Biz editoryal yardım için Jennifer Petrosino ve David DiSilvestro teşekkür etmek istiyorum. Bu araştırma Amerikan Yumurta Kurulu ve Novo Nordisk İlaç gelen Ödülü Numarası 10040042 yanı sıra osu Gıda İnovasyon Merkezi, Ofis Uluslararası İlişkiler, İleri Fonksiyonel Gıdalar Araştırma Merkezi ve Girişimcilik yanı sıra Ödül Numarası 20020728 tarafından desteklenen Ulusal Bilim Vakfı EEC-0914790 (LJL) hibe. Tıbbi Araştırma ve NIH Yol Haritası ile Sağlık (OD) Direktörü, National Institutes Ofisi tarafından finanse edilen ve desteklenen Ulusal Araştırma Kaynakları Merkezi&#39;nden Ödülü Numarası R21OD017244 (OZ) ile UL1RR025755 (OSUCCC) tarafından desteklenmiştir açıklanan proje, NCI P30CA16058. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Ulusal Araştırma Kaynakları Merkezi veya Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmemektedir.

Çalışma protokolü Ohio State Üniversitesi Etik Komiteleri tarafından onaylandı. Hayvan deneyleri IACUC protokolü tarafından kabul edildi. Tüm prosedürler laminer akışı ile seviye 2 biyogüvenlik kabini altında gerçekleştirildi. Biz tüm standart güvenlik şartlarını ve prosedürleri takip etti. mikrokapsüllerin hazırlanması için mikro-kapsülleme tekniği, 18 17 tarif edildiği gibi gerçekleştirilmiştir. Malzemelerin 1. Hazırlıklar <ol><li> Otoklava fizyolojik tuzlu su (su içinde% 0.9 NaCI) içinde% 2 sodyum alginat çözeltisi 10 ml hazırlayın. Fizyolojik tuzlu su içinde% 0.05 PLL çözeltisi hazırlayın. Bir gün önce bu çözümleri hazırlayın ve gece boyunca karıştırın. Kullanmadan önce 0.22 mikron filtre ile çözüm Filtre. </li><li> 50 mM sodyum sitrat ve% 0,9 NaCl solüsyonlarının 100 mM CaCl 2 hazırlayın ve bunları otoklav. </li><li> Tüm solüsyonlar, iğneler, elektrotlar ve beher otoklavlayın. Tıkanmasını önlemek için tel ile iyice temiz iğneler. </li><li> (102 mikrokapsüller kuyunun her cm 2 için gerekli ve kapsül 18 başına 500 hücre yaklaşık vardır edilir) kapsüller için kullanılacak hücrelerin uygun miktarda belirleyin. Iki milyondan hücre başına sodyum aljinat 1 ml kullanın. </li><li> Yüksek glikoz bir &quot;Fibroblast büyüme ortamı&quot; ihtiva eden,% 10 dana serumu, 100 U / ml penisilin / streptomisin hazırlanması (4,500 mg / L glikoz) Dulbecco&#39;nun modifiye edilmiş Eagle ortamı (DMEM). <ol><li> DMEM,% 10 fetal sığır serumu, 10 ug / ml insülin, 1 uM deksametazon, 0.5 mM 3-izobütil-1-metil ksantin ve 100 U / ml penisilin / streptomisin içeren bir &#39;farklılaştırma ortamı I&#39; hazırlayın. </li><li> DMEM içinde &#39;farklılaştırma ortamı II&#39; ihtiva eden,% 10 fetal sığır serumu, 10 ug / ml insülin, 100 U / ml penisilin / streptomisin hazırlayın. </li></ol></li><li> (RI 10 mi Radyo-immünopresipitasyon deney tamponu başına bir proteaz inhibitörü tablet ihtiva eden liziz tamponu hazırlayınPA tamponu). </li></ol> 2. Alginat, mikro-boncuklar hazırlanması (Şekil 1A) <ol><li> Hücre kültürü şişesi eski orta çıkarın. 10 ml PBS ile hücre durulayın. % 0.25 tripsin-EDTA (bir birbirine bağlı T175 şişesi başına 2 mL) ile süspansiyon içinde hücrelerin getirin. </li><li> Hemasitometre kullanarak hücre süspansiyonu hücreleri saymak. Hücre süspansiyonu 10 mi kısım kullanın ve üreticinin talimatlarına göre hücreleri sayın. 5 dakika boyunca oda sıcaklığında 480 x g&#39;de santrifüj ortamda kalan hücreler santrifüj. </li><li> Aşama 1.4 &#39;de tarif edildiği gibi, sodyum alginat hücre pelletini süspanse edin. 5 ml şırıngaya sodyum alginat hücre çözeltisi aktarın. </li><li> Bir 23-gauge iğne ekleme, çözelti içinde hava kabarcıklarını çıkarmak ve havanın 1 ml cep oluşturmak için şırınga ters. </li><li> Kapsülleyicide iğne ağzı altında 100 mM CaCl2 çözeltisi 144 ml içeren, küçük bir beher (180 mi) yerleştirin. Uç approxim ile kapsülleyicide elektrodu takın100 mM CaCl2 çözeltisinin yüzey üzerinde ulaştırılması 2.5 cm. </li><li> Sıkı şırınga pompası sodyum alginat hücre çözeltisi ihtiva eden şırınga yerleştirin. Şırınga açılması kauçuk tüp takın. Sodyum aljinat-hücreli çözüm tüp aracılığıyla yarıya girene kadar pistonu itin. 5.4 kV gerilim ayarlayın. Şırınga pompası 12.06 mm çapında ayarlayın. 