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Mā te morphogenesis o te puku tangata ka whakatūria ngā āhuatanga crypt-villus o te microarchitecture epithelial 3D me te whakaritenga ā-wāhi. E hiahiatia ana tēnei hanganga ahurei hei pupuri i te homeostasis puku mā te tiaki i te niche pūtau take i roto i te crypt basal mai i ngā antigens microbial exogenous me ā rātou metabolites. Hei tāpiri, ko ngā villi puku me te huware huna e whakaatu ana i ngā pūtau epithelial kua rerekē te mahi me te ārai tiaki i runga i te mata mucosal puku. Nō reira, he mea nui te hanga anō i ngā hanganga epithelial 3D mō te hanganga o ngā tauira puku in vitro. He mea nui, ka taea e te mimetic organic gut-on-a-chip te whakaoho i te morphogenesis 3D tūpono o te epithelium puku me ngā mahi tinana me ngā mahi koiora whakarei ake. I konei, ka whakaratohia e mātou he kawa ka taea te whakaputa anō hei whakaoho kaha i te morphogenesis puku i roto i te puku i runga i te maramara microfluidic me roto hoki i te maramara ranu whakauru Transwell. Ka whakaahuahia e mātou ngā tikanga taipitopito mō te hanga taputapu, te whakatipu i ngā pūtau epithelial organoid puku Caco-2 i roto i ngā tautuhinga tikanga me runga i te tūāpapa microfluidic, te whakaoho i te morphogenesis 3D, me te whakaahuatanga o te epithelia 3D kua whakapumautia mā te whakamahi i ngā momo whakaahua maha. Ko tēnei kawa Ka tutuki te whakaoranga o te hanganga moroiti o te puku mā te whakahaere i te rere o te wai basolateral mō te 5 rā. Ka whakamahia e tā mātou tikanga morphogenesis in vitro te ahotea kutikuti e tika ana mō te tinana me te nekehanga miihini, ā, kāore e hiahiatia he hangarau pūtau uaua, he whakarerekētanga rānei, ka nui ake pea te pai o ēnei tikanga i ētahi atu tikanga o nāianei. E whakaaro ana mātou ka whai pānga whānui tā mātou kawa kua whakaarohia ki te hapori rangahau hauora, ka whakarato i tētahi tikanga hei whakaora i ngā paparanga epithelial puku 3D i roto i te vitro mō ngā tono hauora, haumanu, me te rongoā.
E whakaatu ana ngā whakamātautau ka taea e ngā pūtau Caco-2 epithelial puku i whakatōkia ki roto i ngā taputapu microfluidic gut-on-a-chip1,2,3,4,5, bilayer rānei6,7 te hurihanga 3D tūpono i roto i te vitro me te kore e mārama ki te tikanga matua. I roto i tā mātou rangahau tata nei, i kitea e mātou he mea tika te tango i ngā antagonists morphogen i hunaia e te basolateral mai i ngā taputapu ahurea, ā, he rawaka hoki hei whakaoho i te hurihanga 3D epithelial i roto i te vitro, kua whakaaturia e Caco-2 me ngā organoids puku i ahu mai i te tūroro. I whakamanahia ngā pūtau epithelial. I roto i tēnei rangahau, i arotahi mātou ki te whakaputanga pūtau me te tohatoha kukū o tētahi kaitukino Wnt kaha, a Dickkopf-1 (DKK-1), i roto i ngā taputapu microfluidic gut-on-a-chip me ngā taputapu microfluidic kua whakarerekētia kei roto ngā whakaurunga Transwell, e kiia nei ko "Hybrid Chip". E whakaatu ana mātou ko te tāpiri o ngā kaitukino Wnt exogenous (pēnei i te DKK-1, te Wnt repressor 1, te pūmua frizzled-related 1 i tukuna, te Soggy-1 rānei) ki te puku on-chip ka aukati i te morphogenesis, ka whakararuraru rānei i te paparanga epithelial 3D kua whakaritea, e kī ana ko te ahotea kaitukino i te wā o te ahurea te take o te morphogenesis whekau in vitro. Nō reira, ko te huarahi mahi hei whakatutuki i te morphogenesis pakari i te atanga epithelial ko te tango, te pupuri rānei i ngā taumata o ngā kaitukino Wnt i roto i te wāhanga basolateral mā te horoi kaha (hei tauira, i roto i ngā papa gut-on-a-chip, i ngā papa hybrid-on-a-chip rānei) te horapa rānei. Ngā pāpāho basolateral (hei tauira, mai i ngā whakaurunga Transwell ki roto i ngā puna basolateral nui i roto i ngā puna).
I roto i tēnei kawa, ka whakaratohia e mātou he tikanga taipitopito mō te hanga i ngā taputapu iti o te puku-i-runga-i-te-maramara me ngā maramara ranu ka taea te whakauru ki Transwell (ngā taahiraa 1-5) hei whakatipu i ngā pūtau epithelial puku i runga i ngā kiriuhi porous e hangai ana ki te polydimethylsiloxane (PDMS) (ngā taahiraa 6A, 7A, 8, 9) ngā kiriuhi polyester rānei o ngā whakaurunga Transwell (ngā taahiraa 6B, 7B, 8, 9) ā, ka whakaoho i te morphogenesis 3D i roto i te vitro (te taahiraa 10). I tautuhia hoki e mātou ngā āhuatanga pūtau me te ngota e tohu ana i te histogenesis motuhake-kiko me te rerekētanga pūtau e whakawhirinaki ana ki te whakapapa mā te whakamahi i ngā momo whakaahua maha (ngā taahiraa 11-24). Ka whakaoho mātou i te morphogenesis mā te whakamahi i ngā pūtau epithelial puku tangata, pērā i te Caco-2, i ngā organoids puku rānei, i roto i ngā whakatakotoranga ahurea e rua me ngā taipitopito hangarau tae atu ki te whakarerekētanga mata o ngā kiriuhi porous, te hanganga o ngā paparanga kotahi 2D, me te matū koiora puku me te Whakaputa uri o te taiao moroiti koiora. i roto i te vitro. Hei whakaoho i te morphogenesis 3D mai i ngā paparanga kotahi epithelial 2D, i tangohia e mātou ngā kaitukino morphogen i roto i ngā āhua e rua kua whakatipuria mā te rere. te reo ki roto i te wāhanga basolateral o te ahurea. Hei whakamutunga, ka whakaratohia e mātou he whakaaturanga o te whai huatanga o tētahi paparanga epithelial 3D ka taea te whakahou ka taea te whakamahi hei tauira i te tipu epithelial e whakawhirinaki ana ki te morphogen, ngā ahurea tahi o te host-microbiome roa, te mate pathogen, te whara mumura, te ngoikore o te arai epithelial, me ngā maimoatanga e hangai ana ki te probiotic.
