oyiri na iko na plasma oxygen ma ọ bụ ọgwụgwọ corona. Mgbe sterilization nke microfabricated ngwaọrụ jikọtara na silicone tube, ihe nhazi ngwaọrụ dị njikere ịrụ 3D morphogenesis nke epithelium intestinal).
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 (n'aka nri) .The demolded PDMS e bee n'ime iberibe na kpochara maka ọzọ ojiji.b, Foto nke silicon PDMS na-eji akwa akwa akwa na-akwadebe silicon molds. na PDMS porous membrane.d, Usoro foto nke elu na ala PDMS components na kpọkọtara on-chip eriri afọ ngwaọrụ.e, Schematic nke nhazi nke elu, akpụkpọ ahụ, na ala PDMS akụkụ. Nke ọ bụla oyi akwa bụ irreversibly bonded site plasma ma ọ bụ corona ọgwụgwọ.f, Schematic nke ekepụtara gut-on-na-eji microchannel conteda microchannel. chambers.g, Nhazi nke gut-on-a-chip maka omenala microfluidic cell. 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 na hybripd hybripD. chips.Transwell ntinye akwadoro n'adabereghị na omenala 2D monolayers nke eriri afọ epithelial mkpụrụ ndụ na-etinyere n'ime ngwakọ ngwakọ 3D morphogenesis na-eme ka eriri afọ 3D morphogenesis.The media perfused site microchannels n'okpuru cell oyi akwa guzosie ike na Transwell tinye.Scale mmanya, 1 cm.h E bipụtara 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 mkpụrụ ndụ na-emekọrịta ihe (> sk 95% nke Cabet na-eme ihe 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-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-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ ihe na-adịghị mma) 10 na 50) na T-flasks ka a na-egbute iji kwadebe nkwụsịtụ sel ndị na-ekewapụ 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 na-akwado structural microenvironment, morphntogeme dị mkpa nke mejupụtara. R-spondin, na Noggin) na ihe ndị na-eto eto nke a kwadebere dị ka akọwara na Igbe 3 na-agbakwụnye kwa ụbọchị ruo mgbe organoids na-eto eto ~ 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 Transwell ntinye na mgbawa (Igbe 5) .Dịka anyị kwuburu na mbụ, ọ nwere ike ịdị iche iche (3 colitis) dị ka ụdị ọrịa Crown1. ọrịa, ọrịa cancer colorectal, ma ọ bụ onye na-enye onyinye nkịtị), saịtị ọnya (dịka ọmụmaatụ, ọnya na mpaghara na-adịghị ahụkebe) na ebe eriri afọ na traktị (dịka, duodenum, jejunum, ileum, cecum, colon, ma ọ bụ ikensi).Anyị na-enye usoro kachasị mma na Igbe 5 maka ịzụlite colonic organoids (coloids) nke na-achọkarị ihe dị elu karịa morphstin organoids.
a, A na-eji usoro a na-arụ ọrụ maka ntinye nke morphogenesis nke eriri afọ na 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 (nkwadebe mgbawa PD) . A na-ahụta mkpụrụ ndụ akpụkpọ anụ (seeded) na akpụkpọ anụ (seeded). 0 (D0), apical (AP) eruba na-amalite ma nọgide na-enwe maka ụbọchị 2 mbụ (flow, AP, D0-D2) .Basolateral (BL) eruba na-amalitekwa tinyere cyclic ịgbatị mmegharị (agbatị, eruba, AP na BL) mgbe a na-emepụta monolayer 2D zuru ezu. Intestinal 3D morphogenesis nke omenala spontaneous mgbe ụbọchị 5 gasịrị, micromorphin morphogenesis mere spontaneous. D5) .Phase iche oyiyi na-egosi morphology nnọchiteanya nke Caco-2 mkpụrụ ndụ na onye ọ bụla nnwale nzọụkwụ ma ọ bụ oge mgbe ( mmanya graph, 100 µm) .Four schematic eserese na-egosi kwekọrọ ekwekọ cascades nke gut morphogenesis (n'elu aka nri) .The dashed akụ na schematic