3 ml / saat hız ayarlayın. Pompayı başlatın. Kapsülleyicide açın ve 5.4 kV gerilim korumak. </li><li> Kaputun penceresini kapatın ve aljinat mikro oluşumu sonuna kadar gereksiz titreşimi önlemek. Tüm çözeltisi iğne içinden geçtikten sonra, PLL ile kaplamadan önce 20 dakika daha 100 mM CaCl2 çözeltisi içinde aljinat top şeklindeki mikroboncukları katılaşır. </li></ol> PLL ile 3. Kaplama mikro boncuklar (Şekil 1B) <ol><li> Sodyum aljinat hücre top şeklindeki mikroboncukları içeren beher çıkarın ve bu Microb transferi50 ml&#39;lik bir santrifüj tüpüne EADS. Mikroboncuk pelet CaCI2 çözeltisi çıkarın. </li><li> % 0.9 NaCl, 30 ml ekleyerek aljinat-hücresi top şeklindeki mikroboncukları yıkayın. Hafifçe elinizle tüpü çalkalayın. Mikro-yerçekimi tarafından çöktürülmüş sonra 25 ml pipet ile% 0.9 NaCl çıkarın. 3 yıkama olmak üzere toplam iki kez daha tekrarlayın. </li><li> Sodyum alginat çözeltisi, her 1 ml% 0.05 PLL çözeltisi 10 ml kullanın. 10 dakika boyunca dakikada 1.000 devirlerde% 0.05 PLL ve vorteks ekleyin. Not: Genellikle, 10 dakika PLL kaplama için yeterlidir. </li><li> PLL kaplama oluşturulduktan sonra, PLL çözüm kaldırmak ve 3.2 de tarif edildiği gibi kapsülleri 3 kez yıkayın. </li></ol> Aljinat Core 4. Sökme (Şekil 1C) <ol><li> 50 mM sodyum sitrat çözeltisi 30 ml ilave edilir. 5 dakika veya tüm sodyum aljinat çözülür kadar bekleyin. Aşama 3.1.1 tarif edildiği gibi üç kez yıkayın kapsüller. </li><li> % 0.9 NaCl kaldır 50 ml tüp kültür ortamı 20 ml ekleyinve bir hücre kültürü şişesine hücreleri içeren tüm kapsülleri aktarın. Standart hücre kültürü koşullarında 18 altında kapsüllü hücreleri taşıyınız. </li></ol> Vitro Uygulamaları 5. Hücreleri arasında Xenograftlarında ve Host Hücre Etkileşimleri veya metabolit Blake Kinetiği / akışını Eğitim için (Şekil 2) <ol><li> Ko-kültürler için, birbirine karışana kadar 24 oyuklu bir plaka üzerinde kültür konakçı hücreler sunar. Kültürleme preadipositler için &#39;Fibroblast Büyüme Orta&#39; kullanın. </li><li> 24 oyuklu bir plaka ihtiva eden konakçı hücreler konfluent içine aktarın mikrokapsüller. Bir tek tabaka elde etmek için mikro kapsüller (102 mikro / göz cm2) ekleyin. </li><li> Altı gün Farklılaşma Orta II öncesi adiposit Farklılaşma Orta I. ile farklılaşmayı Her 48 saat, değişim ortamını neden olur. RIPA tamponu içinde Hücreleri,. Western blot kullanılarak protein ekspresyonunu analiz etmek için durum başına 50 ug protein kullanın. </li></ol> Suların Vivo Başvurusu 6.Obezite ent (Şekil 3) <ol><li> Anestezi idamesi için oksijen ile anestezi ve% 2 izofluran uyarılması için oksijen ile% 4 izofluran karıştırın. Ayak tutam tarafından yeterli anestezi derinliği onaylayın. <ol><li> Kurumasını kornealar korumak için gözlerinin üzerinde anti-kaşıntı merhem sürün. </li></ol></li><li> Böyle yeşil floresan proteininin (GFP) olarak kalıcı yapay floresan proteini ile etiketlenmiş kapsüllü hücreleri kullanın. </li><li> 3 ml şırınga ve kapsüllü hücreleri enjekte etmek için 20 iğne kullanın. <ol><li> 0.5 x 10 6 hücre Şekil 3&#39;te gösterildiği gibi, bir gonad ve böbrekler intraperitoneal bir alanda yer almaktadır, her Iab yağ depo içine 0.2 ml PBS içinde süspansiyon haline enjekte edilir. Ortalama ağırlığı olan farelerde bu PBS hacmi ve hücre sayısı kullanın 40 gr. Farklı ağırlık ya da doza bağımlı etkileri belirlenmesi için sahip farelerde hücre sayısını ve ses düzeyini ayarlayın. </li></ol></li></ol>

