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Kuumbwa kwedumbu remunhu kunogadzira crypt-villus hunhu hwe3D epithelial microarchitecture uye kurongeka kwenzvimbo. Chimiro ichi chakasiyana chinodiwa kuchengetedza homeostasis yedumbu nekudzivirira niche yemasero ekutanga mu basal crypt kubva kuma antigen e exogenous microbial uye metabolites awo. Pamusoro pezvo, intestinal villi uye secreting mucus zvinopa masero epithelial akasiyana anoshanda ane chidziviriro pamusoro pemucosal yedumbu. Nokudaro, kugadzirazve 3D epithelial structures kwakakosha pakuvaka ma in vitro gut models. Zvinonyanya kukosha, organic mimetic gut-on-a-chip inogona kukonzera 3D morphogenesis inongoerekana yaitika ye intestinal epithelium nemabasa emuviri akawedzerwa uye biomechanics. Pano, tinopa nzira inodzokororwa yekukonzera morphogenesis yedumbu zvakasimba mudumbu pane microfluidic chip pamwe neTranswell embedded hybrid chip. Tinotsanangura nzira dzakadzama dzekugadzira mudziyo, kugadzira Caco-2 kana intestinal organoid epithelial cells munzvimbo dzakajairika pamwe nepa microfluidic platform, induction ye 3D morphogenesis, uye Kutsanangurwa kwe3D epithelia yakagadzirwa uchishandisa nzira dzakawanda dzekufungidzira. Iyi protocol inoita kuti pave nekuvandudzwa kwe functional gut microarchitecture nekudzora kufamba kwemvura yebasolateral kwe5 d. Nzira yedu ye in vitro morphogenesis inoshandisa physiologically related shear stress uye mechanical movement uye haidi complex cell engineering kana manipulation, izvo zvinogona kuita kupfuura dzimwe nzira dziripo. Tinofungidzira kuti protocol yedu yatakasarudza inogona kuva nemigumisiro yakakura kune vezvekutsvagisa biomedical, ichipa nzira yekugadzirisazve 3D intestinal epithelial layers in vitro yekushandiswa kwe biomedical, clinical, uye mishonga.
Kuedza kunoratidza kuti masero eCaco-2 emudumbu anomera mumidziyo yegut-on-a-chip1,2,3,4,5 kana bilayer microfluidic6,7 anogona kuita morphogenesis ye3D inongoerekana yaitika mu vitro pasina kunzwisisa kwakajeka kwemaitiro acho. Mukudzidza kwedu kwazvino, takaona kuti kubviswa kwevanopesana ne basolaterally secreted morphogen kubva mumidziyo yekurima kwakakosha uye kwakakwana kukonzera morphogenesis ye3D epithelial mu vitro, izvo zvakaratidzwa neCaco-2 uye ma intestinal organoids anobva kumurwere. Masero eEpithelial akaongororwa. Muchidzidzo ichi, takanyanya kutarisa pakugadzirwa kwemasero uye kugoverwa kwehuwandu hweWnt antagonist ine simba, Dickkopf-1 (DKK-1), mugut-on-a-chip uye mumidziyo yakagadziriswa microfluidic ine Transwell inserts, inonzi "Hybrid Chip". Tinoratidza kuti kuwedzerwa kweWnt antagonists dzekunze (dzakadai seDKK-1, Wnt repressor 1, yakabudisa frizzled-related protein 1, kana Soggy-1) mugut on-chip kunodzivirira morphogenesis kana kukanganisa prestructured 3D epithelial layer, zvichiratidza kuti antagonist stress panguva yekurima ndiyo inokonzera morphogenesis yemudumbu in vitro. Nokudaro, nzira inoshanda yekuwana morphogenesis yakasimba pa epithelial interface ndeyekubvisa kana kuchengetedza zvishoma huwandu hweWnt antagonists mu basolateral compartment ne active flushing (semuenzaniso, mu gut-on-a-chip kana hybrid-on-a-chip platforms) kana diffusion .Basolateral media (semuenzaniso, kubva kuTranswell inserts mu basolateral reservoirs hombe mu matsime).
Muchirongwa ichi, tinopa nzira yakadzama yekugadzira ma microdevices egut-on-a-chip uye Transwell-insertable hybrid chips (matanho 1-5) ekugadzira masero eepithelial emudumbu pa polydimethylsiloxane (PDMS)-based porous membranes (matanho 6A, 7A, 8, 9) kana polyester membranes yeTranswell inserts (matanho 6B, 7B, 8, 9) uye induced 3D morphogenesis in vitro (nhanho 10). Takaonawo maficha emasero nemamorekuru anoratidza histogenesis chaiyo yetishu uye kusiyana kwemasero kunoenderana nemutsara nekushandisa nzira dzakawanda dzekufungidzira (matanho 11-24). Tinokurudzira morphogenesis tichishandisa masero eepithelial emudumbu wemunhu, akadai seCaco-2 kana intestinal organoids, mumhando mbiri dzekurima dzine ruzivo rwehunyanzvi kusanganisira kugadziriswa kwepamusoro kwe membrane ine porous, kugadzirwa kwe2D monolayers, uye intestinal biochemical uye Kubereka kwe biomechanical microenvironment.in vitro.Kukurudzira 3D morphogenesis kubva ku2D epithelial mamonolayers, takabvisa morphogen antagonists mumhando dzese dzakakura nekuyerera medium muchikamu chepasi chetsika. Chekupedzisira, tinopa mufananidzo wekushandiswa kwe3D epithelial layer inogona kugadziriswa inogona kushandiswa kuratidza kukura kwemorphogen-dependent epithelial, longitudinal host-microbiome co-cultures, hutachiona hwehutachiona, kukuvara kwekuzvimba, kusashanda zvakanaka kwe epithelial barrier, uye probiotic-based therapy Example.influences.