na-anọchi anya ntụziaka nke fluid flowset.b, SEM image na-egosi na-Epi3 elu topology. na-akọwapụta ebe a na-ebuwanye ibu (igbe a na-acha ọcha) na-egosi microvilli emegharịrị na oyi akwa 3D Caco-2 (n'aka nri).c, Anya ihu ihu nke Caco-2 3D guzosie ike, claudin (ZO-1, red) na ahịhịa na-aga n'ihu na-aga n'ihu nke a na-akpọ F-actin (green) na nuclei (acha anụnụ anụnụ) na-eme ka ihe na-eme ka ahụ ghara ịdị na-ahụ maka sel na-eme ka ahụ ghara ịdị na-eme ka ahụ ghara ịdị na-eme ka ahụ ghara ịdị na-ahụ maka sel na-eme ka ahụ ghara ịdị na ya. chips.Arrow na-atụ aka n'etiti schematic na-egosi na ọnọdụ nke focal ụgbọelu maka onye ọ bụla confocal view.d, Time N'ezie nke morphological mgbanwe na organoids cultured on a mgbawa nwetara site frasi iche microscopy na ụbọchị 3, 7, 9, 11, na 13. The inset (n'elu aka nri) na-egosi na elu magnification nke nyere image. iberi were n'ụbọchị 7.f, Onyonyo immunofluorescence kpuchie nke na-egosi akara maka mkpụrụ ndụ stem (LGR5; magenta), sel goblet (MUC2; akwụkwọ ndụ akwụkwọ ndụ), F-actin (isi awọ) na nuclei (cyan) toro na ibe eriri afọ maka ụbọchị 3, n'otu aka ekpe (n'aka ekpe) na ụbọchị iri na atọ (n'etiti) organoids nke LG R. nrịbama na-enweghị akara MUC2.Fluorescence onyoonyo na-egosi epithelial microstructure (n'aka nri) nke 3D organoid epithelium guzosie ike na eriri afọ na mgbawa site n'itinye plasma membrane na CellMask dye (n'aka nri) n'ụbọchị 13 nke omenala. Scale mmanya bụ 50 μm ma ọ bụrụ na e kwuru ọzọ.b Reprinted na ikike site na ntụaka.2. 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 membranes PDMS. Maka ịzụlite mkpụrụ ndụ Caco-2, ntinye elu elu site na ọgwụgwọ UV / ozone naanị zuru ezu iji belata hydrophobicity nke ECM na sel nke CoPDcot. PDMS akpụkpọ ahụ. Otú ọ dị, microfluidic omenala organoid epithelium chọrọ chemical dabeere 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 PDO mgbe ahụ, a na-ewebata protein n'ime PDO. sel na-ejikọta ya, omenala microfluidic cell na-amalite site na ịmịnye naanị ọkara n'ime microchannel elu ruo mgbe mkpụrụ ndụ na-emepụta 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-ekekọta nke dissociated Caco-2 cell (<1 hour) na uko junction mgbochi formation (<1-2 ụbọchị) .To omenala organoids on transwelled organoid, na-agbakwunyere ECM na-eme ka a na-etinye ya n'ime ihe ndị a na-etinye n'ime ihe ndị a na-etinye na ECM. n'elu akpụkpọ ahụ (<3 h) ma nọgide na-enwe ruo mgbe organoids na-etolite monolayer zuru ezu 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 na-asọba na akụkụ basolateral nke oyi akwa epithelial guzosie ike.N'ime eriri afọ na mgbawa, epithelial morphogenesis malitere mgbe a na-agbanye ihe n'ime microchannels elu na nke dị ala (Fig 3a) .Dị ka akọwara na mbụ, ọ dị oké mkpa iwebata mmiri na-asọba na nke dị ala (basolateral na-egbochi morphoge). nye nri zuru oke na serum na mkpụrụ ndụ na-ejikọta na akpụkpọ anụ porous ma na-emepụta nrụgide na-egbuke egbuke, anyị na-etinyekarị ihe abụọ na-asọba na eriri afọ na mgbawa. Na ngwakọ ngwakọ, transwell ntinye nwere epithelial monolayers na-etinye n'ime ngwakọ ibe. Mgbe ahụ, a na-etinye usoro n'okpuru akụkụ basolateral nke porous Transwell ntinye site na microchannel nke microchannel 5. eruba na ma omenala nyiwe.
