Biz akciğerleri sağlamak için Miami Üniversitesi Yaşam Alliance Organ Kurtarma Ajansı teşekkür ederim. Ayrıca Ayrıca Gabriel Gaidosh ederiz Şekil 2B ve 3-D, C Şekil 3A&#39;da deneyler için kullanılan MCherry virüsü Dr. Ben Gerovac ve lisa Novak gösterilen deney için kullanılan DNA hazırlanması için Lisa KUNZI ederiz görüntüleme tesis, Miami Üniversitesi&#39;nde Göz Hastalıkları Anabilim Dalı. Bu çalışma NIH, CF Vakfı ve Dr. Matthias Salathe Uçuş Görevlisi Tıbbi Araştırma Enstitüsü hibe sponsorluğunda edilmiştir.

1. Aşama 1: HEK 293T Kültür <ol><li> Yüksek glikoz Dulbecco Modifiye Eagle Ortamı (DMEM)% 10 (h / h) cenin sığır serumu (FBS) ve% 1 (h / h), penisilin / streptomisin eklenmesi ile (HEK ortam olarak adlandırılır) HEK hücreleri ortamı hazırlar. </li><li> Kat kollajen, kollajen I Çeşitli 30 ul bir damıtılmış su ve 2 ml bir kez 10 cm doku kültürü çanağı. Tabak üzerine yayıldı karışımı 37 ° C de 2 saat ve% 5 CO2 inkübe edilir. </li><li> karışımı çıkarın ve 15 dakika boyunca bir laminer akış kabini bulaşık kurumaya bırakın. </li><li> 10 mi HEK ortamında 1 x 10 6 hücre yoğunluğunda kaplanmıştır tabağa levhalar HEK hücreleri 37 ° C &#39;de inkübe etmek ve% 5 CO2. </li><li> HEK hücreleri (2-3 gün sonra kaplama Şekil 1a)% 50-60 konfluent olduğunda, confluency 2. Görsel değerlendirme yeterlidir sahneye geçin. Hiçbir ölçümü gereklidir. Not: HEK hücreleri (yukarıya bakın) HEK ortamda kültürlenir. Onlar confluency ulaşmak, they bölünmüş (örneğin 1/5) ya da daha sonra kullanılmak üzere aşağı dondurulabilir. </li></ol> 2. Aşama 2: HEK Hücreleri Üretimi ve Lentiviruses (karaciğer hacimleri) Yoğunlaşma Not: protokolleri BSL-2 ABD&#39;de + (gelişmiş BSL-2) koşullar altında kurumsal ve hükümet düzenlemelerine uygun olarak yürütülmelidir. <ol><li> Bir kalsiyum çökeltme prosedürü kullanarak Transfect (lipofektamin ve diğer transfeksiyon ajanları kalsiyum yağış prosedürü kadar verimli değildi). Prosedür aşağıdaki gibi tadil edilmiş olmasına rağmen, ticari bir transfeksiyon kiti kullanılarak yeniden üretilebilirliğinin temini için tavsiye edilir. Oda sıcaklığına Tüm reaktifler getirin. Bu gün 0&#39;dır. </li><li> HEK293 hücrelerinin her 10 cm tabak için, zarf DNA, pMD2-VSVG 4.5 ug, 7.5 ug DNA, pMDLg / pRRE ambalajlar, 3.75 ug DNA, pRSV-rev, lentiviral sentezleme vektörünün 10 ug DNA, 2 M, kalsiyum çözeltisinin 86 ul karıştırın ve 700 ul son hacim olması için 15 ml&#39;lik bir santrifüj tüpüne steril su(Bu sonraki iki çözüm, ticari transfeksiyon kiti temin edilmiştir). </li><li> Bilge-Drop (transfeksiyon kitinde HBS) 2x HEPES tamponlu tuz 700 ul (laminer akış kabini içinde) vorteks ederken eklemek ve 30 dakika (beklerken adım 4 gerçekleştirin) oda sıcaklığında kuluçkaya yatmaktadır. </li><li> Erken inkübasyon süresi boyunca, bir slayt karışımı mevduat 5 ul. Yavaş yavaş yukarı ve aşağı odaklanarak, 10X amacı ile mikroskop altında damlacık kontrol edin. İnce bir çökelti, kalsiyum fosfat çökeltme işlemi teyit görünür