The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

1. Bir Ters Model Sistemde Tohum HIBCPP Hücreleri Hücre Kültürü Filtre Uçlar hazırlayın <ol><li> 5 ug / ml insülin ile takviye edilmiş ön sıcak DMEM / F12 (HAM), 100 U / ml penisilin, 100 ug / ml streptomisin ve% 10 cenin buzağı serumu (FCS). </li><li> Baş aşağı 12 oyuklu plaka (Şekil 1 E) olarak 3 um arasında bir gözenek boyutuna sahip 0,33 cm² büyüme alanı, hücre kültürü filtre malzemeleri yer steril forseps kullanın. </li><li> Hücre kültürü filtre elemanının alt bölmeye (3 mi) ve filtre elemanının üzerine 100 ul içinde orta doldurun. plaka yanı sıra orta alt bölme sel. Filtre elemanının alt bölmesi (Şekil 1 E) dolu kalır şekilde aşırı ortam aspire. Not: adım için bir serolojik pipet kullanılması tavsiye edilir. </li><li> , Kapaklı 12-plaka Kapak ve inkübatör hazırlanan hücre kültürü filtre ekler aktarmak 37 ° C,Hücrelerin ilave edilene kadar% 5 CO2. </li></ol> 2. Yetiştirme ve HIBCPP Hücrelerinin Pasajlanması <ol><li> 5 ug / ml, 100 U / ml penisilin, 100 ug / ml streptomisin ve% 10 FCS ile takviye edilmiş DMEM / F12 (HAM) hazırlayın. </li><li> 37 ° C su banyosunda Pre-sıcak orta ve PBS. şişeye aspire ortamı. şişeye ve girdap 10 ml PBS ekleyin. kez bu adımı yineleyin. </li><li> şişeye ve girdap 3 mi,% 0.25 tripsin-EDTA ekleyin. , Inkübatör içine 37 ° C,% 5 CO2 yerleştirin. </li><li> Yaklaşık 20 dakika sonra inkübatörden balon çıkarın. Hücreler şişenin dibine kopuk olduğundan emin olun ve mikroskop ile incelenen zaman yuvarlak bir şekle görüntüler. Not: hücreler, birbirinden tamamen ayırmak ve bu nedenle sıklıkla topakların bulunurlar. Geçit 38&#39;e kadar hücrelerin kullanımı tavsiye edilir. </li><li> 17 ml orta ekleyin trypsinization durdurmak için. aşağı yukarı pipetleme hücreleri tekrar süspansiyon ve süspansiyonu transfer50 ml&#39;lik bir tüp içine. Oda sıcaklığında 10 dakika boyunca 50 x g&#39;de santrifüjleyin. </li><li> ortamın uygun hacimde bir hücre yeniden süspanse edin ve bir hemositometre kullanılarak hücre sayısı. Not: yeniden süspansiyon haline HIBCPP hücre konsantrasyonu 1 x 10 6 hücre / mL olmalıdır. HIBCPP hücrelerin korunması için, T75-şişeye 10 mi ortam içinde 1-6 x 10 6 hücre bir miktarı aktarmak için önerilmektedir. her ikinci gün orta değiştirin. </li></ol> HIBCPP Hücreleri ile 3. Tohumculuk Ters Hücre Kültürü Filtre ekler <ol><li> (Filtrenin alt tarafına, yani), her bir ters filtre elemanının üzerine hücre süspansiyonuna (yani. 8 x 10 4 hücre) (Şekil 1 E) 80 ul ekle. Not: Hücreler eşit tohumlama önce tersini tüp süspansiyon dağıtılır emin olun. </li><li> inkübatör 12 oyuklu plaka ve transfer kapak, 37 ° C&#39;de,% 5 C ile tohumlanır-hücre kültür filtre ekler KapakO 2. </li><li> İlk gün, 24 oyuklu bir plaka gözlerine 1 mi ortam doldurun. 12 plaka dışına forseps kullanarak hücre kültürü filtre ekler kaldırın, iç orta atmak filtre ekler açmak ve hazırlanan 24 plaka (Şekil 1E) içine standart yönde yerleştirin. </li><li> her ikinci gün taze ortama hücreleri yerleştirin. taze ortam ile 24 kuyu hazırlayın ve buna filtre ekler aktarın. 0.5 mi taze ortam ile uçlar doldurun. 