Bu çalışma Lawrence Livermore Ulusal Laboratuvarı LDRD 18-ERD-062 (C.R.'ye) tarafından finanse edildi. John Muschler'e DgKO ve DgKI hücrelerine verdiği nazik hediye için teşekkürler. Elektron mikroskobu numunesi hazırlama ve veri toplama konusunda tavsiye ve yardım için California Üniversitesi Berkeley Elektron Mikroskobu Laboratuvarı personeline teşekkür ederiz.

1. Çözülme ve alikot laminin-111
NOT: Saflaştırılmış laminin-111 ticari olarak temin edilebilir (tipik olarak Matrigel olarak satılan EHS matrisinden saflaştırılır), ancak tüm kaynaklar bozulmamış, bozulmamış protein sağlamaz.
<ol><li>Ln1 ve çalışan boyut dışlama kromatografisi26 veya poliakrilamid jel elektroforezi26 ile Ln1'in bozulmadığını doğrulayın. Denatüre Ln1, 337 kDa, 197 kDa ve 177 kDa'da bantlara sahip olmalı ve doğal Ln1, 711 kDa'da tek bir banda sahip olmalıdır.</li>
	<li>Ln1'i gece boyunca buzdolabında buz üzerinde çözdürün ve düşük protein bağlayıcı mikrosantrifüj tüplerine (tipik olarak kültür kaplamaları için 100 μg / tüpte veya kaplamalar için 10 μg'da alikot) düşük protein bağlama uçları ve steril teknik kullanarak27. Bitkileri dondurun ve -80 °C'de saklayın.</li>
</ol>2. Düşük pH tamponları kullanarak laminin polimerizasyonu

<ol><li>PolyLM polimerizasyon tamponu yapın: 1 mM CaCl2 ve 20 mM sodyum asetatı ultra saf suda tartın ve karıştırın. Ardından HCl veya asetik asit kullanarak pH'ı 4.0'a ayarlayın. 0,2 μm filtreden steril filtre ve 4 ° C'de saklayın.</li>
	<li>Kontrol polimerizasyon tamponu yapın: 1 mM CalCl2 ve 20 mM Tris'i karıştırın ve gerektiğinde 10 N HCl veya 12 N NaOH kullanarak pH'ı 7.0'a ayarlayın. 0,2 μm filtreden steril filtre ve 4 ° C'de saklayın. NOT: Protokol burada duraklatılabilir.</li>
	<li>Gerekli sayıda Ln-1 alikotunu bir gece buzdolabında çözdürün.</li>
	<li>Steril hücre kültürü tekniğini kullanarak, 100 μg/mL'lik bir nihai konsantrasyonda laminin alikotlara uygun tamponu (adım 2.1'den poliLM polimerizasyon tamponu veya adım 2.2'den kontrol polimerizasyon tamponu) ekleyin (yani 0.5 mL ila 50 μg alikot ekleyin) ve pipetleme ile hafifçe karıştırın.</li>
	<li>Görselleştirme için floresan etiketli laminin kullanıyorsanız, sulandırın ve ağırlıkça 1:50 oranında (örn. 2 μg/100 μg) ekleyin ve pipetleyerek hafifçe karıştırın. NOT: Hücre bağlantısı için substrat olarak lamininler kullanıyorsanız, 2.6-2.8 adımlarını izleyin:</li>
	<li>Uygun tamponda seyreltildikten hemen sonra, lamininleri kültür plastiğine veya cam eşyalara aktarın ve gece boyunca 37 °C'de bir hücre kültürü inkübatöründe inkübe edin. Lamelleri kaplarken, dengeli bir damla oluşturacak kadar büyük bir hacim ekleyin (tipik olarak, 13 mm'lik yuvarlak bir lamel için 50 μL kullanın) ve gece boyunca 37 °C'de nemlendirilmiş bir odada tutun.</li>
	<li>Steril teknik27 kullanarak, 1x fosfat tamponlu salin (PBS) ile yıkayın.</li>
	<li>Sıvıyı dikkatlice çıkarın. Yüzeyin tamamen kurumasına izin vermeyin. NOT: Süt proteini indüksiyonu için hücreleri kaplamak için lamininler kullanıyorsanız, 2.9-2.12 adımlarını izleyin:</li>
	<li>Kültür meme epitel hücreleri. % 2 FBS, 0.01 mg / mL insülin, 0.005 μg / mL EGF, 0.05 mg / mL gentamisin ve 0.05 g / mL normosin ile desteklenmiş DMEM-F12'deki MepG distroglikan knock in (DgKI) veya MepG distroglikan nakavt hücreleri (DgKO) hücreleri.</li>