olmalıdır (Şekil 1b). </li><li> karıştırmak için yukarı ve aşağı HEK hücreleri medya ve pipet 10 ml ilave edilerek 30 dakika sonra çökelmesini durdurun. </li><li> Adım 2,1-2,5 den HEK hücrelerinin çanak alın ve orta (gün 0) çıkarın. yemeğin kenarından dikkatlice ve yavaşça adım 2.5 den çökelti çözüm ekleyin. </li><li> Ertesi gün (gün 1) Açık, kaldırmak ve çökelti içeren orta atmak ve w yerineHEK ortamının i 10 mi. Mikroskop altında ince çökelti olup olmadığını kontrol edin: Bu hücreler (Şekil 1c ve d) arasındaki boş alanlarda tabağına görünür olmalıdır. </li><li> 2. günde,% 40 polietilen glikol, 3.8 mi (PEG) çözeltisi ihtiva eden 15 ml&#39;lik bir santrifüj tüpü içine, bir 0.45 um şırınga filtre ile bir şırınga filtresi ile ortam toplama. (PEG oluşturmak üzere çökeltilmesi, ancak 5 günden fazla için) virüsü koleksiyonları tüm tamamlanıncaya kadar Ters 5x en az 24 saat süre ile 4 ° C&#39;de karıştırın ve saklamak için. </li><li> gün 3 ve 4. günde 4. adımı yineleyin 2.8 HEK orta ile değiştirin ama% 10 çamaşır suyu ile temizleyin ve çanak atmayın. </li><li> 4 ° C&#39;de 20 dakika boyunca 1,650 x g&#39;de santrifüjleyin tüm toplama tüpleri (birlikte, 5. günde genellikle tüm toplama) virüsü peletine. Süpernatantı çıkarın ve atın ve toplamak ve herhangi bir PEG süpernatant kalan çıkarmak için 4 ° C&#39;de 5 dakika boyunca 1.650 xg&#39;de tekrar dönerler. </li><li> Resuspend amfoterisin B 8 olmayan bronşiyal epitel büyüme ortam (Begm) 200 ul her bir tüp pelet. araya havuzuna ve 200 ul tümbölen. -80 ° C&#39;de saklayın virüsü. HIV-1 Gag (P24) antijen ELISA deneyi gerçekleştirmek için ilave bir 10 ul tablet. </li><li> ticari bir ELISA kiti kullanılarak üreticinin protokolüne göre, bir p24 antijen tahlilini gerçekleştirmek. Tahlil pg / ml viral p24 bir tahmin verir. </li></ol> 3. Aşama 3: Kültür İlköğretim HBE Hücreler <ol><li> 10 mi Begm ortam Plaka HBE hücreleri I 10 cm doku kültürü çanağı kaplanmış bir kolajen 1 x 10 6 hücre yoğunluğunda (100 ul amfoterisin B kanalı 0 hücreleri mümkün fungal kirlenmeyi elimine etmek için kullanılması durumunda). 37 ° C&#39;de inkübe edilir ve% 5 CO2. </li><li> Bir sonraki gün, ortam çıkarın ve 4 günlük bir toplam (geçişi için 100 ul amfoterisin B 0 hücreleri) 10 mi Begm ile değiştirin. inkübatör dönmek. </li><li> 4 Gün sonraays, her geçen gün amfoterisin B Değişim medya olmadan sadece Begm kullanın. </li><li> Hücreler% 80-90 ortak akışlı (Şekil 2a; ~ 7 gün sonra kaplama) olduğunda, transdüksiyon devam (transdüksiyon öncesi hazırlık için 3.5 dikkat). Yine, konfluent görsel değerlendirmesi yeterlidir. Hiçbir ölçümü gereklidir. </li><li> transdüksiyon önce, ceket kolajen IV çözeltisi ile geçirgen destek uçlar steril damıtılmış su ile 1/10 seyreltilmiştir. Her ekleme 200 ul ekleyin ve laminer akış kabini kuru gecede izin verin. </li></ol> 4. Aşama 4: HBE Hücreler İletimi <ol><li> Ortamı çıkarın fosfat tamponlu tuzlu su (EDTA / PBS) içinde 1 mM etilendiamintetraasetik asit,% 