Nzira yedu inogona kubatsira kune masayendisiti akasiyana-siyana muzvidzidzo zvekutanga (semuenzaniso, intestinal mucosal biology, stem cell biology, uye developmental biology) uye tsvakiridzo inoshandiswa (semuenzaniso, preclinical drug testing, disease modeling, tissue engineering, uye gastroenterology) broad impact. Nekuda kwekuberekana uye kusimba kwenzira yedu yekukonzera 3D morphogenesis ye intestinal epithelium in vitro, tinofungidzira kuti nzira yedu yehunyanzvi inogona kuparadzirwa kune vateereri vanodzidza nezvekuchinja kwemasero panguva yekukura kwe intestinal, regeneration kana homeostasis. Pamusoro pezvo, nzira yedu inobatsira pakubvunzurudza hutachiona pasi pezvirwere zvakasiyana-siyana zvakaita seNorovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 kana Vibrio cholerae. Vateereri vezvirwere uye pathogenesis zvinobatsirawo. Kushandiswa kwe-on-chip gut microphysiology system kunogona kubvumira longitudinal co-culture 10 uye kuongororwa kwakatevera kwekudzivirira kwemuponesi, mhinduro dzemuviri uye kugadziriswa kwekukuvara kunokonzerwa nehutachiona mudumbu (GI) tract 11. Mamwe matambudziko eGI ane chekuita ne leaky gut syndrome, chirwere checeliac, chirwere cheCrohn, ulcerative colitis, pouchitis, kana irritable bowel syndrome anogona kutevedzera kana 3D intestinal epithelial layers dzagadzirwa uchishandisa 3D intestinal epithelial layers yemurwere, zvirwere izvi zvinosanganisira villous atrophy, crypt shortening, mucosal damage, kana impaired epithelial barrier. Biopsy kana stem cell-derived intestinal organoids12,13. Kuti varatidze zviri nani kuomarara kwenzvimbo yechirwere, vaverengi vanogona kufunga nezvekuwedzera mhando dzemasero anoenderana nechirwere, senge patient peripheral blood mononuclear cells (PBMCs), kumamodeli ane 3D intestinal villus-crypt microarchitectures. tissue-specific immune cells, 5.
Sezvo 3D epithelial microstructure ichigona kugadziriswa uye kuonekwa pasina nzira yekuparadzanisa, vaoni vari kushanda pa spatial transcriptomics uye high-resolution kana super-resolution imaging vanogona kufarira mepu yedu ye spatiotemporal dynamics yemajini nemapuroteni pa epithelial niches. Vanofarira tekinoroji. Mhinduro kune microbial kana immune stimuli. Uyezve, longitudinal host-microbiome crosstalk 10, 14 iyo inobatanidza gut homeostasis inogona kuiswa mu 3D intestinal mucosal layer nekubatsirana kugadzira mhando dzakasiyana dze microbial, microbial communities kana fecal microbiota, kunyanya mu gut-on-a-chip. mupuratifomu. Maitiro aya anokwezva zvakanyanya kune vateereri vari kudzidza nezvekudzivirira muviri mudumbu, gastroenterology, human microbiome, culturomics uye clinical microbiology vari kutsvaga kurima gut microbiota isina kurimwa kare murabhoritari. Kana protocol yedu ye in vitro morphogenesis ichigona kugadziriswa kuti ive nemaitiro ekukura, akadai se multiwell inserts mu 24, 96 kana 384 well plates dzinogara dzichizadza basolateral compartments, protocol inogonawo kuparadzirwa kune avo vari kugadzira mishonga, biomedical kana high-throughput screening kana validation platforms dzeindasitiri yechikafu. Seuchapupu hwepfungwa, takaratidza munguva pfupi yapfuura kuti system ye multiplex high-throughput morphogenesis system inogona kukura kune 24-well plate format. Pamusoro pezvo, zvigadzirwa zvakawanda zve organ-on-a-chip zvakatengeswa16,17,18. Saka, kusimbiswa kwenzira yedu ye in vitro morphogenesis kunogona kukurumidziswa uye kunogona kugamuchirwa nemarabhoritari mazhinji ekutsvagisa, maindasitiri kana hurumende nemasangano ekutonga kuti vanzwisise kugadziridzwa kwemasero kwe in vitro gut morphogenesis padanho re transcriptomic kuti vaedze mishonga kana Kurapa Kwemuviri Kunyudzwa uye kutakurwa kwemishonga inoshandiswa mukurapa kwakaongororwa uchishandisa nzira dze3D gut kana kushandisa nzira dzekutengeserana dze organ-on-a-chip kuti vaone kugona kudzokororwa kwemaitiro emudumbu.
Mamodheru mashoma ekuyedza anoenderana nevanhu akashandiswa kudzidza morphogenesis yemudumbu, kunyanya nekuda kwekushaikwa kwemaprotocol anogona kushandiswa ekukurudzira morphogenesis ye3D mu vitro. Kutaura zvazviri, ruzivo rwakawanda rwemazuva ano nezve morphogenesis yemudumbu rwakavakirwa pazvidzidzo zvemhuka (semuenzaniso, zebrafish20, mbeva21 kana huku22). Zvisinei, anoshanda nesimba uye anodhura, anogona kuva asina chokwadi nezvetsika, uye chinonyanya kukosha, haanyatso sarudza maitiro ekukura kwevanhu. Mamodheru aya akaganhurirwawo mukukwanisa kwawo kuyedzwa nenzira inokwenenzverwa nenzira dzakawanda. Nokudaro, protocol yedu yekugadzirazve 3D tissue structures mu vitro inoshanda kupfuura mamodheru emhuka mu vivo pamwe nemamwe mamodheru etsika e2D static cell culture. Sezvakatsanangurwa kare, kushandisa 3D epithelial structures kwakatibvumira kuongorora nzvimbo yemasero akasiyana mu crypt-villus axis mukupindura kune zvakasiyana-siyana mucousal kana immune stimuli. 3D epithelial layers inogona kupa nzvimbo yekudzidza kuti ma microbial cells anokwikwidzana sei kugadzira nzvimbo dzenzvimbo uye evolution yezvakatipoteredza mukupindura kune zvinhu zvinogamuchira (semuenzaniso, inner versus matinji ekunze, kuburitswa kweIgA uye mapeptide anorwisa utachiona).Uyezve, 3D epithelial morphology inogona kutibvumira kunzwisisa kuti gut microbiota inoronga sei nharaunda dzayo uye inogadzira ma microbial metabolites (semuenzaniso, short-chain fatty acids) ayo anoumba kurongeka kwemasero uye stem cell niches mu basal crypts.Maitiro aya anogona kuratidzwa chete kana 3D epithelial layers dzagadzirwa mu vitro.
Pamusoro penzira yedu yekugadzira 3D intestinal epithelial structures, kune nzira dzakawanda dze in vitro. Intestinal organoid culture inzira yemazuva ano yekugadzira tishu yakavakirwa pakurima masero emudumbu pasi pemamiriro chaiwo e morphogen23,24,25. Zvisinei, kushandiswa kwe3D organoid models yekuongorora kutakurwa kana host-microbiome co-cultures kunowanzo kunetsa nekuti intestinal lumen yakavharirwa mukati me organoid uye, nekudaro, kuunzwa kwezvikamu zve luminal zvakaita se microbial cells kana exogenous antigens kwakaganhurirwa. Kuwana organoid lumens kunogona kuvandudzwa uchishandisa microinjector,26,27 asi nzira iyi inopindira uye inoda basa rakawanda uye inoda ruzivo rwakakosha kuti iite. Uyezve, tsika dzechinyakare dze organoid dzinochengetwa mu hydrogel scaffolds pasi pemamiriro ezvinhu asingachinji hazviratidzi nemazvo biomechanics inoshanda mu vivo.