Tērā pea ka whai hua tā mātou kawa ki te whānuitanga o ngā kaipūtaiao i roto i ngā rangahau taketake (hei tauira, te koiora mucosal whēkau, te koiora pūtau take, me te koiora whanaketanga) me ngā rangahau tono (hei tauira, te whakamātautau rongoā i mua i te haumanu, te whakatauira mate, te hangarau kiko, me te gastroenterology) whānui. Nā te kaha ki te whakaputa me te pakari o tā mātou kawa ki te whakaoho i te morphogenesis 3D o te epithelium whēkau i roto i te vitro, e whakaaro ana mātou ka taea te tohatoha i tā mātou rautaki hangarau ki te hunga whakarongo e ako ana i ngā āhuatanga o te tohu pūtau i te wā o te whanaketanga whēkau, te whakahoutanga, te homeostasis rānei. Hei tāpiri, he whai hua tā mātou kawa mō te uiui i te mate i raro i ngā momo mate urutā pēnei i te Norovirus 8, te Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9, Vibrio cholerae rānei. He whai hua hoki ngā hunga whakarongo ki te mate pukupuku me te pathogenesis. Mā te whakamahi i tētahi pūnaha microphysiology puku i runga i te maramara ka taea te whakatipu tahi roa 10 me te aromatawai i muri mai i te parenga o te manaaki, ngā urupare ārai mate me te whakatikanga whara e pā ana ki te pathogen i roto i te ara gastrointestinal (GI) 11. Ko ētahi atu mate GI e pā ana ki te mate puku turuturu, te mate celiac, te mate Crohn, te colitis ulcerative, te pouchitis, te mate puku irritable rānei ka taea te whakatauira ina whakaritea ngā paparanga epithelial puku 3D mā te whakamahi i ngā paparanga epithelial puku 3D o te tūroro, ko ēnei mate ko te atrophy villous, te whakapoto crypt, te kino mucosal, te ārai epithelial ngoikore rānei. Ko te biopsy, ko ngā organoids puku i ahu mai i te pūtau take rānei12,13. Hei whakatauira pai ake i te uauatanga teitei o te taiao mate, ka whakaaro pea ngā kaipānui ki te tāpiri i ngā momo pūtau e pā ana ki te mate, pērā i ngā pūtau mononuclear toto peripheral tūroro (PBMC), ki ngā tauira kei roto ko ngā microarchitectures villus-crypt puku 3D. ngā pūtau ārai mate motuhake-kiko, 5.
I te mea ka taea te whakatika me te whakaatu i te hanganga iti o te epithelial 3D me te kore e hiahiatia te tukanga wehewehe, ko ngā kaimātakitaki e mahi ana i ngā mahi transcriptomics ā-wāhi me te whakaahua teitei-taumira, teitei-taumira rānei, ka hiahia pea ki tā mātou mahere i ngā whanaketanga ā-wāhi-wā o ngā ira me ngā pūmua i runga i ngā niches epithelial. Kei te hiahia ki te hangarau. Te urupare ki ngā whakaohooho moroiti, ārai mate rānei. Hei tāpiri, ko te whakawhitiwhiti kōrero roa o te manaaki-microbiome 10, 14 e whakarite ana i te homeostasis puku ka taea te whakatū i roto i te paparanga mucosal puku 3D mā te whakatipu tahi i ngā momo moroiti, ngā hapori moroiti, ngā moroiti paru rānei, inā koa i roto i te puku-i-runga-i-te-maramara. i roto i te tūāpapa. He tino ātaahua tēnei huarahi ki te hunga whakarongo e ako ana i te immunology mucosal, te gastroenterology, te microbiome tangata, te culturomics me te microbiology haumanu e rapu ana ki te whakatipu i ngā microbiota puku kāore anō kia whakatipuria i roto i te taiwhanga. Mena ka taea te whakarerekē i tā mātou kawa morphogenesis in vitro ki ngā whakatakotoranga ahurea tauine, pēnei i ngā whakaurunga maha-pūngao i roto i ngā pereti puna 24, 96, 384 rānei e whakakī tonu ana i ngā wāhanga basolateral, ka taea hoki te tohatoha i te kawa ki te hunga e whakawhanake ana i ngā tūāpapa rongoā, koiora, tirotiro-rahi-putanga rānei, whakamana rānei mō te umanga kai. Hei taunakitanga-o-te-kaupapa, i whakaaturia e mātou ināianei te whaihua o tētahi pūnaha morphogenesis maha-putanga teitei ka taea te tauine ki tētahi whakatakotoranga pereti 24-pūngao. Hei tāpiri, kua hokohokohia ngā hua maha-okana-i-runga-i-te-maramara16,17,18. Nō reira, ka taea te tere ake te whakamana i tā mātou tikanga morphogenesis in vitro, ā, ka taea te whakamahi e te maha o ngā taiwhanga rangahau, te ahumahi, te kāwanatanga me ngā tari whakahaere kia mārama ai ki te hōtaka anō pūtau o te morphogenesis puku in vitro i te taumata transcriptomic hei whakamātautau i ngā rongoā, i ngā rongoā koiora rānei. Ko te mimiti me te kawe I aromatawaitia ngā momo rongoā i roto i ngā tauira whakakapi puku 3D, i te whakamahi rānei i ngā tauira okana-i-runga-i-te-maramara ritenga, arumoni rānei, hei aromatawai i te āheinga ki te tāruarua i te tukanga morphogenesis puku.
He iti noa ngā tauira whakamātautau e pā ana ki te tangata kua whakamahia hei ako i te morphogenesis epithelial puku, ko te nuinga nā te kore o ngā tikanga ka taea te whakatinana hei whakaoho i te morphogenesis 3D i roto i te vitro. Inaa, ko te nuinga o ngā mōhiotanga o nāianei mō te morphogenesis puku e ahu mai ana i ngā rangahau kararehe (hei tauira, zebrafish20, kiore21, heihei rānei22). Heoi, he nui te mahi me te utu, he pātai pea mō te matatika, ā, ko te mea nui, kāore e tino whakatau i ngā tukanga whanaketanga tangata. He iti rawa hoki te kaha o ēnei tauira ki te whakamatautauria i roto i te huarahi tauine maha. Nō reira, ko tā mātou tikanga mō te whakaora i ngā hanganga kiko 3D i roto i te vitro he pai ake i ngā tauira kararehe in vivo me ētahi atu tauira ahurea pūtau 2D tūmau tuku iho. E ai ki te whakaahuatanga o mua, mā te whakamahi i ngā hanganga epithelial 3D ka taea e mātou te tirotiro i te rohe ā-wāhi o ngā pūtau kua wehewehea i roto i te tuaka crypt-villus hei whakautu ki ngā momo whakaoho mucosal, ārai mate rānei. Ka taea e ngā paparanga epithelial 3D te whakarato i tētahi wāhi hei ako i te whakataetae a ngā pūtau microbial ki te hanga i ngā niches ā-wāhi me te whanaketanga taiao hei whakautu ki ngā āhuatanga manaaki (hei tauira, ngā paparanga mucus o roto me waho, te huna o IgA me te antimicrobial peptides). Hei tāpiri, mā te āhua o te epithelial 3D ka taea e tātou te mārama ki te hanganga o ngā hapori o te microbiota puku, me te whakaputa tahi i ngā metabolites microbial (hei tauira, ngā waikawa ngako mekameka-poto) e hanga ana i te whakaritenga pūtau me ngā koha pūtau take i roto i ngā crypts basal. Ka taea anake te whakaatu i ēnei āhuatanga ina whakatūria ngā paparanga epithelial 3D i roto i te vitro.
Haunga tā mātou tikanga hanga hanganga epithelial whekau 3D, he maha ngā tikanga in vitro. Ko te ahurea organoid whekau he tikanga hangarau kiko hou e hangai ana ki te whakatipu i ngā pūtau take whekau i raro i ngā āhuatanga morphogen motuhake23,24,25. Heoi, he uaua te whakamahi i ngā tauira organoid 3D mō te tātari kawe, mō ngā ahurea tahi-microbiome rānei nā te mea kei roto i te organoid te lumen whekau, ā, nō reira, he iti te whakaurunga o ngā wāhanga luminal pērā i ngā pūtau microbial, i ngā antigens exogenous rānei. Ka taea te whakapai ake i te urunga atu ki ngā lumen organoid mā te whakamahi i te microinjector,26,27 engari he tikanga whakaeke tēnei, he mahi nui hoki, ā, me whai mātauranga motuhake hei mahi. Hei tāpiri, ko ngā ahurea organoid tuku iho e puritia ana i roto i ngā tūāpapa hydrogel i raro i ngā āhuatanga tūmau, kāore e whakaata tika i ngā mahi biomechanics in vivo.