0.1 tripsin 3 ml ilave edilir ve 37 ° C&#39;de 5 dakika ve% 5 CO2 inkübe edin. tüm hücreleri müstakil olup olmadığını görmek için mikroskop (10X objektif) altında kontrol edin. Aksi takdirde, ilave bir 5 dakika boyunca inkübatör dönmek. </li><li> tüm hücreleri müstakil zaman, 3 eklemeksoya fasulyesi tripsin inhibitörü mi (SBTI 1 mg / ml) karışımı ve 15 ml santrifüj tüpüne transfer. Oda sıcaklığında 5 dakika boyunca 460 x g&#39;de iplik hücreleri Pelet. </li><li> Begm 3 ml içine süpernatant ve tekrar süspansiyon pelet çıkarın ve sayma işlemine devam edin. </li><li> Begm 400 ul (bakınız Tablo 2) &#39;de, bir 12 mm geçirgen destek uç başına 2 x 10 5 hücre oranında yeniden süspanse HBE hücreler (şimdi kanalı 1 ya da P1). transduced edilecek geçirgen destek ekler miktarına göre bu sayıları tahmin. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Yüzey </td><td> Yüzey alanı </td><td> Hücre sayımı </td><td> medya </td><td> Hacim (medya) </td></tr><tr><td> 10 cm çanak </td><td> 52.7 cm2 </td><td> 1 x 10 6 </td><td> begm </td><td> 10 mi </td> &lt;/ Tr&gt; <tr><td> 12 mm filtre </td><td> 1.13 cm2 </td><td> 2 x 10 5 </td><td> begm </td><td> 400 ul </td></tr><tr><td> 24 mm filtre </td><td> 4.52 cm2 </td><td> 8 x 10 5 </td><td> begm </td><td> 800 ul </td></tr></tbody></table> Tablo 2: hücre sayımı başına Medya sınırlandırmaktadır. <ol start="5"><li> 4 enfeksiyon (MOI) faktörünün çok sayıda kullanılarak, hücre süspansiyonunun virüsün uygun hacimde ekleyin. </li><li> 2 ug / ml nihai konsantrasyona heksadimetrin bromit ekleyin. </li><li> apikal bölmeye geçirgen destek insert başına mix (Begm, HBE hücreleri, virüs ve hexadimethrine bromür) 400 ul koyun ve bazolateral bölmeye tek başına Begm 1 ml ekleyin. Kontrol ve test puromisin seçimi varsa da virüs olmadan 37 ° C ve% 5 CO 2. Her zaman plaka HBE hücreleri gecede inkübe. </li><li> Ertesi gün, rem üzerindeove en üst ve taban ortamı, PBS ile yıkayın ve hava-sıvı arayüzü (ALI), medya 8 her iki bölümü değiştirin. </li><li> Hücreler birleştiklerinde, ne olursa olsun, (bir hava-su ara-yüzünün kurulması olarak da bilinir), apikal sıvıyı. onlar tam olgunluğa ulaşana kadar alt bölmeye her gün de orta (ALI) değişen devam edin; genellikle 3 ila 4 hafta sonra. Not: lentiviral vektör, puromisin gibi bir seçim genini ihtiva ise hücreler bir seçme konsantrasyonu eğrisine göre puromisin eklenerek seçilebilir. HBE hücreleri için, 1 ug / ml bir konsantrasyon kalıt aktarımlı hücrelerin istikrarlı seçimi için ideal olduğu tespit edilmiştir. Bununla birlikte, bu konsantrasyon diğer hücrelere veya koşullar için farklı olabilir ve eğrileri öldürerek belirlenmelidir. Medya seçimi geninin tam ifadesini izin sonrası ALI medya ya da (2-3 gün) tarafından değiştirildikten sonra puromycin ilavesi gibi erken bir sürede güne başlayabilirsiniz. bir parçanın bu h puromisin eklemek için emin olunolarak transduced edilmemiştir. Hücreler tamamen farklılaşmış kadar puromycin eklenebilir. </li></ol>

