Preston A. Wells Jr Merkezi Beyin Tümörü Tedavisi için; Bu çalışma Florida Beyin Tümörü Araştırma Merkezi Burslar tarafından desteklenmiştir.

1. Temel GBM Doku toplanması <ol><li> Glioblastoma mutliforme (GBM), tümör, kanser tanısı ve ameliyatı geçiren bir hastanın elde edilir. </li><li> Nöroşirürji takım düzenlemeler yapılmalıdır. Beyin cerrahı, nöral kök hücre (MGK),% 10-15 antibiyotikler (Peniciline / Streptomisin) ile desteklenmiş bazal orta içeren uygun bir boyut tüp rezeke GBM tümör koyacaktır. Soğuk ortamda laboratuarı tarafından ameliyathaneye sağlanmalıdır. Alternatif olarak, soğuk Hepes-tamponlu minimum esansiyel orta (HEM) ve fosfat tamponlu salin (PBS) yüksek antibiyotik konsantrasyonu ile de bu amaçla kullanılabilir. </li><li> Rezeke edilen tümör dokusu buz üzerinde laboratuara teslim edilir ve kaputun altında yerleştirilir. </li></ol> 2. Tek Hücre Süspansiyon içine Primer Tümör Disosiyasyon <ol><li> Orijinal orta / PBS Aşırı şahin tüp çıkarılır ve örnek 5-10m ile 2-3 defa yıkanır.PBS / MGK bazal orta l kan ve enkaz kaldırmak için. PBS / Orta kaldırılır ve GBM tümör dokusu bir Petri kabı yerleştirilir. </li><li> Dokusu küçük parçalar halinde kesilir ve trypsinization işlemi için yüzey alanını artırmak için küçük parçalar halinde 10 numaralı neşter bıçağı ile kıyılmış. Kıyma tümörün büyüklüğüne bağlı olarak 1-3 dakika sürebilir. </li><li> Kıyılmış doku önceden ısıtılmış% 0.05 tripsin-EDTA az 10-15 dakika boyunca 37 ° C su banyosunda 3-5ml tripsinize. Bir elektronik pipet 15 ml Falcon tüp içine küçük tümör parçaları ve tripsin aktarmak için kullanılır. </li><li> Inkübasyon döneminden sonra enzimatik tripsin reaksiyonu durdurmak için eşit miktarda soya tripsin inhibitörü eklenir. </li><li> Birkaç kez aşağı süspansiyon pipetleme ve tripsin inaktivasyon tarafından sağlanmaktadır. Sonra, süspansiyon 5min için 800rpm (110g) santrifüj pelet. </li><li> Süpernatant atılır ve doku parçaları steril MGK ba 1ml yeniden süspansesal orta. Kümeleri hafifçe düzgün bir sütlü tek hücre süspansiyonu elde edilene kadar (3-7 kat) aşağı yukarı pipetleme ve ayrışmış. Pipetleme adımların sayısı kıyılmış doku parçacıklarının büyüklüğü doğrudan bağlıdır. Uzun ve güçlü mekanik ayrılma, hücre ölümü ve kürenin oluşumunda bir azalma meydana gelebilir kaçınılmalıdır. </li><li> Un-ayrışmış parçaları ve enkaz kaldırmak için, 10-15 ml bazal orta tüp eklenir ve hücre süspansiyonu 50ml tüp içine 40 mikron hücre süzgecinden geçirilerek süzülür. </li><li> Süzülmüş süspansiyon 5min 800rpm (110g) santrifüj edilir. Süpernatant daha sonra atılır. </li><li> Pelletted hücreler daha sonra yeniden süspanse hücre sayımı için tam MGK orta 1-2ml. </li></ol> 3. Hücre Sayımı ve Kaplama <ol><li> Hücre süspansiyonu 10 mcL 0.04% Tripan mavi bir 1ml Eppendorf tüp içinde 90 mcL eklenir. Not: diğer uygun hücre dilüsyonuAfin grup de kullanılıyor olabilir. </li><li> Süspansiyonu karıştırmak için pipet ve aşağı yukarı. Hücre yoğunluğu saymak için 10 hücre / tripan mavi karışımı mcL hemasitometre