Ko ētahi atu huarahi e whakamahia ana e ētahi rōpū rangahau e whakamahi ana i ngā tūāpapa hydrogel 3D kua whakaritea hei whakatauira i te hanganga epithelial o te puku mā te whakatipu i ngā pūtau puku tangata motuhake i runga i te mata o te gel. Hangaia ngā tūāpapa hydrogel mā te whakamahi i ngā pokepokea kua tāia 3D, kua mirahia ki te moroiti, kua hangaia rānei ki te lithographic. E whakaatu ana tēnei tikanga i te whakaritenga whaiaro o ngā pūtau epithelial motuhake i roto i te vitro me ngā gradients morphogen e pā ana ki te tinana, e whakatū ana i te hanganga epithelial ōwehenga teitei me te crosstalk stroma-epithelial mā te whakauru i ngā pūtau stromal ki roto i te tūāpapa. Heoi, ko te āhua o ngā tūāpapa kua whakaritea ka ārai pea i te whakaaturanga o te tukanga morphogenetic tūpono. Kāore hoki ēnei tauira e whakarato i te rere luminal, interstitial rānei hihiri, kāore i te ahotea kutikuti wai e hiahiatia ana e ngā pūtau puku kia uru ki te morphogenesis me te whiwhi mahi tinana. I whakamahia e tētahi atu rangahau tata nei ngā tūāpapa hydrogel i roto i te tūāpapa microfluidic me ngā hanganga epithelial puku tauira mā te whakamahi i ngā tikanga whakairo laser. Ka whai ngā organoids puku kiore i ngā tauira whakairo hei hanga i ngā hanganga ngongo puku, ā, ka taea te whakahou i te rere wai intraluminal mā te whakamahi i te kōwae microfluidics. Heoi, kāore tēnei tauira e whakaatu i te tūpono. kāore hoki e uru ana ki ngā tukanga morphogenetic, kāore hoki e uru ana ngā nekehanga mechanobiological o te puku. I taea e ngā tikanga tā 3D mai i te rōpū kotahi te hanga i ngā ngongo puku iti me ngā tukanga morphogenetic tūpono. Ahakoa te hanganga uaua o ngā wāhanga puku rerekē i roto i te ngongo, kāore hoki i roto i tēnei tauira te rere o te wai luminal me te whakarerekētanga miihini. Hei tāpiri, tera pea ka iti te mahi a te tauira, inā koa i muri i te otinga o te tukanga bioprinting, ka whakararuraru i ngā āhuatanga whakamātautau, i ngā taunekeneke pūtau-ki-pūtau rānei. Engari, ko tā mātou kawa kua whakaarohia e whakarato ana i te morphogenesis puku tūpono, te ahotea kutikuti e pā ana ki te tinana, ngā biomechanics e whakaata ana i te nekehanga o te puku, te urunga atu ki ngā wāhanga apical me te basolateral motuhake, me te hanga anō i ngā taiao moroiti koiora uaua o te modularity. Nō reira, ko tā mātou kawa morphogenesis 3D in vitro ka whakarato pea i tētahi huarahi tautoko hei wikitoria i ngā wero o ngā tikanga o nāianei.
Ko tā mātou kawa e aro katoa ana ki te morphogenesis epithelial 3D, me ngā pūtau epithelial anake i roto i te ahurea, kāore he momo pūtau kē atu e karapoti ana pērā i ngā pūtau mesenchymal, ngā pūtau endothelial, me ngā pūtau ārai mate. E ai ki te whakaahuatanga o mua, ko te kaupapa matua o tā mātou kawa ko te whakaoho i te morphogenesis epithelial mā te tango i ngā aukati morphogen i hunaia i te taha basolateral o te reo whakaurua. Ahakoa te kaha o te modularity o tā mātou gut-on-a-chip me te hybrid-on-a-chip ka taea e mātou te hanga anō i te paparanga epithelial 3D undulating, ko ngā uauatanga koiora atu pērā i ngā taunekeneke epithelial-mesenchymal33,34, te whakatakotoranga extracellular Matrix (ECM) 35 me, i roto i tā mātou tauira, ko ngā āhuatanga crypt-villus e kawe ana i ngā niches pūtau take i roto i ngā crypts basal me whai whakaaro anō. Ko ngā pūtau stromal (hei tauira, fibroblasts) i roto i te mesenchyme he mahi nui ki te whakaputa i ngā pūmua ECM me te whakahaere i te morphogenesis puku i roto i te vivo35,37,38. Ko te tāpiri o ngā pūtau mesenchymal ki tā mātou tauira i whakarei ake i te tukanga morphogenetic me te whai huatanga o te piri pūtau. Ko te paparanga endothelial (arā, ngā capillaries, ngā lymphatics rānei) he mahi nui ki te whakahaere i te kawe irahiko39 me te kimi pūtau ārai mate40 i roto i te taiao moroiti puku. Hei tāpiri, ko ngā wāhanga toto ka taea te hono i waenga i ngā tauira kiko he mea nui ina hangaia ngā tauira kiko hei whakaatu i ngā taunekeneke maha-okana. Nō reira, me whakauru pea ngā pūtau endothelial hei whakatauira i ngā āhuatanga ā-tinana tika ake me te whakataunga taumata-okana. He mea nui hoki ngā pūtau ārai mate i ahu mai i te tūroro mō te whakaatu i ngā urupare ārai mate ā-roto, te whakaaturanga antigen, te whakawhitiwhiti ārai mate urutau ā-roto, me te ārai mate motuhake-kiko i roto i te horopaki o te whai i te mate puku.
He māmā ake te whakamahi i ngā maramara ranu i te maramara puku-i-runga-i-te-maramara nā te mea he māmā ake te whakatakotoranga o te taputapu, ā, mā te whakamahi i ngā whakaurunga Transwell ka taea te whakatipu i te epithelium puku kia taea te whakawhanui. Heoi, ko ngā whakaurunga Transwell e wātea ana i te hokohoko me ngā kiriuhi polyester kāore i te pīataata, ā, kāore e taea te whakatauira i ngā nekehanga peristaltic. Hei tāpiri, ko te wāhanga apical o te whakaurunga Transwell i whakanohoia ki roto i te maramara ranu i noho pūmau me te kore he ahotea kutikuti i te taha apical. He mārama, ko ngā āhuatanga pumau i roto i te wāhanga apical he onge te āhei i te whakatipu tahi huakita mō te wā roa i roto i ngā maramara ranu. Ahakoa ka taea e tātou te whakaoho kaha i te morphogenesis 3D i roto i ngā whakaurunga Transwell i te wā e whakamahi ana i ngā maramara ranu, ko te kore o ngā mahi koiora e tika ana mō te tinana me te rere o te wai apical ka whakawhāiti i te whai huatanga o ngā tūāpapa maramara ranu mō ngā tono pea.
Kāore anō kia tino whakapumautia ngā hanganga hou katoa o te tuaka crypt-villus tangata i roto i ngā ahurea gut-on-a-chip me ngā ahurea hybrid-on-a-chip. Mai i te tīmatanga o te morphogenesis mai i te monolayer epithelial, kāore ngā microarchitectures 3D e whakarato i te ōritetanga morphological ki ngā crypts in vivo. Ahakoa i whakaahuahia e mātou te taupori pūtau e tipu haere ana i te taha o te rohe crypt basal i roto i te epithelium 3D microengineered, kāore i te mārama te wehewehe i ngā rohe crypt me villous. Ahakoa ko ngā awa teitei ake o runga i te maramara ka piki ake te teitei o te epithelium microengineered, ko te teitei mōrahi kei te ~300–400 µm tonu. Ko te hohonutanga tuturu o ngā crypts whekau tangata i roto i ngā whekau iti me ngā whekau nui he ~135 µm me ~400 µm, ia, ā, ko te teitei o te villi whekau iti he ~600 µm41.