<ol>
	<li>Ding, B., Kilpatrick, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vector+production,+titration,+and+transduction+of+primary+neurons.">Lentiviral vector production, titration, and transduction of primary neurons.</a> Methods Mol Biol. 1018, 119-131 (2013).</li><li>Sinn, P. L., Sauter, S. L., McCray, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+development+of+improved+lentiviral+and+retroviral+vectors--design,+biosafety,+and+production.">Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors--design, biosafety, and production.</a> Gene Ther. 12, 1089-1098 (2005).</li><li>Cribbs, A. P., Kennedy, A., Gregory, B., Brennan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+production+and+concentration+of+lentiviral+vectors+to+achieve+high+transduction+in+primary+human+T+cells.">Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells.</a> BMC Biotechnol. 13, (2013).</li><li>Delenda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors:+optimization+of+packaging,+transduction+and+gene+expression.">Lentiviral vectors: optimization of packaging, transduction and gene expression.</a> J Gene Med. 6 Suppl 1, S125-S138 (2004).</li><li>Gresch, O., Altrogge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+difficult-to-transfect+primary+mammalian+cells.">Transfection of difficult-to-transfect primary mammalian cells.</a> Methods Mol Biol. 801, 65-74 (2012).</li><li>Jordan, E. T., Collins, M., Terefe, J., Ugozzoli, L., Rubio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+electroporation+conditions+in+primary+and+other+difficult-to-transfect+cells.">Optimizing electroporation conditions in primary and other difficult-to-transfect cells.</a> J Biomol Tech. 19, 328-334 (2008).</li><li>Castillon, N., 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=Regeneration+of+a+well-differentiated+human+airway+surface+epithelium+by+spheroid+and+lentivirus+vector-transduced+airway+cells.">Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells.</a> J Gene Med. 6, 846-856 (2004).</li><li>Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Methods Mol Med. 107, 183-206 (2005).</li><li>Gonzalez-Mariscal, L., 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=Role+of+calcium+in+tight+junction+formation+between+epithelial+cells.">Role of calcium in tight junction formation between epithelial cells.</a> Am J Physiol. 259, C978-C986 (1990).</li><li>Bals, R., 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=Transduction+of+well-differentiated+airway+epithelium+by+recombinant+adeno-associated+virus+is+limited+by+vector+entry.">Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.</a> J Virol. 73, 6085-6088 (1999).</li><li>Ricks, D. M., Kutner, R., Zhang, X. Y., Welsh, D. A., Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+lentiviral+transduction+of+mouse+bone+marrow-derived+mesenchymal+stem+cells.">Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells.</a> Stem Cells Dev. 17, 441-450 (2008).</li><li>Davis, H. E., Morgan, J. R., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polybrene+increases+retrovirus+gene+transfer+efficiency+by+enhancing+receptor-independent+virus+adsorption+on+target+cell+membranes.">Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes.</a> Biophys Chem. 97, 159-172 (2002).