aktarılır. </li><li> Hücreler,% 0.2 heparin (2μg/ml) 20ng/ml EGF, 10 ng bFGF ve 1μl/ml ile desteklenmiş tam MGK orta (MGK bazal, orta ve MGK proliferasyon takviyesi 09:01 oranında bir karışımı) kaplama. uygun doku kültürü gemiler. Orta, 5, 20 ve 40 ml, T25, T80 ve T175 şişeler için kullanılır. Antibiyotikler, kontaminasyon olasılığını azaltmak için 1:100 konsantrasyonda orta eklenebilir. </li><li> Balon, 37 ° C ve% 5 CO 2 inkübatör sette yer almaktadır . </li></ol> 4. GBM elde edilen küre Pasajlanması ve genişleme: <ol><li> Neurospheres ortalama büyüklüğü 150-200 mikron çapında ulaştığında, kültür altkültürü için hazırdır. Her balonun içeriği uygun bir boyut steril Tiss kaldırılır ve yerleştirilirue kültür tüp ve 800 rpm (110 gr) oda sıcaklığında 5 dakika santrifüj. </li><li> Süpernatant kaldırılır ve pelet 1 ml% 0.05 tripsin-EDTA yeniden süspanse. </li><li> Optimal bir trypsinization ulaşmak için, hücre süspansiyonu, 37 ° C&#39;de 2-3 dakika süreyle su banyosunda inkübe edilir. Tripsin faaliyeti durdurmak için soya tripsin inhibitörü eşit hacimli hücre süspansiyonu ilave edilir ve hücre süspansiyonu hafifçe aşağı yukarı pipetlenir ve. </li><li> Hücre süspansiyonu için 800 rpm (110g) 5 dakika santrifüj edilir. Sonra, süpernatantı kaldırılır ve hücrelerin MGK orta 1 ml yeniden süspanse. </li><li> Daha önce açıklandığı gibi bir hücre sayımı yapılır. </li><li> Hücrelerinin büyüme faktörleri ve uygun büyüklükte doku kültürü damarlarda önceki bölümde açıklandığı gibi kaplama ile takviye tam MGK orta 5x10 4 hücre / ml konsantrasyonda kaplanmıştır. </li><li> Nemli bir 37 ° C inkübe Ortaöğretim neurospheres 7-10 gün içinde oluşur% 5 CO 2 ified inkübatör. </li></ol> 5. Temsilcisi Sonuçlar: GBM tümör dokusu hasat tek hücre kaplama sonra, tümör kök hücrelerinin hızla çoğalmasına ve (video ve bkz. Şekil 1), 3-4 gün içinde birkaç hücreden oluşan küçük hücre kümeleri oluşturmak. Bu kümeler büyüdükçe, onlar 150-200 mikron formu (video ve Bakınız Şekil 2) ortalama çapı 7-8 gün böylece daha küresel bir şekil, uygun faz parlak küreler kazanır. Yüksek büyütme, sağlıklı küreler genellikle çevre microspikes göstermektedir. Kültür başlatılması, kültür binging olmayan ayrışmış kümeleri varlığı nedeniyle ve gerçek bir küre gibi karıştırılmamalıdır sonra 1-2 gün içinde kümeler gibi büyük bir küre olması. Primer tümörün küre kültür enkaz miktarı ilk doku kaynağı bağlı olarak değişir ve çevresindeki beyin dokusu içerir olsun ya da olmasın. Uygun doku hazırlama tekniklerienzimatik ve mekanik ayrılma ve örnek sonraki filtrasyon dahil olmak üzere yeterli miktarda orta kültür az enkaz neden olabilir. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Şekil 1" /> Şekil 1. Primer GBM küre kaplama 4 gün sonra kültür. Tümör kök benzeri hücreler çoğalmaya başlar ve 3-4 gün içinde küçük hücre kümeleri oluşturmak. Orijinal büyütme, 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Şekil 2" /> Şekil 2 Passage kaplama 8 gün sonra bir GBM kültür alanında. Orijinal büyütme, 20x.