	<li>Tohum DgKO ve DgKI hücreleri, 10.500 hücre/cm2'de laminin stimülasyonu için kuyucuk plakalarında veya lamel alt tabaklarında</li>
	<li>Seyreltilmiş lamininleri bir hücre kültürü inkübatöründe 37 °C'de 30 dakika inkübe edin.</li>
	<li>PolyLM alikotlarını 20 dakika boyunca 2.000 x g'da santrifüjleyin, ancak protein varlıklarının daha küçük boyutu ve etkili bir şekilde toplamak için daha yüksek hızlara ihtiyaç duyulması nedeniyle LM'yi 20 dakika boyunca 20.000 x g'da nötr pH'da santrifüjleyin.</li>
	<li>Steril teknik27 kullanarak, süpernatanı nazikçe çıkarın ve 3 μg/mL prolaktin, 2.8 μM hidrokortizon ve 5 μg/mL insülin ile desteklenmiş DMEM-F12'nin uygun bir hacimde indüksiyon ortamında yeniden süspanse edin</li>
	<li>Hücreleri uyarmak için laminini hemen hücre kültürü ortamına aktarın. 50-800 μg/mL Ln1 ile hücreleri başarıyla uyardık</li>
</ol>3. İzole edilmiş glikoproteinler kullanılarak laminin polimerizasyonu
<ol><li>Rekombinant nidogen-1 ve Ln1'i gece boyunca buzdolabında buz üzerinde hafifçe çözdürün.</li>
	<li>Düşük protein bağlayıcı uçlar ve tüpler kullanarak ve steril teknik kullanarak hücre kültürü ortamında Ln1'i ağırlıkça 1: 1 oranında nidogen-1 ile karıştırın. Kontrol olarak hücre kültürü ortamında saf Ln1 kullanın.</li>
	<li>Görselleştirme için floresan etiketli laminin kullanıyorsanız, sulandırın ve ağırlıkça 1:50 oranında ekleyin (örn., 2 μg/100 μg). Pipetleme ile yavaşça karıştırın.</li>
	<li>Ln1-nidogen inkübe edin veya Ln1'i bir hücre kültürü inkübatöründe 37 ° C'de 30 dakika inkübe edin.</li>
	<li>Ln1-nidogen kopolimerlerini 2.000 x g'da 30 dakika santrifüjleyin ve kontrol Ln1'i 20.000 x g'de 30 dakika santrifüjleyin.</li>
	<li>Süpernatanı nazikçe çıkarın ve hücre kültürü ortamında 100-800 μg toplam protein / mL'lik bir nihai konsantrasyona kadar yeniden süspanse edin.</li>
	<li>Hücreleri uyarmak için hemen kullanın veya lamellere aktarın ve nemlendirilmiş hücre kültürü inkübatörlerinde 37 ° C'de gece boyunca yapışmasına izin verin.</li>
	<li>Lamellerden kültür ortamını çıkarın, lamelleri 1x PBS ile yıkayın ve ardından oda sıcaklığında 15 dakika boyunca %10 formalin (yani PBS'de %4 formaldehit) ile sabitleyin.</li>
</ol>4. Distroglikan eksprese eden hücreler kullanılarak laminin polimerizasyonu
<ol><li>Kültür meme epitel hücreleri. % 2 FBS, 0.01 mg / mL insülin, 0.005 μg / mL EGF, 0.05 mg / mL gentamisin ve 0.05 g / mL normosin ile desteklenmiş DMEM-F12'deki MepG distroglikan knock in (DgKI) veya MepG distroglikan nakavt hücreleri (DgKO) hücreleri.</li>
	<li>Büyüme oranlarındaki farklılıklar nedeniyle 5.000 hücre/cm2'de DgKI hücrelerini ve 8.000 hücre/cm2'de DgKO hücrelerini tohumlayın. Nemlendirilmiş inkübatörlerde 37 °C'de kültür, her 2-3 günde bir değişir ve titiz bir steril teknik kullanılarak her 4-5 günde bir geçer. Uygun büyüme oranlarını sağlamak için hücreleri bir hemositometre veya otomatik hücre sayacı27 ile dikkatlice sayın.</li>
