Name: isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering
Uploaded: 2017-04-30T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Biz Janine Becker, Caroline Bickelmann ve Ruth Nickels mükemmel teknik yardım için teşekkür ederiz. (- Alman Araştırma Vakfı DFG) - LA 2682 / 7-1 Bu çalışma, Deutsche Forschungsgemeinschaft hibe ile finanse edildi.

Tüm prosedürler deney hayvanlarının kullanımı için Sağlık kılavuzların Ulusal Enstitüsü göre gerçekleştirilmiştir ve kurumsal yönergelere (Landesamt für Soziales, Gesundheit und Verbraucherschutz Abt. Lebensmittel- und Veterinär-, Zentralstelle, Saarbrücken, Almanya) izledi. Cerrahi Araçların hazırlanması 1. <ol><li> % 10 fetal dana serumu (FCS), 100 U / mL penisilin, 0.1 mg / mL streptomisin, hazır diseksiyon makas, cerrahi forseps, küçük hazırlama makas, ince forseps ve 15 ml Dulbecco&#39;nun modifiye edilmiş Eagle ortamında (DMEM ile steril bir Petri kabı tutmak ) epididimal yağ yastıkları hasat için. </li><li> 5 dakika için dezenfekte edici bir çözeltisine cerrahi aletleri Açığa. Seçenek olarak ise, (buhar sterilizasyonu, 121 ° C, 20 dakika) sterilize. </li></ol> 2. Hayvanlar ve Anestezi <ol><li> çalışma ve için belirtildiği gibi dikkatle farelerin zorlanma seç<html> soruşturma altında konu. NOT: Bu çalışmada epididimal yağ hasat için donör olarak erkek vahşi tip C57BL / 6 fareler kullanıldı. Ilgi konusu olan, reklam MVF aynı zamanda transjenik yeşil floresan protein (GFP) pozitif ve donor hayvandan (C57BL / 6-Tg (CAG EGFP) 1Osb / J) 22 izole edilebilir. Bu fragmanlar, GFP-negatif yabani tip farelere 14 implantasyon sonrası GFP immünohistokimyasal boyama ile kolayca tespit edilebilir olan büyük bir avantaj taşımaktadır. </li><li> ksilazin (15 mg / kg) ve ketamin (75 mg / kg) intraperitonal enjeksiyonu ile hayvanlar anestezisi. hayvanlar derinden hiçbir yanıt ile bir ayak parmağı tutam yaparak anestezi emin olun. verici hayvanlar yağ hasattan sonra öldürülür olarak göz yağ belirtilmemektedir. NOT: analjezi ve cerrahi sterilite deneyleri planlanan ülke ve kurumun ilgili kurallarına uyum içindedir emin olun. </li></ol>Epididimal Yağ Pads le &quot;&gt; 3. Hasat <ol><li> Bir işlem tabloya hayvan aktarın. Cerrahi bir stereomikroskop altında yatar pozisyonda hayvan koyun ve ayak Tutam kullanarak derin anestezi onaylayın. </li><li> Bir cerrahi kumaş bantla yapıştırılarak pençelerini hareketsizleştiriniz ve çözüm dezenfekte ile karın dezenfekte. </li><li> diseksiyon makas ile altta yatan kas tabakasının serbest karın cildi ayırın. </li><li> diseksiyon makas ile orta hat laparotomi ve yanal olarak karın duvarı kanatlarını açığa. </li><li> Bilateral testis, epididimis ve ince forseps (Şekil 1) kullanılarak epididimal yağ pedi tanımlar. Yağ yastıklarının dışkı bulaşmasını önlemek için bağırsak yapıları zarar vermeyin. </li><li> stereomikroskop altında küçük hazırlık makas ve ince forseps ile epididimal yağ yastıkları Hasat. Epididim ve yağ arasında birkaç mm&#39;lik bir emniyet marjı tutunKazara epididimal hasat riskini azaltır. NOT: Bu prosedür ayrıca bir stereo olmadan gerçekleştirilebilir. Ancak, bu daha da epididim ve spermatik kord kazara yaralanma riskini artırabilir. </li><li> DMEM 15 mL içeren bir Petri kutusu içine epididimal yağ pedleri transfer hücre laboratuara taşınması için 37 ° C sıcaklıkta önceden ısıtılır. </li><li> abdominal aort ya da servikal bozma çizilmesi ile hayvan kurban. </li></ol> ad-mvf 4. İzolasyonu <ol><li> 15, fosfat tamponlu tuzlu su mL (PBS), steril bir 14-ml polipropilen (PP) tüpler, steril bir 50 ml Erlenmeyer şişesinde, steril 1.5 mL konik mikrosantrifüj tüpleri ve sterilize ince makas üç steril