Çalışmada Shanghai yenilik (1402 H 294300) finansman bilim ve teknoloji Komisyonu, Shanghai Belediyesi (CN) Dr Wen Wang tarafından sponsor tarafından desteklenmiştir. Dr Guangdong Çu (Ulusal doku Mühendisliği merkezi Çin) onun teknik yardım ve bu el yazması için bilimsel tavsiyeler için teşekkür etmek istiyorum. Biz de Bay Huitang Xia (Shanghai dokuzuncu insanların hastane) teşekkür etmek için hayvan refah içinde onun yardım etmek istiyorum.

hayvan konular içeren yordamlar onaylanan yerel kurumsal hayvan bakım ve Etik Komitesi, tarafından hayvan acı en aza indirmek için bir çaba ile. Aşağıdaki iletişim kuralı kurumsal hayvan bakım ve kullanım Komitesi (IACUC) Shanghai dokuzuncu insanlar tarafından kabul edildi &#8217; hastane bağlı Shanghai JiaoTong Üniversitesi Tıp Fakültesi için iletişim kuralı ile s sayı [2017] 063.
 1. Surgically-Induced fare diz osteoartrit manken kurulması <ol><li>için bu cerrahi işlem, kullanım 8-12 hafta yaşlı erkek Sprague Dawley (SD) rats vücut ağırlığı 250 ve 300 g arasında</li>
	<li>40 mg/kg tiletamine ve kas içi enjeksiyon yolu ile 40 mg/kg zolazepam hayvanlarla anestezi.</li>
	<li>Hayvan sol yan pozisyonda bir ısıtmalı sahne alanı üzerine yerleştirin. Sağ diz iyice bir ustura ile tıraş. Cerrahi alan % 70 etanol tarafından takip % 10 providone-iyot solüsyonu ile dezenfekte, iki kez daha tekrarlayın ve sonra % 10 providone-iyot çözüm ile boyayın ve cerrahi bir yastık ile ameliyatsız alanı kapsayacak.</li>
	<li>Cerrahi bir neşter kullanmadan 2 cm kesi jointcapsules ortaya çıkarmak için yanal patellar tendon boyunca olun.</li>
	<li>Cerrahi neşter medial Kollateral bağ transect için kullanın. Menisküs femur doğru yansıtır.</li>
	<li>Steril % 0,9 Sodyum Klorür çözüm eklem kıkırdak yüzeyinde kıkırdak kurumasını önlemek için işlem sırasında damla.</li>
	<li>Medial Menisküs Forseps ile kapmak ve tibial ek bir neşter ( şekil 1A) ile dar noktasında Menisküs ile kesti. Kıkırdak yaralanmasına kaçının.</li>
	<li>4-0 absorbe dikiş ile ek kapsül kapatın.</li>
	<li>Kendine gelmek kadar cerrahi fareler izlemek.</li>
	<li>20 mg/kg gentamisin enfeksiyon ve ameliyat sonrası ağrı gidermek için kas içi bir rota üzerinden 0,05 mg/kg buprenorfin önlemek için enjekte.</li>
	<li>Fareler bir 12 h ışık/karanlık döngüsüne 3-8 hafta boyunca başlangıçlı KOA altında ısı kontrollü hayvan tesiste korumak. Hayvanlar devam ağrısı belirtileri devam ederse buprenorfin (0,05 mg/kg, sohbet) ve gentamisin (20 mg/kg, sohbet), bilhassa ağrı kesici almak.</li>
</ol> 2. Histoloji değerlendirme sıçan KOA Model <ol><li>kurban aşırı CO 2 inhalasyon (hayvan bilinçsizlik yapmak için Ekle Varolan yayına odasında karbon dioksit ile dakikada odası hacmi yaklaşık % 30 bir dolgu oranı ile rats içinde 2-3 dk. korumak CO2 akışı için en az 1 dk sonra solunum durur.) zaman puan (temsilcisi-den sonuçlanmak göstermek--dan 3-8 hafta arasında değişen KOA indüksiyon 8 hafta) sonra ameliyat. belirtilen</li> <li>deri ve kas diz eklemleri etrafında incelemek ve bir neşter ile diz eklemleri kesti.</li>
	<li>3 gün oda sıcaklığında %4 paraformaldehyde (PFA) diz eklem tamir. Daha sonra yaklaşık 4 hafta 4 ° C'de % 20 ethylenediaminetetraacetic asit (EDTA) örneklerinde decalcify ve 3 günde EDTA değiştirin. 
	Dikkat: Paraformaldehyde zehirlidir, uygun kişisel koruyucu donanımları (PPE), eldiven ve gözlük gibi.</li>
	<li>Bir otomatik doku işlemci kullanarak örnekleri kurutmak. Set menü floş bir döngü aşağıdaki gibi başlatın: % 70 etanol için 1 h; o zaman % 80 etanol ve % 95 etanol 1 h için sırasıyla; %2 100 etanol devredir 1s ardından; daha sonra Ksilen 1 h için iki kez her zaman ve her zaman alkol 3 kez 58 ° C'de 1 h için. 
	Dikkat: Ksilen toksik, çok uygun KKE, eldiven ve gözlük gibi giymek. 
	Not: İsteğe bağlı olarak, Coplin kavanozlar için otomatik doku işlemci olarak aynı kurulum ile el ile su kaybı kullanın.</li>
	<li>Olduğu gibi eklem yüzü medial yönünü yerleştirin ve merkezi gömülü dokusuyla parafin blok içine embed. 
