Biz projenin çeşitli aşamalarında yardım, tavsiye ve kritik giriş birçok meslektaşları teşekkür etmek istiyorum. Bu video makalenin Üretim, Eğitim ve Araştırma (BMBF), Diyabet Araştırma (DZD, Alman Merkezi Alman <a href="http://www.dzd-ev.de">Bakanlığı</a> fon http://www.dzd-ev.de), <a href="http://www.imidia.org">IMIDIA</a> (http://www <a href="http://www.imidia.org">imidia.org</a> ) ve Teknoloji Dresden Üniversitesi, Üniversite Hastanesi Carl Gustav Carus. Bu sonuçlara yol açan araştırma hibe anlaşması n ° 115.005 altında Yenilikçi Tıp Girişimi, Avrupa Birliği Yedinci Çerçeve Programı (FP7/2007-2013) fon aldı.

1. Pankreas doku toplama ameliyathanede <ol><li> Cerrah, pankreas kısmi rezeksiyon gerçekleştirir. </li><li> Patolog, buz üzerinde bir kutu içinde pankreas örnek yerleştirdikten sonra, hemen teslim. </li></ol> 2. Adacık izolasyonu için doku seçimi <ol><li> Patolog tanısal amaçlı hasarlı dokuyu koruyarak, yumuşak ve sağlıklı görünen doku seçer. Fibrotik doku ve kullanılabilir dokusunun az 2 g sunan örneklerinden adacık izolasyon dışındadır. </li><li> Euro Collins Çözüm pankreas dokusu daldırın ve laboratuvara buz üzerinde teslim. </li></ol> 3. Insan adacık izolasyonu <ol><li> Pankreas dokusu tartılır, daha sonra 10 cm çanak içine yerleştirin. </li><li> 500 ml cam şişe içinde 150 ml RPMI medya koyun. </li><li> 250 ml cam şişe içinde, 100 mg / ml DNaz ve 5mg/ml Liberase RI 20 ml, 130 ml RPMI medya birleştirerek sindirim enzimi solüsyon hazırlanır. Bu çözümün pankreas dokusu enjekte etmek için bir şırınga içine 10 ml çizin. </li><li> Homojen bu gerilmek amaçlayan sindirim enzimi çözümü, pankreas dokusu içine enjekte edilir. Fibrozis önlenir, sindirim muhtemelen başarısız olacaktır. Daha sonra, yaklaşık 4 mm içine buz üzerinde 3 adet doku kıyma. </li></ol> 4. İnsan pankreas sindirim devre <ol><li> Şekil 1&#39;de gösterildiği gibi sindirim devre birleştirin. Ricordi odasında odasının üst kısmında alt ve dışarı akış yönü. </li><li> Toplam hacmi 300 ml, 500 ml balona kalan sindirim enzim çözeltisi ekleyin ve bir termometre yerleştirin. </li><li> Odasına, pankreas dokusu transfer örgü (gözenek çapı: 600 mikron) yerleştirin ve üç Silikon Nitrür Mermerler (çap: 15 mm), oda kapatın ve 140 ml / dk akış ile pompa start. </li><li> Devre sıcaklığı 37 ulaştığında ° C, zamanlama başlangıç ​​ve periyodik olarak sindirim durdurmak için ne zaman belirlemek için numune almak. Dithizone (2 mg / ml) ile boyanarak sindirilir doku 4 içinde adacıklar mikroskopik görüntüleme sağlar. </li><li> Asiner doku çevresindeki adacıklar ayrılır sonra, buz