4. bölümde açıklandığı gibi, her gün teer edin. </li><li> Hücre kültürü ekler üzerinde HIBCPP ekildi hücrelerinin TEER değerleri aştığında 70 Ω X cm² (yaklaşık olarak 4 gün Tohumlamadan sonra), daha sonra% 1 FCS ve 5 ug / ml insülin içeren bir ortam içinde hücre kültürü devam etmektedir. her bir 1 ml ortam ile 24 oyuklu plakalar hazırlamak. Üst bölmede hazırlanmış kuyular ve döviz ortamına filtre ekler aktarın. Not: konfluent sonra bu serum çekilmesi oluşumuna yol açardaha yüksek bir zar potansiyeli. </li><li> Filtre bölümünden orta aspire, iyi hazırlanmış transfer ve 500 ul orta ile doldurun. kez bu adımı yineleyin. , Inkübatör içine gece boyunca 37 ° C,% 5 CO2 hücreleri koyun. </li></ol> Transepitelyal Elektrik Direnci 4. Ölçümü (TEER) <ol><li> % 80 etanol içinde 15 dakika boyunca epitel doku voltohmmeter bir elektrot uçları bırakın. kaputun altında voltohmmeter aktarın ve bir an için elektrot kurumaya bırakın. dengelenmeye 15 dakika daha hücreler için kullanılan ilgili kültür ortamına içine daldırın. </li><li> filtre elemanı bölmeye kısa kol koyun, alt bölümünün alt her zaman değecek şekilde elektrodun uzun kolu konumlandırarak ölçümleri gerçekleştirmek. Not: Hücre kültürü filtre ekler Direnç değerlerini ortamda hücreler olmadan boş değerler kullanılmalıdır ((ölçülen değer (Ω) - boş değer (Ω)) x filtreYüzey (yani 0.33 cm²)). </li><li> 15 dakika boyunca% 80 etanol içine elektrot yere geri ölçtükten sonra. kuru tüp içinde saklayın. </li></ol> Parasellüler geçirgenliği 5. belirlenmesi <ol><li> 5 ug / ml insülin,% 1 FCS ile takviye edilmiş 200 ml kültür ortamı içinde 1 g FITC inulin eritin. hücrelerin enfeksiyondan önce üst filtre bölmesine çözelti 50 ug / ml uygulanır. </li><li> enfeksiyondan sonra çok İnülin hücre katmanı üzerinden filtre bölmesinden nasıl geçtiğini belirlemek için alt kuyudan orta örnekleri toplamak. </li><li> bir FITC-inulin standart solüsyonu hazırlayın ve 1 gerçekleştirmek: 2 dilüsyonlarla 10 kez (100,% 50,% 25,% 12.5,% 6.25,% 3.13,% 1.56,% 0.78,% 0.39,% 0.2, ve% 0%) . mikroplaka okuyucu Tüm numunelerin ölçülmesi ile floresan belirler. </li></ol> Hücre Kültürü Filtre Eklemeler üzerinde HIBCPP Hücre Enfeksiyon için Bakterilerin 6. hazırlanması <ol><li> Deneyden bir gün önce, sıyrık tO donmuş N. meningitidis Vitox Çikolata Agar üzerinde bir gliserol-stok ve çizgi dışarı kapalı suşları. % 5 CO2 ile 37 ° C &#39;de, bir gece boyunca inkübatör içinde büyür. </li><li> Bir gecelik kültürün 20-30 koloniler ekstrakte edin ve% 0.042 NaHCO 3, 0.01 M MgCI2 ve% 1 Polyvitex ile desteklenmiş 8 mi proteoz pepton ihtiva eden bir tüp içine aktarılır. </li><li> 220 rpm&#39;de, 37 ° C&#39;de 1.25 saat boyunca bakteri çalkalayın. Oda sıcaklığında 10 dakika süre ile 2.684 g&#39;de santrifüje edilerek, bakterinin ufalanması. Süpernatant atılır ve 8 ml serumsuz ortam ile bakteriyel pelletini. Girdap homojen bir süspansiyon haline sağlamak. </li><li> Kültür yoğunluğu belirlemek için, 600 nm&#39;lik bir optik yoğunluğa (OD) olarak 1:10 seyreltme ölçer. OD 0.1 600 bakteri süspansiyonu ayarlayın. Not: Bu süspansiyon yaklaşık içerir. 1 x 10 8 CFU / ml olmuştur. </li><li> Bakteriyel hücre konsantrasyonunu doğrulamak için, bir kadar süspansiyon aşamalı (1:10) seyreltikÇikolata Agar plakaları üzerine 10 -5 seyreltme ve plaka. Not: Benzer seri seyreltme bakteriyel büyüme eğrileri hesaplamak için yapılması gerekir. </li></ol> Çift İmmünofloresan tarafından Bakteriyel Invasion Hücre Kültürü Filtre Eklemeler ve Belirlenmesine İlişkin HIBCPP Hücreleri 7. Enfeksiyon <ol><li> % 1 FCS ve 5 ug / ml insülin içeren bir ortam içinde hücre kültürü devam sonra, epitel doku voltohmmeter ile her gün