Dzimwe nzira dzinoshandiswa nemapoka akati wandei ekutsvagisa dzinoshandisa 3D hydrogel scaffolds dzakagadzirwa kare kuti dzitevedzere chimiro che epithelial yemudumbu nekugadzira masero emudumbu evanhu akaparadzaniswa pamusoro pegel. Gadzira hydrogel scaffolds uchishandisa 3D-printed, micro-milled, kana lithographically yakagadzirwa molds. Iyi nzira inoratidza kurongeka kwemasero epithelial akaparadzaniswa mu vitro ane morphogen gradients dzakakosha mumuviri, zvichigadzira chimiro che epithelial chakakwira uye stroma-epithelial crosstalk nekubatanidza stromal cells muscaffold. Zvisinei, hunhu hwe scaffolds dzakagadzirwa kare hunogona kudzivirira kuratidzwa kweinongoerekana yaitika morphogenetic process pachayo. Aya mamodheru haapewo dynamic luminal kana interstitial flow, asina fluid shear stress iyo masero emudumbu anofanirwa kuita morphogenesis uye kuwana physiological function. Imwe ongororo yazvino yakashandisa hydrogel scaffolds mu microfluidic platform uye patterned intestinal epithelial structures uchishandisa laser-etching techniques. Mouse intestinal organoids dzinotevera etched patterns kuti dzigadzire intestinal tubular structures, uye intraluminal fluid flow inogona inofanira kudzokororwa uchishandisa module ye microfluidics. Zvisinei, modhi iyi hairatidzi maitiro ekuzviparadzanisa emuviri kana kufambisa mafambiro emuviri. Matekiniki ekudhinda e3D kubva muboka rimwe chete akakwanisa kugadzira machubhu madiki emudumbu ane maitiro ekuzviparadzanisa emuviri. Pasinei nekugadzirwa kwakaoma kwezvikamu zvakasiyana zvemudumbu mukati mechubhu, modhi iyi hainawo kuyerera kwemvura yechiedza uye kushanduka kwemuchina. Pamusoro pezvo, kushanda kwemodhi kunogona kushoma, kunyanya mushure mekunge maitiro ekunyora bioprinting apera, zvichikanganisa mamiriro ekuyedza kana kudyidzana kwesero nesero. Pane kudaro, protocol yedu yatinokurudzira inopa morphogenesis yemudumbu inongoerekana yaitika, shear stress inoenderana nemuviri, biomechanics inotevedzera kufamba kwemudumbu, kuwanikwa kwezvikamu zveapical nebasolateral zvakazvimiririra, uye kugadzirwazve kwenzvimbo dzakaoma dzemudumbu dze modularity. Nokudaro, protocol yedu ye in vitro 3D morphogenesis inogona kupa nzira yekuwedzera yekukunda matambudziko enzira dziripo.
Protocol yedu yakatarisana zvachose ne3D epithelial morphogenesis, ine masero epithelial chete ari mutsika uye hapana mamwe marudzi emasero akakomberedza akadai semasero emesenchymal, masero endothelial, uye masero ekudzivirira muviri. Sezvakatsanangurwa kare, musimboti weprotocol yedu kupinza epithelial morphogenesis nekubvisa morphogen inhibitors inoburitswa padivi re basolateral renzvimbo yakaunzwa. Nepo modularity yakasimba yegut-on-a-chip yedu uye hybrid-on-a-chip ichitibvumira kugadzirazve undulating 3D epithelial layer, mamwe ma complexes e biological akadai se epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition35 uye, mumuenzaniso wedu, crypt-villus features dzinoratidza stem cell niches mu basal crypts dzichiri kutarisirwa zvakanyanya. Stromal cells (semuenzaniso, fibroblasts) mu mesenchyme inoita basa guru mukugadzirwa kwemapuroteni eECM uye kutonga intestinal morphogenesis mu vivo35,37,38. Kuwedzerwa kwemasero emesenchymal Kumuenzaniso wedu kwakavandudza maitiro e morphogenetic uye kushanda zvakanaka kwekubatanidza masero. Chikamu che endothelial (kureva, capillaries kana lymphatics) chinoita basa rakakosha mukudzora kutakurwa kwemamorekuru39 uye kuunganidzwa kwemasero ekudzivirira muviri40 munzvimbo yemukati medumbu. Uyezve, zvikamu zveropa zvinogona kubatanidzwa pakati pemhando dzenyama zvinodiwa kana mhando dzenyama dzakagadzirirwa kuratidza kudyidzana kwenhengo dzakawanda. Nokudaro, masero e endothelial angangoda kuiswa kuti aratidze maitiro emuviri akanyatsojeka ane resolution yemazinga enhengo. Masero ekudzivirira muviri anowanikwa nemurwere akakoshawo pakuratidza mhinduro dzemuviri dzekudzivirira muviri, kuratidzwa kweantigen, kutaura kwemuviri kwemuviri kwekudzivirira muviri, uye kusadzivirira kwemuviri kwakanangana nezvirwere zvemudumbu.
Kushandisa ma hybrid chips kuri nyore pane gut-on-a-chip nekuti gadziriro yemuchina iri nyore uye kushandisa Transwell inserts kunobvumira kukura kwe epithelium yemudumbu. Zvisinei, ma Transwell inserts ane polyester membranes anowanikwa kumusika haana kusimba uye haakwanise kutevedzera kufamba kwakaita seperistaltic. Uyezve, apical compartment yeTranswell insert yakaiswa mu hybrid chip yakaramba yakamira isina shear stress kudivi re apical. Zviri pachena kuti static properties mu apical compartment haigone kugonesa kwenguva refu bacterial co-culture muma hybrid chips. Kunyange zvazvo tichigona kukurudzira 3D morphogenesis muTranswell inserts patinenge tichishandisa hybrid chips, kushomeka kwe physiologically related biomechanics uye apical fluid flow zvinogona kuderedza kugona kwema hybrid chip platforms ekushandisa.