üzerinde yerleştirerek ve ısıtma rezistansı devre soğuk yıkama medya 200 ml (900 ml RPMI 1640 5.5 mM glukoz ve% 10 FBS) ekleyerek sindirim hemen durdurmak. </li><li> 250 ml konik tüpler adacık çözüm örnek tüp çıkıyor toplayın. Devre boşalana kadar devam edin. </li></ol><img alt="Şekil 1" src="/files/ftp_upload/2962/2962fig1.jpg" /> Şekil 1. Ve üç silisyum nitrür mermerler (çap: 15 mm): insan pankreas sindirim devresi insan pankreas sindirim devre örgü (600 mikron gözenek çapı) içeren bir Ricordi odasına içerir. Odasından Çıkış peristaltik pompa (140 ml / dk) doku toplama şişesi sürülür. Oklar, akış yönünü gösterir. Enzimatik sindirim için en uygun sıcaklık 37 ° C su banyosunda ısıtma rezistansı çeker tarafından yapılmaktadır. 5. Çamaşır-sindirim sonrası <ol><li> 5 dakika süreyle 1000 rpm&#39;de adacık çözümü, 4 ° C içeren 250 ml konik tüpler santrifüjleyin. </li><li> Süpernatantı atın, (5.5 mM glukoz ve% 10 FBS ile 900 ml RPMI 1640) yıkama medya adacık pelet tekrar süspansiyon ve 50 ml konik tüpler eşit kesirler dağıtmak. (Opsiyonel: yıkama artırmak için ek konik tüpler içine dağıtın) 1000 rpm&#39;de Santrifüj, 4 ° C 5 dk. </li><li> Supernatant atın ve yavaşça el sallayarak pelet gevşetin. </li></ol> 6. Ficoll degrade saflaştırılması <ol><li> Yoğunluğu 1.125 g / ml, pH 7.4 Ficoll medya ile pelletini tekrar ve yavaş yavaş 1.080, 1.060 ve 1.037 g / ml yoğunlukları ile 10 ml alikotları Ficoll medya kaplaması. </li><li> 20 dakika için 4 ° C, 2400 rpm Ficoll gradyanlar santrifüjleyin. </li></ol> 7. Islet Toplama ve Kültür <ol><li> Santrifüj sonra, farklı doku bileşenleri degrade bölümleme tarafından ayırt edilmelidir. </li><li> Yağ ve bağ dokusu oluşan üst tabaka atın. </li><li> 1,037-1,060 g / ml interfaz adacık agregaların ilk katman dikkatlice hasat. Bu tabaka, en saf izole adacıklar içerir. Kesirler verimli izolasyon için ayrı ayrı toplayın. </li><li> 1,060-1,080 g / ml interfaz adacık agrega, ikincisi daha az saf fraksiyon toplayın. </li><li> 50 ml toplam hacmi her konik tüp (5.5 mM glukoz ve% 10 FBS ile 900 ml RPMI 1640) yıkama medya ekleyerek Ficoll degrade sulandırınız. 1000 rpm&#39;de santrifüj, 4 ° C 5 dk. </li><li> Emme ile süpernatantı atın. Pelet Ficoll degrade gevşek bağlı olabileceği gibi dikkatli kullanın. </li><li> Pelet yıkayın with yıkama medya (5.5 mM glukoz ve% 10 FBS 900ml RPMI 1640) ve 1000 rpm az santrifüj, 4 ° C de 5 dakika. Adacık kültür ortamı kullanarak tekrarlayın. </li><li> Kültür ortamı içinde adacıklar süspanse edin ve% 5 CO 2 ile 37 kuvöz koyun ° C daha fazla işlem 24-48 saat önce için. </li></ol>