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No conflicts of interest declared.

Çeşitli yöntemler, kurutma, haddeleme, ve emülsiyonun 19 de dahil olmak üzere hücreleri kapsüllemek için kullanılmıştır. Bu yöntemde, aljinat taneleri daha sonra PLL ile kaplanmış ve aljinat çekirdek kapsülleme tamamlamak için çözülmüş olan, bir iğne aracılığıyla ekstrüde edilir. Bu yöntem yıldır kullanılmaktadır, ancak, arzu edilen boyut ve küresel şekle sahip tanelerin oluşumu hala zordur. kapsüllerin boyutu, sodyum aljinat çözeltisi, ekstruder çapı ve iğne ucu ve ...

Kronik hastalıkların 1 artan insidansı tedavi hücre popülasyonlarının 2 nakli çalışmalar teşvik etmiştir. Sinjenik veya allojenik kök hücreleri, bu uygulamalar 2 için en yaygın olarak kullanılan hücre tipi vardır. Bununla birlikte, bu tedaviler implantasyondan sonra farklılaşması ve kök hücre göçü kontrol izin vermez ve maliyet etkin değildir. Yararlı fonksiyonları ile genetiği değiştirilmiş hücrelerin Transplantasyon birçok hastalığın tedavisini geliştirmek öngörmektedir. Ancak genetik hücre değişiklikleri konağın bağışıklık sistemi tarafından tanınan, bu nedenle, bu tedaviler immünosupresyonu 3 gerektirir. Insülin üreten hücrelerin kapsüllenmesi Chang 4 tarafından geliştirilmiştir. teknik, bir kalsiyum klorür çözeltisi içine daldırılır alginat damlacıklar halinde hücrelerin kapsüllenmesi dayanmaktadır. Aljinat molekülleri (G) (E) manuronik ve guluronik asit oluşur ve Ca ile bağlı olabilir 2+. Jelleşme sonra, taneler, bir poli-L-lisin (PLL) çözeltisi süspanse edilir. Bu adım sırasında, PLL kapsül membran oluşturan alginat moleküllerinde G ve M bağlanır. kapsül, zarın gözeneklilik, M ve PLL konsantrasyonları, inkübasyon süresi, ve sıcaklığı değiştirilerek modüle edilebilir. PLL bağlanmasının, aynı zamanda Çeşidi ve aljinat konsantrasyonuna bağlıdır. Ca + 2 iyonları ile çapraz bağlanmış Alginat matrisler, fizyolojik bir ortamda ya da fosfat ve sitrat iyonları konsantrasyonunun ortak tampon çözeltiler içinde kararlı değildir. Bu tamponlar alginattan Ca 2 + özü ve çekirdeği sıvılaştırılması yapabilirsiniz. Aljinat çekirdeğin Sıvılaşma hücresel hareket ve büyüme için kapsül içindeki alan sağlar. Polikatyonik poli-L-lisin (APL) polianyonik Aljinatta kapsüllenmiş hücreler immünoglobülin için geçirgen olan, ancak toksin besin girişini ve çıkışını sahiptir. Bunlar APL en özellikler uzun vadeli su etkinleştirmekgenetik olarak farklı bilgisayarlar içine naklinden sonra kapsüllü hücrelerin rvival. Elliott ve diğ., Dokuz yıllık implantasyon 5 sonra, bir insan hastada kapsüllenmiş domuz pankreatik hücrelerin saptanmıştır hayatta bildirilmiştir. Kapsülleme teknikleri, mikrokapsülleme (3-800 um) ve macroencapsulation (daha büyük 1000 um) olarak sınıflandırılabilir. Mikrokapsüller macrocapsules 6 daha dayanıklıdır. Dr Chang ve 1964 yılında meslektaşları tarafından keşfedilmesinden bu yana, mikroenkapsülasyon yaygın insülin üreten hücreler anabolik, diğer hormonlar ve biyoaktif moleküllerin 7 enkapsülasyonunda kullanılmıştır. Bu tedaviler fibrozis ve bağışıklık yanıtı 8 olmak üzere konak dokusunda çeşitli zorluklarla karşı karşıya. Başlangıçta, biyopolimerlerin kalitesine ilişkin yan etkiler çözüme kavuşturuldu. Bununla birlikte, anabolik hücrelerinin nakli de hormon overpr bir sonucu olarak, örneğin fibroz gibi yan etkilere, başlatırözel bir bezin dışına oduction. Son yıllarda, obezite ve tip 2 diyabet salgın boyutlarına 9 ulaşmıştır. Yetişkin insanların% 30&#39;dan fazla dünya çapında kilolu ve 10 obez. Artan karın içi (IAB) yağ oluşumu