Kugadziriswazve kwakazara kwe crypt-villus axis yemunhu mu gut-on-a-chip uye hybrid-on-a-chip cultures hakuna kunyatsojeka. Sezvo morphogenesis ichitanga kubva pa epithelial monolayer, 3D microarchitectures hazvireve kuti zvinopa kufanana kwe morphological ne crypts in vivo. Kunyangwe takaratidza huwandu hwemasero ari kuwedzera pedyo ne basal crypt domain mu microengineered 3D epithelium, crypt nenzvimbo dzakaipa hadzina kupatsanurwa zvakajeka. Kunyangwe nzira dzakakwirira dzepamusoro pa chip dzichitungamira kukukwirira kwakawedzerwa kwe microengineered epithelium, kukwirira kwakanyanya kuchiri kungogumira ku ~300–400 µm. Kudzika chaiko kwe crypts yemunhu mudumbu diki neguru i ~135 µm uye ~400 µm, zvichiteerana, uye kukwirira kwe small intestinal villi i ~600 µm41.
Kubva pakuona kwemifananidzo, mifananidzo ye3D microarchitectures inogona kuganhurirwa mudumbu riri pachip, sezvo daro rekushanda rinodiwa kubva pa lens rechinangwa kuenda ku epithelial layer riri pamamirimita mashoma. Kuti dambudziko iri rigadziriswe, chinangwa chiri kure chingadikanwa. Uyezve, kugadzira zvikamu zvitete zvekugadzirira mifananidzo yemifananidzo kwakaoma nekuda kwekusimba kwakanyanya kwePDMS. Uyezve, sezvo layer-by-layer microfabrication yedumbu pachip ichisanganisira kunamira zvachose pakati pe layer yega yega, zvakaoma zvikuru kuvhura kana kubvisa layer yepamusoro kuti uongorore chimiro chepamusoro che epithelial layer. Semuenzaniso, nekushandisa scanning electron microscope (SEM).
Kusapindirana kwePDMS mumvura kwave kuri chinhu chinodzivisa muzvidzidzo zve microfluidic-based zvine chekuita nemamorekuru madiki anopokana nemvura, sezvo PDMS ichigona kupinza mamorekuru akadaro anopokana nemvura. Dzimwe nzira dzePDMS dzinogona kutariswa nezvimwe zvinhu zvepolymeric. Neimwe nzira, kugadziriswa kwePDMS pamusoro (semuenzaniso, kuputira nezvinhu zvinopokana 42 kana poly(ethylene glycol) 43) kunogona kutariswa kuderedza kupindirana kwemamorekuru anopokana nemvura.
Chekupedzisira, nzira yedu haina kunyatsotsanangurwa zvakanaka maererano nekupa kuongorora kwepamusoro-soro kana puratifomu yekuedza "inokodzera zvese" iri nyore kushandisa. Iyi protocol yazvino inoda pombi yesirinji pa microdevice imwe neimwe, iyo inotora nzvimbo muCO2 incubator uye inodzivirira kuyedza kwakakura. Kuganhurirwa uku kunogona kuvandudzwa zvakanyanya nekukwanisa kukura kwemafomati matsva etsika (semuenzaniso, 24-well, 96-well, kana 384-well porous inserts inobvumira kuzadzazve nguva dzose uye kubviswa kwe basolateral media).
Kuti tiite kuti epithelium yemudumbu yemunhu iite 3D iite in vitro iite morphogenesis, takashandisa mudziyo we microfluidic chip intestinal une ma microchannel maviri akafanana uye elastic porous membrane iri pakati kuti tigadzire lumen-capillary interface. Tinoratidzawo kushandiswa kwe single-channel microfluidic device (hybrid chip) inopa continuous basolateral flow pasi pe polarized epithelial layers inokura paTranswell inserts. Mumapuratifomu ese ari maviri, morphogenesis yemasero akasiyana-siyana emudumbu yemunhu inogona kuratidzwa nekushandisa directional manipulation ye flow kubvisa morphogen antagonists kubva mu basolateral compartment. Maitiro ese ekuyedza (Mufananidzo 1) ane zvikamu zvishanu: (i) microfabrication ye gut chip kana Transwell insertable hybrid chip (matanho 1-5; Bhokisi 1), (ii) kugadzirira masero emudumbu epithelial (Caco-2 cells) kana human intestinal organoids; mabhokisi 2-5), (iii) kusimudzirwa kwemasero emudumbu pazvimedu zvemudumbu kana zvimedu zvehybrid (matanho 6-9), (iv) kutangwa kwe3D morphogenesis in vitro (nhanho 10) uye (v) ) kuratidza 3D epithelial microstructure (matanho 11-24). Pakupedzisira, boka rekudzora rakakodzera (rakakurukurwa pazasi) rakagadzirwa kusimbisa kushanda kwe in vitro morphogenesis nekuenzanisa epithelial morphogenesis kune spatial, temporal, conditional, kana procedural controls.
Takashandisa mapuratifomu maviri akasiyana ekugadzira: gut-on-a-chip ine nzira dzakatwasuka kana nzira dzisina kutenderera dzakasanganiswa, kana ma hybrid chips ane Transwell (TW) inserts mu microfluidic device, yakagadzirwa sezvakatsanangurwa muBhokisi 1, uye nhanho 1 -5. "Device Fabrication" inoratidza matanho makuru ekugadzira chip imwe chete kana hybrid chip. "Tsika yeHuman Intestinal Epithelial Cells" inotsanangura kwakabva masero (Caco-2 kana human intestinal organoids) uye maitiro ekugadzira anoshandiswa muprototi iyi. "In vitro morphogenesis" inoratidza matanho ese ayo Caco-2 kana organoid-derived epithelial cells dzinokura pa intestinal chip kana paTranswell inserts ye hybrid chip, zvichiteverwa ne induction ye 3D morphogenesis uye kuumbwa kwechimiro che epithelial chakatsanangurwa. Nhamba yedanho rechirongwa kana nhamba yebhokisi inoratidzwa pasi pemuseve wega wega. Chishandiso chinopa mienzaniso yekuti ma intestinal epithelial layers akasimbiswa anogona kushandiswa sei, semuenzaniso, mukusiyanisa masero, zvidzidzo zve physiology ye gut, kugadzwa kwe host-microbiome ecosystems, uye chirwere. kutevedzera. Mifananidzo yeImmunofluorescence mu "Cell Differentiation" inoratidza nuclei, F-actin uye MUC2 inoratidzwa mu3D Caco-2 epithelial layer inogadzirwa pagut chip. MUC2 signaling iripo mugoblet cells uye mucus inoburitswa kubva mu mucosal surfaces. Mifananidzo yeFluorescent muGut Physiology inoratidza mucus inogadzirwa nekuisa sialic acid neN-acetylglucosamine residues uchishandisa fluorescent wheat germ agglutinin. Mifananidzo miviri inopindirana mu "Host-Microbe Co-Cultures" inoratidza representative host-microbiome co-cultures mugut pa chip. Panel yekuruboshwe inoratidza co-culture yeE. coli expressing green fluorescent protein (GFP) ine microengineered 3D Caco-2 epithelial cells. Panel yerudyi inoratidza localization yeGFP E. coli co-cultured ne3D Caco-2 epithelial cells, ichiteverwa ne immunofluorescence staining neF-actin (tsvuku) uye nuclei (bhuruu). Kutevedzera chirwere kunoratidza hutano kana kubuda kwemudumbu. Zvipembenene zvekuzvimba zviri pasi pedambudziko remuviri nemaantigen ebhakitiriya (semuenzaniso, lipopolysaccharide, LPS) uye masero ekudzivirira muviri (semuenzaniso, PBMC; girini). Masero eCaco-2 akarimwa kuti agadzire 3D epithelial layer. Scale bar, 50 µm. Mifananidzo iri mumutsara wepazasi: "Kusiyana kwemasero" kwakagadziriswa nemvumo kubva kubhuku. 2. Oxford University Press; Yakaburitswazve nemvumo kubva kubhuku. 5. NAS; "Host-Microbe Co-Culture" yakagadziriswa nemvumo kubva kubhuku. 3. NAS; "Disease Modeling" yakagadziriswa nemvumo kubva kubhuku. 5. NAS.