<ol>
	<li>Kahn, S. E., Zraika, S., Utzschneider, K. M., Hull, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+cell+lesion+in+type+2+diabetes:+there+has+to+be+a+primary+functional+abnormality.">The beta cell lesion in type 2 diabetes: there has to be a primary functional abnormality.</a> Diabetologia. 52, 1003-1012 (2009).</li><li>Deng, S., Vatamaniuk, M., Huang, X., Doliba, N., Lian, M. M., Frank, A., Velidedeoglu, E., Desai, N. M., Koeberlein, B., Wolf, B., Barker, C. F., Naji, A., Matschinsky, F. M., Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+islets+isolated+from+type+2+diabetic+subjects.">Structural and functional abnormalities in the islets isolated from type 2 diabetic subjects.</a> Diabetes. 53, 624-632 (2004).</li><li>Ricordi, C., Lacy, P. E., Finke, E. H., Olack, B. J., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+method+for+isolation+of+human+pancreatic+islets.">Automated method for isolation of human pancreatic islets.</a> Diabetes. 37, 413-420 (1988).</li><li>Latif, Z. A., Noel, J., Alejandro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+staining+fresh+and+cultured+islets.">A simple method of staining fresh and cultured islets.</a> Transplantation. 45, 827-830 (1988).</li><li>Ehehalt, F., Knoch, K., Erdmann, K., Krautz, C., J&#228;ger, M., Steffen, A., Wegbrod, C., Meisterfeld, R., Kersting, S., Bergert, H., Kuhlisch, E., Bornstein, S., Bonifacio, E., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin+turnover+in+islets+from+type+2+diabetic+patients.">Impaired insulin turnover in islets from type 2 diabetic patients.</a> Islets. 2, 30-36 (2010).</li><li>Hardt, P. D., Brendel, M. D., Kloer, H. U., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pancreatic+diabetes+(type+3c+diabetes)+underdiagnosed+and+misdiagnosed?.">Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed?.</a> Diabetes Care. 31, S165-S169 (2008).</li><li>Meisterfeld, R., Ehehalt, F., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+disorders+and+diabetes+mellitus.">Pancreatic disorders and diabetes mellitus.</a> Exp Clin Endocrinol Diabetes. 116, S7-S12 (2008).</li><li>Verge, C. F., Gianani, R., Kawasaki, E., Yu, L., Pietropaolo, M., Jackson, R. A., Chase, H. P., Eisenbarth, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+type+I+diabetes+in+first-degree+relatives+using+a+combination+of+insulin,+GAD,+and+ICA512bdc/IA-2+autoantibodies.">Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies.</a> Diabetes. 45, 926-933 (1996).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabet Med. 15, 539-553 (1998).</li><li>Marselli, L., Thorne, J., Ahn, Y. B., Omer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clin. Endocrinol. Metab. 93, 1046-1053 (2008).</li></ol>