Mai i te tirohanga whakaahua, ko te whakaahua tino-taumira in situ o ngā hoahoanga moroiti 3D ka herea pea ki te puku i runga i te maramara, nā te mea ko te tawhiti mahi e hiahiatia ana mai i te arotahi whāinga ki te paparanga epithelial he torutoru mirimita. Hei whakakore i tēnei raruraru, me whakamahi pea i tētahi arotahi tawhiti. Hei tāpiri, he uaua te hanga i ngā wāhanga angiangi mō te whakarite tauira whakaahua nā te tino ngāwari o te PDMS. Hei tāpiri, nā te mea ko te hanganga moroiti paparanga-i-te-paparanga o te puku i runga i te maramara he piri tonu i waenga i ia paparanga, he tino uaua ki te whakatuwhera, ki te tango rānei i te paparanga o runga hei tirotiro i te hanganga mata o te paparanga epithelial. Hei tauira, mā te whakamahi i te karu hiko matawai (SEM).
Ko te ātete wai o te PDMS he mea whakawhāiti i roto i ngā rangahau e pā ana ki te microfluidic e pā ana ki ngā ngota iti ātete wai, nā te mea ka taea e te PDMS te mimiti i aua ngota ātete wai. Ka taea te whakaaro ki ētahi atu rauemi polymeric hei whakakapi i te PDMS. Hei tauira, ka taea te whakaaro ki te whakarerekētanga mata o te PDMS (hei tauira, te paninga ki ngā rauemi lipophilic 42, poly(ethylene glycol) 43 rānei) hei whakaiti i te mimiti o ngā ngota ātete wai.
Hei whakamutunga, kāore anō kia tino whakaahuatia tā mātou tikanga mō te whakarato i tētahi tūāpapa whakamātautau "kotahi te rahi e pai ana mō te katoa" e ngāwari ana ki te whakamahi. Ko te kawa o nāianei e hiahia ana kia whakamahia he papu weronga mō ia pūrere iti, e noho ana i te wāhi i roto i te incubator CO2, ā, ka ārai i ngā whakamātautau nui. Ka taea te whakapai ake i tēnei herenga mā te whakawhanui i ngā whakatakotoranga ahurea auaha (hei tauira, ngā whakaurunga porous 24-puna, 96-puna, 384-puna rānei e āhei ai te whakakī tonu me te tango i ngā pāpāho basolateral).
Hei whakaoho i te morphogenesis 3D o te epithelium whekau tangata i roto i te vitro, i whakamahia e mātou he taputapu whekau maramara microfluidic kei roto e rua ngā microchannels whakarara me te kiriuhi porous elastic i waenganui hei hanga i te atanga lumen-capillary. Ka whakaatuhia hoki e mātou te whakamahinga o tētahi taputapu microfluidic hongere kotahi (he maramara ranu) e whakarato ana i te rere basolateral tonu i raro i ngā paparanga epithelial polarized i whakatipuria i runga i ngā whakaurunga Transwell. I roto i ngā papaaho e rua, ka taea te whakaatu i te morphogenesis o ngā pūtau epithelial whekau tangata mā te whakamahi i te whakahaere ahunga o te rere hei tango i ngā antagonists morphogen mai i te wāhanga basolateral. Ko te tukanga whakamātautau katoa (Pikitia 1) e rima ngā wāhanga: (i) te hanga moroiti o te maramara whekau, te maramara ranu whakauru Transwell rānei (ngā taahiraa 1-5; Pouaka 1), (ii) te whakarite i ngā pūtau epithelial whekau (ngā pūtau Caco-2) ngā organoids whekau tangata rānei; pouaka 2-5), (iii) te whakatipu i ngā pūtau epithelial whekau ki runga i ngā maramara whekau, i ngā maramara ranu rānei (ngā taahiraa 6-9), (iv) te whakaoho i te morphogenesis 3D i roto i te vitro (te taahiraa 10) me te (v)) hei whakaahua i te hanganga moroiti epithelial 3D (ngā taahiraa 11-24). Hei whakamutunga, i hangaia he rōpū whakahaere tika (ka kōrerohia i raro nei) hei whakamana i te whai huatanga o te morphogenesis in vitro mā te whakataurite i te morphogenesis epithelial ki ngā mana whakahaere ā-wāhi, ā-wā, ā-tikanga, ā-tikanga rānei.
E rua ngā tūāpapa ahurea rerekē i whakamahia e mātou: te puku-i-runga-i-te-maramara me ngā awaawa tika, ngā awaawa kore-raina rānei, ngā maramara ranu rānei kei roto ngā whakaurunga Transwell (TW) i roto i tētahi taputapu microfluidic, i hangaia e ai ki te whakaahuatanga i roto i te Pouaka 1, me te taahiraa 1 -5. "Te Hanga Pūrere" e whakaatu ana i ngā mahi matua ki te hanga i tētahi maramara kotahi, i tētahi maramara ranu rānei. "Te Ahurea o ngā Pūtau Epithelial Whekau Tangata" e whakamārama ana i te pūtake pūtau (Caco-2, ngā organoids whēkau tangata rānei) me te tukanga ahurea i whakamahia i roto i tēnei tikanga. "Te morphogenesis in vitro" e whakaatu ana i ngā mahi whānui e whakatipuria ai a Caco-2, ngā pūtau epithelial i ahu mai i te organoid rānei ki runga i tētahi maramara whēkau, ki runga rānei i ngā whakaurunga Transwell o tētahi maramara ranu, ā, whai muri ko te whakaoho i te morphogenesis 3D me te hanganga o tētahi hanganga epithelial kua tohua. Kei raro i ia pere te tau taahiraa kaupapa, te tau pouaka rānei. Ka whakaratohia e te tono ngā tauira o te whakamahinga o ngā paparanga epithelial whēkau kua whakapumautia, hei tauira, i roto i te whakaahuatanga rerekētanga pūtau, ngā rangahau physiology whēkau, te whakatūnga o ngā rauropi manaaki-microbiome, me te whakatauira mate. Ngā whakaahua immunofluorescence i roto i te "Pūtau "Te Whakarerekētanga" e whakaatu ana i ngā karihi, te F-actin me te MUC2 i whakaatuhia i roto i te paparanga epithelial 3D Caco-2 i hangaia i runga i te maramara puku. Kei roto te tohu MUC2 i roto i ngā pūtau goblet me te hūpē i hunaia mai i ngā mata mucosal. Ko ngā whakaahua fluorescent i roto i te Gut Physiology e whakaatu ana i te hūpē i hangaia mā te whakakoi mō te waikawa sialic me ngā toenga N-acetylglucosamine mā te whakamahi i te fluorescent wheat germ agglutinin. Ko ngā whakaahua e rua e taupatupatu ana i roto i te "Host-Microbe Co-Cultures" e whakaatu ana i ngā ahurea manaaki-microbiome i roto i te puku i runga i te maramara. Kei te taha maui te whakaaturanga o te ahurea tahi o te E. coli e whakaatu ana i te pūmua fluorescent matomato (GFP) me ngā pūtau epithelial 3D Caco-2 i hangaia i roto i te microengineered. Kei te taha matau te whakaaturanga o te GFP E. coli i whakatipuria tahitia me ngā pūtau epithelial 3D Caco-2, ā, muri iho ko te whakakoi immunofluorescence me te F-actin (whero) me ngā karihi (kikorangi). Ko te whakatauira mate e whakaatu ana i te puku hauora me te turuturu i roto i ngā maramara mumura puku i raro i te wero ā-tinana me ngā antigens huakita (hei tauira, lipopolysaccharide, LPS) me ngā pūtau ārai mate (hei tauira, (PBMC; kakariki). I whakatōngia ngā pūtau Caco-2 hei whakatū i tētahi paparanga epithelial 3D. Pae tauine, 50 µm. Ngā whakaahua i te rarangi raro: "Te wehewehenga o ngā pūtau" i urutauhia me te whakaaetanga mai i te tohutoro. 2. Oxford University Press; I tāruatia me te whakaaetanga mai i te Tohutoro 5. NAS; "Host-Microbe Co-Culture" i urutauhia me te whakaaetanga mai i te tohutoro 3. NAS; "Desease Modeling" i urutauhia me te whakaaetanga mai i te tohutoro. 5. NAS.
