The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

NOT: plasenta vadeli elektif sezaryen için bir tercih, tekiz gebeliklerde sağlıklı toplanan edilmelidir. Yazılı bilgilendirilmiş onam kendi plasenta toplanması için verilmelidir. Sizin ilgili insan araştırma etik kurul onayı tüm koleksiyon ve insan dokularının kullanılması gerekir. Amniyon Epitel Hücreleri 1. İzolasyon <ol style="margin-left: 40px;"><li> Bir sınıf II biyolojik güvenlik kabini içinde steril bir yüzeye yerleştirin plasentayı. </li><li> Steril Hanks ile dengelenmiş tuz çözeltisi (HBSS) kullanarak, plasenta yüzeyi ve zarlardan mümkün olduğu kadar kan yıkayın. </li><li> Göbek kordonu yukarı bakacak şekilde yerleştirilen plasenta ile, mekanik amniyon ve koryon zarlarının dış kenarını ayrı. </li><li> Ayrıldıktan sonra, elle göbek kordonu doğru, membran kenarında çalışan, plasentanın koryon zarından amniyon zarı şerit. NOT: caref Beul amniyon gelen koryon zarı kirlenmesine parçaları varsa kaldırmak için. </li><li> Steril makas kullanılarak, 250 ml HBSS içeren kabı sızdırmaz bir 500 ml&#39;lik, göbek bağı ve yer tabanından yaklaşık 2 cm amniyon membran kesti. </li><li> Amniyon zarı içeren mühürlü şişeyi sallayarak iyice yıkayın. </li><li> 15 cm Petri kabı içine toplanması steril forseps kullanarak konteyner ve yerden amniyon zarı çıkarın. </li><li> Kanlı veya yırtılmış parçaları atarak, (forseps ile dikey tutulduğunda), yaklaşık 5 cm uzunluğunda parçalar halinde amniyon zarı kesin. </li><li> 500 ml&#39;lik bir yer kabı sızdırmaz şekilde kapatıldı ve 250 ml HBSS ile yaklaşık 3-5 kez yıkama veya amniyon membranı kirlenmeye sebep olan hiçbir kan saydam hale gelinceye kadar. NOT: Herhangi bir kan mevcut çözüm sindirmek enzimatik etkinliğini azaltabilir. </li><li> Dikkat ederek, çözelti sindirmek enzimatik 50 ml ihtiva eden bir kap içine amniyon zarın tüm parçaları transferHBSS taşınmasını en aza indirir. </li><li> Kalan kanı çıkarmak için yavaşça sallanarak 15 dakika için 37 ° C&#39;de su banyosunda veya sıcak bir odada çözüm sindirmek enzimatik olarak amniyon parçaları inkübe. </li><li> Yeni bir steril kap içinde çözüm ve yer sindirmek enzimatik gelen amniyon zarı parçalarını çıkarın. </li><li> Taze enzimatik 100 mi amniyon zara çözeltisi sindirimi ve bir su banyosu ya da yumuşak çalkalama (birinci sindirim) ile sıcak bir odada 60 dakika süre ile 37 ° C&#39;de inkübe ekleyin. </li><li> Amniyon zarı parçalarını çıkarın ve enzimatik zarının her parçasından tahliye çözüm sindirmek izin vermek için yeni konteyner alarak bakım içine yerleştirin. Not: Bu, kabın iç kenarı boyunca amniyon parçaları sürükleyerek elde edilebilir. </li><li> Taze enzimatik 100 mi amniyon membran parçaları çözeltisi sindirimi ve hafif çalkalama (ikinci sindirim) ile 37 ° C&#39;de bir su banyosu ya da sıcak bir odada 60 dakika inkübe ilave edin. </li><li> Hücre asıltı geçmek10 dakika boyunca 1.000 xg&#39;de 70 mikron filtre ve santrifüj yoluyla Adım 1.14 itibaren. </li><li> Çıkarın ve supernatant ve 1 mg / ml soya fasülyesi bazlı bir enzim inhibitörü içeren 4 ml HBSS hücre pelletini. Yavaşça tek bir hücre süspansiyonu elde etmek üzere, 1 ml pipet ile tekrar tekrar pipetle. Not: ikinci sindirilmiş hücre işlem