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

Çıkar çatışması ilan etti.

Normal yetişkin ve fetal beyinleri 1, 2, 3, 4, nöral kök ve progenitör hücreler izole etmek için ve kök gibi, 5 akciğer, prostat 6 7, göğüs ve beyin 8, 9 gibi kanserli dokuların hücreleri neurosphere tahlil da tümör olmuştur sık tercih edilen yöntem olarak kullanılmaktadır. Ex vivo multipotency, implantasyon üzerine yeni tümörler oluşturmak için yeteneği ve kendini yenileme, bu basit ve tekrarlanabilir testi kullanılarak, bir somatik kök hücreler olarak benzer özellikler göstermektedir rezeke edilen tümör dokusu hücreleri belirsiz bir sayıda üretebilirsiniz. Bu hücreler, hücre-hücre etkileşimleri, ve farklılaşma, göç, istila ve hücre ölümü de dahil olmak üzere temel kanser hücre biyolojisi çalışma olabilir. Buna ek olarak, izole tümör kök hücrelerinin tümörleri, form, ilerleme ve nüks ve aynı zamanda çalışma sonunda tedavi için bilgi verebilir altta yatan kaynaklanan hücresel mekanizmaları aydınlatmak için paha biçilmez bir araç sağlar.seçenekleri.

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Tip </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> NeuroCult MGK Bazal Orta (İnsan) </td><td> Orta </td><td> Stem Cell Teknolojileri </td><td> 05750 </td><td></td></tr><tr><td> NeuroCult MGK Silahların Yayılmasını Önleme Takviyeler (İnsan) </td><td> Orta ek </td><td> Stem Cell Teknolojileri </td><td> 05753 </td><td></td></tr><tr><td> % 0.05 tripsin-EDTA </td><td> Reaktif </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td> Reaktif </td><td> Gibco </td><td> 41500-018 </td><td> HEM bileşeni </td></tr><tr><td> * Hepes </td><td> Reaktif </td><td> Sigma </td><td> H4034 </td><td> HEM bileşeni </td></tr><tr><td> * Damıtılmış su </td><td> Reaktif </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNaz I </td><td> Reaktif </td><td> Roche </td><td> 104159 </td><td></td></tr><tr><td> ** Soya tripsin inhibitörü </td><td> Reaktif </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Reaktif </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> No: 10 neşter bıçak </td><td> Cerrahi alet </td><td> BD </td><td> 371610 </td><td></td></tr><tr><td> Petri Dish </td><td> Kültür eşya </td><td> BD Falcon </td><td> 353003 </td><td></td></tr><tr><td> Küçük forseps </td><td> Cerrahi aletler </td><td> Güzel Bilim Araçları </td><td> 11050-10 </td><td></td></tr><tr><td> Hücre süzgeç </td><td> Elek </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 şişesi </td><td> Kültür eşya </td><td> Nalge Nunc uluslararası </td><td> 136196 </td><td></td></tr><tr><td> T80 şişesi </td><td> Kültür eşya </td><td> Uluslararası Nalge Nunc </td><td> 178905 </td><td></td></tr><tr><td> 15 ml tüpler </td><td> Kültür eşya </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml tüpler </td><td> Kültür eşya </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> EGF </td><td> Büyüme faktörü </td><td> Ar-Ge </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Büyüme faktörü </td><td> Ar-Ge </td><td> 3139-FB </td><td></td></tr><tr><td> Heparin </td><td> Büyüme faktörü </td><td> Sigma </td><td> H4784 </td><td> PBS içinde Sulandırılmış </td></tr></tbody></table> * HEM 1M Hepes MEM and160ml 1x10L paket karıştırmak ve hacim distile su kullanılarak 8.75 L getirmek için. 7.4 &#39;e son PH ayarlamad 4 ° C&#39;de saklayın ** Tripsin inhibitörü çözeltisi hazırlamak için, ilk DNaz I çözeltisi (100 mg DNaz 100 ml HEM çözünmüş) 10 ml ve daha sonra DNaz çözüm tripsin inhibitörü 0.14 g ve nihayet HEM kullanarak 1 Litre hacim makyaj. -20 ° C derin dondurucuda son ürün hacimde tutun.

