Name: isolation, culture, and characterization of dental pulp stem cells from human deciduous and permanent teeth
Uploaded: 2024-05-17T13:10:00
Description: Read this Scientific Article Protocol about Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth at JoVE.com

Yazarlar, Thiruvalla'daki Pushpagiri Araştırma Merkezi Müdürü ve Başkanı Dr. Mathew Mazhavancheril'e, Araştırma Merkezi'ndeki prosedürlerin belgelenmesine verdiği destek için minnettardır.

Burada özetlenen protokol, kurumsal insan araştırmaları etik komitesinin (IRB, Pushpagiri Araştırma Merkezi, Kerala) yönergelerine uygundur. Çekilen dişlerin kullanımı, katılımcıların bütünlüğünü, haysiyetini ve haklarını sağlamak için etik standartlara uygun olarak gerçekleştirilmiştir. Bu çalışma için seçilen katılımcılar, ortodontik tedavi için diş çekimi gereken 30 yaşın altındaki sağlıklı bireylerdi. Geniş diş çürüğü veya şiddetli periodontitisi olanlar çalışma...

Protokol, insan süt ve daimi dişlerinden diş pulpası kök hücrelerinin (DPSC'ler) izolasyonunu, kültürünü ve karakterizasyonunu ana hatlarıyla belirtir. Bu hücrelerin depolanması ve çoğalmasının bir açıklamasının yanı sıra in vitro osteoblastlara, adipositlere ve kondrositlere farklılaşma potansiyellerinin değerlendirilmesini içerir35.
Chen ve ark.36, Dental Pulpa Kök Hücrelerinin (DPSC...

