Çalışma Ulusal Sağlık Enstitüsü (R01GM079688, R21RR024439 ve R21GM075838), Ulusal Bilim Vakfı (CBET 0.941.055) ve Mimar Sinan Vakfı tarafından desteklenmiştir.

1. Rat MKH izolasyonu Önceden 1&#39;de açıklandığı gibi, 6-8 hafta eski Sprague-Dawley erkek sıçan, 2 mezenkimal kök hücreleri izole edildi. İzole MKH plastik yüzeyine yapışır ve in vitro kültür sırasında kolayca genişletebilirsiniz. <ol><li> Hayvan anestezi odasına koyun ve yaklaşık beş dakika boyunca anestezi oldu. Anestezi sırasında, yanıp sönen, solunum ve motor aktivite oranı gözlemliyoruz. Motor aktivite durur ve yanıp sönen oranı seyrek olduktan sonra hemen odasından hayvan çıkarın. </li><li> Bir operasyon istasyonunda hayvan uzanın ve servikal dislokasyon hayvan öldürmek. Arka bacaklarda femur ve tibiaları kesilmiş, cilt ve kasların çıkarın. </li><li> Birkaç saniye için% 70 izopropanol disseke femur ve tibiaları koyun ve 1X D-PBS transfer. </li><li> Biyogüvenlik kabini, femur ve tibiaları DMEM içeren bir 10cm çanak transfer edildi. Her kemik, sonra cımbız ile yapıldı ve iki ucu açık bir makas kesilmiştir. 3ml bir enjektöre 22G iğne takın ve DMEM ile doldurun, sonra kemik bir açık ucuna iğne takarak 50ml tüp içine iliği yıkayın. 2 ~ 3 kez her kemik için bu işlemi tekrarlayın. Ilikleri elde edildiğinde, hücreler tekrar süspansiyon ve kemik artıkları ve kan agrega kaldırmak için hücre süspansiyonu 70μm hücre süzgecinden geçirmek. </li><li> 200 gr az hücreleri, 4 ° C 5 dakika aşağı Spin ve aspirasyon ile süpernatantı kaldırmak. 25ml MSC ortamda yeniden süspanse hücreleri (% 10 FBS ve% 1 Kalem-Strep içeren DMEM). 10ml hücre süspansiyonu, toplam iki çanak için her 10cm kültür çanak numaralı seribaşı oldu. 1 ~ 2 hafta boyunca 37 ° C ve% 5 CO2 inkübatör kültür kaplarında saklayın. Orta 2 ~ 3 günde bir değiştirildi. </li></ol> 2. Rat MKH zenginleştirilmesi CD54, CD90, CD73, CD105 ve CD271 3-5 da dahil olmak üzere, MKH zenginleştirmek için bir dizi yüzey proteinleri kullanılmıştır. Bu çalışmada, manyetik hücre sıralayarak MKH zenginleştirmek için işaretleyici olarak kullanılan CD54 ve CD90. <ol><li> Hücreleri confluency 80% ulaştığında, orta aspirat ve her yemek için 4 ~ 5ml tripsin-EDTA ekleyin. Bulaşıkları inkübatör geri koyun ve hücre dekolmanı izin için yaklaşık 5 dakika boyunca inkübe. Hücreleri, müstakil kültür ortamı eşit miktarda tripsin inaktive edildi. 4 ° C, 5 dakika boyunca 200 gr 15ml tüp ve spin hücreleri hücre süspansiyonu toplayın. </li><li> Sonraki adımlar BD IMagnet kullanarak hücre ayrılması için kılavuzuna göre iki yüzey belirteçleri CD54 ve CD90 ile MKH zenginleştirmek için nasıl açıklar. Hücre pelet, 20 milyon hücre / ml hücre boyama tamponu (1X D-PBS içinde% 3 ısı inaktive FBS) yeniden süspanse edildi. Biotinlenmiş CD54 antikor (milyon başına 0.25μg hücreleri) ve biotinlenmiş CD90 antikor (milyon hücre başına 0.15μg) eklenir ve hücre süspansiyonu ile hafifçe karıştırılır. 