Petri kapları hazırlayın. </li><li> bir laminar akış başlığı altında 15 ml PBS ile üç kere Petri tabaklarında yağ pedlerine yıkayın. </li><li> 14 mL&#39;lik bir PP tüpe yağ aktarın. t vasıtasıyla (mL) toplandı yağ dokusu hacminin belirlenmesio boru ölçeği. homojen bir doku süspansiyon elde edilene kadar ince makas mekanik yağ dokusunu inceltmek. </li><li> pipet ölçüm 10 mL vasıtasıyla 50 mL&#39;lik bir Erlenmeyer şişesi içine kollajenaz NB4G (0.5 U / ml PBS) iki hacim ile kıyılmış doku aktarın. Toplam hacim adım 4.3 &#39;de ölçülen yağ dokusu hacim üç kez olur. 37 ° C&#39;de ve% 5 CO2 ile nemlendirilmiş atmosfer koşullarına: Otomatik bir karıştırıcı (25 mm Manyetik bir karıştırma çubuğuna boyutu) ile kuvvetli karıştırma altında 10 dakika için bir kuluçka makinesi içinde doku sindirim gerçekleştirin. </li><li> sindirim durdurulabilir olmadığını yargılama mikroskop altında sindirilmiş doku küçük bir bölümünü (10 uL) dikkate alınmalıdır. Enzimatik sindirim olan (Şekil 2) durdurmak için uygun noktayı gösteren Digestate esas olarak aşağıdaki tek hücre &quot;serbest&quot; ad-MVF içerdiğini teyit edin. NOT: Bu adım prosedürü ile tecrübe gerektirir ve kalite için kritiktirreklam-MVF izole edilmiştir. ad-mvf olmadan tek hücreli süspansiyon yağ sindirimi sonucu uzun süre. </li><li> PBS /% 20 FCS iki hacim enzim nötralize. Toplam hacim aşama 4.4&#39;te hücre-damar süspansiyonu hacmi üç kez olur. Yeni PP tüpler içine arka cep damar süspansiyon aktarın. </li><li> yerçekimi ile yağ kalan reklam-MVF ayırmak için 37 ° C de 5 dakika süre ile süspansiyon inkübe edin. Daha sonra dikkatli bir şekilde 1 mL hassas pipet ile ana yağ süpernatant kaldırmak. Süspansiyon yağsız olması görünene kadar 100 mcL hassas pipet ile bu döngüyü birkaç kez tekrarlayın. </li><li> 50 ml konik santrifüj tüpüne üstüne bir 500 um filtre koyun. yağ pıhtıları kalan çıkarmak için filtre zarı üzerine 14 ml PP tüpler 10 ml ölçüm pipet ile hücre-damar süspansiyon aktarın. tek reklam-MVF sayısına göre yeni bir 14-ml PP tüplere süzüldü süspansiyon aktarın planlanan deneyler için izole eder. NOT: İn vitrmikrovasküler ağı oluşumu odaklanan O deneyleri, ayrıca mikroskopik analiz sırasında görüntüleme kalitesini artırmak için, hücre-damar süspansiyonu saflaştırmak için yararlı olabilir. Bu amaç için, süspansiyon ek olarak, filtre üzerinde toplanan reklam MVF gelen tek hücreleri çıkarmak için, bir 20-mikron filtre ile bir kez filtrelenebilir. </li><li> Reklam-MVF ihtiva eden bir pelet elde etmek için, hücre-damar süspansiyonu (600 x g, 5 dakika, oda sıcaklığı) santrifüjleyin. </li><li> Santrifüj işleminden sonra, ml bırakılır 1 kadar supernatant çıkarın. Bu 1 mL reklam MVF ile pelet yeniden süspanse edin ve 1.5 ml konik bir mikrosantrifüj tüpü içine süspansiyon aktarın. </li><li> bir pelet elde etmek için bir mikrosantrifüj tüpü içinde bir reklam-MVF süspansiyonu (600 x g, 5 dakika) santrifüj. </li><li> PBS /% 20 FCS gereken son hacmine pelet yeniden askıya almak için süpernatantı. NOT: izolatı başına reklam mvf sayısının mikroskobik sayımı ile tespit edilebilir. Nihai hücre vesse bu amaçla, 1/10 İçinl süspansiyonu PBS içinde 1:10 seyreltilir ve bu seyreltme 100 uL, 96 oyuklu bir plaka iyi aktarılır. bir kap benzeri morfolojisi sergileyen reklam MVF tüm sayı daha sonra sayılmış ve bütün izolat için ekstrapole edilir. Gerekli reklam-MVF Konsantrasyon ve saflaştırma tek in vitro veya reklam MVF in vivo uygulamada ilgili uyarlanabilir. Bu mikrovasküler ağ oluşumunun in vitro analizi veya doku defektlerinin içine in vivo implantasyonu için iskeleler reklam-MVF tohumlanmasından kolojen jeller reklam-MVF gömülmesini içerebilir. </li></ol>