Kök hücre araştırmaları, rejeneratif tıp ve doku mühendisliğindeki umut verici uygulamaları nedeniyle biyomedikal biliminde gelişmiştir. Hem insan süt dişlerinin hem de kalıcı dişlerinin pulpa dokusundan elde edilen pulpa kök hücreleri (DPSC'ler), hazır bulunmaları ve multipotent kapasiteleri nedeniyle kök hücre kaynağı olarak büyük ilgi görmüştür 1,2. Bu hücreler, çok sayıda çalışma ile onaylandığı gibi, adipositler, osteoblastlar ve kondrositler dahil olmak üzere çeşitli hücre tiplerine farklılaşma potansiyeline sahiptir3.
Son birkaç on yılda, kök hücrelerin araştırma ve terapötik uygulamaları artmıştır. Kök hücrelerin geniş potansiyeli, elde edildikleri kaynakları çeşitlendirmeyi gerektirir. Seçilen hücrelerin verimliliğini, canlılığını ve köklülüğünü etkileyen çeşitli faktörler vardır. Kemik iliği ve yağ dokuları gibi bilinen çeşitli kök hücre rezervuarlarının varlığına rağmen, bu kaynaklara bağlı invaziv prosedürler, saha morbiditesi ve etik kaygılar sıklıkla araştırılmalarını sınırlar 4,5. Çeşitli kök hücre kaynakları arasında, dental kök hücreler kolay erişilebilirlikleri, yüksek plastisiteleri ve çeşitli potansiyel uygulamaları nedeniyle dikkat çekmiştir. Özellikle insan diş pulpası kök hücreleri, terapötik beklentileri için kapsamlı bir şekilde araştırılmıştır6. Genellikle tıbbi atık olarak atılan dişler, çok sayıda mezenkimal kök hücre içerir7. Bu değerli kök hücre havuzunu korumak, bu kaynakların boşa harcanmamasını sağlamak için hastaların, diş hekimlerinin ve doktorların ortak çabalarını gerektirir ve her bir diş pulpası kök hücresini gelecekteki rejeneratif gereksinimler için kullanılabilir hale getirir.
İnsan yetişkin diş pulpası kök hücreleri (DPSC'ler) ve pul pul dökülmüş insan süt dişlerinden (SHED) elde edilen kök hücreler gibi diş pulpasından türetilmiş kök hücreler, diş pulpasının perivasküler nişinde bulunur. Bu hücrelerin kraniyal nöral krest hücrelerinden kaynaklandığına ve hem mezenkimal kök hücreler (MSC'ler) hem de nöroektodermal kök hücreler için erken belirteçler sergilediğine inanılmaktadır. DPSC'ler ve SHED'ler, çok potansiyelli ve çeşitli doku tiplerini yeniden oluşturma yeteneği göstermiştir8.
Dental kök hücrelerin potansiyel kaynakları sağlıklı, süt ve daimi dişleri kapsar. Kök hücreler, pulpadaki toplam hücre popülasyonunun sadece yaklaşık %1'ini oluşturur ve bu da etkili izolasyon ve genişletme tekniklerinin önemini vurgulamaktadır9. Sonuç olarak, bu kök hücrelerin ekstraksiyonu ve genişletilmesi, DPSC izolasyonunda çok önemli adımlardır10. Çekilmiş veya pul pul dökülmüş dişlerin, fosfat tamponlu salin (PBS) veya Hanks tamponlu salin solüsyonu (HBSS) gibi besin açısından zengin bir taşıma ortamında saklanması gerekir.
Diş pulpasının elde edilmesi, diş tipine bağlı olarak çeşitli yöntemlerle sağlanabilir 7,11. Kökleri emilmiş süt dişleri için kök ucu üzerinden çekim yapılabilir. Benzer şekilde, olgunlaşmamış açık tepeli kalıcı dişlerden pulpa elde etmek için steril dikenli broşlar kullanılabilir. Kökleri tam gelişmiş daimi dişlerde, pulpa odasına erişim, diş kronunu kökten ayırmayı içerir. Bu, sementoenamel kavşağında bir elmas disk kullanılarak dişin kesilmesiyle gerçekleştirilir. Bu kesi, pulpa odasını açığa çıkararak pulpa dokusunun alınmasını sağlar 12,13,14.
Dental pulpa kök hücreleri (DPSC'ler), enzimatik sindirim (ED) veya spontan büyüme olarak da bilinen doku eksplantlarından (OG) büyüme yoluyla izole edilebilir. ED yöntemi, dokuyu tek hücreli süspansiyonlaraayırmak için başta kollajenaz I ve dispas olmak üzere enzimler kullanır 15,16. Daha basit ve daha hızlı olan OG yöntemi, pulpa parçalarının doğranmasını ve doğrudan bir kültür plakasına yerleştirilmesini gerektirir, bu da hücrelerin doku eksplantlarındanbüyümesine izin verir 17. Araştırmacılar, hücre proliferasyon oranlarını, izole kök hücre özelliklerinin korunmasını, farklılaşmayı ve yüzey işaretleyici ekspresyonunu değerlendirmek için her iki tekniği de kullandılar ve karşılaştırdılar18. Yüksek verimlilik ve saplılığa sahip DPSC'lerin elde edilmesi için protokollerin oluşturulması ve standartlaştırılması, etkili ve güvenli tedavilerin önünü açabilir19. Bu protokol, enzimatik sindirim, laboratuvar işleme, koruma ve adipogenez, osteogenez ve kondrogenez için kolorimetrik boyama ile hücre farklılaşması kullanılarak DPSC'lerin çıkarılmasını kapsar.
Bu makalede özetlenen protokol, diş pulpasının dişten ilk izolasyonu ile başlayan, ardından DPSC'lerin laboratuvarda kültürü ve bakımı ile devam eden ve spesifik kök hücre belirteçleri kullanılarak karakterizasyonları ile sonuçlanan adım adım bir prosedür sunmaktadır (Şekil 1). Bu kök hücreleri farklı hücre soylarına indüklemeye yönelik teknikler ve çoklu potansiyellerini vurgulama teknikleri de açıklanmaktadır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-isobuty-l-methyl-xanthine&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>AS001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>RM471</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S staining solution</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>GRM894</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase -Staining kit</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha-MEM, or Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha-MEM, or Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>012-04802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-glycerophosphate&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>G9422-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet-Laminar flow hood</td>
			<td>Labconco Corporation,MO 64132-2696,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD90, CD105, CD73, CD34, CD45, and HLA-DR</td>