aynı seviyede olana kadar bu aşamada hücreler, buz üzerinde bırakılabilir. </li><li> Amniyon zarı parçalarını çıkarın ve enzimatik zarının her parçasından tahliye çözüm sindirmek izin vermek için yeni konteyner alarak bakım içine yerleştirin. Not: Bu, kabın iç kenarı boyunca amniyon parçaları sürükleyerek elde edilebilir. </li><li> 10 dakika boyunca 1.000 xg&#39;de 70 mikron filtre ve santrifüj yoluyla Adım 1.18 hücre süspansiyonu geçirin. </li><li> Çıkarın ve supernatant ve 1 mg / ml soya fasülyesi bazlı bir enzim inhibitörü içeren 4 ml HBSS hücre pelletini. Yavaşça 1 ml p art arda pipettek bir hücre süspansiyonu elde etmek için ipette. </li><li> İki digests veya alternatif Havuz hücreleri hücre sayımları sonrasına kadar ayrılmış hücre digests tutun. NOT: Bu yüksek canlılık hücreleri ile potansiyel olarak düşük canlılığı hücrelerinin karıştırılması önler. </li><li> 10-20 ml&#39;lik bir son hacme HBSS ekleyin ve canlılığını ölçmek için tripan mavisi kullanarak hücreleri sayısını gerçekleştirmek. </li><li> Flow sitometri ile epitel hücre fenotipi onaylayın. Anti-EpCAM-PE kullanılarak uygun antikor kokteyli Makyaj (1: 2 seyreltme), anti-CD90-PeCy5 (1: 250) ve anti-CD105 APC (1: 100), HBSS. </li><li> 25 x 10 6 hücre / ml&#39;lik bir konsantrasyonda bir hücreler ya da antikor kokteyli içinde iki digests bir alt yeniden süspanse edin. </li><li> Flow sitometri, leke izotip kontrolleri içeren antikor kokteylleri ile 1 x 10 6 hücre için (örn., IgG 1 izotop kontrol-PE + Anti-CD90-PeCy5 + Anti-CD105-APC). Set-up akış sitometresi için lekesiz ~ 1 x 10 6 hücre tutun. </li><li> Antikorda hücreleri inkübe60 dakika boyunca 4 ° C&#39;de kokteyller. </li><li> Yıkama akış sitometresi için hücreler, ml başına 5-10.000.000 hücreler HBSS ve tekrar süspansiyon 3x. </li><li> Akış sitometrik ve not sonuçlarını gerçekleştirin. Hücre izolatı (EpCAM pozitif ve CD90 / CD105 negatif hücrelerin yüzdesi) saflığı sırasıyla% 90-95 ve &lt;% 1 olacağı tahmin edilmektedir. </li><li> Nihai uygulama tarafından tespit edildiği üzere, uygun kalite kontrol ve serbest bırakma kriterleri için test edilmesi için bir kenara hücrelerin uygun bir sayısı ayarlayın. NOT: Bu hastalık / viral tarama veya diğer güvenlik veya biyolojik kriterlerini içerebilir. </li><li> Kalan hücreler için, Kriyoprezervasyonun protokolünü takip veya alternatif (kültür adım 3.6 doğrudan) kültür doğrudan yerleştirin. </li></ol> HAECs 2. Kryoprezervasyon <ol style="margin-left: 40px;"><li> Veri veya tripan mavi dışlama manuel sayım flow sitometri kullanılarak hücre sayısı ve canlılığı belirleyin. </li><li> 5 dakika boyunca 700 x g&#39;de santrifüjleyin. </li><li> Süspanse hücrehayvansal ürün içermeyen kriyoprezervasyon medya ml başına 1-10 milyon hücreleri arasındaki s. </li><li> O-halkalı dondurarak saklama şişeleri içine hücrelerin uygun bir hacim pipet ve soğutma yaklaşık olarak meydana geldiği hızı kontrollü dondurma cihazının içine koyun 1 ° C / dakika kadar -80 ° C elde edilir °. </li><li> Uzun süreli depolama için sıvı azot içine şişeleri aktarın. </li></ol> Cyropreserved hAECs 3. erimesi ve Kültür <ol style="margin-left: 40px;"><li> Sıvı azottan dondurarak saklama şişeleri çıkarın ve buz üzerine