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Njem nke afọ mmadụ na-emepụta atụmatụ crypt-villus nke microarchitecture epithelial 3D na nhazi oghere. A chọrọ usoro pụrụ iche a iji nọgide na-enwe homeostasis eriri afọ site na ichekwa niche mkpụrụ ndụ dị na basal crypt site na antigens microbial exogenous na metabolites ha. Na mgbakwunye, villi eriri afọ na mucous na-ewepụta na-enye mkpụrụ ndụ epithelial dị iche iche nke nwere ihe mgbochi nchebe na elu mucosal eriri afọ. Ya mere, imegharị usoro epithelial 3D dị oke mkpa maka iwulite ụdị in vitro gut. Ihe pụrụ iche bụ na organic mimetic gut-on-a-chip nwere ike ịkpalite 3D morphogenesis nke epithelium eriri afọ na-enweghị ọrụ yana ọrụ physiological emelitere na biomechanics. N'ebe a, anyị na-enye usoro a na-emegharị emegharị iji kpalite morphogenesis eriri afọ nke ọma na eriri afọ na obere microfluidic yana na obere Transwell. Anyị na-akọwa ụzọ zuru ezu maka imepụta ngwaọrụ, ịzụlite mkpụrụ ndụ epithelial Caco-2 ma ọ bụ eriri afọ organoid na ntọala ọdịnala yana na ikpo okwu microfluidic, itinye morphogenesis 3D, na njirimara nke 3D edobere. Epithelia site na iji ọtụtụ usoro onyonyo. Usoro a na-enweta mmeghari nke obere ihe owuwu afọ site na ijikwa mmiri mmiri basolateral ruo ụbọchị 5. Usoro in vitro morphogenesis anyị na-eji nrụgide shear na mmegharị igwe dị mkpa na physiological, ọ chọghịkwa injinịa sel dị mgbagwoju anya ma ọ bụ njikwa, nke nwere ike ịka mma usoro ndị ọzọ dị adị. Anyị na-eche na usoro anyị tụrụ aro nwere ike inwe mmetụta sara mbara maka obodo nyocha biomedical, na-enye ụzọ iji mee ka akwa epithelial afọ 3D dị ọhụrụ na vitro maka ngwa biomedical, ụlọ ọgwụ, na ọgwụ.
Nnwale ndị a gosiri na mkpụrụ ndụ Caco-2 nke epithelial nke afọ a zụlitere na ngwa microfluidic gut-on-a-chip1,2,3,4,5 ma ọ bụ bilayer microfluidic6,7 nwere ike ịgafe morphogenesis 3D ozugbo na vitro na-enweghị nghọta doro anya nke usoro dị n'okpuru ya. N'ọmụmụ ihe anyị mere n'oge na-adịbeghị anya, anyị chọpụtara na iwepụ ndị na-emegide morphogen nke basolateral na-apụta site na ngwaọrụ omenala dị mkpa ma zuru oke iji kpalite morphogenesis epithelial 3D na vitro, nke Caco-2 na organoids eriri afọ sitere na onye ọrịa gosipụtara. E mere ka mkpụrụ ndụ epithelial dị irè. N'ọmụmụ ihe a, anyị lekwasịrị anya kpọmkwem na mmepụta mkpụrụ ndụ na nkesa nke onye na-emegide Wnt dị ike, Dickkopf-1 (DKK-1), na ngwa microfluidic emezigharịrị nke nwere ihe ndị Transwell tinyere, nke a na-akpọ "Mkpụrụ Chip Hybrid". Anyị na-egosi na mgbakwunye nke ndị na-emegide Wnt mpụga (dịka DKK-1, Wnt repressor 1, protein frizzled related 1, ma ọ bụ Soggy-1) na eriri afọ na-egbochi morphogenesis ma ọ bụ na-akpaghasị oyi akwa epithelial 3D nke e guzobere, na-atụ aro na nrụgide mmegide n'oge ọdịbendị bụ ihe kpatara morphogenesis eriri afọ na vitro. Ya mere, ụzọ bara uru iji nweta morphogenesis siri ike na njikọ epithelial bụ iwepụ ma ọ bụ idobe ọkwa nke ndị na-emegide Wnt na ngalaba basolateral site na ịsacha ahụ na-arụ ọrụ (dịka ọmụmaatụ, na ikpo okwu gut-on-a-chip ma ọ bụ ngwakọ-na-a-chip) ma ọ bụ mgbasa ozi .Mgbasa ozi basolateral (dịka ọmụmaatụ, site na ntinye Transwell na nnukwu ebe nchekwa basolateral na olulu mmiri).
N'ime usoro a, anyị na-enye usoro zuru ezu maka imepụta ngwaọrụ microdevices na Transwell-insertable hybrid chips (nzọụkwụ 1-5) na mkpụrụ ndụ epithelial eriri afọ na polydimethylsiloxane (PDMS)-based porous membranes (nzọụkwụ 6A, 7A, 8, 9) ma ọ bụ polyester membranes nke Transwell inserts (nzọụkwụ 6B, 7B, 8, 9) ma kpalite 3D morphogenesis in vitro (nzọụkwụ 10). Anyị chọpụtakwara atụmatụ cellular na molekul nke na-egosi histogenesis kpọmkwem anụ ahụ na mgbanwe cellular dabere na usoro site na itinye ọtụtụ ụdị onyonyo (nzọụkwụ 11-24). Anyị na-akpali morphogenesis site na iji mkpụrụ ndụ epithelial eriri afọ mmadụ, dị ka Caco-2 ma ọ bụ organoids eriri afọ, na usoro omenala abụọ nwere nkọwa teknụzụ gụnyere mgbanwe elu nke membranes porous, imepụta monolayers 2D, na biochemical eriri afọ na Mmepụta nke biomechanical microenvironment.in vitro. Iji kpalite 3D morphogenesis site na 2D epithelial monolayers, anyị wepụrụ Ndị na-emegide morphogen n'ụdị abụọ a zụlitere site n'itinye ihe dị n'ime akụkụ basolateral nke ọdịbendị ahụ. N'ikpeazụ, anyị na-enye ihe nnọchianya nke uru nke oyi akwa epithelial 3D a na-emegharị emegharị nke enwere ike iji mee ihe nlereanya uto epithelial dabere na morphogen, omenala ndị ọbịa-microbiome ogologo oge, ọrịa nje, mmerụ ahụ mkpali, nsogbu mgbochi epithelial, na ọgwụgwọ dabere na probiotic Ihe atụ.influences.
Usoro anyị nwere ike ịba uru nye ọtụtụ ndị ọkà mmụta sayensị n'ihe gbasara isi (dịka ọmụmaatụ, bayoloji mucosal eriri afọ, bayoloji mkpụrụ ndụ stem, na bayoloji mmepe) yana nyocha etinyere (dịka ọmụmaatụ, nnwale ọgwụ tupu ọgwụgwọ, ụdị ọrịa, injinịa anụ ahụ, na gastroenterology) mmetụta sara mbara. N'ihi mmụba na ike nke usoro anyị iji kpalite morphogenesis 3D nke epithelium eriri afọ na vitro, anyị na-eche na atụmatụ teknụzụ anyị nwere ike ịgbasa nye ndị na-ege ntị na-amụ mgbanwe nke mgbaàmà mkpụrụ ndụ n'oge mmepe eriri afọ, mmeghari ohuru ma ọ bụ homeostasis. Na mgbakwunye, usoro anyị bara uru maka ịjụ ajụjụ gbasara ọrịa n'okpuru ọtụtụ ihe na-efe efe dịka Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 ma ọ bụ Vibrio cholerae. Ndị na-ege ntị n'ọrịa na ọrịa ndị na-ebute ọrịa bara uru. Ojiji nke sistemụ microphysiology nke eriri afọ nwere ike inye ohere maka mmepe ogologo oge 10 na nyocha ọzọ nke nchekwa ndị ọbịa, nzaghachi mgbochi ahụ na mmezi mmerụ ahụ metụtara nje na eriri afọ (GI) 11. Enwere ike ịme ihe atụ nke nsogbu GI ndị ọzọ metụtara ọrịa afọ na-agbapụta, ọrịa celiac, ọrịa Crohn, ulcerative colitis, pouchitis, ma ọ bụ ọrịa eriri afọ na-ewe iwe mgbe a na-akwadebe akwa epithelial eriri afọ 3D site na iji akwa epithelial eriri afọ 3D nke onye ọrịa, ọrịa ndị a gụnyere atrophy villous, mkpụmkpụ crypt, mmebi mucosal, ma ọ bụ mgbochi epithelial mebiri emebi. Biopsy ma ọ bụ stem cell organoids eriri afọ sitere na mkpụrụ ndụ 12,13. Iji mee ka gburugburu ọrịa ahụ dị mgbagwoju anya nke ọma, ndị na-agụ akwụkwọ nwere ike ịtụle ịgbakwunye ụdị sel ndị metụtara ọrịa, dị ka mkpụrụ ndụ mononuclear ọbara nke onye ọrịa (PBMCs), na ụdị nwere microarchitectures 3D intestinal villus-crypt. mkpụrụ ndụ mgbochi nke anụ ahụ kpọmkwem, 5.