Magut-on-chip nemahybrid chips zvakagadzirwa uchishandisa maPDMS replicas akabviswa kubva musilicon molds ne soft lithography1,44 uye akaiswa mapatani neSU-8. Dhizaini yema microchannels mu chip yega yega inoonekwa nekufunga nezve hydrodynamics dzakadai se shear stress uye hydrodynamic pressure1,4,12. Dhizaini yekutanga ye gut-on-a-chip (Extended Data Fig. 1a), iyo yaive nema juxtaposed parallel straight microchannels maviri, yakashanduka kuita complex gut-on-a-chip (Extended Data Fig. 1b) inosanganisira ma curved microchannels maviri kuti awedzere nguva yekugara kwemvura, nonlinear flow patterns, uye multiaxial deformation yemasero akakurira (Fig. 2a–f) 12. Kana biomechanics dzakaoma dzegut dzichida kugadzirwazve, complex gut-on-a-chips dzinogona kusarudzwa. Takaratidza kuti convoluted Gut-Chip inokonzerawo zvakanyanya 3D morphogenesis munguva yakafanana ine degree yakafanana ye epithelial growth zvichienzaniswa nepakutanga. Gut-Chip, zvisinei nerudzi rwemasero akakurira. Saka, kuti zvikonzere 3D morphogenesis, magadzirirwo egut ane mutsara uye akaomarara e-on-chip anogona kuchinjana. PDMS replicates dzakarapwa pasilicon molds neSU-8 patterns dzakapa zvinhu zvisina kunaka mushure mekubviswa (Mufananidzo 2a). Kuti vagadzire ura pachip, PDMS layer yepamusoro yakagadzirwa yakasungirirwa kufirimu rePDMS rine maburi uye yakazobatanidzwa nePDMS layer yepasi nekubatanidza kusingadzoreki uchishandisa corona treater (Mufananidzo 2b–f). Kuti vagadzire ma hybrid chips, ma PDMS replicas akarapwa akasungirirwa kuma glass slides kuti vagadzire single-channel microfluidic devices dzinogona kugamuchira Transwell inserts (Mufananidzo 2h uye Extended Data Fig. 2). Maitiro ekubatanidza anoitwa nekurapa pamusoro pePDMS replica negirazi ne oxygen plasma kana corona treatment. Mushure mekuvhiyiwa kwechigadzirwa che microfabricated chakasungirirwa pa silicone tube, mudziyo wakagadzirirwa kuita 3D morphogenesis ye intestinal epithelium (Mufananidzo 2g).
a, Mufananidzo wechimiro chekugadzirira zvikamu zvePDMS kubva muzviumbwa zvesilicon zvakagadzirwa neSU-8. Mhinduro yePDMS isina kurapwa yakadururwa pasilicon mold (kuruboshwe), yakanatswa pa60 °C (pakati) ndokubviswa (kurudyi). PDMS yakabviswa yakachekwa kuita zvidimbu ndokucheneswa kuti ishandiswezve.b, Mufananidzo wesilicon mold yakashandiswa kugadzirira PDMS upper layer.c, Mufananidzo wesilicon mold inoshandiswa kugadzira PDMS porous membrane.d, Mifananidzo yakatevedzana yezvikamu zvePDMS zvepamusoro nepazasi uye mudziyo wepain-chip wakaunganidzwa.e., Mufananidzo wekugadzirisa zvikamu zvePDMS zvepamusoro, zvepazasi. Chikamu chimwe nechimwe chinosungirirwa zvisingadzoreki ne plasma kana corona treatment.f, Mufananidzo wemudziyo wegut-on-a-chip wakagadzirwa une superimposed convoluted microchannels uye vacuum chambers.g, Kugadzwa kwegut-on-a-chip ye microfluidic cell culture. Gut yakagadzirwa pachip yakaunganidzwa ne silicone tube ne syringe yakaiswa pa coverlip. Chigadzirwa chechip chakaiswa pachivharo che Dhishi rePetri re150 mm rekugadzirisa. Chinosunga chinoshandiswa kuvhara chubhu yesilicone.h, Mifananidzo yekuona yekugadzira chip yakasanganiswa uye 3D morphogenesis uchishandisa ma hybrid chips. Transwell inserts dzakagadzirirwa dzakazvimiririra kuti dzikure. 2D monolayers yemasero epithelial emudumbu akaiswa mu hybrid chip kuti aite 3D morphogenesis yemudumbu. Nzvimbo yacho inoyerera kuburikidza nema microchannels ari pasi pe cell layer yakaiswa paTranswell insert.Scale bar, 1 cm.h Yakadhindwazve nemvumo kubva kune reference.4. Elsevier.