hücreler ölçer. Hücreler yaklaşık 500 Ω x cm² bir teer ulaşmış ise, enfeksiyon yürütmek. </li><li> Zaman, belirtilen süre boyunca% 5 CO2 ile 37 ° C &#39;de, enfeksiyon 10 deposu inkübatör enfekte hücreleri (MOI) bir çokluğunda hazırlanmış bakteriyel süspansiyonun hücreleri enfekte etmektedir. Not: İçişleri Bakanlığı izdiham (1.21 x 10 6 hücre / cm2) de dikkate hücre kültürü filtre elemanının başına hücre sayısını alarak hesaplanabilir. </li><li> Infect durdur% 1 sığır serum albümini (BSA) ihtiva eden 500 ul serumsuz ortam maddesi ile üç kez (SFM) yıkayarak iyonu alt bölme için, filtre bölmesine 1 ml uygulanmıştır. </li><li> 500 ul SFM filtre bölmesi içinde% 1 BSA tampon içeren ve spesifik olmayan bağlanma sitelerinin antikorların yapışmasını önlemek için, oda sıcaklığında 20 dakika boyunca alt bölme 1 ml ile bloklayın. </li><li> 100 ul primer antikor anti N. inkübe Filtre bölümü ve oda sıcaklığında 20 dakika boyunca alt bölmeye 500 ul: meningitidis α-OMP (200 1). Not: Gerekirse, antikor konsantrasyonu titre edilmesi gerekmektedir. </li><li> Filtre bölümünden orta aspire hücreleri yıkayın,% 1 BSA içeren bir iyi hazırlanmış 1 ml SFM filtre elemanını aktarmak ve% 1 BSA içeren 500 ul SFM ile boşaltılmış filtre bölmesi doldurun. İki kez bu adımı yineleyin. </li><li> Filtre iştigal 500 ul% 4 formaldehit hücreleri saptamakrtment, oda sıcaklığında 10 dakika boyunca alt bölme 1 mi. </li><li> Filtre bölümünden orta aspire hücreleri yıkayın, 1 ml PBS ile bir hazırlanan oyuğa filtre elemanını aktarmak ve 500 ul PBS ile boşaltılmış filtre bölmesi doldurun. kez bu adımı yineleyin. Not: Numuneler 4 ° C&#39;de PBS içinde gece boyunca saklanabilir. </li><li> Kesme sabit hücre kültürü ekler üzerinden filtre eder ve 250 ul PBS,% 1 BSA ihtiva eden ile yıkayın. hücre dışı bakteriler leke: etiketli 250 ul floresan (emisyon dalga boyu 594 nm) ikincil antikor, tavuk anti tavşan (1: 500) ile, oda sıcaklığında 15 dakika boyunca inkübe hücreleri. Not: Gerekirse, antikor konsantrasyonu titre edilmesi gerekmektedir. </li><li> 250 ul PBS, oda sıcaklığında 1 saat boyunca% 1 BSA ve% 0.5 Triton X-100 ihtiva eden hücreleri geçirgenliği. 250 ul PBS% 1 BSA içeren hücreleri üç kez yıkayın. </li><li> 250 ul primer antikor anti N. inkübe meningitidis </em&gt; α-OMP (1: 200), 30 dakika boyunca ekstra ve hücre içi bakteri leke. 250 ul PBS% 1 BSA içeren hücreleri üç kez yıkayın. </li><li> Floresan etiketli (emisyon dalga boyu 660 nm) Phalloidin (1: 250) ve 4&#39;, 6-diamidino-2-fenilindol dihidroklorür (DAPI) (: floresan etiketli (emisyon dalga boyu 488 nm) ikincil antikor (1: 500), 250 ul uygulayın 1: 50,000), oda sıcaklığında 1 saat boyunca ekstra ve hücre içi bakteri, aktin hücre iskeleti ve çekirdekleri leke. Not: Gerekirse, antikor konsantrasyonu titre edilmesi gerekmektedir. </li><li> 250 ul PBS% 1 BSA içeren hücreleri üç kez yıkayın. mikroskop aracılığıyla inceleme kadar 4 ° C&#39;de orta ve mağaza montaj hücreleri gömün. </li><li> Önceden tanımlanmış alan başına işgal bakteri sayısını belirler. Filtre membranı başına 20 alanlar sayarak bunu yapın. işgal bakteri yüzdesini hesaplayın. Not: Bir alan coeffic 20 mikroskobik alanları ortalama bakteri sayısı çarpınsağ- lasa. Sonuç 0.33 cm² hücre kültürü filtre elemanının içinde bulunan toplam bakteri miktarını ifade eder. enfeksiyon süresince ortam yetişen bakteri miktarı ile değerine bölünür. </li></ol>