Ebe ọ bụ na enwere ike idozi ma gosipụta usoro epithelial 3D na-enweghị usoro nkewa, ndị na-ekiri ihe na-arụ ọrụ na transcriptomics na onyonyo dị elu ma ọ bụ nke dị elu nwere ike inwe mmasị na nhazi anyị nke usoro spatiotemporal nke mkpụrụ ndụ ihe nketa na protein na niches epithelial. Nwere mmasị na teknụzụ. Mmeghachi omume na mkpali microbial ma ọ bụ immunodeficiency. Ọzọkwa, enwere ike ịmepụta longitudinal host-microbiome crosstalk 10, 14 nke na-ahazi homeostasis eriri afọ na oyi akwa mucosal eriri afọ 3D site na ijikọ aka na ịzụlite ụdị microbial dị iche iche, obodo microbial ma ọ bụ microbiota fecal, karịsịa na gut-on-a-chip. n'elu ikpo okwu. Usoro a na-adọrọ mmasị karịsịa nye ndị na-agụ akwụkwọ na-amụ banyere mgbochi ahụ ike nke mucosal, gastroenterology, microbiome mmadụ, culturomics na microbiology ụlọ ọgwụ na-achọ ịzụlite microbiota eriri afọ a na-akọbeghị na mbụ n'ime ụlọ nyocha. Ọ bụrụ na enwere ike ịgbanwe usoro morphogenesis in vitro anyị ka ọ bụrụ usoro omenala a na-agbanwe agbanwe, dị ka ntinye multiwell na efere olulu 24, 96 ma ọ bụ 384 nke na-ejupụta akụkụ basolateral mgbe niile, enwere ike kesaa usoro ahụ nye ndị na-emepụta nyiwe ọgwụ, biomedical ma ọ bụ nyocha dị elu ma ọ bụ nkwenye maka ụlọ ọrụ nri. Dịka ihe akaebe nke ụkpụrụ, anyị gosipụtara n'oge na-adịbeghị anya na o kwere omume nke sistemụ morphogenesis dị elu nke multiplex nke a na-agbanwe agbanwe ruo na usoro efere olulu 24. Na mgbakwunye, ejirila ọtụtụ ngwaahịa organ-on-a-chip mee ahịa 16, 17, 18. Ya mere, enwere ike ime ngwa ngwa nke usoro morphogenesis in vitro anyị ma nwee ike ịnabata ya site na ọtụtụ ụlọ nyocha nyocha, ụlọ ọrụ ma ọ bụ gọọmentị na ụlọ ọrụ nchịkwa iji ghọta mmegharị sel nke morphogenesis in vitro na ọkwa transcriptomic iji nwalee ọgwụ ma ọ bụ biotherapeutics. a tụlekwara mbufe nke ndị na-achọ ọgwụ site na iji ọgwụ ndị ọzọ e ji 3D mee ma ọ bụ site na iji ụdị organ-on-a-chip eme ihe ma ọ bụ nke azụmahịa iji chọpụta otú usoro morphogenesis afọ si agbanwe.
E jirila ọnụọgụgụ nnwale dị iche iche nke mmadụ mee ihe iji mụọ gbasara epithelial morphogenesis n'ime eriri afọ, nke bụ isi n'ihi enweghị usoro ndị a ga-etinye n'ọrụ iji kpalite morphogenesis 3D na vitro. N'ezie, ọtụtụ ihe ọmụma dị ugbu a gbasara morphogenesis n'ime eriri afọ dabere na ọmụmụ anụmanụ (dịka ọmụmaatụ, zebrafish20, òké21 ma ọ bụ ọkụkọ22). Agbanyeghị, ha na-arụsi ọrụ ike ma na-efu ego, enwere ike ịjụ ajụjụ n'ụzọ omume, nke kachasị mkpa, anaghị ekpebi usoro mmepe mmadụ kpọmkwem. Ụdị ndị a dịkwa oke oke n'ikike ha nwere ike ịnwale n'ụzọ dị iche iche. Ya mere, usoro anyị maka imeghari nhazi anụ ahụ 3D na vitro na-aka mma na ụdị anụmanụ dị ndụ yana ụdị omenala 2D ndị ọzọ. Dịka akọwara na mbụ, iji usoro epithelial 3D nyere anyị ohere inyocha ebe mkpụrụ ndụ dị iche iche dị na axis crypt-villus na nzaghachi na mkpali mucosal ma ọ bụ mgbochi ahụ. 3D epithelial oyi akwa nwere ike inye ohere ịmụ otu mkpụrụ ndụ microbial si asọmpi ịmepụta niche oghere na evolushọn gburugburu ebe obibi na nzaghachi na ihe ndị ọbịa (dịka ọmụmaatụ, oyi akwa mucous dị n'ime megide mpụta, mwepụ nke IgA na peptides nje bacteria). Ọzọkwa, ọdịdị epithelial 3D nwere ike inye anyị ohere ịghọta otu microbiota eriri afọ si ahazi obodo ya ma na-emepụta metabolites nje bacteria (dịka ọmụmaatụ, obere abụba acids) nke na-akpụzi nhazi sel na niche sel dị na basal crypts. A pụrụ igosipụta atụmatụ ndị a naanị mgbe e guzobere oyi akwa epithelial 3D na vitro.
Na mgbakwunye na ụzọ anyị si emepụta usoro epithelial afọ 3D, enwere ọtụtụ ụzọ in vitro. Omenala organoid afọ bụ usoro injinia anụ ahụ ọgbara ọhụrụ dabere na ịkọ mkpụrụ ndụ eriri afọ n'okpuru ọnọdụ morphogen kpọmkwem23,24,25. Agbanyeghị, ojiji nke ụdị organoid 3D maka nyocha njem ma ọ bụ co-cultures host-microbiome na-esiri ike mgbe ụfọdụ n'ihi na lumen eriri afọ dị n'ime organoid, ya mere, iwebata ihe ndị dị ka mkpụrụ ndụ microbial ma ọ bụ antigens exogenous nwere oke. Enwere ike imeziwanye ohere ịnweta organoid lumens site na iji microinjector,26,27 mana usoro a na-awakpo ma na-arụsi ọrụ ike ma chọọ ihe ọmụma pụrụ iche iji rụọ ọrụ. Ọzọkwa, omenala organoid ọdịnala nke a na-edobe na hydrogel scaffolds n'okpuru ọnọdụ static anaghị egosipụta nke ọma biomechanics na-arụ ọrụ na vivo.