15 dakika boyunca buz üzerinde inkübasyondan sonra, işaretli hücrelerin aşırı 1X BD IMAG tampon hacmi ile yıkandı. Etiketli hücreler 5 dakika boyunca, 4 ° C 200g aşağı bükülmüş. </li><li> Vortex BD IMAG streptavidin parçacıkları iyice, her 10 milyon hücre 40μl parçacıkları ekleyin. Parçacık etiketli hücreleri ile iyice karıştırın ve hücrelerin ~ 12 ° C&#39;de 30 dakika süreyle 6 inkübe. Bu streptavidin parçacıklar bağlamak için sırasıyla CD54 ve CD90 yüzey proteinlerine bağlı olduğu, biotinlenmiş anti-CD54 ve anti-CD90. </li><li> Inkübasyon süresi boyunca, olumlu bir kısmını toplamak için yuvarlak bir alt test tüpü etiket. Inkübasyondan sonra, 20 milyon hücre / ml 1X BD IMAG tampon ile etiketleme ses getirmek ve etiketli hücreleri pozitif fraksiyon toplanması tüp transferi. BD IMagnet üzerine yerleştirin ve pozitif fraksiyonu tüp kalmasına izin, 6 dakika sonra BD IMagnet hala olumlu bir fraksiyonu tüp ile bir bardak Pasteur pipeti ile süpernatant kaldırmak. </li><li> BD IMagnet pozitif fraksiyonu tüp çıkarın ve buz üzerine yerleştirin. 1ml buz 1X BD IMAG tampon ve nazik karıştırılarak tekrar süspansiyon hücreleri ekleyin. Tüp BD IMagnet üzerine yerleştirin ve 2 ~ 4 dakika kalmasına izin. Yeni bir cam Pasteur pipeti ile süpernatantı. 2.5 bir kez daha açıklandığı gibi yıkama işlemi tekrarlayın. </li><li> BD IMagnet ve kültür ortamında tekrar süspansiyon hücreleri, tohum hücreleri ve flow sitometri için 10cm çanak korumak için 75cm 2 balon tüp çıkarın. </li></ol> 3. Yüzey Marker İfade Sitometrisi ile doğrulanması Flow sitometri analizi ifade CD54 ve CD90 elde edilen hücreler doğrulamak için yapıldı. HSC işaretleyici CD45 MKH HKH&#39;lerin mahrum olduklarını teyit etmek için kullanılmıştır. <ol><li> Hücreleri ~% 80 konfluent hale geldiğinde, tripsin-EDTA ile hücreleri trypsinize ve 15ml bir tüp içine hücreleri toplamak. 200g, 4 ° C 5 dakika hücreleri toplamak için santrifüjleyin. </li><li> Süpernatant aspire.h kez 1X D-PBS ile hücreler. Son bir konsantrasyon 5 ~ 10 milyon hücre / ml ve buz üzerinde hücrelerin tutmak hücre boyama tampon yeniden süspanse hücreleri (1X D-PBS% 2 ve% 0.05 sodyum azid FBS içeren). (2 izotip kontrolü IgG2a;.. 3 İzotip kontrolü IgG1, 4 CD45; 5 CD54;.. 6 CD90 1 hücreleri sadece) altı etiketli tüpler içine kısım 100μl hücre süspansiyonu. </li><li> (:: 0.5μg milyon başına hücreleri; CD54: 0.25μg milyon başına hücreleri; CD90: CD45 hücreleri 20μl milyon başına IgG2a ve IgG1 0.15μg milyon başına hücreleri) uygun konsantrasyonları izotip kontrol ve primer antikor ekleyin ve 4 ° C&#39;de inkübe 30 dakika. </li><li> 100μl hücre boyama tampon 1X D-PBS ile iki kez ve daha sonra tekrar süspansiyon hücreleri hücrelerinin yıkayın. SA-PE (streptavidin fikoeritrin milyon hücre başına 0.15μg kullanın) ekleyin ve 4 ° C&#39;de 30 dakika karanlıkta. </li><li> Etiketli hücreleri 1X D-PBS ile iki kez yıkayın. 400μl hücre boyama tampon hücrelerin yeniden süspanse edin ve flow sitometri analizi için bir şahin tüp transferi. </li></ol> 4. MKH Türetilmiş Tek koloni ayrılması MKH farklı hücre şekli, büyüme oranı gibi farklılaşma yeteneğinin 6 farklı alt popülasyonlar oluşan heterojen bir nüfus. Ancak, tüm alt popülasyonlar bilinen MSC belirteçlerinin ifade etme ve bu nedenle bu alt popülasyonlar ayrı işaretleri kullanmak mümkün değildir. Bu nedenle, tek hücre oluşturduğu koloni farklı alt popülasyonlar, ayırmak için uygulanan klonlama silindir. <ol><li> ~ 10cm çanak başına yaklaşık 50 100 hücre Plaka hücreleri. 