			<td>BioLegend, Inc.CA 92121,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m )</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP025</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>REMI Elektrotechnik Limited (REMI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>1101 | 1102</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp. NJ 08701, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp. NJ 08701, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Growth Medium</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>AT006</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Conical tubes (15 or 50 )</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td>546021P/546041P</td>
			<td>15 mL and 50 mL</td>
		</tr>
		<tr>
			<td>Cryo freezing container</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>15-350-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryolabels</td>
			<td>Label India:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial storage boxes&#160;</td>
			<td>Cryostore Storage Boxes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials (1.8 mL)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>PW1282</td>
			<td>Self standing</td>
		</tr>
		<tr>
			<td>Culture flask (25 cm&#178;)</td>
			<td>Corning Inc.NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCG4/TCG6</td>
			<td>T25/T75</td>
		</tr>
		<tr>
			<td>Culture Plates</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP129/TCP008</td>
			<td>60 mm/100 mm</td>
		</tr>
		<tr>
			<td>Dental Diamond Discs</td>
			<td>Komet SC 29730, USA</td>
			<td></td>
			<td>Komet&#160;</td>
		</tr>
		<tr>
			<td>Dental Spoon Excavator</td>
			<td>Brasseler,GA 31419,USA</td>
			<td>5023591U0</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethosone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethosone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TC185</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche Diagnostics,Mannheim,Germany.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche Diagnostics GmbH, Mannheim,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>MB106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol -70%</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>Extraction forceps&#160;</td>
			<td>Dentsply Sirona, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>RM9954</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin-coated tissue culture plate</td>
			<td>Corning Inc.Corning, NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences,CA 95131,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry buffer</td>
			<td>BD Biosciences,CA 95131,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slip 22 x 22 mm</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Lonza Group Ltd,4002 Basel, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed dental handpiece&#160;</td>
			<td>NSK Ltd,Tokyo 8216, Japan</td>
			<td></td>
			<td>Ti-Max Z series</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX, or 3-isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Pfizer Inc. NY 10017,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>I5523</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>I5523</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS) premix</td>
			<td>Corning Incorporated,MA 01876,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus Corp.,Tokyo 163-0914,Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (60% )</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>MB063</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine mixed with epinephrine</td>
			<td>DENTSPLY,NC 28277,USA</td>
			<td></td>
			<td>Citanest</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Air Liquide,75007 Paris,France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen storage tank</td>
			<td>Cryo Scientific Systems Pvt. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf AG,22339 Hamburg,Germany</td>
			<td>30020</td>
			<td>Accupipet-2-20 &#181;L</td>
		</tr>
		<tr>
			<td>Mini tissue grinder</td>
			<td>Bio-Rad Lab, Inc. CA 94547,USA</td>
			<td></td>
			<td>ReadyPrep mini grinders</td>
		</tr>
		<tr>
			<td>Minus 80 freezer</td>
			<td>Blue Star Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld Superior,arktheidenfeld,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oil red O stain</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>1024190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Osteogenic Differentiation Medium (ODM)&#160;</td>
			<td>STEMCELL Technologies Inc.Vancouver, BC, V5Z 1B3,Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL119</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution (PBS) without Ca++ and Mg++</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TS1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific,MA, USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td>P3813-1PAK</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Proline&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade Size 15&#160;</td>
			<td>Swann-Morton Ltd, Sheffield, S6 2BJ,UK</td>
			<td>BDF-6955C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>AS102</td>
			<td>4% w/v solution</td>
		</tr>
		<tr>
			<td>Sterile centrifuge tubes</td>
			<td>Tarsons Products Pvt. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile container -20 mL</td>
			<td>3M Center, MN 55144-1000,USA</td>
			<td></td>
			<td>3 M</td>
		</tr>
		<tr>
			<td>Sterile phosphate-buffered saline (PBS)</td>
			<td>Sigma Aldrich, USA</td>
			<td>P3813-1PAK</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Sterile pipettes (2, 5, and 10 mL )</td>
			<td>Eppendorf AG,22339 Hamburg,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes and tips</td>
			<td>Eppendorf India Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade Handle</td>
			<td>Becton, Dickinson and Co.,NJ,USA</td>
			<td>371030</td>
			<td>BP Handle 3</td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>BSW-01D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture, and characterization of dental pulp stem cells from human deciduous and permanent teeth