yerleştirin. NOT: Yalnızca transferi için - bir sonraki adıma geçin hızla. </li><li> Buz kalır, sadece küçük bir parça (~ 5 mm x 5 mm) kadar 37 ° C de hızlı bir şekilde dondurarak saklama şişeleri çözülme. NOT: Bu hücre çözümü dondurulması medyada DMSO toksik etkilerini azaltmak için sıcak olmadığı önemlidir. </li><li> Soğuk (~ 4 ° C), serum içermeyen ortam ile dondurarak saklama Ortam 10 kat seyreltilir, damla damla uygun bir boyut halinde ilaved tüp veya kap. </li><li> Oda sıcaklığında 5 dakika boyunca 700 x g&#39;de santrifüjleyin. </li><li> Ml başına 1.000.000 hücre bir konsantrasyonda için, serum barındırmayan ortam içinde yeniden süspanse edin. Tripan mavi dışlama ile otomatik veya manuel sayım kullanarak hücre sayısı ve canlılığı belirleyin. Beklenen canlılık sonrası çözülme öncesi kriyokoruma canlılığı% 5-10 daha az olacaktır. </li><li> Plaka standart hücre kültürü-işlenmiş plastik eşya üzerine hücreler, veya alternatif kullanmak Tip I kat kültür yüzeylere kollajen. </li><li> Tip I Kollajen kaplama için aşağıdaki protokolü takip <ol><li> İnsan plasentasından (25 ml deiyonize su ve 50 ul asetik asit, 15 mg, kolajen) &#39;ye ait asit çözünebilir Tip I Kolajen hazırlayın. </li><li> Parafilm ile beher Kapak ve kolajen iplikler eriyene kadar 37 ° C&#39;de orta karıştırın. </li><li> 0.22 um filtre kullanılarak steril filtre kolajen çözeltisi. </li><li> Deiyonize su ile filtrelenmiş kollajen stok 01:10 sulandırmak. Bu seyreltilmiş hisse çalışma conce olduğunuKaplama, plastik ve zar yüzeylerin ntration çözeltisi. </li><li> Kollajen kat plastik, 37 ° C&#39;de, oda sıcaklığında 2 saat için cam ve zar yüzeylerin ya da O / N, 2-8 ° C&#39;de ilave edildi. </li><li> Kaplanmış yüzey aşırı sıvı kaldırmak, ve O / N kurumasını bekleyin. </li><li> Hücreleri ve orta tanıtan önce steril doku kültürü dereceli su veya HBSS ile durulayın. </li></ol></li><li> Levha serumsuz ortam içinde 25,000 hücre / cm2 yoğunluğunda hAECs ve ek 72-96 saat boyunca oluşmasına izin verir. </li><li> Ek sonra, dikkatli bir şekilde aspire edilmesiyle ve serumsuz ortamda uygun bir hacim, her 2-3 günde bir ile değiştirerek ortamı değiştirmek. </li><li> Aşağıdaki protokolü kullanarak Passage hücreleri <ol><li> Aspire hücre kültürü ortamı ve steril HBSS 1x hacmi ile yıkayın </li><li> Hücreler, hücre kültürü yüzeyinden ayırma gözlenen kadar enzimatik 1x hacmi ekleyin çözüm sindirmek ve 5-15 dakika inkübe, ya da. </li><li> Çözelti, hücreleri ihtiva eden sindirimi ce sağlanması enzimatik toplamakrf defalarca hücre kültürü yüzeyine karşı çözüm sindirmek enzimatik pipetleme hücre kültürü yüzeyinden ayrılır </li><li> 5 dakika boyunca 700 x g&#39;de santrifüjleyin. </li><li> 1 mg / ml soya fasülyesi bazlı bir enzim inhibitörü ihtiva eden HBSS uygun bir hacimde hücre pelletini. Yavaşça tek bir hücre süspansiyonu elde etmek için bir 1ml pipet ile art arda pipetle. </li><li> 1.000.000 hücre / ml&#39;lik bir konsantrasyonda serum içermeyen ortam ekleyin. Tripan mavi dışlama ile otomatik veya manuel sayım kullanarak hücre sayısı ve canlılığı belirleyin. </li><li> Levha serumsuz ortam içinde 25,000 hücre / cm2 yoğunluğunda hAECs, standart kültür yöntemleri ile devam eder. </li></ol></li></ol>