Ụzọ ndị ọzọ ọtụtụ ndị otu nyocha na-eji eme ihe na-eji ihe mkpuchi hydrogel 3D e guzobere ahazi iji mee ka ọ dị ka usoro epithelial afọ site n'ịkọpụta mkpụrụ ndụ eriri afọ mmadụ dịpụrụ adịpụ n'elu gel. Jiri ihe mkpuchi hydrogel nke e biri ebi 3D, nke e ji micro-milled, ma ọ bụ nke e ji lithographic mee. Usoro a na-egosi nhazi onwe onye nke mkpụrụ ndụ epithelial dịpụrụ adịpụ na vitro yana gradients morphogen dị mkpa na physiologically, na-eguzobe usoro epithelial dị elu na stroma-epithelial crosstalk site na itinye mkpụrụ ndụ stromal na ihe mkpuchi ahụ. Agbanyeghị, ọdịdị nke ihe mkpuchi e guzobere ahazi nwere ike igbochi ngosipụta nke usoro morphogenetic na-enweghị onwe ya. Ụdị ndị a anaghịkwa enye mmụba ọkụ ma ọ bụ interstitial na-agbanwe agbanwe, na-enweghị nrụgide mmiri mmiri nke mkpụrụ ndụ eriri afọ kwesịrị ịgafe morphogenesis ma nweta ọrụ physiological. Ọmụmụ ihe ọzọ na-adịbeghị anya jiri ihe mkpuchi hydrogel na ikpo okwu microfluidic na nhazi epithelial eriri afọ nke e ji usoro laser-etching mee ihe. Organoids eriri afọ òké na-agbaso ụkpụrụ e ji kpụọ iji mepụta usoro tubular eriri afọ, enwere ike ịmegharị mmiri mmiri intraluminal site na iji modul microfluidics. Agbanyeghị, ụdị a egosipụtaghị Usoro morphogenetic na-apụtaghị ìhè ma ọ gụnyekwara mmegharị mechanobiological nke eriri afọ. Usoro mbipụta 3D sitere na otu otu ahụ nwere ike ịmepụta obere tubes eriri afọ nwere usoro morphogenetic na-apụtaghị ìhè. N'agbanyeghị mmepụta dị mgbagwoju anya nke akụkụ eriri afọ dị iche iche n'ime tube ahụ, ụdị a enweghịkwa mmiri ọkụ na mgbanwe mechanical. Na mgbakwunye, ọrụ nlereanya nwere ike ịnwe oke, karịsịa mgbe usoro bioprinting gwụchara, na-akpaghasị ọnọdụ nnwale ma ọ bụ mmekọrịta sel-site-cell. Kama nke ahụ, usoro anyị tụrụ aro na-enye morphogenesis eriri afọ na-enweghị isi, nrụgide shear dị mkpa na physiological, biomechanics nke na-eṅomi mmegharị afọ, nnweta nke ngalaba apical na basolateral onwe ha, na imepụtaghachi gburugburu microbio dị mgbagwoju anya nke modularity. Ya mere, usoro morphogenesis 3D anyị na vitro nwere ike inye ụzọ mgbakwunye iji merie ihe ịma aka nke usoro ndị dị ugbu a.
Usoro anyị lekwasịrị anya kpamkpam na morphogenesis epithelial 3D, naanị mkpụrụ ndụ epithelial na ọdịbendị na enweghị ụdị mkpụrụ ndụ ndị ọzọ gbara ya gburugburu dịka mkpụrụ ndụ mesenchymal, mkpụrụ ndụ endothelial, na mkpụrụ ndụ ji alụso ọrịa ọgụ. Dịka akọwara na mbụ, isi ihe dị na usoro anyị bụ mkpali nke morphogenesis epithelial site na iwepụ ihe mgbochi morphogen nke e wepụtara n'akụkụ basolateral nke usoro ewebatara. Ọ bụ ezie na modularity siri ike nke gut-on-a-chip anyị na hybrid-on-a-chip na-enye anyị ohere ịmepụta oyi akwa epithelial 3D na-agbaze, ihe mgbagwoju anya ndị ọzọ dị ka mmekọrịta epithelial-mesenchymal33,34, extracellular Matrix (ECM) deposition 35 na, na ụdị anyị, atụmatụ crypt-villus nke na-ebunye niche mkpụrụ ndụ na basal crypts ka a ka ga-atụlekwu. Mkpụrụ ndụ Stromal (dịka ọmụmaatụ, fibroblasts) na mesenchyme na-arụ ọrụ dị mkpa na mmepụta nke protein ECM na nhazi nke morphogenesis eriri afọ na vivo35,37,38. Mgbakwunye nke mkpụrụ ndụ mesenchymal na ụdị anyị mere ka usoro morphogenetic na njikọ mkpụrụ ndụ dịkwuo mma. arụmọrụ. Akwa endothelial (ya bụ, capillaries ma ọ bụ lymphatic) na-arụ ọrụ dị mkpa n'ịchịkwa njem molekul39 na nchịkọta mkpụrụ ndụ ji alụso ọrịa ọgụ40 na gburugburu afọ. Ọzọkwa, ihe ndị dị na vasculature nke enwere ike ijikọ n'etiti ụdị anụ ahụ bụ ihe dị mkpa mgbe emebere ụdị anụ ahụ iji gosipụta mmekọrịta dị n'etiti akụkụ ahụ. Ya mere, enwere ike itinye mkpụrụ ndụ endothelial iji gosipụta njirimara physiological ziri ezi karịa na mkpebi ọkwa akụkụ ahụ. Mkpụrụ ndụ ji alụso ọrịa ọgụ sitere na onye ọrịa dịkwa mkpa maka igosipụta nzaghachi mgbochi ọrịa ebumpụta ụwa, ngosipụta antigen, njikọta mgbochi ọrịa ebumpụta ụwa, na mgbochi ọrịa nke anụ ahụ kpọmkwem n'ihe gbasara iṅomi ọrịa eriri afọ.
Ojiji nke chips ngwakọ dị mfe karịa eriri afọ n'ihi na ntọala ngwaọrụ dị mfe ma ojiji nke ihe ntinye Transwell na-enye ohere maka ọdịbendị epithelium eriri afọ. Agbanyeghị, ihe ntinye Transwell dị n'ahịa nwere akpụkpọ polyester anaghị agbanwe agbanwe ma enweghị ike ịme ka mmegharị dị ka peristaltic. Ọzọkwa, akụkụ apical nke ihe ntinye Transwell nke etinyere na chip ngwakọ ahụ nọgidere na-eguzo ọtọ na-enweghị nrụgide shear n'akụkụ apical. N'ụzọ doro anya, ihe ndị na-anaghị agbanwe agbanwe na ngalaba apical anaghị ekwe ka ọdịbendị nje bacteria ogologo oge na chips ngwakọ. Ọ bụ ezie na anyị nwere ike ịkpalite 3D morphogenesis nke ọma na ihe ntinye Transwell mgbe anyị na-eji chips ngwakọ, ụkọ nke biomechanics na mmiri mmiri apical metụtara physiological nwere ike igbochi ohere nke ikpo okwu chips ngwakọ maka ojiji nwere ike.
Nhazigharị zuru oke nke axis crypt-villus mmadụ na omenala gut-on-a-chip na hybrid-on-a-chip eguzobebeghị nke ọma. Ebe ọ bụ na morphogenesis na-amalite site na epithelial monolayer, microarchitectures 3D anaghị enye myirịta morphological na crypts na vivo. Ọ bụ ezie na anyị kọwara ọnụọgụgụ mkpụrụ ndụ na-amụba nso na ngalaba crypt basal na microengineered 3D epithelium, akọwapụtaghị mpaghara crypt na villous nke ọma. Ọ bụ ezie na ọwa elu dị elu na chip na-eduga na mmụba nke epithelium microengineered, elu kachasị elu ka nwere oke na ~300–400 µm. Ezigbo omimi nke crypts eriri afọ mmadụ na obere na nnukwu eriri afọ bụ ~135 µm na ~400 µm, n'otu n'otu, na elu nke obere eriri afọ bụ ~600 µm41.