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USA. 94, 3094-3099 (1997).</li><li>Vandenhaute, E., Stump-Guthier, C., Lasierra Losada, M., Tenenbaum, T., Rudolph, H., Ishikawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus+may+be+an+underestimated+site+of+tumor+invasion+to+the+brain:+an+in+vitro+study+using+neuroblastoma+cell+lines.">The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines.</a> Cancer Cell Int. , 15-102 (2015).</li></ol>

The authors have nothing to disclose.

SP&#39;nin epitel hücreleri, kan 2,3 dan CSF ayıran BCSFB oluştururlar. Son zamanlarda BCSFB bir fonksiyonel insan model olarak HIBCPP hücre hattı kuruldu. Hücreler, yüksek membran potansiyelinin geliştirilmesi, makromoleküller için düşük geçirgenlik, aynı zamanda TJs 5 sürekli elyaf varlığı da dahil vitro BCSFB arasında önemli bir engel fonksiyonlarını gösterir. TJ proteinler hücrelerin bazolateral / apikal kutuplarına katkıda bulunur. kutup yüzeyi reseptörler...

Kan-beyin-omurilik sıvısı bariyeri (BCSFB) kan ve beyin 1 arasında üç engelleyici yerlerden biridir. Morfolojik korelasyon koroid pleksus (SP) 2,3 epitel hücreleri, güçlü bir vaskülarize ve beyin ventriküller bulunan bir endotelyal ve epitelyal bükülü. CP serebrospinal akışkan (CSF) üretimi için hem de kan ikinci ayırma görevini görür. Bariyer fonksiyonunu elde etmek için, CP epitel hücreleri, düşük pinositoz aktivite gösteren spesifik taşıyıcıları ifade eder ve yoğun sıkış kesişmeler (TJs) -2,3 sürekli bir ağ ile birleştirilir. Japon kadın 4 kötü huylu bir koroid pleksus papillomu türetilen insan koroid pleksus papillomu (HIBCPP) hücreleri, BCSFB in vitro model fonksiyonel oluşturmak için kullanıldı. HIBCPP hücreleri TJ oluşumu gibi bir fonksiyonel BCSFB özellikleri bir çift göstermektedirşeritler, transepitelyal elektrik direnci (TEER) gibi belirlenebilir yüksek transepitelyal membran potansiyeli geliştirilmesi ve Makromoleküller için az geçirgenlik. Ayrıca, HIBCPP hücreleri iyonik mikro düzenleyen hizmet ve bazolateral / apikal polarite 5,6,7 gösterebilir karakteristik taşıyıcıları, ifade eder. BCSFB merkezi sinir sistemi (MSS) 8 içine patojen (bakteriler, virüsler ve mantarlar) için bir giriş yeri olarak işlev gösterilmiştir. Neisseria meningitidis (N. meningitidis), Gram-negatif bakteri de dahil olmak üzere patojenlerin, işgali menenjit gibi ciddi hastalıklara neden olabilir. Bu CP koruyucu epitel engelinin üstesinden gelecek, kanıtlar meningokok hastalığı damar ve CP epitel hücreleri 9,10 meningokoklara artan miktarlarda sergileyen hastalarda histopatolojik gözlemler ile desteklenir. konakçı hücreler ba içine girmeyecteria genellikle aracılık ettiği veya konakçı hücreler üzerinde bulunan spesifik yüzey reseptörleri tarafından tetiklenen endositoksik mekanizmaları kaçırma. Bu reseptörler ile patojenlerin etkileşimleri türler olabilir çünkü belirli 11, hayvan modelleri sadece kısıtlı ölçüde konsülte edilebilir. HIBCPP hücre hattı işgali sürecinin yanı sıra bir insan modeli sisteminde altta yatan moleküler mekanizmaları incelemek için fırsat sağlar. Hücre kültürü ekler istihdam iki ayrı hücre taraftan konakçı hücreleri ile patojenlerin etkileşimleri analiz etmemizi sağlar. Bir çok bakteri, aşağıdakileri içeren N. meningitidis enfeksiyonu deneyleri sırasında yerçekimi etkisi güçlü bir şekilde bağlıdır. deneyleri sırasında HIBCPP hücreleri ile patojenlerin optimum etkileşim, bakteriler, başlangıçta hücre kültürü filtre elemanı sisteminin üst bölmeye eklenir. Sırasıyla, in vitro sistemde iki varyasyonları olmuştur esta apikal veya bazolateral hücre tarafından enfeksiyonu etkinleştirmek içinblished: Standart sistemde HIBCPP hücreleri mikroorganizmalar CSF-tarafında yer alan ve hücreler (Şekil 1A, C), apikal