	Not: İsterseniz, forseps parafin blok içine el ile örnekleri katıştır kullanın.</li>
	<li>Döner microtome tarafından 30 µm aralıkları ile 2 seri bölümler için 5 µm sagittal bölümleri medial kenar boşluğundan eklem kesmeye başlayın.</li>
	<li>Bölümleri cam slaytlarda dağ ve Ksilen içindeki slaytları deparaffinize. 2 seri bölümler arasındaki bir slayt Hematoksilen ile leke &#38; Eozin (H &#38; E) Boyama: % 0,1 Hematoksilen 15 dakika, % 1 Asetik asit çözüm için 10 s ve % 0,5 Eozin 10 dk için. Ve Safranin O/hızlı yeşil boyama ile diğer slayt leke: % 0,1 Hematoksilen 15 dk, %0.02 için hızlı yeşil 3 dk, % 1 Asetik asit çözüm 10 s ve %0,1 Safranin O 3 dakika süreyle 
	Dikkat: Ksilen toksik, eldiven, gözlük gibi uygun KKE giymek.</li>
	<li>Fareler 6 ' osteoartrit Araştırma Derneği International (OARSI) histopatoloji değerlendirme sistemi kullanarak lekeli slaytlar Puan edinildi.</li>
</ol> 3. Bir insan Mezenkimal Kök hücreleri floresan boya yaptım ile etiketleme <ol><li>hücre etiketleme çözüm % 100 etanol, 1 mM bir konsantrasyon hazırlamak yaptı. Hücre etiketleme çözüm ışıktan korumak ve 6 ay için oda sıcaklığında saklayın.</li>
	<li>HaMSCs Dulbecco Standart hücre kültür yordamlarda ile büyümek &#39; s modifiye kartal &#39; % 1 penisilin ve streptomisin ve %10 10 cm petri kabına içinde fetal buzağı serum (FCS) ile takıma s orta (DMEM).</li>
	<li>HaMSCs 2 mL % 0.25 tripsin ile 1 mM EDTA öncesinde yaklaşık %80 izdiham ile ayırmak.</li>
	<li>Tripsin 4 mL kültür orta ile etkisiz hale getirmek ve hücreleri 3 dakika oda sıcaklığında 800 x g de spin.</li>
	<li>Hücreleri 1 x 10 6 hücre/mL 5 mL serum serbest kültür Orta yoğunluk itibariyle askıya alma.</li>
	<li>Hücreleri 50 ekleyerek etiket µL yaptım 5 mL serum serbest kültür orta 50 dk. 37 ° C'de çözümde hücre etiketleme</li>
	<li>3 dakika süreyle 800 x g de etiketli hücreleri spin</li>
	<li>Hücreleri iki kez daha 5 mL fosfat tampon serum (PBS) vasıl 800 x g 3 dakika yıkayın.</li>
	<li>Cy5 filtre seti ile floresan mikroskop kullanarak etiketli haMSCs kontrol edin.</li>
	<li>Trypan mavi boyama ile hücreleri saymak ve hücrelerin içinde 100 µL PBS enjeksiyon için 10 6 x 2,5 elde.</li>
</ol> 4. Vivo Floresan parça haMSCs için Imaging <ol><li>sekiz hafta sonra ameliyat (Adım 1), anestezi KOA fareler tarafından kas içi enjeksiyon-25 mg/kg tiletamine ve 25 mg/kg zolazepam.</li>
	<li>Sağ diz iyice ortak alan ortaya çıkarmak için bir ustura ile tıraş. % 70 etanol ile ortak alanı dezenfekte.</li>
	<li>26 G şırınga iğne kullanarak enjekte 2.5 x 10 100 µL PBS üçgen alanının merkezinde askıya hücreleri etiketli 6 oluşan medial patellar bağ, medial femoral düzgün ve medial tibial düzgün (kenarında şekil 1B).</li>
	<li>Görüntüleme yazılımını açın ve görüntüleme sistemi içinde vivo bioluminescence başlatılamıyor.</li>
	<li>Pozisyon Rat sırtüstü pozisyon görüntüleme sistemi Merkezi aşamasında ve odasının kapısını kapatın.</li>
	<li>Kontrol &#34; floresan &#34; onay kutusunu ve Seç floresan filtre ayarlar 640 nm için uyarma ve 680 nm için emisyon ( şekil 2A).</li>
	<li>Select alan ö ayarla piksel binning orta (8), görüş, durdurmak F/2 ve pozlama zaman otomatik olarak. Optimum korumak Pozlama süresi tüm çalışma ( şekil 2A) arasında karşılaştırılabilir sonuçlar elde etmek için tutarlı.</li>
	<li>Anestezi kurtarma için monitör ve aşamada hayvan kaldırmak. 
	Not: Yer hayvan bir Isıtma yastığı üzerinde 2-3 sarılma katmanlarla kaplı.</li>
	<li>Floresan ( şekil 2B) ölçümü için radyant verimliliğini birimleri seçin. Faiz (ROI) analiz için bölgeleri seçin ve her resim ( şekil 2C) floresan sinyallerini ölçmek.</li>
	<li>İn vivo içinde düşsel sırayla haMSCs boyuna saklama eklemde çalışmaya her fare için yordamı yineleyin. 
	Not: Biz hayvanlar görüntüleme öneririz &#39; floresan sinyalleri tarafından içinde vivo görüntüleme sistemi her iki günde enjeksiyon sonra ilk hafta ve bir kez her hafta kadar sinyali tespit edilemez.</li>
</ol>