Çıkar çatışması ilan etti.

Bizim protokolünü kullanarak, insan adacıklar kısmi pankreatektomi alınan pankreas dokusundan izole edilebilir. Bu protokolün başarı birkaç kritik noktaları sırasında alınan bakım dayanır. Beta-hücre canlılığını korumak için, numune laboratuvara buz üzerinde hızla taşınması olması esastır. Ayrıca, doku sindirim süresi, doku fibrozis ve medya enzimatik aktivite derecesine göre ampirik olarak optimize edilmiş olmalıdır. Bu Ricordi odasına elle uygulanan mekanik kuvvet derecesi için de geçerlidir. Böylece iyi bir adacık verimi elde etmek için, protokol en iyi özel bir bilim adamı ya da teknik yardımcısı tarafından yapılır. Takım zaten adacık izolasyonu yaşanan sürece İlk başarısızlık norm. Adacık izolasyon protokolü ve standart insan adacık izolasyon yöntemi arasında önemli farklılıklar vardır: 1) cerrahi işlem sırasında birkaç saat iskemi maruz pankreas dokusu kullanmasına rağmen, hemen sitesinde işlenir. Bu tahsisi ve adacık izolasyonu tesisi teslim sırasında birkaç saat için iskemik kalan pankreas / adacık nakli için beyin-ölü donörler, eksplante pancreata aksine. Standart bir protokol pankreatik kanal içine demlenmeye 2) ise, doğrudan pankreas dokusu içine enjekte kollajenaz. 3) Biz COBE hücre işlemci kullanarak sürekli bir Ficoll degrade adacıklar toplama yerine süreksiz Ficoll degrade kullanarak adacıklar ayırın. Cerrahi numune çok fibrotik veya yeterli sayıda adacık izole etmek için kıt olduğu durumlarda, doku hala lazer yakalama mikrodiseksiyon (LCM) 10 tarafından alınacak. Bu gen ekspresyonu verileri adacık izolasyon başarısız ya da verim çok düşük olsa bile, hemen hemen her numune tahsil edilecek sağlar. Ne yazık ki, LCM fonksiyonel çalışmalar için canlı hücreler üretmek değildir ve bunların tutarı proteomik analiz için genellikle yetersiz kalmaktadır. Böylece, adacıklar almak için kollajenaz sindirim protokolü ile paralel olarak LCM kullanarak cerrahi örnekleri işlemek için en etkili yolu olabilir. Kısmi pancreatectomies adacık izolasyonu için doku toplarken, hastanın klinik öykü ve metabolik durumunu dikkatle incelemek için önemlidir. Kısmen pancreatectomized bir hasta tip 3c diyabet yani şeker hastalığı, cerrahi 6 yol açan pankreas bozukluğu ikincil tarafından etkilenmiş olabilir . Kliniğimizde 2010 yılında bu ameliyat geçiren 43 katılan hastalar arasında, 32 non-diyabetik, 5 tip 2 diyabet ve 6 etkilendi tipi 3c diyabet vardı. Bu veriler, daha önceki çalışmaları ile bozulmuş glikoz metabolizması ve diyabet pankreas kanser veya kronik pankreatit 6 muzdarip hastaların büyük bir kısmını işaret kabul etmiş sayılırsınız. Biz birincil kökenli 7 pankreas ameliyatı olan semptomların başlangıcından önce en az bir yıl tanısı kondu diyabet olarak kabul edilecek. Adacık otoantijenler karşı antikor düzeyleri de potansiyel bir diyabet 8 otoimmün kökenli değerlendirmek için ölçülmelidir. Pankreatektomi geçiren bir hastanın, tanı konmamış diyabet muzdarip veya glukoz hoşgörüsüz olabileceğinden, tüm non-diyabetik hastalarda oral glukoz tolerans testi önce 9 pankreatektomi verilmelidir .