I hangaia ngā maramara puku-i-runga-maramara me ngā maramara ranu mā te whakamahi i ngā tārua PDMS i tangohia mai i ngā pokepokea ai te silicon mā te whakamahi i te lithography ngohengohe1,44 me te tauira ki te SU-8. Ka whakatauhia te hoahoa o ngā awa iti i roto i ia maramara mā te whakaaro ki ngā āhuatanga waiora pēnei i te ahotea kutikuti me te pēhanga waiora1,4,12. Ko te hoahoa puku-i-runga-maramara taketake (Raraunga Whānui Pikitia 1a), i titoa e rua ngā awa iti tika whakarara e hono tahi ana, kua whanake hei awa-i-runga-maramara matatini (Raraunga Whānui Pikitia 1b) kei roto ko te takirua o ngā awa iti piko hei whakaoho i te wā noho wai kua piki ake, ngā tauira rere kore-raina, me te whakarerekētanga maha-tuaka o ngā pūtau kua whakatipuria (Pikitia 2a–f) 12. Ina hiahiatia kia hangaia anō ngā mahi koiora puku uaua ake, ka taea te whiriwhiri i ngā awa-i-runga-maramara matatini. Kua whakaaturia e mātou ko te Wawa-maramara matatini ka whakaoho kaha hoki i te morphogenesis 3D i roto i te wā rite me te nui o te tipu epithelial rite ki te taketake. Maramara-Puku, ahakoa te momo pūtau i whakatipuria. Nō reira, hei whakaoho i te morphogenesis 3D, ka taea te whakawhiti i ngā hoahoa puku raina me te matatini i runga i te maramara. Ko ngā tauira PDMS i rongoatia ki runga i ngā pokepokea silicon me ngā tauira SU-8 i whakarato i ngā āhuatanga kino i muri i te tangohanga (Pikitia 2a). Hei hanga i te puku ki runga i te maramara, ko te paparanga PDMS o runga kua whakaritea i honoa ā-raupapa ki te kiriata PDMS porous, kātahi ka whakaritea ki te paparanga PDMS o raro mā te hononga kore e taea te whakakore mā te whakamahi i te corona treater (Pikitia 2b–f). Hei hanga maramara ranu, ko ngā tauira PDMS kua rongoatia i honoa ki ngā kiriata karāhe hei hanga i ngā taputapu microfluidic kotahi-hongere ka taea te whakauru i ngā whakaurunga Transwell (Pikitia 2h me te Raraunga Whānui Pikitia 2). Ka mahia te tukanga hononga mā te rongoā i ngā mata o te tauira PDMS me te karāhe ki te plasma hāora, ki te maimoatanga corona rānei. I muri i te whakahoromata o te taputapu hanga-iti e piri ana ki te ngongo silicone, kua rite te whakatakotoranga taputapu ki te mahi i te morphogenesis 3D o te epithelium puku (Pikitia 2g).
a, Whakaahua hoahoa o te whakarite i ngā wāhanga PDMS mai i ngā pokepokea ai he tauira SU-8 mō te silicon. I ringihia te otinga PDMS kāore anō kia rongoatia ki runga i te pokepokea ai he silicon (maui), i rongoatia i te 60 °C (waenganui) ā, i wetekina (matau). I tapahia te PDMS wetekina kia mongamonga, ā, i horoia mō ētahi atu whakamahinga. b, Whakaahua o te pokepokea ai he silicon i whakamahia hei whakarite i te paparanga o runga o te PDMS. c, Whakaahua o te pokepokea ai he silicon i whakamahia hei hanga i te kiriuhi porous PDMS. d, He raupapa whakaahua o ngā wāhanga PDMS o runga me raro me te taputapu puku i runga i te maramara kua huihuia. e, Whakaahua hoahoa o te whakaritenga o ngā wāhanga PDMS o runga, o te membrane, me raro. Ka herea ia paparanga e te maimoatanga plasma, corona rānei, kāore e taea te hoki whakamuri. f, Whakaahua hoahoa o te taputapu puku-i-runga-i-te-maramara kua hangaia me ngā hongere iti kua whakakikoruatia me ngā rūma korehau. g, Tatūnga o te puku-i-runga-i-te-maramara mō te ahurea pūtau microfluidic. I whakatakotoria te puku i hangaia i runga i te maramara kua huihuia me te ngongo silicone me te syringe ki runga i te uhi. I whakatakotoria te taputapu maramara ki runga i te taupoki o te Petri 150 mm rihi mō te tukatuka. Ka whakamahia te here hei kati i te ngongo silicone. h, Ngā whakaahua tirohanga o te hanganga maramara ranu me te morphogenesis 3D mā te whakamahi i ngā maramara ranu. I whakaurua ngā whakaurunga Transwell i whakaritea motuhake hei whakatipu i ngā paparanga 2D o ngā pūtau epithelial whekau ki roto i te maramara ranu hei whakaoho i te morphogenesis 3D whekau. Ka tukuna te reo mā roto i ngā awa iti i raro i te paparanga pūtau i whakatūria ki runga i te whakaurunga Transwell. Pae tauine, 1 cm.h I tāia anō me te whakaaetanga mai i te tohutoro. 4. Elsevier.
I roto i tēnei tikanga, i whakamahia te raina pūtau Caco-2 me ngā organoid whekau hei pūtake epithelial (Pikitia 3a). I whakatipuria motuhaketia ngā momo pūtau e rua (Pouaka 2 me te Pouaka 5) ā, i whakamahia hei whakatō i ngā awa iti kua pania ki te ECM o te puku i runga i te maramara, i ngā whakaurunga Transwell rānei. Ina huihui ngā pūtau (>95% te kapi i roto i ngā ipu), ka hauhaketia ngā pūtau Caco-2 kua whakatipuria auautia (i waenganui i ngā ara 10 me te 50) i roto i ngā ipu-T hei whakarite i ngā whakatārewatanga pūtau kua wehea e te wai trypsinization (pouaka 2). I whakatipuria ngā organoid whekau tangata mai i ngā biopsies whekau, i ngā tangohanga pokanga rānei ki roto i ngā domes scaffold Matrigel i roto i ngā pereti 24-puna hei tautoko i te taiao moroiti hanganga. I tāpirihia ngā reo kei roto ngā morphogens nui (pērā i te Wnt, R-spondin, me te Noggin) me ngā tauwehe tipu i whakaritea e ai ki te whakaahuatanga i roto i te Pouaka 3 i ia rā anō tae noa ki te tipu o ngā organoid ki te ~500 µm te whānui. Ka hauhaketia ngā organoid kua tino tipu, ā, ka wehea ki roto i ngā pūtau takitahi hei whakatō ki runga i te puku. ngā whakaurunga Transwell rānei ki runga i tētahi maramara (Pouaka 5). E ai ki tā mātou i kōrero ai i mua, ka taea te wehewehe i runga i te momo mate12,13 (hei tauira, te mate puku ulcerative, te mate a Crohn, te mate pukupuku colorectal, te kaituku noa rānei), te wāhi whara (hei tauira, te wāhi whara me te wāhi kāore i whara) me te wāhi o te puku i roto i te ara (hei tauira, te duodenum, te jejunum, te ileum, te cecum, te koroni, te rectum rānei). Ka whakaratohia e mātou he kawa kua arotauhia i roto i te Pouaka 5 mō te whakatipu i ngā organoid koroni (coloids) e hiahia ana ki ngā kukū teitei ake o ngā morphogens i ngā organoid whekau iti.