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Meng Li, Ming Hao ve Wen Wang geçerli çalışanları ve hücresel Biyomedikal grup hisse senedi opsiyonu sahipleri vardır (Nasdaq: CBMG). Diğer yazarlar hiçbir çıkar çatışması beyan ederim.

Başucu için tezgah KOA rejeneratif kök hücre tedavisinde getirebiliriz önce güvenlik standartları ve kök hücre tedavisi biodistribution çalışmaları acilen ihtiyaç vardır. Ancak, hastalığın patolojik ortam sebat ve Transplante haMSCs10biodistribution önemli bir rol oynar. Son zamanlarda, bizim grup enjeksiyonları8normal şartlar altında daha haMSCs intra artiküler enjeksiyon artık patolojik bir KOA ortamında kalıcı gösterdi. Enflamatuar ve dejenera...

Diz Osteoartriti (KOA) eklem kıkırdak kaybı ve dünya1çevresinde Yaşlılarda büyük kronik bir hastalık haline gelmiştir ilerici iltihap kaynaklanan bir dejeneratif hastalığıdır. Ancak, geçerli terapiler anti-inflamatuar ilaçlar, fiziksel takviyeleri ve cerrahi işlemler kullanarak yalnızca semptomatik ağrı2için geçici bir rahatlama sağlayabilir.
İnsan yağ elde edilen Mezenkimal Kök hücre (haMSCs) onların multipotent farklılaşma kıkırdak rejenerasyon ve immunomodulatory özellikleri3için potansiyel sayesinde diz osteoartrit için umut verici bir rejeneratif ilaç haline gelmiştir, 4. Eylem içinde vivomekanizmaları araştırmak için farmakolojik yolları ile karşılaştırıldığında, küçük KOA hayvan modellerinde canlı haMSCs izleme için gerekçe ve fizibilite haMSC terapi klinik uygulamadan önce kurmak için şu anda öğreticidir. Preklinik test etmek için medial Menisektomi (MM) ile tutarlı tekrarlanabilirlik5nispeten uygun bir model sağlayan KOA de, fareler, ikna etmek için eklem mekanik yük oynattığını. MM tarafından indüklenen KOA başlangıcı ön çapraz bağ transeksiyon tek başına veya kombine ile kısmi medial Menisektomi6daha öncedir. Bu nedenle, enjekte haMSCs KOA patolojik microenvironment ile uzun vadeli Hofstede kez MM7,8tarafından indüklenen Sıçanlarda değerlendirilir.
HaMSCs tedavi edici etkinliği yaygın olarak bildirilen, ilgili bilgi üzerinde olmasına rağmen intra artiküler (IA) enjeksiyon yolu ile implante haMSCs in vivo sebat kıt9,10' dur. Bu nedenle, çeşitli hücresel etiketleme yöntemleri, immunohistology11, luciferase12, yeşil flüoresan protein13 transfection, demir oksit manyetik rezonans görüntüleme (MRG)14 etiketleme de dahil olmak üzere geliştirilmiştir. ve çok sayıda hücre floresan boyalar8,15,16. Emek yoğun Histoloji analizleri ile karşılaştırıldığında, vivo non-invaziv görüntüleme bulmak belgili tanımlık gerçek zamanlı dağıtım ve floresan sinyallerini10ile,17etiketli hücre dinamiği için Optik aygıtları kullanır. Fonksiyonel canlı hücre görüntüleme için cytocompatible floresan etiketlemeyi hücresel aktiviteler kök hücre nakli18sonra ortaya çıkarmak için bir gelişmiş izleme radyoaktif ücretsiz bir tekniktir. Ayrıca, çok renkli floresan lipofilik boyalar hidrofilik amino-reaktif boyalar veya floresan proteinler, onların gelişmiş hücre geçirgenliği ve gelişmiş Floresans kuantum sayıları19gibi avantajlara sahip.