Muchirongwa ichi, Caco-2 cell line uye intestinal organoids zvakashandiswa se epithelial sources (Mufananidzo 3a). Marudzi ese emasero akarimwa ega (Bhokisi 2 neBhokisi 5) uye akashandiswa kudyara ma microchannels akaputirwa neECM e on-chip gut kana Transwell inserts. Kana masero akabatana (>95% kufukidza mumaflasks), masero eCaco-2 akarimwa nguva dzose (pakati pema passages 10 ne50) muT-flasks anokohwewa kugadzirira dissociated cell suspensions ne trypsinization fluid (bhokisi 2). Human intestinal organoids kubva mu intestinal biopsies kana surgical resections akarimwa muMatrigel scaffold domes muma 24-well plates kutsigira structural microenvironment. Medium containing essential morphogens (senge Wnt, R-spondin, naNoggin) uye growth factors dzakagadzirwa sezvakatsanangurwa muBhokisi 3 dzaiwedzerwa mazuva ese kusvika organoids dzakura kusvika ~500 µm mu dhayamita. Organoids dzakakura zvakakwana dzinokohwewa uye yakakamurwa kuita masero ega ega kuti idyare mudumbu kana Transwell inserts pa chip (Bhokisi 5). Sezvatakambotaura, inogona kusiyaniswa zvichienderana nerudzi rwechirwere 12,13 (semuenzaniso ulcerative colitis, chirwere cheCrohn, kenza yemudumbu, kana donor yakajairika), nzvimbo yechironda (semuenzaniso, chironda kana nzvimbo isina maronda) uye nzvimbo yemudumbu mugwara (semuenzaniso, duodenum, jejunum, ileum, cecum, colon, kana rectum). Tinopa nzira yakagadziriswa muBhokisi 5 yekurima colonic organoids (coloids) inowanzoda huwandu hwakawanda hwe morphogens kupfuura small intestinal organoids.
a, Kufamba kwebasa rekusimudzira morphogenesis yemudumbu mugut chip. Caco-2 human intestinal epithelium uye intestinal organoids zvinoshandiswa muprototype iyi kuratidza morphogenesis ye3D. Masero eepithelial akaparadzaniswa akadyarwa mumudziyo wakagadzirwa wegut-on-a-chip (kugadzirira chip). Kana maseru adyarwa (kudyarwa) uye akabatanidzwa (akabatanidzwa) kuPDMS porous membrane pazuva re0 (D0), apical (AP) flow inotanga uye inochengetwa kwemazuva maviri ekutanga (flow, AP, D0-D2). Basolateral (BL) flow inotangwawo pamwe chete nekufamba kwecyclic stretching motions (stretch, flow, AP uye BL) kana 2D monolayer yakazara yaumbwa. Intestinal 3D morphogenesis yakaitika yoga mushure memazuva mashanu e microfluidic culture (morphogenesis, D5). Phase contrast images inoratidza morphology inomiririra yeCaco-2 cells padanho rega rega rekuyedza kana nguva (bar graph, 100 µm). Mana schematic diagrams anoratidza cascades dzinoenderana yekuchinja kwemudumbu (kumusoro kurudyi). Miseve ine maburi mumufananidzo inomiririra gwara rekuyerera kwemvura.b, mufananidzo weSEM unoratidza topology yepamusoro pe3D Caco-2 epithelium (kuruboshwe). Chikamu chinotaridza nzvimbo yakakura (bhokisi rine maburi machena) chinoratidza microvilli yakagadziriswazve pa3D Caco-2 layer (kurudyi).c, Kutarisa kwakatwasuka kweCaco-2 3D yakasimwa, claudin (ZO-1, tsvuku) uye membrane yemuganhu webhurashi inoenderera mberi yakanyorwa kuti F-actin (girini) uye nuclei (bhuruu). Immunofluorescence confocal visualization yemasero epithelial pamachipisi emudumbu. Miseve inonongedzera kumufananidzo wepakati inoratidza nzvimbo yenzvimbo yekutarisa yemaonero ega ega e confocal.d, Nguva yekuchinja kwechimiro muma organoids akakurira pa chip yakawanikwa ne phase contrast microscopy pamazuva 3, 7, 9, 11, uye 13. Chikamu chinotaridza (kumusoro kurudyi) chinoratidza kukura kwakanyanya kwemufananidzo wakapihwa.e, DIC photomicrograph ye organoid 3D epithelium yakasimwa Mudumbu pachikamu chakatorwa pazuva rechinomwe.f, Mifananidzo ye immunofluorescence yakafukidza inoratidza zviratidzo zvemasero emuviri (LGR5; magenta), masero egoblet (MUC2; girini), F-actin (grey) uye nuclei (cyan) zvakakurira pamachipisi emudumbu kwemazuva matatu, zvichiteerana (Kuruboshwe) uye 13-day (pakati) organoids pa epithelial layer.Onawo Extended Data Figure 3, iyo inoratidza LGR5 signaling isina MUC2 signaling.Mifananidzo ye fluorescence inoratidza epithelial microstructure (kurudyi) ye 3D organoid epithelium yakamiswa mudumbu pachip nekuisa plasma membrane neCellMask dye (kurudyi) pazuva regumi nematatu re culture.Scale bar i50 μm kunze kwekunge zvataurwa neimwe nzira.b Yakadhindwazve nemvumo kubva ku reference.2. Oxford University Press; c Yakagadziriswa nemvumo kubva ku Reference.2. Oxford University Press; e uye f yakagadziriswa nemvumo ne reference.12 Pasi pe Creative Commons License CC BY 4.0.
Mudumbu pane chip, zvinodikanwa kugadzirisa pamusoro pe hydrophobic ye PDMS porous membrane kuti ECM coating ibudirire. Muchirongwa ichi, tinoshandisa nzira mbiri dzakasiyana kugadzirisa hydrophobic ye PDMS membranes. Pakugadzira Caco-2 cells, kushanda pamusoro ne UV/ozone treatment chete kwaive kwakakwana kuderedza hydrophobic ye PDMS surface, kufukidza ECM uye kubatanidza Caco-2 cells kuPDMS membrane. Zvisinei, microfluidic culture ye organoid epithelium inoda chemical-based surface functionalization kuti iwane kuiswa zvakanaka kwemapuroteni eECM nekushandisa polyethyleneimine (PEI) uye glutaraldehyde kuPDMS microchannels. Mushure mekugadzirisa surface, ECM proteins dzakaiswa kuti dzifukidze functionalized PDMS surface uye dzozopinzwa mu isolated organoid epithelium. Mushure mekunge masero abatanidzwa, microfluidic cell culture inotanga nekungoisa medium mu upper microchannel kusvika masero agadzira monolayer yakazara, nepo microchannel yakaderera ichichengetedza static conditions. Iyi nzira yakagadziriswa yekushanda pamusoro uye ECM coating inobvumira kusungirirwa kwe organoid. epithelium yekukonzera 3D morphogenesis pamusoro pePDMS.