<ol>
	<li>Langer, R., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering.">Tissue engineering.</a> Science. 260 (5110), 920-926 (1993).</li><li>Khademhosseini, A., Langer, 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+decade+of+progress+in+tissue+engineering.">A decade of progress in tissue engineering.</a> Nat Protoc. 11 (10), 1775-1781 (2016).</li><li>Novosel, E. C., Kleinhans, C., Kluger, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+is+the+key+challenge+in+tissue+engineering.">Vascularization is the key challenge in tissue engineering.</a> Adv Drug Deliv Rev. 63 (4-5), 300-311 (2011).</li><li>Rouwkema, J., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+and+Angiogenesis+in+Tissue+Engineering:+Beyond+Creating+Static+Networks.">Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks.</a> Trends Biotechnol. 34 (9), 733-745 (2016).</li><li>Laschke, M. W., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+in+tissue+engineering:+angiogenesis+versus+inosculation.">Vascularization in tissue engineering: angiogenesis versus inosculation.</a> Eur Surg Res. 48 (2), 85-92 (2012).</li><li>Sarker, M., Chen, X. B., Schreyer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+vascularisation+within+tissue+engineering+constructs.">Experimental approaches to vascularisation within tissue engineering constructs.</a> J Biomater Sci Polym Ed. 26 (12), 683-734 (2015).</li><li>Frueh, F. S., Menger, M. D., Lindenblatt, N., Giovanoli, P., Laschke, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+emerging+vascularization+strategies+in+skin+tissue+engineering.">Current and emerging vascularization strategies in skin tissue engineering.</a> Crit Rev Biotechnol. 20, 1-13 (2016).</li><li>Utzinger, U., Baggett, B., Weiss, J. A., Hoying, J. B., Edgar, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+time+series+microscopy+of+neovessel+growth+during+angiogenesis.">Large-scale time series microscopy of neovessel growth during angiogenesis.</a> Angiogenesis. 18 (3), 219-232 (2015).</li><li>Laschke, M. W., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevascularization+in+tissue+engineering:+Current+concepts+and+future+directions.">Prevascularization in tissue engineering: Current concepts and future directions.</a> Biotechnol Adv. 34 (2), 112-121 (2016).</li><li>Baiguera, S., Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelialization+approaches+for+viable+engineered+tissues.">Endothelialization approaches for viable engineered tissues.</a> Angiogenesis. 16 (1), 1-14 (2013).</li><li>Wagner, R. C., Kreiner, P., Barrnett, R. J., Bitensky, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+and+cytochemical+localization+of+a+catecholamine-sensitive+adenylate+cyclase+in+isolated+capillary+endothelium.">Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium.</a> Proc Natl Acad Sci U S A. 69 (11), 3175-3179 (1972).</li><li>Wagner, R. C., Matthews, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+culture+of+capillary+endothelium+from+epididymal+fat.">The isolation and culture of capillary endothelium from epididymal fat.</a> Microvasc Res. 10 (3), 286-297 (1975).</li><li>Laschke, M. W., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+microvascular+fragments:+natural+vascularization+units+for+regenerative+medicine.">Adipose tissue-derived microvascular fragments: natural vascularization units for regenerative medicine.</a> Trends Biotechnol. 33 (8), 442-448 (2015).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+of+porous+scaffolds+is+improved+by+incorporation+of+adipose+tissue-derived+microvascular+fragments.">Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments.</a> Eur Cell Mater. 24, 266-277 (2012).</li><li>Frueh, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+microvascular+fragments+improve+vascularization,+lymphangiogenesis+and+integration+of+dermal+skin+substitutes.">Adipose tissue-derived microvascular fragments improve vascularization, lymphangiogenesis and integration of dermal skin substitutes.</a> J Invest Dermatol. 137 (1), 217-227 (2017).</li><li>McDaniel, J. S., Pilia, M., Ward, C. L., Pollot, B. E., Rathbone, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+multilineage+potential+of+cells+derived+from+isolated+microvascular+fragments.">Characterization and multilineage potential of cells derived from isolated microvascular fragments.