Site n'echiche onyonyo, onyonyo dị elu nke microarchitectures 3D nwere ike ịdị naanị na eriri afọ na mgbawa, ebe ọ bụ na anya ọrụ achọrọ site na lensị ebumnuche ruo na oyi akwa epithelial dị na nha milimita ole na ole. Iji merie nsogbu a, enwere ike ịchọ ihe mgbaru ọsọ dị anya. Ọzọkwa, ime akụkụ dị gịrịgịrị maka nkwadebe ihe nlele onyonyo na-esiri ike n'ihi oke ike nke PDMS. Ọzọkwa, ebe ọ bụ na nhazi microfabrication nke afọ na mgbawa na-agụnye ịrapara na-adịgide adịgide n'etiti oyi akwa ọ bụla, ọ na-esiri ike nke ukwuu imepe ma ọ bụ wepụ oyi akwa elu iji lelee nhazi elu nke oyi akwa epithelial. Dịka ọmụmaatụ, site na iji igwe nyocha elektrọn (SEM).
Mmetụta nke PDMS abụrụla ihe na-egbochi ọmụmụ ihe ndị dabere na microfluidic nke na-emeso obere molekul hydrophobic, ebe ọ bụ na PDMS nwere ike itinye molekul hydrophobic ndị dị otú ahụ n'ụzọ na-abụghị kpọmkwem. Enwere ike ịtụle ihe ndị ọzọ maka PDMS na ihe ndị ọzọ polymeric. N'aka nke ọzọ, enwere ike ịtụle mgbanwe elu nke PDMS (dịka ọmụmaatụ, mkpuchi na ihe lipophilic 42 ma ọ bụ poly(ethylene glycol) 43) iji belata nnabata nke molekul hydrophobic.
N'ikpeazụ, a kabeghị ụzọ anyị nke ọma n'ihe gbasara inye nyocha dị elu ma ọ bụ ikpo okwu nnwale "otu nha dabara na nke niile" nke dabara adaba maka onye ọrụ. Usoro a dị ugbu a chọrọ mgbapụta sirinji maka ngwaọrụ microdevice ọ bụla, nke na-ewe ohere na incubator CO2 ma gbochie nnwale buru ibu. Enwere ike imeziwanye mmachi a nke ukwuu site na nhazi nke ụdị omenala ọhụrụ (dịka ọmụmaatụ, ntinye oghere 24, olulu 96, ma ọ bụ oghere oghere 384 nke na-enye ohere ka a na-emegharị ma wepụ mgbasa ozi basolateral mgbe niile).
Iji kpalite 3D morphogenesis nke epithelium eriri afọ mmadụ na vitro, anyị jiri ngwaọrụ eriri afọ microfluidic chip nke nwere microchannels abụọ yiri ya na akpụkpọ ahụ na-agbanwe agbanwe n'etiti iji mepụta njikọ lumen-capillary. Anyị na-egosikwa ojiji nke ngwaọrụ microfluidic otu-channel (mkpụrụ ndụ ngwakọ) nke na-enye mmiri basolateral na-aga n'ihu n'okpuru akwa epithelial polarized toro na ntinye Transwell. Na nyiwe abụọ ahụ, enwere ike igosipụta morphogenesis nke mkpụrụ ndụ epithelial eriri afọ mmadụ dị iche iche site na itinye njikwa ntụziaka nke mmiri iji wepụ ndị na-emegide morphogen site na ngalaba basolateral. Usoro nnwale dum (Foto 1) nwere akụkụ ise: (i) microfabrication nke eriri afọ ma ọ bụ Transwell insertable hybrid chip (nzọụkwụ 1-5; Igbe 1), (ii) nkwadebe nke mkpụrụ ndụ epithelial eriri afọ (mkpụrụ ndụ Caco-2) ma ọ bụ organoids eriri afọ mmadụ; igbe 2-5), (iii) ọdịbendị nke mkpụrụ ndụ epithelial eriri afọ na ibe eriri afọ ma ọ bụ ibe ngwakọ (nzọụkwụ 6-9), (iv) mkpali nke morphogenesis 3D na vitro (nzọụkwụ 10) na (v)) iji mara microstructure epithelial 3D (nzọụkwụ 11-24). N'ikpeazụ, e mere otu njikwa kwesịrị ekwesị (a tụlere n'okpuru) iji kwado irè nke morphogenesis in vitro site na iji tụnyere morphogenesis epithelial na njikwa oghere, oge, ọnọdụ, ma ọ bụ usoro.
Anyị jiri usoro omenala abụọ dị iche iche: eriri afọ-na-chip nwere ọwa kwụ ọtọ ma ọ bụ ọwa ndị na-abụghị ahịrị gbagọrọ agbagọ, ma ọ bụ ibe ngwakọ nwere ihe ntinye Transwell (TW) na ngwaọrụ microfluidic, nke e mepụtara dịka akọwara na Igbe 1, na nzọụkwụ 1 -5. "Mmepụta Ngwaọrụ" na-egosi usoro ndị bụ isi n'ime otu mgbawa ma ọ bụ mgbawa ngwakọ." Omenala nke Mkpụrụ Epithelial Intestinal Mmadụ" na-akọwa isi iyi mkpụrụ ndụ (Caco-2 ma ọ bụ organoids intestinal mmadụ) na usoro omenala ejiri na usoro a. "In vitro morphogenesis" na-egosi usoro niile e ji akụ mkpụrụ ndụ Caco-2 ma ọ bụ epithelial sitere na organoid na mgbawa eriri afọ ma ọ bụ na ntinye Transwell nke mgbawa ngwakọ, na-esote mmụba nke morphogenesis 3D na nhazi nke nhazi epithelial a maara. Nọmba nzọụkwụ mmemme ma ọ bụ nọmba igbe dị n'okpuru akụ ọ bụla. Ngwa ahụ na-enye ihe atụ nke otu esi eji akwa epithelial intestinal guzobere, dịka ọmụmaatụ, na njirimara nkewa mkpụrụ ndụ, ọmụmụ ihe gbasara ahụ ike afọ, ntọala nke usoro microbiome host-microbiome, na ụdị ọrịa. Foto immunofluorescence na "Mkpụrụ ndụ Ihe dị iche iche na-egosi nuclei, F-actin na MUC2 nke a na-egosipụta na oyi akwa epithelial 3D Caco-2 nke e mepụtara na eriri afọ. MUC2 na-egosi na mkpụrụ ndụ goblet na imi sitere na elu mucosal. Foto fluorescent na Gut Physiology na-egosi imi nke a na-emepụta site na iji agba sialic acid na N-acetylglucosamine residues site na iji fluorescent wheat germ agglutinin. Foto abụọ ahụ na-ekpuchi na "Host-Microbe Co-Cultures" na-egosi nnọchi anya host-microbiome co-cultures na eriri afọ na chip. Ogwe aka ekpe na-egosi mmekorita nke E. coli na-egosipụta protein fluorescent green (GFP) yana microengineered 3D Caco-2 epithelial cells. Ogwe aka nri na-egosi ebe GFP E. coli jikọtara ya na mkpụrụ ndụ epithelial 3D Caco-2, nke na-esochi immunofluorescence staining na F-actin (uhie) na nuclei (acha anụnụ anụnụ). Nhazi ọrịa na-egosi ahụike na eriri afọ na-agbapụta na mgbawa afọ n'okpuru ihe ịma aka physiological na antigens bacterial (dịka ọmụmaatụ, lipopolysaccharide, LPS) na mkpụrụ ndụ ndị na-alụso ọrịa ọgụ (dịka ọmụmaatụ, PBMC; akwụkwọ ndụ akwụkwọ ndụ). E mepụtara mkpụrụ ndụ Caco-2 iji guzobe oyi akwa epithelial 3D. Ogwe nha, 50 µm. Foto ndị dị n'ahịrị ala: "Mgbanwe nke mkpụrụ ndụ" emere site na ikike sitere na ntụaka.2. Oxford University Press; Emegharịrị ya site na ikike sitere na Ref.5. NAS; "Ọdịnala Ndị ọbịa-Microbe Co-Culture" emere site na ikike sitere na ref.3. NAS; "Ịmepụta Ọrịa" emere site na ikike sitere na ntụaka.5. NAS.