Kukura kwemasero eTranswell kunodawo kuputirwa kweECM vasati vadyara masero; zvisinei, kuumbwa kwemasero eTranswell hakudi matanho akaoma ekurapa kuti kuite kuti pamusoro pemasero ane maburi ashande. Pakukura kwemasero eCaco-2 paTranswell inserts, kuputirwa kweECM pamasero ane maburi kunokurumidza kunamatira kwemasero eCaco-2 akaparadzaniswa (
Hunhu hwema microengineered 3D epithelial layers hunogona kuongororwa nekushandisa nzira dzakasiyana dzekufungidzira, kusanganisira phase contrast microscopy, differential interference contrast (DIC) microscopy, SEM, kana immunofluorescence confocal microscopy (Mifananidzo 3 ne4). Phase contrast kana DIC imaging inogona kuitwa nyore nyore chero nguva panguva yekurima kuti iongorore chimiro uye kubuda kwe3D epithelial layers. Nekuda kwekujeka kwePDMS nemafirimu epolyester, mapuratifomu egut-on-a-chip uye hybrid chip anogona kupa real-time in situ imaging pasina chikonzero chekuparadzanisa kana kuparadzira mudziyo. Paunenge uchiita immunofluorescence imaging (Mifananidzo 1, 3c, f na 4b, c), masero anowanzo gadziriswa ne4% (wt/vol) paraformaldehyde (PFA), ichiteverwa neTriton X-100 uye 2% (wt/vol)) bovine serum albumin (BSA), zvakateerana. Zvichienderana nerudzi rwesero, fixatives dzakasiyana, Zvinodzivirira masero, uye zvinodzivirira zvinogona kushandiswa. Ma antibodies ekutanga anotarisa mutsara-unoenderana nemasero kana nzvimbo zviratidzo zvinoshandiswa kuratidza masero asina kufamba-famba ari panzvimbo iri pa chip, zvichiteverwa nema antibodies echipiri pamwe chete ne counterstain dye inotarisa nucleus (semuenzaniso, 4′, 6-diamidino-2-phenylene) indole, DAPI) kana F-actin (semuenzaniso, phalloidin yakanyorwa fluorescently). Kufungidzira kwe fluorescence-based live kunogonawo kuitwa panzvimbo iri kuti kuone kugadzirwa kwemachubhu (Mufananidzo 1, "Kusiyanisa kwemasero" uye "Gut physiology"), kuunganidzwa kwema microbial cells (Mufananidzo 1, "Host-microbe co-culture"), kuunganidzwa kwemasero ekudzivirira muviri (Mufananidzo 1, 'Disease Modeling') kana contours ye 3D epithelial morphology (Mufananidzo 3c, f na 4b, c). Pakugadzirisa gut pa chip kuti iparadzanise chikamu chepamusoro kubva kune chikamu chezasi che microchannel, sezvakatsanangurwa mu ref.Sezvinoratidzwa muMufananidzo 2, 3D epithelial Maumbirwo pamwe chete ne microvilli iri pamuganhu we apical brush zvinogona kuratidzwa neSEM (Mufananidzo 3b). Kuratidzwa kwezviratidzo zvekusiyanisa kunogona kuongororwa nekuita quantitative PCR5 kana single-cell RNA sequencing. Muchiitiko ichi, 3D layers ye epithelial cells inokura muma gut chips kana hybrid chips inokohwewa ne trypsinization uye yozoshandiswa kuongorora mamorekuru kana majini.
a, Kufamba kwebasa rekusimudzira morphogenesis yemudumbu mu hybrid chip. Caco-2 uye intestinal organoids zvinoshandiswa muprototi iyi kuratidza 3D morphogenesis mu hybrid chip platform. Dissociated epithelial cells dzakadyarwa mu prepared Transwell inserts (TW prep; ona mufananidzo uri pazasi). Kana masero adyarwa (mbeu) uye akabatanidzwa ku polyester membranes muTranswell inserts, masero ese akarimwa pasi pemamiriro ezvinhu asingachinji (TW culture). Mushure memazuva manomwe, Transwell insert imwe chete ine 2D monolayer ye epithelial cells yakabatanidzwa mu hybrid chip kuti iunze basolateral flow (Flow, BL), izvo zvakazoita kuti pave ne3D epithelial layer (morphogenesis). Phase contrast micrographs inoratidza morphological features ye human organ epithelial cells inobva kune normal donor (C103 line) ascending colon padanho rega rega re experiment kana nguva. Schematics mu upper layers inoratidza experimental configuration ye entry ye one step.b, Hybrid chips (left schematic) inogona kutungamira ku 3D morphogenesis ye organoid epithelial masero ane maonero epamusoro-pasi e confocal microscopy akatorwa panzvimbo dzakasiyana dzeZ (kumusoro, pakati, uye pasi; ona mitsetse yekurudyi ine schematic uye inoenderana nemadotsi). yakaratidza hunhu hwakajeka hwe morphological.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled view) yemasero e epithelial anobva ku organoid akakurira mu static Transwell (TW; inset mukati mebhokisi jena rakacheneswa) zvichienzaniswa ne hybrid chip (largest full shot) ichienzanisa 2D zvichienzaniswa ne 3D morphology, zvichiteerana. Peya ye 2D vertical cross-cut views (inset mukona yepamusoro yekurudyi; "XZ") inoratidzawo 2D ne 3D features.Scale bar, 100 µm.c Yakadhindwazve nemvumo kubva kune reference.4. Elsevier.
Kudzora kunogona kugadzirwa nekugadzira masero akafanana (Caco-2 kana masero epithelial epithelial emukati) kuita maviri-dimensional monolayers pasi pemamiriro etsika akajairwa. Zvinonyanya kukosha, kupera kwezvinovaka muviri kunogona kukonzerwa nekukwanisa kushoma kwe microchannels (kureva ~4 µL muchikamu chepamusoro pane dhizaini yekutanga yegut-chip). Saka, chimiro che epithelial chisati chashandiswa uye mushure mekushandiswa kwe basolateral flow chinogonawo kuenzaniswa.
Maitiro e soft lithography anofanira kuitwa muimba yakachena. Pachikamu chimwe nechimwe chiri pa chip (zvikamu zvepamusoro nepazasi uye membranes) uye ma hybrid chips, ma photomasks akasiyana akashandiswa uye akagadzirwa pa silicon wafers dzakasiyana nekuti kukwirira kwema microchannels kwakanga kwakasiyana. Kureba kwenzvimbo dzepamusoro nepazasi dzemudumbu pa chips i500 µm uye 200 µm, zvichiteerana. Kureba kwenzvimbo dze hybrid chip i200 µm.
Isa wafer yesilicon ine 3-inch mundiro ine acetone. Peta ndiro zvinyoro nyoro kwemasekonzi makumi matatu, wobva waomesa wafer nemhepo. Isa wafer mundiro ine IPA, wozotenderedza ndiro kwemaminitsi makumi matatu kuti icheneswe.