1 ~ 2 hafta boyunca 37 ° C ve% 5 CO2 inkübatör inkübe edin. Bu dönemde, ters bir mikroskop ile iyi izole koloniler inceleyin. Koloniler yeterince büyük boyut (her koloni 100&#39;den fazla hücre daha iyi) ulaştığında, çanak alt kısmında bir sharpie koloniler işaretleyin. Işaretlenir koloni almak zaman aldı birinin yakınında herhangi bir çevre koloniler olmadığından emin olun. </li><li> Aspire orta ve çanak 1X D-PBS ile bir kez yıkayın. Steril bir forseps ile steril bir klonlama silindir Pick up ve nazikçe işaretlenmiş koloni çevresinde yer. Işaretli tüm koloniler üzerine yerleştirilen bir klonlama silindir kadar bu işlemi tekrarlayın. Aldı koloniler her klonlama silindir sadece bir koloni içerdiğini birbirlerine uzak olmalıdır. </li><li> 100μl tripsin-EDTA her klonlama silindir ekleyin ve ~ 5 dakika süreyle geri inkübatör çanak koymak. 5 dakika sonra, onlar kadar yuvarlama olup olmadığını görmek için mikroskop altında hücrelerin kontrol edin. Hücreler yukarı kaldırdı, tripsin inaktive kültür ortamı eşit miktarda ekleyebilirsiniz. 200μl micropipettor hücre süspansiyonu karıştırın ve 60mm çanak 3ml prewarmed kültür ortamı içeren bir hücre süspansiyonu aktarmak. Bulaşıkları düzgün Etiket ve kuvöz içine geri koymak. Önümüzdeki birkaç gün içinde morfolojik değişim izleme. </li></ol> 5. Temsilcisi Sonuçlar Sıçan MKH izolasyonu için kısmen açıklanan protokole göre, plastik yapışık MKH kaplama sonraki gün görünür olmalıdır. Hücrelerin sayısı hızla artmaya devam ederken, konfluent hücreleri Şekil 1A gösterilen hücreler gibi görünmelidir. Hücrelerine ula geldiğinde ~ confluency% 80 (Şekil 1B), alt kültüre yürütülen olabilir. Altkültürü sırasında, tripsin-EDTA hücreleri ayırmak için kullanılan ve Şekil 1C gösterildiği gibi kaldırdı hücreleri küçük ve yuvarlak. <img src="/files/ftp_upload/1852/1852fig1.jpg" alt="Şekil 1" /> Şekil 1. Sıçan MKH Faz kontrastlı görüntüler. (A) konfluent MKH. Hücrelerin büyük çoğunluğu mili gibi ya da yıldız gibi. (B) MKH yaklaşık% 80 confluency. (C) trypsinization sonra kaldırdın hücreleri küçük ve yuvarlak. Bir kez MKH manyetik hücre sıralama ile zenginleştirilmiş, flow sitometri analizi, yüzey işaretleyici ifadeleri doğrulamak için yapılır. Zenginleştirme iyi ise, hücreler HSC marker CD45 (Şekil 2) karşı olumlu MSC belirteçlerinin CD54 ve CD90 karşı boyama ancak olumsuz göstermek gerekir. İzotip denetimleri IgG2a ve IgG1 negatif kontrol olarak kullanılır. <img src="/files/ftp_upload/1852/1852fig2.jpg" alt="Şekil 2" /> Şekil 2. Yüzey belirteçleri için MKH Flow sitometri analizi. MKH IgG2a (izotip kontrolü 1), IgG1 (izotip kontrolü 2), CD45, CD54 ve CD90 karşı antikorlar ile etiketlenir. MKH CD54 ve CD90 ifade değil CD45. Hücreleri uygun klonal yoğunlukta numaralı seribaşı zaman, koloniler tek hücre çıkmalıdır. Şekil 3A, tek bir hücrenin oluşturduğu bir koloni temsil eder. Klonlama silindir sömürgelerden elde edilen koloniler ve hücreler ayrı ayrı kültürü ayrı kullanılabilir. Şekil 3B ve 3C iki ayrı koloniler elde edilen hücreler temsil eder. Koloni 2 türetilen hücreler (Şekil 3C) yuvarlak ise koloni 1 türetilen hücreleri gibi mili (Şekil 3B). <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1852/1852fig3.jpg" alt="Şekil 3" /> Şekil 3. MKH ve tek koloni elde edilen hücreler tarafından Colony oluşumu. MKH klonal yoğunluğa sahip bir form tek tek koloniler kültüre . Bu koloniler ayrı kültüre klonlama silindir ve farklı koloniler hücreleri tarafından ayrılabilir. Klonal yoğunluğu kaplama (A) temsilcisi koloni MKH tarafından oluşturulmuştur. (B) bir koloni elde edilen hücreler, Mil gibi. (C) Yuvarlak hücreleri başka bir koloni türetilmiştir.