</a> J Surg Res. 192 (1), 214-222 (2014).</li><li>Nakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+autotransplantation+of+microvessel+fragments+on+experimental+random-pattern+flaps+in+the+rat.">Effect of autotransplantation of microvessel fragments on experimental random-pattern flaps in the rat.</a> Eur Surg Res. 30 (3), 149-160 (1998).</li><li>Nakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+autotransplantation+of+microvessel+fragments+into+the+rat+heart.">Successful autotransplantation of microvessel fragments into the rat heart.</a> Eur Surg Res. 31 (3), 240-248 (1999).</li><li>Shepherd, B. R., Hoying, J. B., Williams, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+transplantation+after+acute+myocardial+infarction.">Microvascular transplantation after acute myocardial infarction.</a> Tissue Eng. 13 (12), 2871-2879 (2007).</li><li>Pilia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+perfusion+of+microvascular+fragments+in+a+rodent+model+of+volumetric+muscle+loss+injury.">Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury.</a> Eur Cell Mater. 28, 11-23 (2014).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+microvascular+fragments+from+aged+donors+exhibit+an+impaired+vascularisation+capacity.">Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity.</a> Eur Cell Mater. 28, 287-298 (2015).</li><li>Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Green+mice'+as+a+source+of+ubiquitous+green+cells.">'Green mice' as a source of ubiquitous green cells.</a> FEBS Lett. 407 (3), 313-319 (1997).</li><li>Honek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+age-related+insulin+sensitivity+by+VEGF-dependent+vascular+plasticity+in+adipose+tissues.">Modulation of age-related insulin sensitivity by VEGF-dependent vascular plasticity in adipose tissues.</a> Proc Natl Acad Sci U S A. 111 (41), 14906-14911 (2014).</li><li>Cho, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+role+of+LYVE-1-positive+macrophages+in+adipose+tissue.">Angiogenic role of LYVE-1-positive macrophages in adipose tissue.</a> Circ Res. 100 (4), e47-e57 (2007).</li><li>Han, S., Sun, H. M., Hwang, K. C., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-Derived+Stromal+Vascular+Fraction+Cells:+Update+on+Clinical+Utility+and+Efficacy.">Adipose-Derived Stromal Vascular Fraction Cells: Update on Clinical Utility and Efficacy.</a> Crit Rev Eukaryot Gene Expr. 25 (2), 145-152 (2015).</li><li>Chen, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Differentiation+of+Adipose-Derived+Stem+Cells+from+Porcine+Subcutaneous+Adipose+Tissues.">Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues.</a> J Vis Exp. (109), e53886 (2016).</li><li>Guillaume-Jugnot, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+adipose-derived+stromal+vascular+fraction+in+patients+with+systemic+sclerosis:+12-month+follow-up.">Autologous adipose-derived stromal vascular fraction in patients with systemic sclerosis: 12-month follow-up.</a> Rheumatology (Oxford). 55 (2), 301-306 (2016).</li><li>Tissiani, L. A., Alonso, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Prospective+and+Controlled+Clinical+Trial+on+Stromal+Vascular+Fraction+Enriched+Fat+Grafts+in+Secondary+Breast+Reconstruction.">A Prospective and Controlled Clinical Trial on Stromal Vascular Fraction Enriched Fat Grafts in Secondary Breast Reconstruction.</a> Stem Cells Int. , 2636454 (2016).</li><li>Calcagni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+treatment+of+SVF-enriched+fat+grafting+for+painful+end-neuromas+of+superficial+radial+nerve.">The novel treatment of SVF-enriched fat grafting for painful end-neuromas of superficial radial nerve.</a> Microsurgery. , (2016).</li><li>Hoying, J. B., Boswell, C. A., Williams, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+potential+of+microvessel+fragments+established+in+three-dimensional+collagen+gels.">Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.</a> In Vitro Cell Dev Biol Anim. 32 (7), 409-419 (1996).</li><li>Kirkpatrick, N. D., Andreou, S., Hoying, J. B., Utzinger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+collagen+remodeling+during+angiogenesis.">Live imaging of collagen remodeling during angiogenesis.</a> Am J Physiol Heart Circ Physiol. 292 (6), H3198-H3206 (2007).</li></ol>

Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Bu çalışmada reklam mvf izolasyonu için köklü bir protokol mevcut. fare adipoz dokudan reklam MVF edinme birkaç kritik adımlarla basit bir işlemdir. Fareler farklı subkutanöz ve karın içi yağ birikintilerini sergiler. Daha önce fareler için tarif edildiği gibi, reklam MVF izolasyonu için en uygun yağ kaynağı, daha büyük kan damarlarının 11 ile boyut, homojen bir yapı ve en az kirlenme, 12 epididimal yağ pedleri. Bunun aksine, farelerde...

Doku mühendisliği, in vivo meslektaşları 1, 2 çalışamaz işlevini geri ya da takviye iddia ettikleri doku ve organ ikamelerinin üretim odaklanmaktadır. Mühendislik doku yapıları kaderi önemlisi yeterli vaskülarizasyon 3 bağlıdır. Bu yapılara içinde Mikrovasküler ağlar hiyerarşik alıcının damarsal 4&#39;e kaynaştırma sonra verimli kan perfüzyonunu sağlamak için Arterioller, kapiller ve venüllerden ile düzenlenmelidir. Bu tür ağların nesil doku mühendisliğinde temel zorluklardan biridir. Bu amaçla, deneysel vaskülarizasyon stratejileri geniş spektrumlu son yirmi yılda 5, 6 üzerinde tanıtıldı. Anjiojenik yaklaşımlar mühendislik Tiss içine alıcı mikrodamarlar içeri büyümesini teşvikBu tür büyüme dahil 7 faktörleri olarak yapısal ya da fiziko-kimyasal iskele modifikasyonu vasıtasıyla evet. Bununla birlikte, büyük bir üç boyutlu yapıları vaskülarizasyonu için, anjiyogenez-bağımlı stratejiler belirgin mikrodamarlar gelişmekte olan 8 yavaş büyüme oranları ile sınırlıdır. Doku yerlefltirilifl 9 öncesinde inşa dahilinde aksine, prevascularization kavramı fonksiyonel mikrovasküler ağların nesil için hedefliyor. Geleneksel prevascularization bu iskeleler olan endotel hücreleri, duvar hücreleri veya kök hücreler 10 gibi kap oluşturan hücre, ko-kültür içerir. mikrovasküler ağ oluşturulduktan sonra prevascularized yapıları daha sonra doku defektlerinin implante edilebilir. Karmaşık ve zaman alıcı in vitro temel aldığı için dikkate değer, bu prevascularization yaklaşım, klinik ortamda uygulamak zordur &lt;/ em&gt; önemli düzenleyici engeller 9 kısıtlandığı prosedürleri,. Bu duruma göre, geniş bir klinik uygulama için daha müsait olan yeni prevascularization stratejilerinin geliştirilmesi için hala bir ihtiyaç vardır. Bu tür bir prevascularization stratejisi adipoz doku türevli mikrovasküler parçalarının (reklam MVF) uygulanması olabilir. Reklam-MVF sıçanlarda 11, 12 ve fareler 13 yağ dokusundan büyük miktarlarda hasat edilebilir kuvvetli vaskülarizasyon birimini temsil eder. Bunlar arteriyoler, kapiler ve bir lümen içeren bir fizyolojik mikrodamar morfolojisi sergileyen venüler damar bölümü ve stabilize edici perivasküler hücreleri 14, 15 oluşmaktadır. Bu benzersiz özellik Bakterinin yetişmesinden önceki süreye olmadan doku defektleri içine reklam MVF seribaşı iskelelerinin hemen implantasyonu verir. Orada, ad-MVF hızla içinde yeniden birleştirmekFonksiyonel mikrovasküler ağlara. Ayrıca, reklam MVF ilave olarak kendi çarpıcı rejeneratif kapasitesine katkıda mezenkimal kök hücrelerin 16, zengin bir kaynak oluşturmaktadır. Buna uygun olarak, reklam MVF artan doku mühendisliği 14, 15, 17, 18, 19, 20, 21 farklı alanlarında kullanılmaktadır. Ad-mvf izolasyonu aslen 12 sıçanlarda 11 kurulmuştur. Bu yazıda, epididimal yağ pedleri gelen fare reklam mvf standardize izolasyonunu sağlayan bir protokol, açıklar. Bu transgenik fare modelleri kullanılarak reklam MVF fonksiyonu altında yatan moleküler mekanizmaların daha başka bilgi sağlayabilir.