a, Rerenga mahi mō te whakaoho i te morphogenesis puku i roto i te maramara puku. Ka whakamahia te epithelium puku tangata Caco-2 me ngā organoids puku i roto i tēnei kawa hei whakaatu i te morphogenesis 3D. I whakatōngia ngā pūtau epithelial motuhake ki roto i te taputapu puku-i-runga-i-te-maramara kua whakaritea (te whakarite maramara). Kia whakatōngia (whakatōngia) ngā pūtau me te piri (whakapiri) ki te membrane porous PDMS i te rā 0 (D0), ka tīmatahia te rere apical (AP) me te pupuri mō ngā rā tuatahi e 2 (rerenga, AP, D0-D2). Ka tīmatahia hoki te rere Basolateral (BL) me ngā nekehanga totoro huringa (toto, rere, AP me te BL) ina hangaia he paparanga 2D katoa. I puta noa te morphogenesis 3D puku i muri i te 5 rā o te ahurea microfluidic (morphogenesis, D5). E whakaatu ana ngā whakaahua rerekētanga wāhanga i te morphology tohu o ngā pūtau Caco-2 i ia taahiraa whakamātautau, i ia wā rānei (kauwhata pae, 100 µm). E whā ngā hoahoa kauwhata e whakaatu ana i ngā rerenga ōrite o te morphogenesis puku (matau o runga). Ko te tohu Ko ngā pere i roto i te hoahoa e tohu ana i te ahunga o te rere o te wai. b, Whakaahua SEM e whakaatu ana i te āhua o te mata o te epithelium 3D Caco-2 kua whakatūria (maui). Ko te whakaurunga e whakaatu ana i te rohe whakanuia (pouaka ma kua pītitia) e whakaatu ana i ngā microvilli kua whakahoutia i runga i te paparanga 3D Caco-2 (matau). c, Tirohanga whakapae o mua o te Caco-2 3D kua whakatūria, te claudin (ZO-1, whero) me ngā kiriuhi rohe paraihe tonu kua tohua ko te F-actin (kakariki) me ngā karihi (kikorangi). Whakaaturanga confocal immunofluorescence o ngā pūtau epithelial i runga i ngā maramara whekau. Ko ngā pere e tohu ana ki te hoahoa waenga e tohu ana i te wāhi o te papa arotahi mō ia tirohanga confocal. d, Te roanga wā o ngā huringa morphological i roto i ngā organoids i whakatipuria ki runga i te maramara i whiwhihia e te karuārai ā-wāhanga i ngā rā 3, 7, 9, 11, me te 13. Ko te whakaurunga (runga matau) e whakaatu ana i te whakanui teitei o te whakaahua i whakaratohia. e, Whakaahua whakaahua DIC o te epithelium 3D organoid kua whakatūria ki roto i te puku i runga i te poro i tangohia i te rā 7. f, Ngā whakaahua immunofluorescence kua whakakikoruatia e whakaatu ana i ngā tohu mō ngā pūtau take (LGR5; magenta), ngā pūtau kapu (MUC2; kakariki), F-actin (hina) me ngā karihi (cyan) i whakatipuria ki runga i ngā maramara puku mō ngā rā e 3, ia (Mauī) me ngā organoid 13-rā (waenganui) i runga i te paparanga epithelial. Tirohia hoki te Pikitia Raraunga Whānui 3, e whakaatu ana i te tohu LGR5 me te kore tohu MUC2. Ngā whakaahua fluorescence e whakaatu ana i te hanganga moroiti epithelial (matau) o te epithelium organoid 3D i whakatūria i roto i te puku i runga i te maramara mā te whakakoi i te kiriuhi plasma ki te tae CellMask (matau) i te rā 13 o te ahurea. Ko te pae tauine he 50 μm ki te kore e kīia tētahi atu. b I tāia anō me te whakaaetanga mai i te tohutoro. 2. Oxford University Press; c I whakarerekētia me te whakaaetanga mai i te Tohutoro. 2. Oxford University Press; e me f i whakarerekētia me te whakaaetanga mā te tohutoro. 12 I raro i te Raihana Creative Commons CC BY 4.0.
I roto i te puku i runga i te maramara, me whakarerekē te mata hydrophobic o te membrane porous PDMS kia angitu ai te paninga ECM. I roto i tēnei kawa, e rua ngā tikanga rerekē ka whakamahia e mātou hei whakarerekē i te hydrophobicity o ngā membrane PDMS. Mō te whakatipu i ngā pūtau Caco-2, ko te whakahohenga mata mā te maimoatanga UV/ozone anake he rawaka hei whakaiti i te hydrophobicity o te mata PDMS, hei pani i te ECM me te tāpiri i ngā pūtau Caco-2 ki te membrane PDMS. Heoi, ko te ahurea microfluidic o te epithelium organoid me whai mahi mata matū hei whakatutuki i te whakatakotoranga whai hua o ngā pūmua ECM mā te tono i te polyethyleneimine (PEI) me te glutaraldehyde ki ngā microchannels PDMS. Whai muri i te whakarerekētanga mata, i whakatakotoria ngā pūmua ECM hei hipoki i te mata PDMS kua mahihia, kātahi ka whakaurua ki roto i te epithelium organoid motuhake. Whai muri i te whakapiringa o ngā pūtau, ka tīmata te ahurea pūtau microfluidic mā te whakauru noa i te reo ki roto i te microchannel o runga tae noa ki te hanga e ngā pūtau he monolayer katoa, ko te microchannel o raro e mau tonu ana i ngā āhuatanga pumau. Mā tēnei tikanga kua arotauhia mō te whakahohenga mata me te paninga ECM ka taea te whakapiri i te epithelium organoid ki te whakaoho i te morphogenesis 3D ki runga i te PDMS. mata.
Me pania hoki ngā ahurea Transwell ki te ECM i mua i te whakatōnga pūtau; heoi, kāore e hiahiatia he mahi maimoatanga uaua i mua i ngā ahurea Transwell hei whakahohe i te mata o ngā whakaurunga porous. Mō te whakatipu pūtau Caco-2 i runga i ngā whakaurunga Transwell, ka tere ake te whakapiri o ngā pūtau Caco-2 kua wehea (<1 hāora) me te hanganga arai hononga piri (<1-2 rā) mā te paninga ECM i runga i ngā whakaurunga porous. Hei whakatipu organoids i runga i ngā whakaurunga Transwell, ka whakatōngia ngā organoids motuhake ki runga i ngā whakaurunga kua pania ki te ECM, ka tāpirihia ki te mata o te membrane (<3 hāora) ā, ka puritia kia hanga rā anō ngā organoids i te paparanga kotahi me te hanganga arai. Ka mahia ngā ahurea Transwell i roto i ngā pereti 24-pū me te kore e whakamahi i ngā maramara ranu.