kısmı ile temas içine almak da durum taklit filtre elemanının üst bölmesine ekilir. Buna karşılık, bir ters hücre kültürü filtresi elemanı sisteminde HIBCPP hücreleri kullanılarak bakteri kan dolaşımına girmiş koşullarını yansıtmaktadır. Mikroorganizmalar bazolateral tarafı (Şekil 1B, D) kan ve karşılaşma CP epitel hücrelerinde yaymak. Dikkate değer olarak, bu model sistemde bakteri bazolateral hücre tarafı 5,7 arasında, özellikle polar bir şekilde HIBCPP hücreleri istila gösterilmiştir. Daha sonra CP enfeksiyon, işgal patojenler örüntü tanıma reseptörleri ligasyon yoluyla doğuştan gelen bağışıklık sistemi (PRR) tarafından kabul edilebilir. PRR ve iyi tanımlanmış üye Toll-benzeri reseptör (TLR) ailesine aittir. TLR can binVadeli patojenle ilişkili moleküler yapılar (PAMPS) olan bulaşıcı mikroorganizmaları genel karakteristik özellikleri, d. Reseptör bağlama da BCSFB 13,14 boyunca bağışıklık hücrelerinin göçünde uyaran sitokinler ve kemokinlerin 12 ekspresyonunu tetikleyen konakçı hücre aktivasyonu sinyal iletim yol açar. Hücreler, birkaç HIBCPP mRNA düzeyindeki TLR ve N ile birlikte bu enfeksiyon ifade gösterilmiştir meningitidis CXCL1-3, IL6, IL8 ve TNFa 15,16 dahil olmak üzere çeşitli sitokinler ve kemokinlerin, salgılanması ile sonuçlanır. Burada, BCSFB taklit eden bir ters hücre kültürü uç sisteminde yetiştirme ve insan hücre hattı HIBCPP enfeksiyonunu tarif eder. Bu model sistemi, in vivo ilgili bazolateral hücre tarafı hem de daha sonra, hücresel yanıt patojenlerin etkileşimleri incelemek için olanak sağlar.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>4&amp;acute;,6 diamidino-2-phenylindole (DAPI)</td><td>Life Technologies</td><td>D1306</td><td></td></tr><tr><td>12-well plates</td><td>Starlab</td><td>CC7682-7512</td><td></td></tr><tr><td>24-well plates</td><td>Starlab</td><td>CC7682-7524</td><td></td></tr><tr><td>Anti Neisseria meningitidis &amp;alpha;-OMP</td><td></td><td></td><td>This antibody was a gift from Drs. H. Claus and U. Vogel (University of W&amp;uuml;rzburg, Germany)</td></tr><tr><td>Alexa Fluor 488 (chicken anti rabbit)</td><td>Invitrogen</td><td>A21441</td><td></td></tr><tr><td>Alexa Fluor 594 (chicken anti rabbit)</td><td>Invitrogen</td><td>A21442</td><td></td></tr><tr><td>Alexa Fluor 660 Phalloidin</td><td>Invitrogen</td><td>A22285</td><td></td></tr><tr><td>Bovine serum albumine (BSA)</td><td>Calbiochem</td><td>12659</td><td></td></tr><tr><td>Chocolate agar plates</td><td>Biomerieux</td><td>43109</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma</td><td>C8273</td><td></td></tr><tr><td>DMEM/F12 + L-Glut + 15 mM HEPES</td><td>Gibco</td><td>31330-095</td><td></td></tr><tr><td>DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred</td><td>Gibco</td><td>11039-047</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma</td><td>F3272</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>19278</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>2393</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>55761</td><td></td></tr><tr><td>PBS + Mg + Ca</td><td>Gibco</td><td>14040-174</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>1670049</td><td></td></tr><tr><td>Polyvitex</td><td>Biomerieux</td><td>55651</td><td></td></tr><tr><td>Proteose peptone</td><td>BD</td><td>211684</td><td></td></tr><tr><td>Serum-free medium</td><td>Gibco</td><td>10902-096</td><td></td></tr><tr><td>Thincert cell culture inserts for 24-well plates, pore size 3 &amp;micro;m</td><td>Greiner</td><td>662630</td><td></td></tr><tr><td>Tissue culture flask 75 cm&amp;sup2; red cap sterile</td><td>Greiner</td><td>658175</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode</td><td>Millipore</td><td>MERSSTX00</td><td></td></tr></tbody></table>