Rejeneratif tıp ve terapötik uygulamalar alanında, kök hücre araştırmaları hızla ilgi görmektedir. Hem süt dişlerinde hem de kalıcı dişlerde bulunan pulpa kök hücreleri (DPSC'ler), erişilebilirlikleri, uyum sağlayabilmeleri ve doğuştan gelen farklılaşma yetenekleri nedeniyle hayati bir kök hücre kaynağı olarak ortaya çıkmıştır. DPSC'ler, özellikle rejeneratif amaçlar için etkileyici çok yönlülük ve potansiyel sergileyen, hazır ve bol miktarda bulunan bir mezenkimal kök hücre rezervuarı sunar. Vaatlerine rağmen, ana engel, diş pulpası hücreleri içinde çok küçük bir fraksiyon olarak temsil edildikleri göz önüne alındığında, DPSC'lerin etkili bir şekilde izole edilmesi ve karakterize edilmesinde yatmaktadır. Aynı derecede önemli olan, bu paha biçilmez hücresel kaynağın uygun şekilde korunmasıdır. DPSC izolasyonu için iki baskın yöntem, enzimatik sindirim (ED) ve genellikle spontan büyüme olarak adlandırılan doku eksplantlarından (OG) büyümedir. Bu protokol, öncelikle DPSC izolasyonu için enzimatik sindirim yaklaşımına odaklanır ve ekstraksiyon, laboratuvar içi işleme ve hücre korumayı kapsayan adımları karmaşık bir şekilde detaylandırır. Ekstraksiyon ve korumanın ötesinde, protokol DPSC'lerin farklılaşma becerisini araştırıyor. Spesifik olarak, bu kök hücreleri adipositlere, osteoblastlara ve kondrositlere farklılaşmaya teşvik etmek için kullanılan prosedürleri ana hatlarıyla belirtir ve multipotent özelliklerini sergiler. Kolorimetrik boyama tekniklerinin daha sonra kullanılması, başarılı farklılaşmanın doğru bir şekilde görselleştirilmesini ve onaylanmasını kolaylaştırır, böylece izole edilmiş DPSC'lerin kalibresini ve işlevselliğini doğrular. Bu kapsamlı protokol, diş pulpası kök hücre ekstraksiyonu, yetiştirilmesi, korunması ve karakterizasyonunun tüm spektrumunu kapsayan bir plan işlevi görür. DPSC'lerin barındırdığı önemli potansiyelin altını çiziyor, kök hücre araştırmalarını ilerletiyor ve gelecekteki rejeneratif ve terapötik atılımlar için umut vaat ediyor.