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Kök-benzeri hücrelerin tümörleri, meme, akciğer, kolon, prostat ve beyin gibi izole edilmiştir. Kritik bir konu, özellikle glioblastoma mutliforme (GBM), tüm bu tümörler, tümör oluşumu, ilerleme ve nüks rollerini araştırmak için tümör başlatarak hücre popülasyonu (lar) belirlemek ve yalıtmak için. Anlama tümör hücre popülasyonlarının başlatarak bu tümörler için etkili tedavi yaklaşımları bulmak için ipuçları sağlayacaktır. Sadeliği ve tekrarlanabilirlik nedeniyle neurosphere assay (NSA), bu tümör hücrelerinin birçok izolasyon ve yayılması için tercih edilen bir yöntem olarak kullanılmaktadır. Bu protokol, cerrahi rezeksiyon insan GBM tümör dokusu kök benzeri hücreler izole etmek ve genişletmek için neurosphere kültür yöntemi göstermektedir. Prosedürleri bir başlangıç ​​kimyasal sindirim ve tümör dokusu mekanik ayrılma, ve daha sonra NSA kültürü ortaya çıkan tek bir hücre süspansiyonu kaplama içerir. 150-2, 7-10 gün sonra birincil neurospheresÇapı 00 mikron gözlenen ve daha Pasajlanması ve genişleme için hazır olabilir.

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Bu video protokolü neurosphere tahlil kültürü yöntemi kullanılarak insan glioblastoma mutliforme cerrahi rezeksiyon (GBM) tümör dokusu hücreleri gibi izolasyon ve kök genişlemesi göstermektedir.

Neurosphere Testi İnsan Glioblastoma Multiforme Tümör Hücreleri İzolasyon ve Genişleme

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

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Research

JoVE Journal

Neuroscience

29.6K Görüntüleme.  University of Florida. Bu video protokolü neurosphere tahlil kültürü yöntemi kullanılarak insan glioblastoma mutliforme cerrahi rezeksiyon (GBM) tümör dokusu hücreleri gibi izolasyon ve kök genişlemesi göstermektedir.

Video: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Neurosphere Testi Yetişkin Fare Nöral Kök Hücre İzolasyonu ve Genişleme

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Nörobilim

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere Testi kullanarak Embriyonik Fare Nöral Kök Hücre Kültürü oluşturulması

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

İnsan Mantar şeklinde Lezzet papilla hücrelerinin izolasyonu ve Kültürü

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Kaynaklı Nöronlar içine Yetişkin İnsan Beyninin perisit türetilmiş Hücreler Lineage yeniden programlama

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

F9 NADİR-LacZ Hücre tabanlı Muhabir Test kullanma E8.5 Embriyolar ve Neurosphere Kültürleri Bağıl Retinoik Asit Düzeylerinin Kantitatif Ölçümü

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Mikroenjeksiyon kullanarak ınsan hela hücrelerinde Spliceosomal snRNA yerelleştirme Analizi

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

İnsan İndüklenen Pluripotent Kök Hücrelerden Beyin Mikrovasküler Endotel Hücrelerinin Derivasyonu, Genişlemesi, Kriyoprezervasyonu ve Karakterizasyonu

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Yetişkin Zebra Balığı Kullanarak Öğrenme ve Bellek Çalışmalarında Bilişsel Değerlendirme için İlişkisel Bir Öğrenme Aracı Olarak Mekik Kutusu Tahlil

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

İnsan Sünnet Derisinden Primer Schwann Hücrelerinin, Keratinositlerin ve Fibroblastların İzolasyonu, Kültürü ve Karakterizasyonu

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

İnsan Mikroglia Benzeri Hücreler: İnsan Sinaptozomları Kullanılarak İndüklenmiş Pluripotent Kök Hücrelerden ve In Vitro Canlı Hücre Fagositoz Analizinden Farklılaşma

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...