E ji ihe ndị e ji PDMS mepụta ma chips n'ime eriri afọ na nke ngwakọ nke e si na silicon ebupụta site na lithography dị nro1,44 ma jiri SU-8 mee ihe. A na-ekpebi nhazi nke microchannels dị na mgbawa ọ bụla site n'ịtụle hydrodynamics dị ka nrụgide shear na nrụgide hydrodynamic1,4,12. Nhazi mbụ nke eriri afọ na-agbagharị agbagharị (Foto data 1a), nke nwere microchannels abụọ kwụ ọtọ, agbanweela ghọọ eriri afọ dị mgbagwoju anya (Foto data agbatịgharị 1b) nke gụnyere ụzọ abụọ nke microchannels gbagọrọ agbagọ iji kpalite mmụba oge obibi mmiri, ụkpụrụ mmiri na-abụghị nke ahịrị, na mgbanwe multiaxial nke mkpụrụ ndụ a zụlitere (Foto 2a-f) 12. Mgbe ọ dị mkpa ka emegharị biomechanics eriri afọ ndị ọzọ mgbagwoju anya, enwere ike ịhọrọ eriri afọ dị mgbagwoju anya. Anyị egosila na eriri afọ na-agbagharị agbagharị na-akpalitekwa morphogenesis 3D nke ukwuu n'otu oge ahụ yana oke uto epithelial yiri ya ma e jiri ya tụnyere nke mbụ. Ngụkọta-Igwe, n'agbanyeghị ụdị mkpụrụ ndụ a zụlitere. Ya mere, iji kpalite morphogenesis 3D, nhazi eriri afọ dị larịị na nke dị mgbagwoju anya nwere ike ịgbanwe. Ihe ndị e ji mee PDMS agwọ na ebu silicon nwere ụkpụrụ SU-8 nyere njirimara na-adịghị mma mgbe e mechasịrị ha (Foto 2a). Iji mepụta eriri afọ na mgbawa, a na-ejikọta oyi akwa PDMS dị elu nke a kwadebere na ihe nkiri PDMS nwere oghere wee jikọta ya na oyi akwa PDMS dị ala site na ijikọta ihe na-agbanwe agbanwe site na iji ihe na-agwọ corona (Foto 2b–f). Iji mepụta ibe ngwakọ, a na-ejikọta ihe ndị e ji mee PDMS agwọ na slide iko iji mepụta ngwaọrụ microfluidic otu ọwa nke nwere ike ịnabata ihe ntinye Transwell (Foto 2h na Extended Data Fig. 2). A na-eme usoro njikọ ahụ site n'ịgwọ elu nke ihe ndị e ji mee PDMS na iko na plasma oxygen ma ọ bụ ọgwụgwọ corona. Mgbe emechara ka ngwaọrụ microfacbricated nke ejikọtara na tube silicone dị na ya, ntọala ngwaọrụ ahụ dị njikere ime morphogenesis 3D nke epithelium eriri afọ (Foto 2g).
a, Ihe atụ nke nkwadebe nke akụkụ PDMS site na ebu silicon SU-8 a kpụrụ akpụ. A wụsara ihe ngwọta PDMS a na-agwọbeghị n'elu ebu silicon (aka ekpe), gwọọ ya na 60 °C (etiti) ma gbazee ya (n'aka nri). A bepụrụ PDMS a gbajiri agbaji ma hichaa ya maka ojiji ọzọ.b, Foto nke ebu silicon ejiri kwadebe oyi akwa elu PDMS.c, Foto nke ebu silicon eji emepụta membrane porous PDMS.d, Usoro foto nke ihe mejupụtara PDMS dị elu na ala na ngwaọrụ eriri afọ a chịkọtara.e, Nhazi nke nhazi nke ihe mejupụtara PDMS dị elu, membrane, na ala. A na-ejikọta oyi akwa ọ bụla site na plasma ma ọ bụ ọgwụgwọ corona.f, Nhazi nke ngwaọrụ gut-on-a-chip e mepụtara na microchannels convoluted na vacuum chambers.g, Ntọala nke gut-on-a-chip maka ọdịbendị mkpụrụ ndụ microfluidic. A na-etinye eriri afọ e mepụtara na chip e jikọtara ya na tube silicon na sirinji na mkpuchi mkpuchi. E tinyere ngwaọrụ chip na mkpuchi nke Nri Petri nke dị 150 mm maka nhazi. A na-eji ihe njide ahụ mechie tube silicone.h, Foto anya nke mmepụta chip ngwakọ na morphogenesis 3D site na iji chips ngwakọ. Ntinye Transwell nke a kwadebere n'onwe ya iji mepụta 2D monolayers nke mkpụrụ ndụ epithelial eriri afọ na chip ngwakọ iji kpalite 3D morphogenesis eriri afọ. A na-agbaze ihe ahụ site na obere ọwa dị n'okpuru oyi akwa sel nke edobere na ntinye Transwell. Ogwe nha, 1 cm.h E bipụtaghachiri ya na ikike site na ntụaka.4. Elsevier.
N'ime usoro a, ejiri eriri mkpụrụ ndụ Caco-2 na organoids eriri afọ mee ihe dị ka isi iyi epithelial (Foto 3a). E mepụtara ụdị mkpụrụ ndụ abụọ ahụ n'adabereghị onwe ha (Igbe nke 2 na Igbe nke 5) ma jiri ha kụọ obere ọwa mmiri nke eriri afọ ma ọ bụ ihe ndị e tinyere na Transwell nke ejiri ECM kpuchie. Mgbe mkpụrụ ndụ jikọtara ọnụ (ihe karịrị 95% mkpuchi na karama), a na-ewe mkpụrụ ndụ Caco-2 a na-akọkarị (n'etiti paseeji 10 na 50) na karama T iji kwadebe nkwụsịtụ mkpụrụ ndụ ndị kewara ekewa site na mmiri trypsinization (igbe nke 2). E tinyere organoids eriri afọ mmadụ site na biopsies eriri afọ ma ọ bụ nkwụsịtụ ịwa ahụ na domes Matrigel scaffold na efere olulu 24 iji kwado gburugburu ebe obibi nhazi. A na-agbakwunye morphogens dị mkpa (dị ka Wnt, R-spondin, na Noggin) na ihe ndị na-eto eto dị ka akọwara na Igbe nke 3 kwa ụbọchị ruo mgbe organoids toro ruo ~500 µm na dayameta. A na-ewe organoids ndị toro eto zuru oke ma kewaa ha n'ime Mkpụrụ ndụ otu maka ịgha mkpụrụ n'ime eriri afọ ma ọ bụ ihe e tinyere na Transwell na mgbawa (Igbe nke 5). Dịka anyị kọrọ na mbụ, enwere ike ịmata ọdịiche dị na ya dabere na ụdị ọrịa 12,13 (dịka ọmụmaatụ ọnya afọ, ọrịa Crohn, kansa eriri afọ, ma ọ bụ onye na-enye onyinye nkịtị), ebe mmerụ ahụ (dịka ọmụmaatụ, mmerụ ahụ ma ọ bụ mpaghara na-abụghị mmerụ ahụ) na ebe eriri afọ dị na tract (dịka ọmụmaatụ, duodenum, jejunum, ileum, cecum, colon, ma ọ bụ rectum). Anyị na-enye usoro kachasị mma na Igbe nke 5 maka ịzụlite organoids colonic (coloids) nke na-achọkarị oke morphogens karịa obere organoids eriri afọ.