Ana hatlarıyla belirtilen protokolün başarılı bir şekilde uygulanması, çok soylu farklılaşma yeteneğine sahip dental pulpa kök hücreleri (DPSC'ler) verdi ve bu da çok potansiyelli olduklarını gösterdi.
Canlılık testleri DPSC'lerin yaşayabilirliği, çeşitli zaman noktalarında bir Tripan Blue dışlama testi kullanılarak değerlendirildi. Sonuçlar, kültür dönemi boyunca sürekli olarak yüksek canlılık (%95'ten fazla) göstererek izolasyon ve kü...

Read this Scientific Article Protocol about Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth at JoVE.com

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

Bu protokol, multipotent kök hücrelerin enzimatik sindirim yoluyla diş pulpasından ekstraksiyonunu, kültürünü ve korunmasını vurgular. Ek olarak, osteoblastlara, adipositlere ve kondrositlere farklılaşma potansiyellerini göstererek süreçte hassasiyet ve tutarlılığın önemini vurgular.

İnsan Süt ve Daimi Dişlerinden Dental Pulpa Kök Hücrelerinin İzolasyonu, Kültürü ve Karakterizasyonu

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

Çıkarılan insan dişini diş forsepsleri ile yerine sabitleyerek başlayın. Ardından, bir diş el aletine bağlı bir elmas disk ve bir su soğutucusu kullanarak dişi kesin. Bir diş ekskavatörü kullanarak, pulpa dokusunu dişten çıkarın.Ardından, pulpa dokusunu dikkatlice küçük parçalara ayırmak için steril bir cerrahi bıçak kullanın. Daha sonra bu parçaları homojen bir karışım oluşturmak için PBS'li mini bir doku öğütücüye yerleştirin. Kıyılmış doku parçalarını 15 mililitrelik bir tüpe aktarın ve bunları bir tip kollajenaz karışımı kullanarak sindirin ve boşaltın.Enzim karışımındaki dokuyu iki saat boyunca 37 santigrat derecede inkübe edin. İnkübasyondan sonra, enzimleri nötralize edin. Daha sonra, sindirilmiş dokuyu 15 mililitrelik bir tüpe aktarın ve oda sıcaklığında beş dakika boyunca 300 G'de döndürün.Daha sonra süpernatanı dikkatlice atın ve peleti% 10 ila 20 FBS ile desteklenmiş taze DMEM'de yeniden süspanse edin. Hücre kültürü için, hücre süspansiyonunu dikkatlice 25 santimetre karelik bir kültür şişesine aktarın. Hücreleri 37 santigrat derecede% 5 karbondioksit içinde inkübe edin.İki ila üç gün sonra, steril bir serolojik pipet kullanarak, ortamı dikkatlice aspire edin ve yeni bir ortamla değiştirin. Daha sonra, DPSC'lerin karakterizasyonu için akış sitometresini açın ve izole edilen hücrelerin mezenkimal kök hücre yapısını onaylayın. DPSC'lerin osteoblastlara, adipositlere ve kondrositlere başarılı bir şekilde farklılaşması, DPSC'lerin çok etkili doğasını doğrular.DPSC'lerin iki katına çıkma süresi, mezenkimal kök hücreler için bildirilenle tutarlıydı, bu da sağlıklı ve proliferatif bir hücre popülasyonu olduğunu düşündürüyordu. Akış sitometrisi analizi, DPSC'lerin çoğunluğunun CD-90, CD-73 ve CD-105 için pozitif olduğunu gösterdi ve mezenkimal kök hücreler olarak kimliklerini doğruladı. Ayrıca, hücrelerin% 2'sinden azı CD-34, CD-45 ve HLADR için pozitifti ve bu da önemli hematopoietik veya endotel hücre kontaminasyonunun olmadığını doğruladı.

isolation-culture-characterization-dental-pulp-stem-cells-from-human

Research

JoVE Journal

Bioengineering

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Kemik Rejenerasyon için İnsan perivasküler Kök Hücre kullanın

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Biyomühendislik

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Geliştirme, Genişleme ve<em&gt; In vivo</em&gt; İnsan Embriyonik Kök Hücreleri (hESC) den İnsan NK Hücrelerinin İzleme ve ve Bağlı Pluripotent Kök Hücre (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Verimli üretimi ve besleyici-Alerjik IPSCs CRISPR-Cas9 sistemini kullanarak insan pankreas hücreleri düzenleme

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Yalıtım insan alveoler kapaklarini ve Osteojenik etkinlik kapaklarini türetilmiş hücre D vitamini etkileri Mezenkimal kök hücrelerin

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Erişkin İnsan Dermal Fibroblastlarının Abdominal Deriden İzolasyon ve İndüklenen Pluripotent Kök Hücrelerin Indüklenen Alacalı Kök Hücrelerin Indükleyici Olmayan Yöntemle Üretilmesi

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

İnsan Pluripotent Kök Hücrelerinden Olgun Serebellar Organoidlerin Ölçeklenebilir Üretimi ve İmmünboyama ile Karakterizasyonu

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Sessiz İnsan Hematopoetik Kök Hücrelerini Korumak İçin Bir Kültür Yöntemi

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

Tüp bebek pankreas soylarına doğru İnsan Diş Pulpası Kök Hücrelerinin İndüksiyonu

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Mikro Yerçekiminde Diş ve Solunum Hücre ve Organlarının Yayılımı

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...