Böylece, bir kırmızı burada dahil iletişim kurallarını kullanmak lazer ile lipofilik carbocyanines etiketli hücreleri heyecanlandırmak için mi (DilC18(5)), hangi bir far-red floresan Dil (dialkylcarbocyanines) analog20' dir. Bir kırmızı kaymıştır uyarma ve emisyon spectra autofluorescent girişim önler ve derin doku canlı hayvan8uzun bir süre görüntüleme sağlar. Bu yöntem izleme ile etiketli vivo içinde hücreleri mi anlamak ve geçerli kök hücre Yenileyici tedavisi geliştirmek için gerekli olduğu gibi haMSCs, hayvan modellerinde transplante kök hücre izlemek için geçerlidir.

<table><tbody><tr><td>Matrx VMR animal anesthesia system</td><td>Midmark</td><td>VIP3000</td><td></td></tr><tr><td>4-0 suture</td><td>Shanghai Jinhuan</td><td>KC439</td><td></td></tr><tr><td>Razor</td><td>Pritech</td><td>LD-9987</td><td></td></tr><tr><td>Gentamicin</td><td>Zhejiang Jindakang Animal Health Product Co., Ltd.</td><td>None</td><td></td></tr><tr><td>0.9% Sodium chloride solution</td><td>Hunan Kelun Pharmaceutical Co., Ltd.</td><td>H43020455</td><td></td></tr><tr><td>Penicillin</td><td>Shanghai Kangfu chemical pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Buprenorphine</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>16005</td><td>Dilute to final concentration of 10% in PBS</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>Dilute to final concentration of 20% in PBS</td></tr><tr><td>0.1% Hematoxylin Solution, Mayer&amp;rsquo;s</td><td>Sigma-Aldrich</td><td>MHS16</td><td></td></tr><tr><td>0.5% Eosin Y solution, alcoholic</td><td>Sigma-Aldrich</td><td>HT110116</td><td></td></tr><tr><td>Safranin O</td><td>Sigma-Aldrich</td><td>S8884</td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td></td></tr><tr><td>Shandon Excelsior ESTM Tissue Processor</td><td>Thermo Fisher</td><td>A78400006</td><td></td></tr><tr><td>Shandon Histocentre&amp;trade; 3 Tissue Embedding Center</td><td>Thermo Fisher</td><td>B64100010</td><td></td></tr><tr><td>Fully Automated Rotary Microtome</td><td>Leica</td><td>RM2255</td><td></td></tr><tr><td>DiD</td><td>Molecular Probes, Life&lt;br/&gt; Technologies</td><td>V-22887</td><td></td></tr><tr><td>D-MEM High Glucose</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>PBS</td><td>GIBCO, Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-114</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>Corning</td><td>V118877</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>Optima MAX-TL</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>0.4% Trypan Blue solution</td><td>Sigma-Aldrich</td><td>93595</td><td></td></tr><tr><td>Titetamme</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Zolazepam</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Sterile hyposermic syringe for single use 26G</td><td>Shanghai Misawa Medical Industry</td><td>None</td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>PerkinElmer</td><td>124262</td><td></td></tr><tr><td>Living Imaging 4.0 software</td><td>PerkinElmer</td><td>None</td><td></td></tr></tbody></table>

in vivo tracking of human adipose-derived mesenchymal stem cells in a rat knee osteoarthritis model with fluorescent lipophilic membrane dye