<table border="1"><tbody><tr><th> Isim </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><th> Ekipman </th><th></th><th></th></tr><tr><td> 1 ml Serolojik pipet (CELLSTAR) </td><td> greiner biyo-bir </td><td> 604181 </td></tr><tr><td> 10 ml Serolojik pipet (CELLSTAR) </td><td> greiner biyo-bir </td><td> 607180 </td></tr><tr><td> 25 ml Serolojik pipet (CELLSTAR) </td><td> greiner biyo-bir </td><td> 760180 </td></tr><tr><td> 10 ml Şırınga </td><td> BD (Becton Dickinson ve Şirket) </td><td> 300912 </td></tr><tr><td> 50 ml Şırınga </td><td> BD (Becton Dickinson ve Şirket) </td><td> 300865 </td></tr><tr><td> 5 ml Şırınga </td><td> BD (Becton Dickinson ve Şirket) </td><td> 309050 </td></tr><tr><td> 18 G 1,1 / 2 İğne Microlance 3 </td><td> BD (Becton Dickinson ve Şirket) </td><td> 304622 </td></tr><tr><td> 27 GX1 / 2 İğne </td><td> Braun </td><td> 4658300 </td></tr><tr><td> 27 GX3 / 4 İğne Microlance 3 </td><td> BD (Becton Dickinson ve Şirket) </td><td> 302200 </td></tr><tr><td> 3 cm hücre kültürü çanak 2x2 mm ızgara </td><td> Nunc </td><td> 174926 </td></tr><tr><td> 20 cm Doku kültürü çanak </td><td> BD (Becton Dickinson ve Şirket) </td><td> 353003 </td></tr><tr><td> 6 cm Kolay tutuş petri </td><td> BD (Becton Dickinson ve Şirket) </td><td> 351016 </td></tr><tr><td> 150 ml şişe depolama </td><td> Corning Incorporated </td><td> 431175 </td></tr><tr><td> 250 ml&#39;lik şişeler Erlenmeyer </td><td> Schott Duran </td><td> 21 217 36 </td></tr><tr><td> 500 ml&#39;lik şişeler Erlenmeyer </td><td> Schott Duran </td><td> 21 217 44 </td></tr><tr><td> 250 ml Vidalı Kapak Konik Alt Santrifüj Tüpü </td><td> Corning Incorporated </td><td> 430776 </td></tr><tr><td> 50 ml Falcon konik tüp </td><td> BD (Becton Dickinson ve Şirket) </td><td> 352070 </td></tr><tr><td> 260 ml Easyflask Sigara tedavi </td><td> Nunc </td><td> 156800 </td></tr><tr><td> Millex GV Filtre Ünitesi Duropore (0,22 mm) </td><td> Millipore </td><td> SLGV033RB </td></tr><tr><td> Filtre Şişe En Vakum (PES 70 mm Çap Membran, 0,22 mikron gözenek boyutu, 45 mm boyun boyutu, 500 ml hacim) </td><td> Corning Incorporated </td><td> 431118 </td></tr><tr><td> Dansitometre (Densito 30px) LWE37463 </td><td> Mettler Toledo </td><td> 51324450 </td></tr><tr><td> Hızlı PES Filtre Ünitesi 0,2 mikron </td><td> Nalgene </td><td> 569-0020 </td></tr><tr><td> Isıtma Bataryası </td><td> BioRep Technologies Inc </td><td> HC-02 </td></tr><tr><td> Multifuge 4 KS-R </td><td> Heraeus </td><td> 75015680 </td></tr><tr><td> Hidrolik Typ PD5206 </td><td> Heidolph </td><td> 523-52060-00-2 </td></tr><tr><td> Ricordi kamara (600 mikron gözenek çapı metal örgü) </td><td> BioRep Technologies Inc. </td><td> Model 50-U-01, Seri 0503-001 </td></tr><tr><td> Hazırlık Standı </td><td> BioRep Technologies Inc. </td><td> 50-PS-01 </td></tr><tr><td> O-Ringler </td><td> BioRep Technologies Inc. </td><td> YA-50-U-01 </td></tr><tr><td> Silikon Nitrür Mermerler (15 mm set 9) </td><td> BioRep Technologies Inc. </td><td> SN-01 </td></tr><tr><td> Silikon Boru </td><td> Tygon </td><td> R 3603 8,0 X4, 0 mm </td></tr><tr><td> Cerrahi forseps </td><td> Braun </td><td> BD168R </td></tr><tr><td> Cerrahi makaslar (Aesculap) </td><td> Braun </td><td> BC273R </td></tr><tr><td> Su banyosu, 200 EKK </td><td> Hibe </td><td> EKK 200 </td></tr><tr><th> Reaktifler </th><th></th><th></th></tr><tr><td> Kollajenaz -&gt; Liberase RI (100 mg) </td><td> Roche </td><td> 11815032001 </td></tr><tr><td> D-Glukoz (+) </td><td> Merck </td><td> 1.08337.1000 </td></tr><tr><td> Dimethylsülfoksit (DMSO) </td><td> Merck </td><td> 1.16743.1000 </td></tr><tr><td> Diphenylthiocarbazone (DTZ) </td><td> Sigma Aldrich </td><td> D5130 </td></tr><tr><td> DNaz I (100 mg) </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> Dulbeccos 1xPBS </td><td> PAA Laboratuvarları </td><td> H15-002 </td></tr><tr><td> Electrolytsolution E154 </td><td> Serumwerk </td><td> 00509 </td></tr><tr><td> Fetal sığır serumu (FBS) </td><td> PAA Laboratuvarları </td><td> A15-101 </td></tr><tr><td> Ficoll 400 </td><td> Sigma Aldrich </td><td> F9378 </td></tr><tr><td> Fetal Sığır serum (FBS) </td><td> Gibco </td><td> 10270-106 </td></tr><tr><td> 2 - (4 - (2-hidroksietil) - 1-piperazinyl) ethansulfons ‰ ure (Hepes) </td><td> Roth </td><td> 9105,3 </td></tr><tr><td> 5 M NaOH </td><td> klinik eczane, hastane </td><td> - </td></tr><tr><td> Penisilin / Streptomisin (100x) </td><td> PAA Laboratuvarları </td><td> P11-010 </td></tr><tr><td> Pipettaid (AÇIK) </td><td> BD (Becton Dickinson ve Şirket) </td><td> 357591 </td></tr><tr><td> Potassiumchlorid </td><td> Fluka </td><td> 60132 </td></tr><tr><td> Potassiumdihydrogenphospate susuz </td><td> JT Baker </td><td> 0241 </td></tr><tr><td> Potassiummonohydrogenphosphate </td><td> JT Baker </td><td> 3246-01 </td></tr><tr><td> RPMI Orta 1640 [-] D-Glukoz [+] L-Glutamine </td><td> Gibco </td><td> 11879-020 </td></tr><tr><td> RPMI Orta 1640 [+] L-Glutamine </td><td> PAA Laboratuvarları </td><td> E15-840 </td></tr><tr><td> Sodiumhydrogencarbonat </td><td> Merck </td><td> 1.06329.0500 </td></tr></tbody></table> Medya ve Çözümler Dithizone Çözüm <ul><li> 10 ml DMSO 100 mg DTZ çözülür ve 40 ml PBS içinde sulandırmak </li><li> Steril filtre solüsyonu (0.22 mikron) </li></ul> Enzim Çözüm <ul><li> 100 mg 20 ml RPMI 1640 medya Liberase RI (takviyesi olmadan) çözülür </li><li> (Takviyesi olmadan), 1 ml RPMI 1640 medya 100 mg DNaz çözülür </li><li> Steril filtre solüsyonu (0.22 mikron) ve enzim buz üzerinde tutmak </li></ul> Yıkama medya (1000ml/5.5mM Glikoz) <ul><li> 100 ml FBS (ısı inaktive) çözülür </li><li> 450 ml RPMI 1640 (glikoz) </li><li> 450 ml RPMI 1640 (11 mM glikoz) </li><li> Steril filtre solüsyonu (0.22 mikron) </li></ul> Islet kültür ortamı (500ml / 5,5 mM Glukoz) <ul><li> 10 ml 1M Hepes (pH, 7.4 &#39;) çözülür. </li><li> 50 ml FBS ekle </li><li> 5 ml penisilin / streptomisin (100x) </li><li> 2.75 ml 1 M Glikoz Çözüm ekle </li><li> 432,25 ml RPMI 1640 (glikoz) </li><li> Steril filtre solüsyonu (0.22 mikron) </li></ul> Euro Collins Çözümü <ul><li> 4083 g potasyum hidrojenfosfat </li><li> 70,0 g glukoz </li><li> 2237 g potasyum klorür </li><li> 14,80 g potasyum monohydrogenphosphate </li><li> 1.680 g sodyum hidrojen karbonat </li><li> 40 ml elektrolit solüsyonu (E 154) </li><li> pH: 7.4 </li><li> 2000 ml distile su ekleyin </li><li> Steril filtre solüsyonu (0.22 mikron) </li></ul> Ficoll Stok Çözümü <ul><li> 500 g Ficoll Euro Collins Çözüm 1000 ml </li><li> 8.94 g Hepes ekle </li><li> Ficoll çözüldü kadar gecede karıştırın. </li><li> pH: 7.4 </li><li> Tedbir yoğunluğu </li></ul> Ficoll Degradeler <ul><li> Yoğunluğuna göre FBS miktarı, Euro Collins Çözüm ve Ficoll Stok Çözümü hesaplayın </li><li> 3 çözümler birleştirin ve bir dansitometresi ile yoğunluk ölçümü </li><li> Yoğunluğu ayarlanır kadar Euro Collins Çözüm ya da Ficoll Stok Çözüm </li><li> 4 Steril filtre çözümü (0.22 mm) ve mağaza ° C </li></ul>