<ol>
	<li>de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+glial+glutamate+transporters+in+adult+mesenchymal+stem+cells.">Induction of glial glutamate transporters in adult mesenchymal stem cells.</a> J Neurochem. 91, 155-166 (2004).</li><li>Porter, R. M., Huckle, W. R., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dexamethasone+withdrawal+on+osteoblastic+differentiation+of+bone+marrow+stromal+cells.">Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells.</a> J Cell Biochem. 90, 13-22 (2003).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25, 2739-2749 (2007).</li><li>Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells:+from+basic+biology+to+clinical+applications.">Human mesenchymal stem cells: from basic biology to clinical applications.</a> Gene Ther. 15, 109-116 (2008).</li><li>Erica, L., Herzog, L. C., Diane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krause+Plasticity+of+marrow-derived+stem+cells.">Krause Plasticity of marrow-derived stem cells.</a> Blood. 102, 3483-3493 (2003).</li><li>Colter, D. C., Sekiya, I., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+subpopulation+of+rapidly+self-renewing+and+multipotential+adult+stem+cells+in+colonies+of+human+marrow+stromal+cells.">Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.</a> Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).</li></ol>

Bu protokol, izole etmek ve zenginleştirmek MKH anlatılmaktadır. Tek koloni elde edilen hücreleri ayırmak için bir yöntem de dahil edilmiştir. Başarılı bir izolasyon, zenginleştirme ve koloni ayrımı için önemli olan birkaç adım vardır. Hücre izolasyonu sıçan yaparken, bir hücre süzgeç ya da kan pıhtılaşması ve kemik enkaz kurtulmak için benzer büyüklükte bir steril naylon örgü süzülmeye tavsiye edilir. Gecede hücreleri kaplama sonra, birçok ölü hücreleri orta yüzen olacak ve ö...