a, Usoro ọrụ maka mkpali nke morphogenesis eriri afọ na eriri afọ. A na-eji Caco-2 eriri afọ mmadụ na organoids eriri afọ na usoro a iji gosipụta morphogenesis 3D. A kụrụ mkpụrụ ndụ epithelial dịpụrụ adịpụ na ngwaọrụ a kwadebere na eriri afọ (nkwadebe chip). Ozugbo a kụrụ mkpụrụ (mkpụrụ) ma jikọta (ejikọta) na akpụkpọ ahụ porous PDMS na ụbọchị 0 (D0), a na-amalite ma na-edobe usoro apical (AP) maka ụbọchị abụọ mbụ (flow, AP, D0-D2). A na-amalitekwa usoro basolateral (BL) yana mmegharị cyclic (stretch, flow, AP na BL) mgbe e guzobere monolayer 2D zuru oke. Usoro 3D nke eriri afọ mere na mberede mgbe ụbọchị ise nke ọdịbendị microfluidic (morphogenesis, D5). Foto dị iche iche nke usoro na-egosi ụdị morphology nke mkpụrụ ndụ Caco-2 na usoro nnwale ọ bụla ma ọ bụ oge (graf bar, 100 µm). Ihe osise eserese anọ na-egosi cascades kwekọrọ na afọ morphogenesis (n'elu aka nri). Àkwá ndị a gbagọrọ agbagọ n'usoro ihe osise ahụ na-anọchite anya ntụziaka nke mmiri mmiri.b, onyonyo SEM na-egosi elu elu nke epithelium Caco-2 3D e guzobere (n'aka ekpe). Isi ihe dị n'ime ya na-egosi mpaghara ahụ a gbagọrọ agbagọ (igbe ọcha a gbagọrọ agbagọ) na-egosi microvilli e weghachiri ọhụrụ na oyi akwa Caco-2 3D (n'aka nri).c, Echiche ihu kwụ ọtọ nke Caco-2 3D e guzobere, claudin (ZO-1, uhie) na akpụkpọ ahịhịa na-aga n'ihu nke akpọrọ F-actin (akwụkwọ ndụ akwụkwọ ndụ) na nuclei (acha anụnụ anụnụ) Immunofluorescence confocal visual nke mkpụrụ ndụ epithelial na ibe eriri afọ. Àkwá ndị na-atụ aka na usoro etiti na-egosi ebe oghere focal dị maka echiche confocal ọ bụla.d, Oge mgbanwe morphological na organoids a zụlitere na mgbawa enwetara site na microscopy nkebi dị iche na ụbọchị 3, 7, 9, 11, na 13. Isi ihe dị n'ime ya (n'elu aka nri) na-egosi mmụba dị elu nke onyonyo enyere.e, DIC photomicrograph nke organoid 3D epithelium e guzobere na afọ na mpekere e sere n'ụbọchị nke 7.f, Foto immunofluorescence kpuchie nke na-egosi ihe nrịbama maka mkpụrụ ndụ stem (LGR5; magenta), mkpụrụ ndụ goblet (MUC2; akwụkwọ ndụ akwụkwọ ndụ), F-actin (isi awọ) na nuclei (cyan) toro na mpekere afọ ruo ụbọchị atọ, n'otu n'otu (Ekpere) na organoids ụbọchị iri na atọ (etiti) na oyi akwa epithelial. Lee kwa Foto Data Gbasara 3, nke na-egosipụta akara LGR5 na-enweghị akara MUC2. Foto fluorescence na-egosi microstructure epithelial (n'aka nri) nke epithelium organoid 3D nke e guzobere n'afọ na mpekere site na iji agba CellMask (n'aka nri) mee ka akpụkpọ ahụ plasma tee ya na ụbọchị nke 13 nke omenala. Ogwe nha bụ 50 μm ọ gwụla ma e kwuru ihe ọzọ.b E bipụtaghachiri ya na ikike site na ntụaka.2. Oxford University Press; c Emegharịrị ya na ikike site na ntụaka.2. Oxford University Press; e na f emegharịrị ya na ikike site na ntụaka.12 N'okpuru Creative Commons License CC BY 4.0.
N'ime afọ na mgbawa, ọ dị mkpa ịgbanwe elu hydrophobic nke akpụkpọ ahụ porous PDMS maka mkpuchi ECM nke ọma. Na usoro a, anyị na-eji ụzọ abụọ dị iche iche gbanwee hydrophobic nke akpụkpọ ahụ PDMS. Maka ịzụlite mkpụrụ ndụ Caco-2, naanị mkpali elu site na ọgwụgwọ UV/ozone zuru ezu iji belata hydrophobicity nke elu PDMS, kpuchie ECM ma tinye mkpụrụ ndụ Caco-2 na akpụkpọ ahụ PDMS. Agbanyeghị, ọdịbendị microfluidic nke epithelium organoid chọrọ ọrụ elu dabere na kemịkalụ iji nweta nchekwa dị mma nke protein ECM site na itinye polyethyleneimine (PEI) na glutaraldehyde n'usoro na microchannels PDMS. Mgbe mgbanwe elu gasịrị, etinyere protein ECM iji kpuchie elu PDMS arụ ọrụ wee tinye ya na epithelium organoid dịpụrụ adịpụ. Mgbe etinyere mkpụrụ ndụ ahụ, ọdịbendị microfluidic cell na-amalite site na ịgbasa naanị ihe dị n'ime microchannel elu ruo mgbe mkpụrụ ndụ ahụ ga-etolite monolayer zuru oke, ebe obere microchannel na-ejigide ọnọdụ kwụ ọtọ. Usoro a kachasị mma maka ịgbalite elu na mkpuchi ECM na-eme ka njikọ nke organoid na-eme ka ọ dị mma. epithelium iji kpalite morphogenesis 3D n'elu PDMS.
Omenala Transwell chọkwara mkpuchi ECM tupu a kụọ mkpụrụ ndụ; agbanyeghị, omenala Transwell anaghị achọ usoro ọgwụgwọ dị mgbagwoju anya iji mee ka elu nke ihe ndị nwere oghere rụọ ọrụ. Maka itolite mkpụrụ ndụ Caco-2 na ihe ndị dị na Transwell, mkpuchi ECM na ihe ndị nwere oghere na-eme ka njikọ nke mkpụrụ ndụ Caco-2 kewara ekewa (<1 awa) na nhazi ihe mgbochi njikọ siri ike (<1-2 ụbọchị) dịkwuo ngwa ngwa. Iji mepụta organoids na ihe ndị dị na Transwell, a na-akụ mkpụrụ organoids ndị dịpụrụ adịpụ na ihe ndị e ji ekpuchi ECM, na-ejikọ ya na elu akpụkpọ ahụ (<3 awa) ma na-edobe ya ruo mgbe organoids ga-etolite monolayer zuru oke nwere iguzosi ike n'ezi ihe mgbochi. A na-eme omenala Transwell na efere olulu mmiri 24 na-ejighị ibe ngwakọ.
Enwere ike ịmalite morphogenesis In vitro 3D site n'itinye mmiri mmiri na akụkụ basolateral nke oyi akwa epithelial guzobere. N'ime afọ na mgbawa, epithelial morphogenesis malitere mgbe etinyere mmiri mmiri n'ime obere ọwa elu na ala (Foto 3a). Dịka akọwara na mbụ, ọ dị mkpa iwebata mmiri mmiri na ngalaba dị ala (basolateral) maka iwepụ ihe mgbochi morphogen nke na-egbochi ntụziaka na-aga n'ihu. Iji nye mkpụrụ ndụ ndị jikọtara na akpụkpọ ahụ oghere zuru oke ma mepụta nrụgide shear luminal, anyị na-etinyekarị mmiri mmiri abụọ n'afọ na mgbawa. Na iberibe ngwakọ, etinyere ihe ntinye Transwell nwere epithelial monolayers n'ime iberibe ngwakọ. Mgbe ahụ, etinyere ihe mgbasa ozi n'okpuru akụkụ basolateral nke ihe ntinye Transwell oghere site na microchannel. Njem morphogenesis n'ime eriri afọ mere ụbọchị 3-5 mgbe mmalite nke mmiri basolateral na nyiwe ọdịbendị abụọ ahụ.