</li><li>Horani, A., Nath, A., Wasserman, M. G., Huang, T., Brody, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-associated+protein+kinase+inhibition+enhances+airway+epithelial+Basal-cell+proliferation+and+lentivirus+transduction.">Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction.</a> Am J Respir Cell Mol Biol. 49, 341-347 (2013).</li><li>Manzanares, D., 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=Functional+apical+large+conductance,+Ca2+-activated,+and+voltage-dependent+K++channels+are+required+for+maintenance+of+airway+surface+liquid+volume.">Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume.</a> J Biol Chem. 286, 19830-19839 (2011).</li><li>Schmid, A., 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=Real-time+analysis+of+cAMP-mediated+regulation+of+ciliary+motility+in+single+primary+human+airway+epithelial+cells.">Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells.</a> J Cell Sci. 119, 4176-4186 (2006).</li><li>Unwalla, H. J., Ivonnet, P., Dennis, J. S., Conner, G. E., Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta1+and+cigarette+smoke+inhibit+the+ability+of+beta2-agonists+to+enhance+epithelial+permeability.">Transforming growth factor-beta1 and cigarette smoke inhibit the ability of beta2-agonists to enhance epithelial permeability.</a> Am J Respir Cell Mol Biol. 52, 65-74 (2015).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> BMC Biotechnol. 6, (2006).</li><li>Schneiderman, S., Farber, J. L., Baserga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+decreasing+the+toxicity+of+polyethylene+glycol+in+mammalian+cell+hybridization.">A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.</a> Somatic Cell Genet. 5, 263-269 (1979).</li><li>Schmid, A., 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=Soluble+adenylyl+cyclase+is+localized+to+cilia+and+contributes+to+ciliary+beat+frequency+regulation+via+production+of+cAMP.">Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP.</a> J Gen Physiol. 130, 99-109 (2007).</li><li>Manzanares, D., 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=Airway+Surface+Dehydration+by+Transforming+Growth+Factor+beta+(TGF-beta)+in+Cystic+Fibrosis+Is+Due+to+Decreased+Function+of+a+Voltage-dependent+Potassium+Channel+and+Can+Be+Rescued+by+the+Drug+Pirfenidone.">Airway Surface Dehydration by Transforming Growth Factor beta (TGF-beta) in Cystic Fibrosis Is Due to Decreased Function of a Voltage-dependent Potassium Channel and Can Be Rescued by the Drug Pirfenidone.</a> J Biol Chem. 290, 25710-25716 (2015).</li><li>Manzanares, D., 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=IFN-gamma-mediated+reduction+of+large-conductance,+Ca2+-activated,+voltage-dependent+K++(BK)+channel+activity+in+airway+epithelial+cells+leads+to+mucociliary+dysfunction.">IFN-gamma-mediated reduction of large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction.</a> Am J Physiol Lung Cell Mol Physiol. 306, L453-L462 (2014).</li><li>Chen, X., 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=A+soluble+adenylyl+cyclase+form+targets+to+axonemes+and+rescues+beat+regulation+in+soluble+adenylyl+cyclase+knockout+mice.">A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.</a> Am J Respir Cell Mol Biol. 51, 750-760 (2014).</li><li>Ransford, G. A., 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=Pannexin+1+contributes+to+ATP+release+in+airway+epithelia.">Pannexin 1 contributes to ATP release in airway epithelia.</a> Am J Respir Cell Mol Biol. 41, 525-534 (2009).</li></ol>