kronik inflamasyon insidansını artırır ve tip 2 diyabet, kardiyovasküler hastalık, bazı kanserler ve diğer morbidite 11-13 teşvik etmektedir. Çeşitli türden kanıtlar IAB yağ ile ilişkili patogenez belirli adipositlerce önlenebilir önerdi. Son çalışmalar metabolizma artırmak ve in vivo 14 kemirgenlerde obezite ve insülin direnci azaltabilir IAB bölgeye deri altı adipositlere bu nakli göstermiştir. Obezite ve insülin direnci etkili azalma, ısı 15,16 şeklinde enerji dağıtma yeteneğine sahip termojenik adipositler ile ilişkilendirilmiştir. Adipositlerin termojenik modifikasyonu kararlı transfeksiyonu ile elde edilebilirBu tür eşleşmemiş protein 1 (UCP1) veya UCP1 ve diğer termojenik genlerin 15,16 ekspresyonunu düzenleyen genlerin mitokondriyal proton uncoupling, katılan genlerin. Bizim son çalışmalar aldehit dehidrojenaz 1 a1 (Aldh1a1) yetersizlik bu farelerde 17,18 obezite ve insülin direnci azaltır IAB yağ termojenik yeniden yol açtığını gösterdi. Özellikle, termojenik Aldh1a1 eksikliği kapsüllenmesi (Aldh1a1 - / -) Preadipositler Iab yağ 18 tedavisi için yeni bir terapötik fırsat düşündüren obez vahşi tip farelerde Iab yağ aynı terapötik etkisine aracılık eder. Deneysel ayarlarında, kapsüllü hücreler maliyet etkin bir şekilde 19 özel hücre popülasyonlarının etkilerini incelemek için araştırmacılar sağlar. Burada obezite bir fare modelinde termojenik katabolik hücre çizgisinin kapsüllenmesi ve laboratuar ve terapötik uygulama yöntemini tartışır. protokol t açıklarmikrokapsül üretiminde (Şekil 1) için HREE fazlar: alginat mikro (Şekil 1A), polikatyonik poli-L-lisin (PLL), mikro yüzeyi (Şekil 1B) ilgili membranlar, oluşumu ve çıkarılması oluşumu alginat çekirdekler (Şekil 1C).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Encapsulation device (VAR V1)</td>
			<td>Nisco</td>
			<td>LIN-0042</td>
			<td>None</td>
		</tr>
		<tr>
			<td>KD scientific syringe pump</td>
			<td>KD scientific</td>
			<td>780100Y</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Olympus microscope&#160;</td>
			<td>Olympus Optical</td>
			<td>IX70-S8F2</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Sigma</td>
			<td>MKBP8122V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Poly-l-lysine hydrobromide (PLL)</td>
			<td>Sigma</td>
			<td>020M5006V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>SLBJ2662V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma</td>
			<td>030M0200</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>SLBD2595V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Gels</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td>None</td>
		</tr>
		<tr>
			<td>ATGL primary antibody (from rabbit)</td>
			<td>Cell Signaling</td>
			<td>2138S</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Secondary anti body (anti rabbit)</td>
			<td>LI-COR</td>
			<td>926-68071</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Radio-Immunoprecipitation Assay (RIPA) buffer</td>
			<td>Boston BioProducts</td>
			<td>D25Y6Z</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>RNBD2893</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Cortizone 10 anti-itch ointment</td>
			<td>Cortizone 10</td>
			<td>C4029138</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Newborn calf serum (CS)</td>
			<td>Sigma</td>
			<td>N4762</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td>None</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I0516</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Insulin (bovine)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