Ka taea te tīmata i te morphogenesis 3D in vitro mā te whakamahi i te rere wai ki te taha basolateral o te paparanga epithelial kua whakapumautia. I roto i te puku i runga i te maramara, i tīmata te morphogenesis epithelial i te wā i tukuna ai te reo ki roto i ngā microchannels o runga me raro (Pikitia 3a). E ai ki te whakaahuatanga o mua, he mea nui kia whakauruhia te rere wai ki te wāhanga o raro (basolateral) hei tango tonu i ngā aukati morphogen huna aronga. Hei whakarato i ngā matūkai me te serum e ranea ana ki ngā pūtau e herea ana ki ngā kiriuhi porous me te whakaputa i te ahotea kutikuti luminal, ka whakamahia e mātou te rere takirua i roto i te puku i runga i te maramara. I roto i ngā maramara ranu, i whakauruhia ngā whakaurunga Transwell kei roto ngā paparanga epithelial ki roto i ngā maramara ranu. Kātahi, ka whakamahia te reo i raro i te taha basolateral o te whakaurunga Transwell porous mā roto i te microchannel. I puta te morphogenesis puku 3-5 ngā rā i muri i te tīmatanga o te rere basolateral i roto i ngā papa ahurea e rua.
Ka taea te tātari i ngā āhuatanga āhua o ngā paparanga epithelial 3D i hangaia mā te whakamahi i ngā momo whakaahua, tae atu ki te microscopy rerekētanga ā-wāhanga, te microscopy rerekētanga rerekētanga (DIC), te SEM, te microscopy confocal immunofluorescence rānei (Pikitia 3 me te 4). Ka taea te mahi ngāwari i te whakaahua rerekētanga ā-wāhanga, te whakaahua DIC rānei i ngā wā katoa i te wā ahurea hei aroturuki i te āhua me te putanga mai o ngā paparanga epithelial 3D. Nā te māramatanga whatu o ngā kiriata PDMS me te polyester, ka taea e ngā papaaho gut-on-a-chip me ngā papaaho hybrid te whakarato whakaahua in situ wā-tūturu me te kore e hiahiatia te wehewehe, te wetewete rānei i te taputapu. I te wā e mahi ana i te whakaahua immunofluorescence (Pikitia 1, 3c, f me te 4b, c), ka whakapūmauhia ngā pūtau ki te 4% (wt/vol) paraformaldehyde (PFA), ā, ka whai ake ko Triton X-100 me te 2% (wt/vol) ) bovine serum albumin (BSA), kia rite ai. I runga i te momo pūtau, ka taea te whakamahi i ngā fixatives rerekē, ngā permeabilizers, me ngā kaihoko aukati. Ngā paturopi tuatahi e aro ana ki ngā pūtau e whakawhirinaki ana ki te whakapapa, ki te Ka whakamahia ngā tohu rohe hei whakaatu i ngā pūtau kua mau tonu ki runga i te maramara, ā, ka whai ake ko ngā paturopi tuarua me te tae counterstain e aro ana ki te karihi (hei tauira, 4′,6-diamidino-2-phenylene) indole, DAPI) F-actin rānei (hei tauira, phalloidin kua tohua ki te fluorescence). Ka taea hoki te mahi whakaahua ora e hangai ana ki te fluorescence i roto i te wāhi hei kite i te hanga huware (Pikitia 1, "Te rerekētanga pūtau" me te "Pūtaiao puku"), te koroni matapōkere o ngā pūtau moroiti (Pikitia 1, "Te ahurea tahi a te kaihautū-moroiti"), te kohikohinga o ngā pūtau ārai mate (Pikitia 1, 'Te Whakatauira Mate') ngā āhua rānei o te āhua epithelial 3D (Pikitia 3c,f me te 4b,c). I te whakarerekētanga o te puku i runga i te maramara hei wehe i te paparanga o runga mai i te paparanga microchannel o raro, e ai ki te whakaahuatanga i roto i te tohutoro. E ai ki te whakaaturanga i roto i te Pikitia 2, ka taea te tiro i te āhua epithelial 3D me te microvilli i runga i te rohe paraihe apical mā te SEM (Pikitia 3b). Ko te whakaaturanga o te rerekētanga Ka taea te aromatawai i ngā tohu mā te mahi i te PCR5 tauanga, te raupapatanga RNA pūtau-kotahi rānei. I tēnei wā, ka hauhakehia ngā paparanga 3D o ngā pūtau epithelial i whakatipuria i roto i ngā maramara puku, i ngā maramara ranu rānei mā te trypsinization, kātahi ka whakamahia mō te tātari ira, i te tātari ira rānei.
a, Rerenga mahi mō te whakaoho i te morphogenesis whekau i roto i te maramara ranu. Ka whakamahia a Caco-2 me ngā organoids whekau i roto i tēnei kawa hei whakaatu i te morphogenesis 3D i roto i te tūāpapa maramara ranu. I whakatōngia ngā pūtau epithelial wehea ki roto i ngā whakaurunga Transwell kua whakaritea (TW prep; tirohia te pikitia i raro nei). I muri i te whakatōngia (whakatōngia) o ngā pūtau me te piri ki ngā kiriuhi polyester i roto i ngā whakaurunga Transwell, i whakatōngia ngā pūtau katoa i raro i ngā āhuatanga tūmau (ahurea TW). I muri i ngā rā e 7, i whakauruhia he whakaurunga Transwell kotahi kei roto he paparanga kotahi 2D o ngā pūtau epithelial ki roto i tētahi maramara ranu hei whakauru i tētahi rere basolateral (Flow, BL), i te mutunga ka arahina ki te whakaputanga o tētahi paparanga epithelial 3D (morphogenesis). Ngā micrograph rerekētanga wāhanga e whakaatu ana i ngā āhuatanga morphological o ngā pūtau epithelial whekau tangata i ahu mai i te kaituku noa (raina C103) koroni piki i ia taahiraa whakamātautau, i ia wā rānei. Ko ngā mahere i roto i ngā paparanga o runga e whakaatu ana i te whirihoranga whakamātautau mō ia taahiraa. b, Ka taea e ngā maramara ranu (te mahere maui) te arahi ki te morphogenesis 3D o ngā pūtau epithelial organoid me ngā tirohanga microscopy confocal o runga-ki raro i tangohia i ngā tūranga Z rerekē (runga, waenganui, me raro; tirohia te kauwhata matau me ngā rārangi iraira e rite ana). i whakaatu i ngā āhuatanga āhuahanga mārama.F-actin (cyan), nucleus (hina).c, Ngā maikoropiko confocal fluorescence (tirohanga koki 3D) o ngā pūtau epithelial i ahu mai i te organoid i whakatipuria i roto i te Transwell tūmau (TW; whakaurunga i roto i te pouaka ma) me te maramara ranu (te whakaahua katoa nui rawa atu) e whakataurite ana i te āhuahanga 2D me te 3D, ia.E whakaatu ana hoki he takirua o ngā tirohanga tapahi poutū 2D (whakaurunga i te kokonga matau o runga; "XZ") i ngā āhuatanga 2D me te 3D.Pae tauine, 100 µm.c I tāia anō me te whakaaetanga mai i te tohutoro.4. Elsevier.
Ka taea te whakarite i ngā mana whakahaere mā te whakatipu i ngā pūtau ōrite (Caco-2, ngā pūtau epithelial organoid rānei o te puku) ki roto i ngā paparanga kotahi-ahu-rua i raro i ngā tikanga whakatipu pūmau tikanga. He mea nui, ka puta pea te whakahekenga matūkai nā te iti o te kaha rōrahi o ngā hongere iti (arā, ~4 µL i roto i te hongere o runga i te hoahoa maramara puku taketake). Nō reira, ka taea hoki te whakarite i te āhua o te epithelial i mua me muri i te whakamahinga o te rere basolateral.
Me mahi te tukanga whakakōrero ngohengohe i roto i tētahi rūma ma. Mō ia paparanga o te maramara (ngā paparanga o runga me raro me ngā kiriuhi) me ngā maramara ranu, i whakamahia ngā kopare whakaahua rerekē, ā, i hangaia ki runga i ngā anga silicon motuhake nā te mea he rerekē te teitei o ngā awaawa iti. Ko ngā teitei whainga o ngā awaawa iti o runga me raro o te puku i runga i te maramara he 500 µm me te 200 µm, ia. Ko te teitei whainga o te awaawa o te maramara ranu he 200 µm.