The authors have nothing to disclose.

Burada özetlenen protokol% 100 iletim verimliliği yakın sağlar. Bununla birlikte, bu hedefe ulaşmak için önemli olan işlem sırasında adım vardır. Örneğin, transfeksiyon işlemi sırasında, transdüksiyon karışımı çökeltilerin varlığı (damla) veya çanak transdüksiyon (Şekil 1b ve d) gün sonra iyi bir transfeksiyon için uygun hem de. bir çökelti ya da topakların bulunması olmaması transfeksiyon reaktifleri birinin bir eksiklik, bir pH sorun, ya da kötü ...

Replikasyon-kusurlu, pseudotyped Lentivirüslerden gelişimi (LV) in vitro 1 transgenlerin sunmak için güvenli ve etkin bir yöntemle araştırmacılar sağlamıştır. Nedeniyle uzun vadeli ifade, bu AG, aynı zamanda, in vivo 2,3 terapötik maddelerin uygulanması için de kullanılabilir. LV üretimi birçok plazmid ile insan embriyonik böbrek (HEK) hücrelerinin ko-transfeksiyon için gereken: transfer vektörü, ambalaj gag / pol, ambalaj rev ve veziküler stomatit virüsü glikoproteini (VSV-G) plazmidler zarf. REV geni, böylece bir replikasyon yetkili lentivirüs üretilmesi için rekombinasyon olasılığını azaltarak ayrı bir ambalaj plasmid üzerinde kodlandığı için üçüncü nesil paketleme sistemleri ikinci kuşak sistemler daha güvenli olarak kabul edilir. İkinci nesil ambalaj sistemleri beş kat daha fazla toplam iletim üniteleri verim rağmen, orijinal viral genomun bölünmüş oluşturmak içinÜçüncü nesil sistem sadece minimal 3,4 transdüksiyon seviyesini azalmıştır. Hücre hatları daha kolay ve verimli bir transduced edilebilir olsa da, bu in vivo daha iyi temsil 5,6 dokular, çünkü, araştırmacılar, birincil hücreler kendi yoğunlaştırdığını var. Bununla birlikte, birincil hücreler transdüksiyona dirençli bulunmaktadır. Farklılaşmış hava yolu epitel hücrelerinin transdüksiyonu rapor edilmiş olmasına rağmen, genel verimlilik% 15 7 aşmamıştır. yüksek titre LVS üretimine olanak sağlayan bir protokol aşağıda tarif edilmiştir. Bir adım-adım yaklaşımı birincil HBE hücrelerine neredeyse% 100 transdüksiyon verim elde etmek için belirtilmiştir. Daha spesifik olarak, protokol 3 başarılı olmak için enstrümantal optimum kültür koşullarını tanımlamaktadır. Kısaca HEK 293T hücreleri gelişmiş yeşil floresan EF-1 promotörü tahrik ifade ile lentiviral ifade vektörü pCDH kullanılarak transfekte edilirprotein (EGFP) ya MCherry kırmızı flöresanlı protein ve bir üçüncü nesil ambalajlama sistemi. ortama salınan LVs sonra 24 saat 72 toplanır. Virüs partikülleri, p24 antijen enzim bağlı immünosorbent deneyi (ELISA) kiti kullanılarak titre tahmin önce, polietilen glikol (PEG), viral konsantrasyona uygun ve kolay bir yöntem ile konsantre edilmiştir. 4 (MOI) bir faktör bir enfeksiyon çokluğunda (tripsinize sonra) takarken sonra, diferansiyasyonlu olmayan primer HBE hücreleri (heksadimetrin bromit ekleme ve 16 saat inkübe, proliferasyon ortam (bronşiyal epitel büyüme ortam ya Begm) 8 transdüksiyona bir gecede). Hücreler daha sonra ayırt etmek için izin verilir. Viral transgen (tartışma uygun promotör kullanılarak) uzun vadeli ifade edildiğinden, bu ifade bir yalancı solunum yolu epitelinde farklılaşma boyunca muhafaza edilir veya farklılaşmadan sonra (indüklenebilir bir promotör kullanılarak) indüklenebilir. <p cleşek = &quot;jove_content&quot;&gt; Bu protokol hücre hatları yerine birincil HBE hücrelerini kullanmak istediğiniz araştırmacıların geniş ilgi ve hücre tiplerini transduce diğer zor adapte olabilir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Sigma</td>
			<td>12306C</td>
			<td>use at 10%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x)</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD Biosciences</td>
			<td>354231</td>
			<td>dilute 1:75 in distilled water</td>
		</tr>
		<tr>
			<td>Calphos</td>
			<td>Clontech</td>
			<td>631312</td>
			<td>Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O</td>
		</tr>
		<tr>
			<td>Polythylene glycol 8000</td>
			<td>VWR scientific</td>
			<td>101108-210</td>
			<td>stock of 40%</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A9528</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell 12mm</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C7521</td>
			<td>dilute 1:10 in distilled water</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>stock of 2mg/ml</td>
		</tr>
		<tr>
			<td>Puromycin (10.000x)</td>
			<td>Thermo Scientific</td>
			<td>A11138-03</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Alliance HIV-1 p24 ELISA</td>
			<td>PerkinElmer</td>
			<td>NEK050001KT</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Thermo Scientific</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDH-EF1-MCS-IRES-Puro</td>
			<td>System Biosciences</td>
			<td>CD532A-2</td>
			<td>lentiviral expression vector</td>
		</tr>
		<tr>
			<td>pMD2-VSVG</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pRSV-rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td>packaging DNA</td>
		</tr>
	</tbody>
</table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