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) .Nleba anya nkebi ma ọ bụ DIC imaging nwere ike ịrụ ọrụ nke ọma n'oge ọ bụla nke ọdịdị ọdịdị 3 n'oge ọ bụla protruD. Layers.Due ka anya transparency nke PDMS na polyester films, ma gut-on-a-chip na ngwakọ mgbawa nyiwe nwere ike na-enye ezigbo oge na ọnọdụ imaging na-enweghị mkpa nke sectioning ma ọ bụ disassembly nke ngwaọrụ. Mgbe na-arụ immunofluorescence imaging (Figures 1, 3c, f na 4b) paraparat sel (wt / 4) na-emekarị nke na-edozi. (PFA), sochiri Triton X-100 na 2% (wt / vol) ) bovine serum albumin (BSA), n'usoro. Dabere na ụdị cell, dị iche iche fixatives, permeabilizers, na igbochi agents nwere ike iji.A na-eji ọgwụ mgbochi mbụ na-ezubere cellage-based cell ma ọ bụ mpaghara akara akara iji gosipụta sel ndị na-adịghị agbanwe agbanwe na situ na mgbanaka nchikota nke ihe mgbochi di iche iche, na-esote ya. (dịka ọmụmaatụ, 4',6-diamidino-2-phenylene) indole, DAPI) ma ọ bụ F-actin (dịka ọmụmaatụ, fluorescently labeled phalloidin) .Fluorescence dabeere ndụ imaging nwekwara ike na-eme na ọnọdụ iji chọpụta mucus mmepụta (Fig. 1, "Cell iche iche" na "Gut physiology"), random cell colonization (Fig. co-omenala") , mbanye nke dịghịzi mkpụrụ ndụ (Fig. 1, 'Disease Modeling') ma ọ bụ contours nke 3D epithelial morphology (Fig. 3c, f na 4b, c) .Mgbe modifying eriri afọ na mgbawa ikewapụ elu oyi akwa si ala microchannel oyi akwa, dị ka akọwara na ref.As2, nke ọma na Fig. na apical brush ókè nwere ike ịhụ anya site SEM (Fig. 3b) .Nkwupụta nke ihe nrịbama dị iche iche nwere ike nyochaa site n'ịrụ quantitative PCR5 ma ọ bụ otu-cell RNA sequencing. N'okwu a, 3D oyi akwa nke epithelial sel ndị toro na gut ibe ma ọ bụ ngwakọ ibe na-egbute site trypsinization na wee jiri ya mee ihe maka molekul.
a, A na-eji usoro a na-arụ ọrụ maka ntinye nke intestinal morphogenesis 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. omenala n'okpuru ọnọdụ static (omenala TW) .Mgbe ụbọchị 7 gasịrị, otu ntinye Transwell nwere 2D monolayer nke mkpụrụ ndụ epithelial na-ejikọta ya n'ime ngwakọ ngwakọ iji webata mmiri nke basolateral (Flow, BL), nke mechara mee ka ọgbọ nke 3D epithelial Layer (morphogenesis) .Phase contrast micrographs na-egosi morphological atụmatụ nke mkpụrụ ndụ epitheli3 nkịtị nke Donor organ deceli1. colon na ọ bụla nnwale nzọụkwụ ma ọ bụ oge point. The schematics na elu n'ígwé na-egosi na nnwale nhazi maka onye ọ bụla nzọụkwụ.b, Ngwakọ 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 nri dotted ahịrị na kwekọrọ ekwekọ). gosiri doro anya morphological e ji mara.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 na-asọpụrụ 2D versus. nlegharị anya n'ofe (itinye na akuku aka nri elu; "XZ") na-egosikwa atụmatụ 2D na 3D. Ogwe ọnụ ọgụgụ, 100 µm.c Edegharịrị ya na ikike site na ntụaka.4. Elsevier.
Enwere ike ịkwadebe njikwa site n'ịmepụta 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, ụkọ nri nwere ike ịkpata n'ihi oke ike nke microchannels (ie ~ 4 µL na n'elu ọwa na mbụ gut-chip design). eruba nwekwara ike 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 nke dị ala na membranes) na ngwakọ ngwakọ, a na-eji fotomask dị iche iche mee ihe na silicon wafers dị iche iche n'ihi na elu nke microchannels dị iche iche. Ebe dị elu nke elu na elu microchannels nke eriri afọ na mgbawa bụ 500 µm n'ịdị elu. ngwakọ mgbawa bụ 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.
Ngwọta piranha (ngwakọta nke hydrogen peroxide na sulfuric acid gbadoro anya, 1: 3 (vol/vol)) nwere ike iji nhọrọ 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 na iji mkpuchi mkpuchi na-agba ọsọ. µm dị elu maka akwa akwa nke eriri afọ na mgbawa; lee "Nzọụkwụ dị oke mkpa" n'okpuru) setịpụrụ 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, spin mkpuchi na nro ime nzọụkwụ nke Igbe a (nzọụkwụ 7 na 8) na-sequentially ugboro ugboro (lee nzọụkwụ 9) na-mepụta abụọ n'ígwé nke 250 µm A oké oyi akwa nke SU-8, nke nwere ike layered na isonyere 1 a µ oyi akwa nke 5 UV oyi akwa na nzọụkwụ 5. elu.
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 ihe mkpuchi iko n'ime. Enwere ike idobe 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 ọnụ ụlọ dị ala na wafers n'ime ụlọ elu.Drop 100 μL nke trichloro (1H, 1Hroo)Hrooctane ngwọta na slide 2H, 1H, 1Hrooctane na ihe ngwọta slide sil. oghere maka silanization.
Kpoo vial nke mkpụrụ ndụ Caco-2 oyi kpọnwụrụ n'ime mmiri ịsa mmiri 37ºC, 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.