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The authors have nothing to disclose.

Bu metodolojinin başarısında önemli bir etkiye sahip birkaç kritik parametreler vardır. HAECs izolasyonundan önce 3 saat süre ile plasenta veya amniyon depolanması ancak doku mümkün olan en kısa zamanda işlem önerilmektedir, lojistik ve programlama amacıyla arzu edilebilir. Doku saklanacaksa, bu depolama amniyon membran diseksiyon ve yıkama sonrası yapılması tavsiye edilir. Amniyon sıvısı 4 ° C&#39;de steril HBSS içeren antibiyotik saklanabilir ancak hücre canlılığı, uzun depolama süresi il...

Plasenta gibi perinatal kaynaklardan, plasental membranlar, göbek kordonu ve amniyotik sıvıdan elde edilen hücreler rejeneratif tıp 1,2 için hücrelerin bir potansiyel kaynağı olarak araştırmacılar ve klinisyenler dikkatini çekmiştir. Bu ilginin nedeni, bu hücre tipleri, tüm plastisite ve bağışıklık yeteneği 3, potansiyel terapötik uygulamalar için temel olan bazı özellikleri derecesine sahip olmasıdır. hAECs rejeneratif hücre malzemeden bir miktarda potansiyel kaynağı temin terimi ya da önceden dönem amniyon zar 4 elde edilebilir heterojen epitel nüfus bulunmaktadır. hücresel bir tedavi olarak çekici hAECs yapmak özellikleri kendi multipotency, düşük imünojenite ve anti-inflamatuar özelliklerini içerir. hAECs (yüksek ölçüde, in vitro ve in vivo olarak multipotent mezodermal soy içine türetme yeteneğinde olduğu kardiyomiyopati bulunmuşturyocytes, myocytes, osteositler, adiposit), Endodermal soy (pankreas hücreleri, karaciğer hücreleri, akciğer hücreleri) ve ektodermal soyları (saç, cilt, sinir hücreleri ve astrositler) 5-10. Reassuringly kendi multipotency hAECs ya bir şekilde tümörlere görünür ya da in vivo olarak tümör gelişimini teşvik etmemesine karşın. Ayrıca, hAECs de sınıf II insan lökosit antijenleri (HLA&#39;lar) 8 düşük seviyelerde ifade, ayrıcalıklı bağışıklık bulunmaktadır. Bu özellik muhtemelen bağışıklık yetkili maymunlar, tavşan, kobay, sıçan, ve domuzlar 11-13 kullanıldığı çalışmalarda gösterildiği gibi, allojenik ve ksenojenik transplantasyon sonrası immün ret kaçınmak için kendi yeteneğini temelini oluşturmaktadır. hAECs güçlü immünomodülatör ve immünosüpresif özellikler gösterir ve böylece otoimmün hastalığın tedavisinde potansiyel klinik uygulamalar için önemli bir pratik avantaj sağlar. hAECs hem doğal ve adaptif bağışıklık sistemi üzerinde bağışıklık fonksiyonlarını uygulamak inanılmaktadır. Üzerindeimmunomodülator salgılanması 14 faktörleri ile öne mekanizmaların e. Klinik öncesi hayvan hastalık modellerinde hAECs Mevcut uygulamalar felç, multipl skleroz, karaciğer hastalığı, şeker hastalığı ve kronik ve akut akciğer hastalıkların tedavisi bulunmaktadır. Araştırmacılar nedeniyle benzersiz özellikleri inme sonrası beyin iltihabı tedavi etmek hAECs kullanarak ilgi gösterdi. HAECs, onlar aşılamak olabilir kan beyin bariyerini kadar 60 gün boyunca hayatta, nöronlar içine ayırt, inflamasyonu azaltmak ve nörolojik hastalıkların 