Hava yolu hücre farklılaşması ve fonksiyon işlemlerini incelemek için yararlı bir model hava-sıvı arayüzü koşulları sağlar kullanılarak primer insan bronş epitel (HBE) hücrelerin in vitro kültüründe. Son birkaç yıl içinde, transgen teslimat için lentiviral vektörleri kullanımı yaygın bir uygulama haline gelmiştir. belli olmayan HIV sözde yazılan Lentivirüslerden tamamen farklılaşmış solunum yolu epitel hücreleri iletimi raporlar varken, genel transdüksiyon verimliliği genellikle% 15&#39;den az olduğunu. Burada sunulan protokol güvenilir ve etkili bir metot lentivirüsler üretmek ve primer insan bronş epitel hücreleri nakletmek için içerir. Hücreler takmak da 4 enfeksiyonun faktörü bir çoğu ile, farklılaşmamış bronşiyal epitel hücreleri, bronşiyal epitel büyüme ortamında transdüksiyonunu kullanılarak% 100&#39;e yakın verimlilik sağlar. Bu protokol, adım-adım, yüksek titre lentiviral vektörler ve th hazırlanması ve konsantrasyon açıklare transdüksiyon süreci. Bu primer insan bronş epitel hücrelerinin yüksek verimli transductions elde etmek için uygun kültür koşulları tespit deneyleri anlatılır.

Şekil 1 1a Şekil. Transfeksiyon sürecinde hücreleri ve çözümleri değerlendirmek farklı anahtar adımları göstermektedir önce virüs üretimi için transfeksiyon için HEK293T hücrelerinin optimum confluency gösterir. Hücreler çanak boyunca eşit olarak dağıtılmış olması önemlidir. 1b parlak bir alan optik ve 10X objektif kullanılarak transfeksiyon karışımı bir damla çökelti ortaya Şekil. Da...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Optimal lentivirüs üretimi ve Hücre Kültürü Koşullar Gerekli Başarıyla transduce İlköğretim İnsan Bronş Epitel Hücreleri

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

optimal-lentivirus-production-cell-culture-conditions-necessary-to

Research

JoVE Journal

Biology

19.1K Görüntüleme.  University of Miami, Miller School of Medicine. Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Video: Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Donmuş Hücreler itibaren Kültür: İnsan Embriyonik kök hücrelerin

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Biyoloji

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Lentivirüs Üretim

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Eksplantlar gelen Yetiştirilen İlköğretim İnsan Bronşiyal Epitel Hücreleri

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Kültür İlköğretim İnsan Fallop Tüp Epitel Hücreleri

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Biyogüvenlik Geliştirilmiş Polimer complexed Ecotropic lentivirüs İnsan Hücreleri, İletimi

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Sıçan Adrenokortikal hücreleri ve deneyleri Steroidogenic fonksiyonların birincil kültür

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Tehlike Değerlendirmesi İçin Epitel Hücreleri ve Primer İmmün Hücrelerden Oluşan Çok Hücreli İnsan Alveoler Modeli

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Organoid Kültür için İnsan Akciğer Epitel Progenitör Hücrelerinin İzolasyonu ve Zenginleştirilmesi

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...