Whakatakotoria he anga silicon 3-inihi ki roto i tētahi rihi kua whakakiia ki te acetone. Āta hurihurihia te pereti mō te 30 hēkona, kātahi ka whakamarokehia ki te hau. Whakawhitihia te anga ki tētahi pereti kua whakakiia ki te IPA, kātahi ka hurihia te pereti mō te 30 hēkona kia horoia.
Heoi anō, ka taea te whakamahi i te otinga piranha (he ranunga o te hauwai peroxide me te waikawa whanariki kukū, 1:3 (vol/vol)) hei tango i ngā toenga rauropi mai i te mata o te papa silicon.
He tino waikura te wairewa Piranha, ā, ka puta he wera. Me whai i ētahi atu mahi ārai haumaru. Mō te whakakorenga para, tukua te wairewa kia whakamatao, kātahi ka whakawhiti ki tētahi ipu para ma, maroke. Whakamahia ngā ipu tuarua, ā, tāpirihia he tapanga tika ki ngā ipu para. Me whai i ngā aratohu haumaru o te whare mō ētahi atu tikanga taipitopito.
Whakamaroketia ngā papa angiangi mā te whakatakoto ki runga i te pereti wera 200 °C mō te 10 meneti. I muri i te whakamaroketanga, ka rūrūtia te papa angiangi kia rima ngā wā i te hau kia whakamatao.
Ringihia te ~10 karamu o te photoresist SU-8 2100 ki waenganui o te papa whakarewa silicon kua horoia. Whakamahia he tweezers hei hora i te photoresist kia ōrite ki runga i te papa whakarewa. I ētahi wā, whakatakotoria te papa whakarewa ki runga i te pereti wera 65°C kia kore ai e piri te photoresist, kia māmā ake ai te hora. Kaua e whakatakoto tika te papa whakarewa ki runga i te pereti wera.
I tohatohahia te SU-8 ki runga i te anga mā te whakahaere i te paninga hurihuri. Whakahōtakahia he hurihanga hou o te SU-8 mō te 5–10 hēkona kia horapa i te 500 rpm i te whakaterenga o te 100 rpm/s. Whakatakotoria te hurihuri matua mō te tauira matotoru 200 µm i te 1,500 rpm, ka whiwhi rānei i te matotoru 250 µm (kia 500 µm te teitei mō te paparanga o runga o te puku i runga i te maramara; tirohia "Ngā taahiraa nui" i raro nei) i te whakaterenga o te 300 rpm/s 30 hēkona i te 1,200 rpm.
Ka taea te whakarite i te tere hurihuri matua kia rite ki te matotoru ūnga o te tauira SU-8 i runga i te papa silicon.
Hei hanga i ngā tauira SU-8 he 500 µm te teitei mō te paparanga o runga o te puku i runga i te maramara, i tāruatia ngā takahanga paninga hurihuri me te tunu ngohengohe o tēnei Pouaka (ngā takahanga 7 me te 8) i ia wā (tirohia te takahanga 9) kia puta ai he paparanga e rua o te 250 µm te matotoru o te SU-8, ka taea te whakatakoto paparanga me te hono atu mā te hihi UV i te takahanga 12 o tēnei pouaka, kia 500 µm te teitei o te paparanga.
Tunua mārire ngā papa angiangi kua pania ki te SU-8 mā te whakatakoto mārire i ngā papa angiangi ki runga i te pereti wera i te 65 °C mō te 5 meneti, kātahi ka huri i te tautuhinga ki te 95 °C ka whakatōkia mō ētahi atu 40 meneti.
Hei whakatutuki i te teitei 500 μm o te tauira SU-8 i te microchannel o runga, tāruatia ngā taahiraa 7 me te 8 hei whakaputa i ngā paparanga SU-8 e rua, 250 μm te matotoru.
Mā te whakamahi i te UV Mask Aligner, whakamātauria te rama kia rite ki ngā tohutohu a te kaihanga hei tatau i te roa o te wā e whakaatuhia ana te anga. (te roa o te whakaaturanga, ms) = (te horopeta whakaaturanga, mJ/cm2)/(te mana o te rama, mW/cm2).
I muri i te whakatau i te wā whakaatu, whakatakotoria te kopare whakaahua ki runga i te pupuri kopare o te whakarārangi kopare UV, ka whakatakotoria te kopare whakaahua ki runga i te anga kua pania ki te SU-8.
Whakatakotoria te mata kua tāia o te kopare whakaahua ki te taha kua pania ki te SU-8 o te anga silicon hei whakaiti i te mararatanga UV.
Whakatakotoria poutū te angaanga kua pania ki te SU-8 me te kopare whakaahua ki te 260 mJ/cm2 o te mārama UV mō te wā whakaaturanga kua whakaritea (tirohia te taahiraa 10 o tēnei pouaka).
I muri i te pānga ki te hihi UV, i tunua ngā papa angiangi kua pania ki te SU-8 i te 65°C mō te 5 meneti me te 95°C mō te 15 meneti i runga i ia pereti wera hei hanga tauira me te teitei o te 200 μm. Whakaroahia te wā tunu i te 95 °C ki te 30 meneti hei hanga tauira me te teitei o te 500 µm.
Ka ringihia te kaiwhakawhanake ki roto i tētahi rihi karāhe, ā, ka whakatakotoria te wafer tunua ki roto i te rihi. Ka rerekē pea te rahinga o te kaiwhakawhanake SU-8 i runga i te rahi o te pereti karāhe. Kia tino whakamahia he kaiwhakawhanake SU-8 kia nui ai te tango i te SU-8 kāore anō kia kitea. Hei tauira, ina whakamahi ana i tētahi rihi karāhe 150 mm te whānui me te kaha 1 rita, whakamahia te ~300 mL o te kaiwhakawhanake SU-8. Whakawhanakehia te pokepoke mō te 25 meneti me te huri mārie i ētahi wā.
Horoia te pokepokea ai kua whanaketia ki te ~10 mL o te kaiwhakawhanake hou, ā, muri iho ka rehuhia te wai mā te whakamahi i te pipette ki te IPA.
Whakatakotoria te anga ki roto i te kaihoroi parāuma ka tukuna ki te parāuma hāora (hau hauātea, pēhanga ūnga 1 × 10−5 Torr, mana 125 W) mō te 1.5 meneti.
Whakatakotoria te angaanga ki roto i tētahi mīhini whakamaroke korehau me tētahi kiriata karāhe i roto. Ka taea te whakanoho i ngā angaanga me ngā kiriata taha ki taha. Mena ka wehea te mīhini whakamaroke korehau kia maha ngā paparanga e tētahi pereti, whakatakotoria ngā kiriata ki te rūma o raro, me ngā angaanga ki te rūma o runga. Ringihia te 100 μL o te otinga trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane ki runga i tētahi kiriata karāhe, ka whakamahia te korehau hei whakamaroke.
Whakarewahia tētahi pounamu o ngā pūtau Caco-2 tio ki roto i tētahi kaukau wai 37°C, kātahi ka whakawhitia ngā pūtau kua rewa ki tētahi pounamu T75 kei roto 15 mL o te wai Caco-2 kua whakamahanatia i mua i te 37°C.
Hei tuku i ngā pūtau Caco-2 i te ~90% huihuinga, tuatahi, whakauruhia he reo Caco-2 mahana, PBS, me te 0.25% trypsin/1 mM EDTA ki roto i te kaukau wai 37°C.
Mimia te wai mā te tango korehau. Horoia ngā pūtau kia rua ngā wā ki te 5 mL o te PBS mahana mā te tango korehau anō me te tāpiri i te PBS hou.