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<tr>
<td>1X D-PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14040-133</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>BD Imagnet</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552311</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG2a</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555572</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG1</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555747</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD54</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL054B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD90</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL005B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD45</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL001B</td>
<td>&nbsp;</td>
<tr>
<td>BD Imag buffer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552362</td>
<td>&nbsp;</td>
<tr>
<td>Round-bottom tube</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin particles</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>557812</td>
<td>&nbsp;</td>
<tr>
<td>SA-PE</td>
<td>&nbsp;</td>
<td>R&amp;D systems</td>
<td>F0040</td>
<td>&nbsp;</td>
<tr>
<td>Cloning cylinder</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2059</td>
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isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs

MKH, yetişkin kök hücrelerin tedavi edici uygulamalar için umut verici bir kaynak bir nüfus vardır. Bu hücreler, kemik iliğinden izole edilebilir ve plastik bağlılık nedeniyle hematopoietik kök hücreler (HKH&#39;lerin) kolaylıkla ayrılabilir. Bu protokol, sıçan femur ve tibiaları MKH izole etmek için nasıl açıklamaktadır. Izole hücreler manyetik hücre sıralayarak iki MKH yüzey belirteçleri CD54 ve CD90 karşı daha da zenginleştirilmiş. CD54 ve CD90 yüzey belirteçleri İfade sonra akış sitometri analizi ile teyit edildi. HSC işaretleyici CD45 sıralaması MKH HKH&#39;lerin tüketilmiş olup olmadığını kontrol etmek için de yer aldı. MKH doğal olarak oldukça heterojen. Farklı şekiller, proliferasyon ve farklılaşma yetenekleri hücrelerin alt popülasyonlar vardır. Bu alt popülasyonlar, bilinen tüm MKH belirteçlerinin ifade ve farklı alt popülasyonlar için hiçbir benzersiz marker henüz tespit edilmiştir. Bu nedenle, farklı alt popülasyonlar ayırmak için alternatif bir yaklaşım tek koloni elde edilen hücreler ayrı klonlama silindir kullanıyor. Tek koloniler elde edilen hücreler daha sonra kültür ve ayrı olarak değerlendirilir.

Watch this Scientific Journal Video about Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs at JoVE.com

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MKH, femur ve tibiaları izole edilmiş ve daha sonra manyetik hücre sıralama ile zenginleştirilmiş. Sıralama hücreler flow sitometri ile yüzey belirteçlerinin ifade teyit edildi. Bu hücreler de klonal yoğunluğu tek koloniler oluşturmak için kültüre edildi ve daha sonra bu kolonilerin klonlama silindir ile ayrıldı.

İzolasyon ve Rat Mezenkimal Kök Hücreler zenginleştirilmesi (MKH) ve MKH Türetilmiş Tek koloni Ayırma

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

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Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

28.4K Görüntüleme.  City of Hope Cancer Center. Rat MKH, femur ve tibiaları izole edilmiş ve daha sonra manyetik hücre sıralama ile zenginleştirilmiş. Sıralama hücreler flow sitometri ile yüzey belirteçlerinin ifade teyit edildi. Bu hücreler de klonal yoğunluğu tek koloniler oluşturmak için kültüre edildi ve daha sonra bu kolonilerin klonlama silindir ile ayrıldı.

Video: Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH&#39;lerin)

Biyoloji

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

İnsan Göbek kordonu İzolasyon ve Hayvan Serum Genişleme Mezenkimal stromal hücreler (MKH) ve endotel Koloni Oluşturan Progenitör Hücreler (ECFCs) Türetilmiş

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Hoechst İzolasyonu ve Karakterizasyonu Düşük CD45 Negatif Fare Akciğer Mezenkimal Kök Hücre

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

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

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

İnsan Lipoaspirates den Yağ-türetilen Kök Hücre Manuel İzolasyon

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Kas Uydu Hücreleri İzolasyon, Kültür ve Transplantasyon

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146 İzolasyonu<sup&gt; +</supSıçan Akcigerlerinden&gt; Resident Akciğer Mezenkimal Stromal hücreler

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

İzolasyon ve Rat Mezenkimal Kök Hücreler zenginleştirilmesi (MKH) ve MKH Türetilmiş Tek koloni Ayırma

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İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH'lerin)