<table><tbody><tr><td>1.5-mL conical microcentrifuge tube</td><td>VWR, Kelsterbach, Germany</td><td>700-5239</td><td></td></tr><tr><td>100-&amp;micro;L precision pipette</td><td>Eppendorf, Hamburg, Germany</td><td>4920000059</td><td></td></tr><tr><td>10-mL measuring pipette</td><td>Costar, Corning Inc., New York, USA</td><td>4488</td><td></td></tr><tr><td>14-mL PP tubes</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>187261</td><td></td></tr><tr><td>1-mL precision pipette</td><td>Eppendorf, Hamburg, Germany</td><td>4920000083</td><td></td></tr><tr><td>500-&amp;micro;m filter (pluriStrainer 500 &amp;micro;m)</td><td>HISS Diagnostics, Freiburg, Germany</td><td>43-50500-03</td><td></td></tr><tr><td>50-mL conical centrifuge tube</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>227261</td><td></td></tr><tr><td>50-mL Erlenmeyer flask</td><td>VWR, Kelsterbach, Germany</td><td>214-0211</td><td></td></tr><tr><td>96-well plate</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>65518</td><td></td></tr><tr><td>cell detachment solution (Accutase)</td><td>eBioscience, San Diego, CA USA</td><td>00-4555-56</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Charles River, Cologne, Germany</td><td>027</td><td></td></tr><tr><td>C57BL/6-Tg(CAG-EGFP)1Osb/J mice</td><td>The Jackson Laboratory, Bar Harbor, USA</td><td>003291</td><td></td></tr><tr><td>CD117-FITC</td><td>BD Biosciences, Heidelberg, Germany</td><td>553373</td><td></td></tr><tr><td>CD31-PE</td><td>BD Biosciences, Heidelberg, Germany</td><td>553354</td><td></td></tr><tr><td>Collagenase NB4G&amp;nbsp;</td><td>Serva Electrophoresis GmbH, Heidelberg, Germany</td><td>17465.02</td><td>Lot tested by manufacturer for enzymatic activity</td></tr><tr><td>Dissection scissors</td><td>Braun Aesculap AG &amp;amp;CoKG, Melsungen, Germany</td><td>BC 601</td><td></td></tr><tr><td>DNA-binding dye (Bisbenzimide H33342)</td><td>Sigma-Aldrich, Taufkirchen, Germany</td><td>B2261</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle medium (DMEM)&amp;nbsp;</td><td>PAN Biotech, Rickenbach, Germany</td><td>P04-03600</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Biochrom GmbH, Berlin, Germany</td><td>S0615</td><td></td></tr><tr><td>Fine forceps</td><td>S&amp;amp;T AG, Neuhausen, Switzerland</td><td>FRS-15 RM-8</td><td></td></tr><tr><td>Fine scissors</td><td>World Precision Instrumets, Sarasota, FL, USA</td><td>503261</td><td></td></tr><tr><td>Dermal skin substitute (Integra)</td><td>Integra Life Sciences, Sain Priest, France</td><td>62021</td><td></td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Serumwerk Bernburg AG, Bernburg, Germany</td><td>7005294</td><td></td></tr><tr><td>M-IgG2akAL488&amp;nbsp;&amp;nbsp;</td><td>eBioscience, San Diego, CA USA</td><td>53-4724-80</td><td></td></tr><tr><td>Octeniderm (disinfecting solution)</td><td>Sch&amp;uuml;lke &amp;amp; Mayer, Norderstedt, Germany</td><td>118211</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Biochrom, Berlin, Germany</td><td>A2213</td><td></td></tr><tr><td>Petri dish</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>664160</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Lonza Group, Basel, Switzerland</td><td>17-516F</td><td></td></tr><tr><td>pluriStrainer 20-&amp;micro;m (20 &amp;micro;m filter)</td><td>HISS Diagnostics, Freiburg, Germany</td><td>43-50020-03</td><td></td></tr><tr><td>Rat-IgG2akFITC</td><td>BD Biosciences, Heidelberg, Germany</td><td>553988</td><td></td></tr><tr><td>Rat-IgG2akPE</td><td>BD Biosciences, Heidelberg, Germany</td><td>553930</td><td></td></tr><tr><td>Small preparation scissors</td><td>S&amp;amp;T AG, Neuhausen, Switzerland</td><td>SDC-15 R-8S</td><td></td></tr><tr><td>Surgical forceps</td><td>Braun Aesculap AG &amp;amp;CoKG, Melsungen, Germany</td><td>BD510R</td><td></td></tr><tr><td>Tape (Heftpflaster Seide) 1.25 cm</td><td>Fink &amp;amp; Walter GmbH, Mechweiler, Germany</td><td>1671801</td><td></td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Bayer Vital GmbH, Leverkusen, Germany</td><td>1320422</td><td></td></tr><tr><td>&amp;alpha;-SMA-AL488</td><td>eBioscience, San Diego, CA USA</td><td>53-9760-82</td><td>Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722)</td></tr></tbody></table>

isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering

Fonksiyonel bir mikrovasküler ağ hayatta kalma ve mühendislik doku yapıları entegrasyonu için önemli bir önem taşımaktadır. Bu amaçla, çeşitli anjiyogenik ve prevascularization stratejileri kurulmuştur. Bununla birlikte, bir çok hücre-bazlı yaklaşımlar mikrovasküler ağı oluşumu için zaman alıcı, in vitro aşamaları içermektedir. Bu nedenle, bu operasyon sırasında tek aşamalı yöntemler için uygun değildir. Adipoz doku türevli mikrovasküler fragmanları (reklam MVF) umut vadeden vaskülarizasyon birimini temsil eder. Kolayca yağ dokusundan izole edilmiş ve bir işlevsel mikrodamar morfoloji sergiler edilebilir. Dahası, hızla in vivo implantasyonu sonrası yeni mikrovasküler ağlara yeniden birleştirmek. Ayrıca, reklam-MVF lemfanjiojenezi neden olduğu gösterilmiştir. Son olarak, daha da yüksek vaskülarizasyon etkisine de katkıda mezenkimal kök hücrelerin zengin bir kaynaktır. Daha önceki çalışmalarda biz olağanüstü vascularizati göstermiştirmühendislik kemik ve cilt ikame reklam-mvf kapasitesine. Bu çalışmada, biz fare yağ dokusundan reklam mvf enzimatik izolasyonu için standart bir protokol rapor.