Enwere ike inyocha atụmatụ ọdịdị nke akwa epithelial 3D microengineered site na iji usoro onyonyo dị iche iche, gụnyere microscopy dị iche iche, microscopy differential nnyonye anya (DIC), SEM, ma ọ bụ immunofluorescence confocal microscopy (Foto 3 na 4). Enwere ike ime ntụgharị nke usoro ma ọ bụ onyonyo DIC ngwa ngwa n'oge ọ bụla n'oge ọdịbendị iji nyochaa ọdịdị na mpụta nke akwa epithelial 3D. N'ihi nghọta anya nke PDMS na ihe nkiri polyester, ma nyiwe chip-on-a-chip na ngwakọ nwere ike inye onyonyo n'oge na-adịghị anya na-enweghị mkpa maka nkewa ma ọ bụ iwepụ ngwaọrụ ahụ. Mgbe ị na-eme onyonyo immunofluorescence (Foto 1, 3c, f na 4b, c), a na-edozi mkpụrụ ndụ na paraformaldehyde 4% (wt/vol) (PFA), nke Triton X-100 na 2% (wt/vol) ) bovine serum albumin (BSA) na-esochi, n'usoro. Dabere na ụdị sel, ihe ndozi dị iche iche, permeabilizers, na igbochi. Dabere na ụdị sel, ihe ndozi dị iche iche, permeabilizers, na mgbochi Enwere ike iji ihe ndị na-eme ka ihe dị ndụ pụta ìhè. A na-eji ọgwụ mgbochi mbụ na-elekwasị anya na mkpụrụ ndụ ma ọ bụ ihe nrịbama mpaghara iji gosipụta mkpụrụ ndụ ndị na-adịghị arụ ọrụ na mpaghara ahụ na chip, wee soro ọgwụ mgbochi nke abụọ yana agba mgbochi nke na-elekwasị anya na nucleus (dịka ọmụmaatụ, 4′, 6-diamidino-2-phenylene) indole, DAPI) ma ọ bụ F-actin (dịka ọmụmaatụ, phalloidin a na-akpọ fluorescent). Enwere ike ime onyonyo ndụ dabere na fluorescence na situ iji chọpụta mmepụta mucous (Fig. 1, "Nhazi mkpụrụ ndụ" na "Ọdịdị afọ"), nhazi nke mkpụrụ ndụ microbial na-enweghị usoro (Fig. 1, "Ọdịiche nke Ọrịa") ma ọ bụ nhazi nke 3D epithelial morphology (Fig. 3c, f na 4b, c). Mgbe a na-agbanwe eriri afọ na chip ahụ iji kewaa oyi akwa elu na oyi akwa microchannel dị ala, dịka akọwara na ref. Dịka egosiri na Fig. 2, ọdịdị epithelial 3D yana microvilli na Enwere ike iji SEM hụta ókè ahịhịa apical (Foto 3b). Enwere ike inyocha ngosipụta nke ihe nrịbama dị iche iche site na ime nhazi PCR5 ma ọ bụ otu mkpụrụ ndụ RNA. N'okwu a, a na-egbutu akwa 3D nke mkpụrụ ndụ epithelial a kụrụ na eriri afọ ma ọ bụ chips ngwakọ site na trypsinization wee jiri ya mee nyocha molekul ma ọ bụ mkpụrụ ndụ ihe nketa.
a, Usoro ọrụ maka mkpali nke morphogenesis eriri afọ na mgbawa ngwakọ. A na-eji Caco-2 na organoids eriri afọ na usoro a iji gosipụta morphogenesis 3D na ikpo okwu mgbawa ngwakọ. A na-akụ mkpụrụ ndụ epithelial kewara ekewa na ntinye Transwell akwadoro (nkwadebe TW; lee foto dị n'okpuru). Ozugbo a kụrụ mkpụrụ (mkpụrụ) ma jikọta mkpụrụ ndụ na akpụkpọ polyester na ntinye Transwell, a na-akọ mkpụrụ ndụ niile n'okpuru ọnọdụ static (omenala TW). Mgbe ụbọchị 7 gasịrị, a na-etinye otu ntinye Transwell nwere monolayer 2D nke mkpụrụ ndụ epithelial n'ime mgbawa ngwakọ iji webata mmiri basolateral (Flow, BL), nke mechara duga na mmepụta nke oyi akwa epithelial 3D (morphogenesis). Micrographs dị iche na-egosi atụmatụ morphological nke mkpụrụ ndụ epithelial akụkụ mmadụ sitere na onye nyere onyinye nkịtị (ahịrị C103) na-arịgo colon na nzọụkwụ nnwale ọ bụla ma ọ bụ oge. Usoro dị na akwa elu na-egosi nhazi nnwale maka nzọụkwụ ọ bụla.b, Ibe ngwakọ (usoro aka ekpe) nwere ike ibute morphogenesis 3D nke mkpụrụ ndụ epithelial organoid nwere confocal elu-ala Echiche mikroskopi e sere n'ọnọdụ dị iche iche nke Z (elu, etiti, na ala; lee akara eserese aka nri na ahịrị ntụpọ kwekọrọ). gosiri njirimara morphological doro anya. F-actin (cyan), nucleus (isi awọ).c, Fluorescence confocal micrographs (echiche akụkụ 3D) nke mkpụrụ ndụ epithelial sitere na organoid nke a zụlitere na Transwell static (TW; inset n'ime igbe ọcha dashboard) megide chip ngwakọ (ose zuru oke kachasị) na-atụnyere 2D megide 3D morphology, n'otu n'otu. Otu ụzọ nke echiche obe kwụ ọtọ 2D (inset n'akụkụ aka nri elu; "XZ") na-egosikwa atụmatụ 2D na 3D. Ogwe nha, 100 µm.c E bipụtaghachiri ya na ikike site na ntụaka.4. Elsevier.
Enwere ike ịkwadebe njikwa site n'ịkọpụta otu mkpụrụ ndụ ahụ (Caco-2 ma ọ bụ mkpụrụ ndụ epithelial nke eriri afọ) ka ọ bụrụ monolayer abụọ n'okpuru ọnọdụ omenala nkịtị. Ihe pụrụ iche bụ na mbelata nri nwere ike ibute n'ihi oke olu nke obere ọwa mmiri (ya bụ, ~4 µL na ọwa elu na nhazi mbụ nke eriri afọ). Ya mere, enwere ike iji morphology epithelial tupu na mgbe etinyere ya na mmiri basolateral.
A ga-eme usoro lithography dị nro n'ime ụlọ dị ọcha. Maka oyi akwa ọ bụla dị na chip (elu na ala oyi akwa na membranes) na chips ngwakọ, ejiri ma kpụọ ihe mkpuchi foto dị iche iche na wafer silicon dị iche iche n'ihi na elu nke microchannels dị iche. Elu ebumnuche nke microchannels elu na ala nke eriri afọ dị na chip bụ 500 µm na 200 µm, n'otu n'otu. Ogologo ebumnuche nke ọwa nke chip ngwakọ bụ 200 µm.
Tinye wafer silicon nke dị sentimita atọ n'ime efere nwere acetone. Jiri nwayọ tụgharịa efere ahụ ruo sekọnd iri atọ, wee kpoo wafer ahụ n'ikuku. Tinye wafer ahụ na efere nwere IPA, wee tụgharịa efere ahụ ruo sekọnd iri atọ ka ọ dị ọcha.