isolation of human islets from partially pancreatectomized patients

, Tip 2 diyabet ve Langerhans arıza 1 adacık patogenezinde soruşturmalar organ bağışına 2 tip 2 diyabetik adacıklar sınırlı sayıda engel var . Burada adacıklar tip 2 diyabetik ve non-diyabetik hastalarda farklı pankreas hastalıkları (iyi veya kötü huylu pankreas tümörleri, kronik pankreatit ve koledok veya duodenal tümörlerin) nedeniyle kısmi pankreatektomi geçirmiş elde İnsan pankreas dokusundan izole etmek için protokol paylaşmak . Katılan tüm hastalara aynı zamanda yerel etik komitesi tarafından onaylanmış olan bu çalışma, onların rızasını verdi. Cerrahi örneklerin hemen tanısal amaçlı hasarlı doku istinat yumuşak ve sağlıklı görünmesini pankreatik doku, adacık izolasyonu için seçilen patolog teslim edildi. 1.000 &#39;den fazla adacıkları izole için, pankreas dokusunun en az 2 g ile başlamak zorundaydık. Ayrıca bizim protokol gerekli enzim içeren medya enjekte gözle görülür bir doku gerilmek ve daha sonra yüzey alanını artırarak sindirime yardımcı olmak için kıyma oldu. İnsan pankreas dokusu büyük miktarda (&gt; 15g) kullanılabilir olduğu zaman zaman durum dahil etmek için protokol uygulanabilirliği genişletmek için, doku sindirmek için bir Ricordi odası (50 ml). Sindirim sırasında, elle numune ile doku fibrozis seviyesine göre değişen bir yoğunlukta Ricordi kamara 3 salladı. Bir süreksiz Ficoll degrade sonra asiner doku adacıkları ayırmak için kullanılır oldu. Biz doku pelet kadar küçük homojen Ficoll ortamda 1.125 g / ml &#39;lik bir yoğunluk ile yeniden süspanse olması gerektiğini kaydetti. Izolasyondan sonra, biz onların fonksiyonel iyileşmeyi kolaylaştırmak için en az 48 saat süreyle% 5 CO 2 ile 37 ° stressiz koşulları (hiçbir sallayarak veya rotasyon) ° C altında adacıklar kültür. Protokol ve gelecekteki gelişme yaygın uygulama, tip 2 diyabet araştırma büyük ölçüde ilerleyen, diyabetik insan adacık büyük miktarlarda zamanında hasat ve non-diyabetik hastalarda klinik olarak uyumlu sağlayabilir.

Bu büyük ölçüde fibrozis ve kollajenaz aktivitesi büyük ölçüde farklılıklar nedeniyle hazırlıklar arasında değişmektedir rağmen protokol, pankreas dokusu gram başına ~ 500 adacık ortalama vermiştir. Daha sonra 24 saat sonra izolasyon Ayıklama, doku işleme sırasında Dithizone ilk boyama ile&gt;% 90 adacık saflık elde etti. Arındırılmış adacıklar kalitesini belirlemek için, 25 mM glukoz ile uyarılmış insülin salgılanması için 2 saat boyunca durağan koşullar altında test edilmiştir. Biz ECIT adacık izolasyonu ve organ nakli merkezleri elde adacık insülin salgılanması Benzer bulundu. Olarak kısmen pancreatectomized pancreata izole adacıklar adacık nakli, kritik bir faktör olarak kabul edilmez toplam iEQ değerlendirilmesi anlamına gelmez. Önemlisi, yöntem bize, 2005 ve 2008 5 arasında 25 kısmi pancreatectomized hastalarda fonksiyonel çalışmalar için adacıkları izole etmek için etkin . Bu adacıklar batı blot ve immünohistokimya glukoz ile uyarılmış insülin salgılanması ve özel protein ekspresyonu analiz edilmiştir. Kısmen pancreatecomized pancreata izole adacıklar da gen ve protein ekspresyon profilleri, ultrastrüktürel görünüm ve non-diyabetik ve diyabetik adacıklar fonksiyonel yanıtları karşılaştırmak için kullanılan olabilir. Daha fazla hasta organ bağışına daha kısmi pankreatektomi geçiren olduğundan, protokol yeterince örnekler ve veriler sağlam bir istatistiksel analiz için toplanan izin verebilir.