Bu çalışmada, 12 aylık erkek, vahşi tip C57BL / 6 farelerinin (: 35 ± 1 g vücut ağırlığı ortalaması) 7- yağ dokusu ile altı reklam-MVF izolasyon prosedürleri gerçekleştirildi. Şekil 1, daha sonra, mekanik ve enzimatik reklam-MVF yalıtımlı murin epididimal yağ pedleri hasat göstermektedir. yağ toplama için gereken süre 30 dakika olmuştur ve reklam MVF izolasyonu için 120 dakika idi. Toplam olarak, prosedür, 150 dakika sürdü. <p class="j...

Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Bu umut verici vaskülarizasyon birimlerini temsil adipoz doku türevli mikrovasküler fragmanları izole etmek için bir protokol mevcut. Bunlar hızlı bir şekilde izole edilebilir, bu nedenle, doku mühendisliği farklı alanlarında tek aşamalı prevascularization için kullanılabilmektedir, nitro işleme gerektirmez ve yoktur.

Doku Mühendisliği vaskülarizasyon Birimleri olarak Kemirgen Yağ Dokusu kaynaklı Mikrovasküler Parçalarının İzolasyonu

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

isolation-murine-adipose-tissue-derived-microvascular-fragments-as

Research

JoVE Journal

Bioengineering

12.4K Görüntüleme.  Saarland University.  Bu umut verici vaskülarizasyon birimlerini temsil adipoz doku türevli mikrovasküler fragmanları izole etmek için bir protokol mevcut. Bunlar hızlı bir şekilde izole edilebilir, bu nedenle, doku mühendisliği farklı alanlarında tek aşamalı prevascularization için kullanılabilmektedir, nitro işleme gerektirmez ve yoktur. 

Video: Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Elastomerik PGS Arter Doku Mühendisliği Yapı iskeleleri

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Biyomühendislik

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Doku Mühendisliği için elektrospinning Liflerin Postprodüksiyon İşleme

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Elektrik Stimülasyonu murin myoblast Progenitör Hücreler ve Uygulama Mühendislik İskelet Kası Dokular

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Mikrovasküler Fragmanlardan Vaskülarize Termojenik Yağ Dokusunun Üç Boyutlu Kültürü

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

Hücre Fenotimi ve Sekretom Karakterizasyonu için İnsan Yağ Dokusu Mikro-parçalanma

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Biyoloji

Murin CD34+ Kök Hücrelerinin Damar Duvarından İzolasyonu ve Manyetik Olarak Ayrılması

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Yazar Spot Işığı: Manyetik Boncuk Tarama-Birleştirilmiş Akış Sitometrisi Kullanılarak Vasküler Duvarda Yerleşik Murin CD34+ Kök Hücrelerinin Gelişmiş İzolasyonu ve Karakterizasyonu

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Farelerden Vasküler Duvarda Yerleşik CD34+ Kök Hücrelerin İmmünomanyetik İzolasyonu

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Tıp

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vasküler Endotel Hücrelerinin Aşağı Akış Uygulamaları için Farklı Yağ Depolarından İzolasyonu ve Tanımlanması

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Murin Beyaz Yağ Dokusunun Yarı Otomatik İzolasyonu

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Yazar Spotlight: Murin Beyaz Yağ Dokusundan Stromal Vasküler Fraksiyonun Bir Doku Ayrıştırıcı Kullanılarak Yarı Otomatik İzolasyonu  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

Bir Doku Dissosiyatör Kullanarak Stromal Vasküler Fraksiyonun Murin Beyaz Yağ Dokusundan Yarı Otomatik İzolasyonu

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Doku Mühendisliği vaskülarizasyon Birimleri olarak Kemirgen Yağ Dokusu kaynaklı Mikrovasküler Parçalarının İzolasyonu

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Bir Doku Dissosiyatör Kullanarak Stromal Vasküler Fraksiyonun Murin Beyaz Yağ Dokusundan Yarı Otomatik İzolasyonu