Enwere ike iji ngwọta piranha (ngwakọta hydrogen peroxide na acid sulfuric concentrated, 1:3 (vol/vol)) mee ihe iji wepụ ihe fọdụrụ n'ime ihe ndị sitere n'okike n'elu silicon wafer.
Ngwọta Piranha na-emebi ihe nke ukwuu ma na-emepụta okpomọkụ. Ihe ndị ọzọ dị mkpa maka nchekwa dị mkpa. Maka mkpofu ihe mkpofu, hapụ ihe mkpofu ahụ ka ọ jụọ oyi ma bufee ya na akpa mkpofu dị ọcha ma kpọọ nkụ. Jiri akpa ndị ọzọ ma denye akara nke ọma na akpa mkpofu. Biko soro ntuziaka nchekwa nke ebe ahụ maka usoro zuru ezu.
Mmiri mmiri dị n'ime wafer ndị ahụ site n'itinye ha n'elu efere ọkụ nke 200 °C ruo nkeji iri. Mgbe mmiri kpọrọ nkụ gasịrị, a na-amagharị wafer ahụ ugboro ise n'ikuku ka ọ jụọ oyi.
Wụsa ihe dịka 10 g nke fotoresist SU-8 2100 n'etiti wafer silicon a kpụchara akpụcha. Jiri tweezers gbasaa photoresist ahụ n'ụzọ zuru oke na wafer ahụ. Mgbe ụfọdụ, tinye wafer ahụ n'elu efere ọkụ 65°C iji mee ka photoresist ahụ ghara ịrapara ma dịkwa mfe ịgbasa. Etinyela wafer ahụ kpọmkwem n'elu efere ọkụ ahụ.
E kesara SU-8 n'otu aka ahụ n'elu wafer ahụ site n'iji mkpuchi ntụgharị. Hazie ntụgharị SU-8 na-abata maka sekọnd 5-10 iji gbasaa na 500 rpm na ọsọ nke 100 rpm/s. Tọọ ntụgharị isi maka nhazi ọkpụrụkpụ 200 µm na 1,500 rpm, ma ọ bụ nweta ọkpụrụkpụ 250 µm (na-eme ka elu 500 µm dị elu maka oyi akwa elu nke eriri afọ na mgbawa ahụ; lee "Nzọụkwụ dị mkpa" n'okpuru) nke edobere na ọsọ nke 300 rpm/s sekọnd 30 na 1,200 rpm.
Enwere ike ịgbanwe ọsọ ntụgharị isi dịka ọkpụrụkpụ ebumnuche nke ụkpụrụ SU-8 dị na wafer silicon.
Iji mepụta usoro SU-8 nke dị 500 µm elu maka akwa elu nke afọ na chip ahụ, a na-emegharị mkpuchi ntụgharị na nzọụkwụ achịcha dị nro nke Igbe a (nzọụkwụ 7 na 8) n'usoro (lee nzọụkwụ 9) iji mepụta akwa abụọ nke 250 µm A akwa dị arọ nke SU-8, nke enwere ike itinye ma jikọta ya site na ikpughe UV na nzọụkwụ 12 nke igbe a, na-eme ka akwa ahụ dị 500 µm elu.
Jiri nwayọ mee achịcha wafer ndị a kpuchiri na SU-8 site n'itinye wafer ndị ahụ n'elu efere ọkụ na 65°C maka nkeji ise, wee gbanwee ntọala ahụ ka ọ bụrụ 95°C wee tinye ya na incubator maka nkeji iri anọ ọzọ.
Iji ruo 500 μm nke ụkpụrụ SU-8 n'ime obere ọwa elu, megharịa nzọụkwụ 7 na 8 iji mepụta oyi akwa SU-8 abụọ dị 250 μm n'obosara.
Jiri UV Mask Aligner mee nnwale oriọna dịka ntuziaka onye nrụpụta si dị iji gbakọọ oge mkpughe nke wafer ahụ. (oge mkpughe, ms) = (ọnụọgụ mkpughe, mJ/cm2)/(ike oriọna, mW/cm2).
Mgbe ị chọpụtachara oge ikpughe, tinye ihe mkpuchi ihu na ihe mkpuchi ihu nke ihe mkpuchi ihu UV ma tinye ihe mkpuchi ihu na wafer a kpụchara akpụ nke SU-8.
Debe elu e biri ebi nke ihe mkpuchi ihu ahụ kpọmkwem n'akụkụ SU-8 nke silicon wafer iji belata mgbasa UV.
Kpuchie wafer na ihe mkpuchi foto nke SU-8 n'ụzọ kwụ ọtọ ruo 260 mJ/cm2 nke ìhè UV maka oge ekpughere ya (lee nzọụkwụ 10 nke igbe a).
Mgbe e kpughechara UV, a na-esi achịcha wafer silicon a kpụchara akpụcha SU-8 na 65°C maka nkeji ise na 95°C maka nkeji iri na ise na efere ọkụ ọ bụla iji mepụta ụkpụrụ dị elu nke 200 μm. Gbasaa oge mgbe emechara achịcha na 95°C ruo nkeji iri atọ iji mepụta ụkpụrụ dị elu nke 500 µm.
A na-awụsa onye mmepụta ihe n'ime efere iko, a na-etinyekwa wafer e ji eme achịcha n'ime efere ahụ. Olu nke onye mmepụta SU-8 nwere ike ịdị iche dabere na nha efere iko ahụ. Jide n'aka na ị na-eji onye nrụpụta SU-8 zuru oke iji wepụ SU-8 na-enweghị ihe ọ bụla. Dịka ọmụmaatụ, mgbe ị na-eji efere iko nke dayameta ya bụ 150 mm nwere ikike 1 L, jiri ihe nrụpụta SU-8 nke dị ihe dịka 300 mL. Mepụta ebu ahụ maka nkeji iri abụọ na ise na ntụgharị dị nro mgbe ụfọdụ.
Jiri ihe dị ka 10 mL nke ihe nrụpụta ọhụrụ sachaa ebu ahụ e mepụtara, wee jiri IPA fesa ya site n'iji pipette fesa ya.
Debe wafer ahụ n'ime ihe nhicha plasma ma tinye ya na plasma oxygen (gas ikuku, nrụgide ebumnuche 1 × 10−5 Torr, ike 125 W) maka nkeji 1.5.
Debe wafer ahụ n'ime ihe na-ekpo ọkụ nke nwere ihe na-ekpo ọkụ nke nwere ihe na-ekpo ọkụ nke nwere iko slide n'ime ya. Enwere ike itinye wafer na slide n'akụkụ ibe ha. Ọ bụrụ na e kewaa ihe na-ekpo ọkụ nke nwere ihe na-ekpo ọkụ n'ime ọtụtụ oyi akwa site na efere, tinye slide ahụ n'ime ụlọ dị ala, tinye wafer ahụ n'ime ụlọ dị elu. Debe 100 μL nke ihe ngwọta trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane n'elu ihe na-ekpo ọkụ nke nwere ...
Mee ka otu karama nke mkpụrụ ndụ Caco-2 kpọnwụrụ akpọnwụ n'ime mmiri ịsa ahụ nke 37°C, wee bufee mkpụrụ ndụ ndị ahụ gbazere agbaze na karama T75 nke nwere 15 mL nke 37°C nke a na-ekpo ọkụ na Caco-2.
Iji gafere mkpụrụ ndụ Caco-2 na ~90% confluency, buru ụzọ kpoo Caco-2 ọkara, PBS, na 0.25% trypsin/1 mM EDTA n'ime mmiri ịsa ahụ nke 37°C.
Jiri vacuum aspiration saa mkpụrụ ndụ ahụ ugboro abụọ. Jiri 5 mL nke PBS ọkụ saa mkpụrụ ndụ ahụ site n'ịkwagharị vacuum aspiration ma tinye PBS ọhụrụ.