a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier to study bacterial infection from the basolateral side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

Burada ters bir hücre kültürü insert sisteminde kültürlenmesini ve HIBCPP hücrelerin enfeksiyonunu tanımlar. Bu model bize (Şekil 1) yayma ve kan dolaşımı yoluyla epitel hücreleri giren bakteri fizyolojik durumunu üreten, bazolateral hücre taraftan işgali mekanizmaları ve altında yatan moleküler sinyal yolları çalışma sağlar. HIBCPP hücreleri asgari düzeye moleküler değişimi kısı...

Watch this Scientific Journal Video about A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side at JoVE.com

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

İnsan Kan BOS Bariyer A Koroid Pleksus Epitel Hücre bazlı Modeli bazolateral Side Bakteriyel Enfeksiyon Eğitim için

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

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14.1K Görüntüleme.  Heidelberg University. The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Video: A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Yöntem kan-beyin bariyerini bir insan hücre tabanlı bir İletişim Modelinin Hazırlanması için Geliştirilmiş

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Biyomühendislik

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

bir<em&gt; İn Vitro</em&gt; Empedans Spektroskopisi kullanarak kan-beyin bariyerinin Model: T Hücre-endotelyal hücre Etkileşim Üzerine Bir Odak

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

İmmünoloji ve Enfeksiyon

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Bir kullanma Lenfosit Ekstravazasyonunun analizi<em&gt; İn Vitro</em&gt; İnsan Kan-beyin bariyeri Model

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Yalıtım taze insan beyin dokusundan beyin kapiller

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Nörobilim

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

Mikrovasküler Endotel Hücreleri Ile Mikrobiyal Traversal ile Kan-Beyin Bariyerinin Geçirgenliğinin Analizi

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

Bariyer geçişleri patojenler veya ilaçlar ve onların etkileşim beyin ile çalışmak için bir insan kan-beyin arabirim modeli

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Neisseria meningitidis İndüklenmiş Pluripotent Kök Hücre Kaynaklı Beyin Endotel Hücrelerinin Enfeksiyonu

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

Fare Koroid Pleksusunun Mikrodiseksiyonu ve Tüm Montajlı Taramalı Elektron Mikroskobu Görselleştirmesi

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Programlanabilir Çıkış Frekansı Ile Kendi Kendine Yapılmış Volt-Ampermetre Kullanarak TEER Ölçümlerini Gerçekleştirmek Için Basit Bir Yaklaşım

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Biyoloji

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

İnsan Kan-Beyin Bariyerini Modelleyen Üçlü Kültür Hücre Sistemi

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

İnsan Kan BOS Bariyer A Koroid Pleksus Epitel Hücre bazlı Modeli bazolateral Side Bakteriyel Enfeksiyon Eğitim için

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