Mhinduro yepiranha (musanganiswa wehydrogen peroxide ne concentrated sulfuric acid, 1:3 (vol/vol)) inogona kushandiswa zvakanyanya kubvisa zvasara zve organic kubva pamusoro pesilicon wafer.
Mushonga wePiranha unokuvadza zvakanyanya uye unokonzera kupisa. Mamwe matanho ekuchengetedza anodiwa. Pakuraswa kwemarara, rega mushonga utonhore wobva wauisa mumudziyo wemarara wakachena, wakaoma. Shandisa mimwe midziyo woisa mazita emidziyo yemarara zvakanaka. Ndokumbirawo mutevedzere nhungamiro dzekuchengetedza dzenzvimbo iyi kuti muwane mamwe maitiro akadzama.
Sungunusai mvura mumawafers nekuisa pa 200 °C inopisa kwemaminitsi gumi. Mushure mekushaya mvura, wafer yakazununguswa kashanu mumhepo kuti itonhore.
Dururirai ~10 g ye photoresist SU-8 2100 pakati pe silicon wafer yakacheneswa. Shandisa ma tweezers kuparadzira photoresist zvakaenzana pa wafer. Dzimwe nguva isa wafer pa 65°C hot plate kuti photoresist isanyanya kunamira uye ive nyore kuparadzira. Usaise wafer zvakananga pa hot plate.
SU-8 yakagoverwa zvakaenzana pawafer nekushandisa spin coating. Ronga kutenderera kunouya kweSU-8 kwemasekonzi mashanu kusvika gumi kuti ipararire pa500 rpm nekukurumidzisa kwe100 rpm/s. Isa main spin patterning yeukobvu hwe200 µm pa1,500 rpm, kana kuwana ukobvu hwe250 µm (zvichiita kuti kukwirira kwe500 µm kwechikamu chepamusoro chegumbu pachip; ona "Matanho akakosha" pazasi) akaiswa pakukurumidzisa kwe300 rpm/s nemasekonzi makumi matatu pa1,200 rpm.
Kumhanya kukuru kwekutenderera kunogona kugadziriswa zvichienderana neukobvu hweSU-8 pateni iri pasilicon wafer.
Kuti vagadzire mapatani eSU-8 ane urefu hwe500 µm echikamu chepamusoro chedumbu chiri pachipu, kupeta kwespin uye matanho ekubika akapfava eBhokisi iri (matanho 7 ne8) zvakadzokororwa zvakatevedzana (ona danho 9) kuti pave nezvikamu zviviri zve250 µm. Chikamu chakakora cheSU-8, chinogona kubatanidzwa nekuratidzwa kweUV muchikamu 12 chebhokisi iri, zvichiita kuti chikamu chikwire che500 µm.
Bika zvinyoro nyoro mawafers akaputirwa neSU-8 nekuisa mawafers acho zvakanaka padhishi rinopisa pa65 °C kwemaminitsi mashanu, wobva wachinja mamiriro acho kuita 95 °C woaisa kwemamwe maminitsi makumi mana.
Kuti uwane kukwirira kwe500 μm kweSU-8 pattern mu microchannel yepamusoro, dzokorora matanho 7 ne8 kuti ugadzire 250 μm SU-8 layers dzakakora.
Uchishandisa UV Mask Aligner, ita bvunzo yerambi zvichienderana nemirairo yemugadziri kuti uverenge nguva yekubuda kwewafer. (nguva yekubuda, ms) = (dose yekubuda, mJ/cm2)/(simba remwenje, mW/cm2).
Mushure mekuona nguva yekubuda, isa photomask pachibati chemasiki cheUV mask aligner woisa photomask paSU-8 coated wafer.
Isa pamusoro pemask yakadhindwa zvakananga padivi reSU-8 rakaputirwa nesilicon wafer kuderedza kupararira kweUV.
Isa wafer yakaputirwa neSU-8 uye photomask yakatwasuka kusvika pa260 mJ/cm2 yechiedza cheUV kwenguva yakatarwa yekuratidzwa (ona danho rechi10 rebhokisi rino).
Mushure mekuvhenekerwa neUV, mawafer esilicon akaputirwa neSU-8 akabikwa pa65°C kwemaminitsi mashanu uye 95°C kwemaminitsi gumi nemashanu pahot plate yega yega kuti vagadzire mapatani ane hurefu hwe200 μm. Wedzera nguva yekubheka mushure mekubika pa95°C kusvika pamaminitsi makumi matatu kuti vagadzire mapatani ane hurefu hwe500 µm.
Mugadziri anodururwa mundiro yegirazi, uye wafer yakabikwa inoiswa mundiro. Huremu hwemugadziri weSU-8 hunogona kusiyana zvichienderana nehukuru hwendiro yegirazi. Iva nechokwadi chekuti washandisa mugadziri weSU-8 wakakwana kuti ubvise zvachose SU-8 isina kusviba. Semuenzaniso, kana uchishandisa dhishi regirazi re150 mm dhayamita rine simba re1 L, shandisa ~300 mL yemugadziri weSU-8. Gadzira mold kwemaminitsi makumi maviri nemashanu uchitenderedza zvinyoro nyoro.
Sukurudza chakuvhuvhu chakagadzirwa ne ~10 mL yemuchina mutsva unogadzirwa uchizoteverwa neIPA nekupfapfaidza mushonga uchishandisa pipette.
Isa wafer mu plasma cleaner woiisa mu oxygen plasma (mhepo ine gasi, target pressure 1 × 10−5 Torr, simba 125 W) kwemaminitsi 1.5.
Isa wafer mu vacuum desiccator ine girazi rinotsveyama mukati. Mawafer nemasiraidhi zvinogona kuiswa padivi nepadivi. Kana vacuum desiccator yakakamurwa kuita zvikamu zvakasiyana neplate, isa masiraidhi mukamuri repasi uye mawafer mukamuri repamusoro. Donhedza 100 μL ye trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane solution pane girazi rinotsveyama woisa vacuum kuti silanization ishande.
Nyungudutsa bhodhoro remasero eCaco-2 akaomeswa nechando muchirongo chemvura che37°C, wobva waendesa masero acho akanyungudika mubhodhoro reT75 rine 15 mL ye37°C inodziya Caco-2 medium.
Kuti masero eCaco-2 apfuure panosvika 90% yekubatana, tanga waisa Caco-2 medium inodziya, PBS, uye 0.25% trypsin/1 mM EDTA mumvura inodziya ye37°C.
Fukidza masero kaviri ne5 mL yePBS inodziya nekudzokorora kufukidzwa kwevacuum uye kuwedzera PBS itsva.
Nguva yekutumira: Chikunguru-16-2022