encapsulation thermogenic preadipocytes for transplantation into adipose tissue depots

Hücre kapsülleme yarı geçirgen zarlar içinde canlı hücre tutmak üzere geliştirilmiştir. engrafted kapsüllü hücreler uzun süreli sağkalım elde etmek için tedavi edilen hastaya dokularında düşük molekül ağırlıklı metabolitleri alışverişi yapabilirsiniz. yarı-geçirgen zar, bağışıklık sistemi tarafından reddini önlemek için kapsüllenmiş hücrelerin engrafted sağlar. Kapsülleme işlemi insülin gibi biyolojik olarak aktif bileşiklerin, başka hormonlar ve sitokinler kontrollü bir şekilde serbest bırakılmasını sağlayacak şekilde tasarlanmıştır. Burada obez farelerin karın içi adipoz dokusunda ısı üretimi ve enerji tüketimine (termogenez) için lipitler tüketen katabolik hücrelerin kapsüllenmesi için bir yöntem tarif eder. Termojenik katabolik hücrelerin kapsüllenmesi obezitenin önlenmesi ve tedavisi için potansiyel olarak uygulanabilir ve tip 2 diyabet olabilir. Katabolik hücrelerin başka potansiyel uygulama alkoller veya diğer toksik metabolitler ve çevresel kirleticiler gelen detoksifikasyon içerebilir.

Şekil 1 mikro-üretimin her aşaması mikroskop altında kontrol edilebilir olduğunu göstermektedir. Şekil 2A co-kültür nasıl adipositleri kapsüllü hücreleri tek tabaka ile gösterir. Şekil 2B adiposit / mikro kapsüller ko-kültürlerini kullanarak kantitatif çalışma temsili bir örnek olduğunu adipositler Western blot ile analiz edilmiştir bölüm 5. Lisatlar tarif edilmiştir. Kapsüllenmiş hücrelerin Bu deneyde analiz edilmemiştir. 1000 seyreltme:...

Watch this Scientific Journal Video about Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots at JoVE.com

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Yağ Dokusu Depolarında içine Nakli Enkapsülasyon termojenik Preadipositler

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Research

JoVE Journal

Medicine

9.2K Görüntüleme.  The Ohio State University. Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Video: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS Saflaştırılmış Fotoreceptör Öncü Hücrelerinin Yetişkin Fare Retinasına Subretinal Transplantasyonu

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Tıp

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Heterotopik Kalp Nakli bir Fare Modeli Kullanarak Pulmoner Vana Nakli

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Interskapular kahverengi adipoz dokuda arasında Çerenkov Lüminesans Görüntüleme

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Yerelleştirme, Kimlik ve Eksizyon Kemirgen Yağ depolarının

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

İnsan Brown Yağ Dokusu Depolar otomatik Pozitron Emisyon Tomografi / Bilgisayarlı Tomografi tarafından Parçalı ve Manyetik Rezonans Görüntüler Kayıtlı

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Fare Yumurtalık Yağ Pad içine Transplantasyon

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Bir teknik subkutan karın yağ doku biyopsisi diyatermi sigara yöntemi ile için

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

İnsan yağ dokusu mikrovasküler işlevini kullanarak Videomicroscopy değerlendirilmesi

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Bir sıçan modelinde kısmi kolektomi sonra kaçağı azaltmak için Yağ dokusundan elde edilen kök hücre sayfasının nakli

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Yağ dokusundan elde edilen Stromal vasküler kesir için potansiyel kıkırdak rejenerasyon üreten klinik protokol

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...