15 hayvan modellerinde hasarlı santral sinir sistemi dokusunun yenilenmesini teşvik edebilir dair kanıtlar vardır. hAECs hedef ve multipl skleroz gelişmesi ve ilerlemesine katkıda birden patolojik yollar ters yeteneği sunuyoruz. Örneğin, klinik öncesi hayvan çalışmalarında kaynaklanan hAECs kuvvetle bağışıklık olduğunu göstermektedir vepotansiyel çevresel immün tolerans neden ve devam eden inflamatuar yanıtları tersine çevirebilirsiniz. hAECs in vivo sinir hücrelerine dönüştürmek ve nörotropik geniş bir dizi 16 faktörleri salgılayarak endojen sinir sistemini geliştirmek için kapasitesine sahip olduğu gösterilmiştir. İnsan ve kemirgen amniyon epitel hücreleri, hayvan modellerinde karaciğer hastalığının tedavisi için terapötik etkinliğini göstermiştir. Düşük hepatosit apoptoz eşliğinde karaciğer hastalığı, karaciğer canlı hAECs nakledilmesiyle için haec nakli kurşun, karbon tetraklorür hasar indüksiyon modelinde ve hepatik inflamasyon ve fibrozis 17 azalmıştır. hAECs insülin ve glükoz taşıyıcıları içeren ifade pankreas faktörlere stimüle edilebilir. Çeşitli çalışmalar, diyabetik farelerde 18 kan şekeri seviyelerinin normale dönmesi için hAECs potansiyelini araştırdık. Farelerde almahAECs, her iki hayvan vücut ağırlığı ve kan glukoz seviyeleri, hücre enjeksiyonunun ardından, normal seviyelere düşmüştür. Bu çalışmalar, diyabet tedavisi için hAECs kullanımı için güçlü bir sunulmaktadır. hAECs yetişkin ve yenidoğan modelleri 19 hem de önlenmesi ve deneysel akut ve kronik akciğer hasarı tamir kanıtlanmış rolü vardır. Bu çalışmalar, hAECs Kistik fibroz membran geçirgenliğinin düzenleyicisi (CFTR), kistik fibroz 20 hastalarda mutasyona uğramış, ve iyon kanalının de dahil olmak üzere birden fazla akciğer ilişkili proteinler ifade eden fonksiyonel akciğer epitel hücrelerine in vitro olarak ayırt bulundu. HAECs yaralı yetişkin ve yeni doğan akciğer teslim edilir, ayrıca, bunların nötrofil, makrofajlar ve lenfositler 21-23 dahil olmak üzere akciğer lökosit işe, azaltılması, konak bağışıklık hücrelerinin modülasyonu yoluyla onarım etkilerini uygularlar. Onların bolluğu göz önüne alındığında,Emniyet kaydı ve birden hastalıklar için kanıtlanmış klinik uygulamalar, hAECs kullanarak klinik çalışmalarda kaçınılmazdır. Klinik denemeler içine haec tedavilerin çeviri hızlandırılması amacı ile, biz mevcut iyi üretim uygulamaları (cGMP) kurallarına uygun olarak hayvansal ürün içermeyen reaktifler kullanılarak, klinik çalışmalar için uygun bir şekilde kültür hAECs, izole cryopreserve ve yöntemler geliştirdi . Biz bu protokolünü biz hayvansal kaynaklı reaktifler 6 kullanarak hAECs izole başarıyla kullandığınız bir önceden yayınlanmış bir protokol tabanlı. Biz hayvansal ürün içermeyen reaktif ile hayvansal kaynaklı ürünler yerine orijinal protokol değişmiş ve sonraki optimizasyon hücre verimi, canlılığı ve saflık optimize etmek yapıldı. Amacımız, insan klinik denemeler için hücre üretimi için düzenleyici standartlara uygun bir protokol geliştirmekti.