İnsan yağ elde edilen Mezenkimal Kök hücre (haMSC) tedavisinin klinik uygulama diz osteoartrit (KOA) desteklemek için hücre sebat etkinliği ve haMSCs hayvan modellerinde biodistribution inceledi. Lipofilik floresan boya ile haMSCs hücre membran etiketlemek için bir yöntem gösterdi. Daha sonra intra artiküler enjeksiyon cerrahi müdahale ile indüklenen KOA fareler etiketli hücre dinamik olarak görüntüleme sistemi bir vivo içinde tarafından izlenen. Lipofilik istihdam carbocyanines yaptım (DilC18 (5)), hangi üzerinden dokuları çevreleyen doğal yeşil autofluorescence uyarma önlemek için kırmızı bir lazer kullanmak bir far-red floresan Dil (dialkylcarbocyanines) analog,. Ayrıca, kırmızı kaymıştır emisyon spectra ile izin derin doku görüntüleme içinde canlı hayvan ve etiketleme işlemi sitotoksik efekt veya haMSCs fonksiyonel hasar yok neden. Bu yaklaşım bir verimli izleme yöntemini haMSCs bir sıçan KOA modeli için olmak gösterilmiştir. Bu yöntemin uygulama da en uygun yönetim rota ve MSCs önceden klinik çalışmalarda diğer kaynaklardan doz belirlemek için kullanılabilir.

KOA modeli ikna etmek için MM SD fareler (şekil 3) sağ diz eklemi gerçekleştirildi. Sekiz hafta sonra ameliyat fareler kurban ve diz eklemleri seri bölümlerini her iki H ile değerlendirildi &#38; E ve Safranin O/hızlı yeşil boyama (şekil 4). H için &#38; E boyama, eklem kıkırdak yüzeyinde daha ameliyat olmadan normal eklem cerrahisi diz kaba sınırları sergiledi. Safranin-O/hızlı yeşil, boyama için gözlenen...

Watch this Scientific Journal Video about In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye at JoVE.com

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Vivo İnsan yağ elde edilen Mezenkimal kök hücrelerin sıçan diz osteoartrit modelinde floresan lipofilik membran boya ile izleme

Bu iletişim kuralı tarafından far-red floresan intra artiküler (IA) enjeksiyon yoluyla bir sıçan diz osteoartrit (KOA) modelinde etiketleme hücre sebat ve biodistribution insan yağ elde edilen Mezenkimal kök hücrelerin (haMSCs) izlemek için etkili bir yöntem açıklanır.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

in-vivo-tracking-human-adipose-derived-mesenchymal-stem-cells-rat

Research

JoVE Journal

Developmental Biology

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

8.9K Görüntüleme.  Cellular Biomedicine Group, China. Bu iletişim kuralı tarafından far-red floresan intra artiküler (IA) enjeksiyon yoluyla bir sıçan diz osteoartrit (KOA) modelinde etiketleme hücre sebat ve biodistribution insan yağ elde edilen Mezenkimal kök hücrelerin (haMSCs) izlemek için etkili bir yöntem açıklanır.

Video: In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biyoloji

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Küçük Hayvan Kas Aşı dinamiği ile Takip In Vivo Floresan Görüntüleme

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

Floresan SapC-DOPS Nanovesicles kullanma Beyin Tümörleri ve Artrit Vivo Optik Görüntüleme

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Tıp

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Fare Kemik İliği monositlerin Takip<em&gt; İn Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

İmmünoloji ve Enfeksiyon

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

için Femur Pencere Odası Modeli<em&gt; İn Vivo</emFare Kemik İliği içinde&gt; Hücre Takibi

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Atçılık tendinopatisinde Kemik İliği Mezenkimal Kök Hücreler Etiketli Teknesyum- 99m Vivo Görüntüleme ve Takip

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Ultrason destekli İntrakardiyak enjeksiyon bağırsak için giriş için deneysel iltihabi bağırsak hastalıkları fare modellerinde kullanım artırmak için insan Mezenkimal Kök hücre

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Dönem Yenidoğan Sıçanlarda Kök Hücrelerin İntratrakeal Aşılanması

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Görselleştirme ve miktar Mezenkimal hücre Adipojenik farklılaşma potansiyeli bir Lineage belirli Marker ile

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...

Vivo İnsan yağ elde edilen Mezenkimal kök hücrelerin sıçan diz osteoartrit modelinde floresan lipofilik membran boya ile izleme

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