Watch this Scientific Journal Video about Isolation of Human Islets from Partially Pancreatectomized Patients at JoVE.com

Isolation of Human Islets from Partially Pancreatectomized Patients

Araştırma için tip 2 diyabetik adacık kaynağı yetersiz. Burada kısmi pankreatektomi geçiren hastalardan izole adacıklar için protokol paylaşın. Bu yaklaşım, tip 2 diyabetik adacıklar elde etmek için eşsiz bir mekan temsil eder ve temel ve klinik çalışmalar için yeterli sayıda non-diyabetik hastalarda klinik olarak uyumlu.

Kısmen Pancreatectomized hastalar İnsan Islets izolasyonu

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

isolation-of-human-islets-from-partially-pancreatectomized-patients

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.9K Görüntüleme.  University Hospital Carl Gustav Carus, University of Technology Dresden. Araştırma için tip 2 diyabetik adacık kaynağı yetersiz. Burada kısmi pankreatektomi geçiren hastalardan izole adacıklar için protokol paylaşın. Bu yaklaşım, tip 2 diyabetik adacıklar elde etmek için eşsiz bir mekan temsil eder ve temel ve klinik çalışmalar için yeterli sayıda non-diyabetik hastalarda klinik olarak uyumlu.

Video: Isolation of Human Islets from Partially Pancreatectomized Patients

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

İnsan Pankreas Adacıklar için boyama Protokoller

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Tıp

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Cerrahi Örnekler kaynaktan İnsan pankreas adacıklarının Lazer Diseksiyon için Geliştirilmiş Protokolü

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

Optik nörit Hastalarda demiyelinizasyona ardından Görsel Hasar ve Onarım belirlemek ve ölçmek için dinamik Görsel Testler

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Taze Cerrahi Örneklerden İzolasyon ve İnsan Ventriküler kardiyomiyositlerin Fonksiyonel Karakterizasyonu

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Hastalar ve İnsan endotel hücrelerinden türetildiği Prostat dolaşımdaki tümör hücrelerinin arasında E-selektin aracılı Etkileşimleri gözlemleyin vitro Yöntemi

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Tip 2 Diyabet Fare Modeli: Bozulmamış Islets Of Pankreas Adacık İzolasyonu ve Canlı hücre 2-Foton Görüntüleme

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

İzolasyon ve profil MicroRNA içeren İnsan Bile dan ekzozomlar

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Full-root Aortic Valve Replacement by Stentless Aortic Xenografts in Patients with Small Aortic Roots

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial valve might not meet the functional needs. The implantation of a too-small stented pericardial valve, leading to an effective orifice area indexed to a body surface area less than 0.85 cm2/m2, is regarded as prosthesis-patient mismatch (PPM). A PPM negatively affects the regression of left ventricular hypertrophy and thus the normalization of left ventricular function and the alleviation of symptoms. Persistent left ventricular hypertrophy is associated with an increased risk of arrhythmias and sudden cardiac death. In the case of predictable PPM, there are three options: 1) accept the PPM resulting from the implantation of a stented pericardial valve when comorbidities of the patient forbid the more technically demanding operative technique of implanting a larger prosthesis, 2) enlarge the aortic root to accommodate a larger stented valve substitute, or 3) implant a stentless biological valve or a homograft. Compared to classical aortic valve replacement with stented pericardial valves, the full-root implantation of stentless aortic xenografts offers the possibility of implanting a 3-4 mm larger valve in a given patient, thus allowing significant reduction in transvalvular gradients. However, a number of cardiac surgeons are reluctant to transform a classical aortic valve replacement with stented pericardial valves into the more technically challenging full-root implantation of stentless aortic xenografts. Given the potential hemodynamic advantages of stentless aortic xenografts, we have adopted full-root implantation to avoid PPM in patients with small aortic roots necessitating an aortic valve replacement. Here, we describe in detail a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses. Limitations of this technique and alternative options are discussed.

Full-root aortic valve replacement by stentless aortic xenograft is a viable option in patients with small aortic roots. We describe, a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses, and discuss its limitations and alternative options.

Küçük Aort Köklü Hastalarda Stentless Aortik Xenograftlarla Tam Kök Aort Kapak Replasmanı

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Dönem Placentae Fetal membranlar birincil insan Decidual hücre izolasyon

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...