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			<td style="width:119px;">13-361B</td>
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			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
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			<td>S17329&#160;</td>
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			<td>19-014-643&#160;</td>
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			<td style="width:119px;">S-15374-M</td>
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			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
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			<td height="21" style="height:21px;">General purpose face mask</td>
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			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
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			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
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			<td style="width:119px;">T3449</td>
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			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
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			<td style="width:119px;">T6522</td>
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			<td height="21" style="height:21px;">Cryostor CS5</td>
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			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
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			<td height="21" style="height:21px;">anti-EpCam-PE</td>
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			<td style="width:119px;">130 - 091-253</td>
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			<td style="width:119px;">130-098-845</td>
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			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
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			<td>356551</td>
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			<td>356543</td>
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			<td height="21" style="height:21px;">50mL falcon tubes</td>
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			<td>352070</td>
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			<td height="21" style="height:21px;">15mL falcon tubes</td>
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			<td>352096</td>
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			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
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			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
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			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
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			<td style="width:119px;">02-707-401</td>
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			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
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			<td>14-245-97&#160;</td>
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			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
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			<td>02-202-102&#160;</td>
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			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
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			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
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			<td></td>
		</tr>
	</tbody>
</table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

Bu prosedür doğru devam edildiğinde, 120,000,000 hAECs bir ortalama verim 80-160,000,000 hücre tipik aralık, beklenmelidir. Bu verimi kaynaktan, 83 ±% 4 arasında bir ortalama canlılığı beklenebilir. Klinik yöntemde artan ortalama verimi ve biraz daha düşük bir canlılık bağlı olarak serum proteinleri olmaması belki de, hayvan türevi ürün daha yüksek bir tripsin aktivitesi nedeniyle olabilir ve. İzole hAECs &lt;% 1, CD90, CD105, mezenkimal geni taşıyan hücreler ile% 92 EpCAM pozitif hücreleri ...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K Görüntüleme.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

Video: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

İnsan Fetal Pankreas Hücre Kümeleri İzolasyon, Kültür ve Görüntüleme

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

İnsan Hipofiz Adenom Hücreleri üç boyutlu Aljinat-boncuk Kültür

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Köpek arter ve venlerin Primer Endotel Hücreleri İzolasyon ve Kültür

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

İnsan böbrek Perivasküler Stromal hücreler için bir roman klinik sınıf izolasyon yöntemi

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

İnsan Nazal Epitel Organoidlerinin Kültürü ve Görüntülenmesi

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

İnsan Tedavisi için Yenilikçi Bir Ksenojenik İçermeyen Yöntemle İnsan Adipoz Türevi Kök Hücrelerin İzolasyonu ve Kültürü

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

İnsan Kök Hücre Kaynaklı Retina Pigment Epitel Hücrelerinin Kriyo Depolamasının İlerletilmesi

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Yazar Spotlight: Retina Pigment Epitel Hücre Dondurma için Verimli ve Uygulanabilir Bir Yöntemin Geliştirilmesi

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

İnsan Embriyonik Kök Hücre Kaynaklı Retina Pigment Epitel Hücrelerinin Optimal Aşamada Dondurularak Saklanması

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...