	<li>Negatif kontroller olarak fraktal Ln1 ve DgKO hücreleri oluşturmak için DgKI hücrelerini kullanarak, 2 gün boyunca kuyucuk plakalarında veya lamel alt tabaklarında ve kültürde 10.500 hücre/cm2'de laminin stimülasyonu için tohum hücreleri. Ln-1'i bir buzdolabında buz üzerinde gece boyunca hafifçe çözdürün ve ardından 3 μg / mL prolaktin, 2.8 μM hidrokortizon ve 5 μg / mL insülin ile desteklenmiş DMEM-F12'nin indüksiyon ortamı ile karıştırın. 35 mm'lik bir tabak için, 50-800 μg Ln1 içeren 1 mL indüksiyon ortamı kullanın.</li>
	<li>Floresan Ln1 kullanıyorsanız, ağırlıkça 50:1 oranında karıştırın.</li>
	<li>Ortamı hücrelerden çıkarın ve 3 μg/mL prolaktin, 2.8 μM hidrokortizon ve 5 μg/mL insülin ile desteklenmiş DMEM-F12'nin laminin ve indüksiyon ortamıyla kaplayın</li>
	<li>Hücreleri 2 gün boyunca kültürleyin ve daha sonra% 10 formalin ile sabitleyin.</li>
</ol>5. Laminin mikroyapısının optik mikroskopi ile karakterizasyonu
<ol><li>Sabit numuneleri toplayın ve oda sıcaklığında 5 dakika boyunca 1x PBS ile 3 kez yıkayın.</li>
	<li>Nemlendirilmiş bir odada oda sıcaklığında 1 saat boyunca %0.5 Triton X-100, %3 sığır serum albümini, %10 keçi serumu ve 40 μg/mL anti-fare bloke edici antikor fragmanı ile 1x PBS'de numuneleri bloke edin ve geçirgenleştirin. NOT: Triton X-100 tehlikelidir. Eldiven ve göz koruması ile tutun ve uygun şekilde atın.</li>
	<li>Anti-laminin antikorunu 1:100 oranında 1x PBS ile %0.5 Triton X-100 ve %3 BSA ile karıştırın ve numunelere oda sıcaklığında 2 saat veya gece boyunca nemlendirilmiş bir odada 4 °C'de ekleyin (tipik olarak altta ıslak bir laboratuvar mendili olan uç kutuları kullanın).</li>
	<li>Birincil antikoru çıkarın ve% 0.5 Triton X-100 ve% 3 BSA ile 1x PBS ile 3 kez yıkayın.</li>
	<li>% 0.5 Triton X-100 ve% 3 BSA ile 1x PBS'deki örneklere 1:500 seyreltmede uygun bir ikincil antikor ekleyin ve oda sıcaklığında karanlıkta nemlendirilmiş bir odada inkübe edin.</li>
	<li>İkincil antikoru çıkarın ve% 0.5 Triton X-100 ve% 3 BSA ile 1x PBS ile 3 kez yıkayın.</li>
	<li>Hücre içeren numuneler için, 45 dakika boyunca 6 μM phalloidin ekleyin, ardından% 0.5 Triton X-100 ve% 3 BSA ile PBS ile 3 yıkama ve ardından PBS ile 3 yıkama yapın. Daha sonra PBS'de 0.1 μg/mL DAPI ile 5 dakika boyayın.</li>
	<li>Uygunsa numuneleri monte edin.</li>
	<li>Yüksek çözünürlüklü konfokal mikroskopi kullanan görüntü ln-1 yapısı. Daldırma objektifleri ile donatılmış bir lazer tarama mikroskobu kullanın (suya daldırma Plan Apochromat 40X/1.1NA veya yağa daldırma Plan Apochromat 63X/1.4NA).</li>
</ol>6. Kutu sayma boyutu ile laminin yapılarının karakterizasyonu
<ol><li>Matlab araç kutularında veya ImageJ'de bulunan kutu sayma boyut algoritmalarını kullanarak fraktal özellikleri analiz edin. Bu yöntemler, laminin eşik görüntülerini ve bir log-log üzerine çizer, kutuların boyutuna kıyasla Ln1 içeren kutu sayısını çizer. Bu parametreler arasındaki ilişkinin eğimi kutu sayma boyutudur: fraktal olmayan geometrilerin boyutu 0, 1 veya 2 iken, fraktal geometrilerin tamsayı olmayan boyutu28 olacaktır. NOT: Asitle muamele edilmiş, glikoprotein ile muamele edilmiş ve DgKI hücresine bağlı Ln1'in tümü ~1.7'lik bir kutu sayma fraktal boyutuna sahipken, kontrol Ln1'in fraktal boyutu 2.0'dır.</li>
</ol>7. Elektron mikroskobu ile laminin yapılarının karakterizasyonu
<ol><li>Hücre kültürü ortamında lamininleri yeniden süspanse edin ve nemli 37 °C ortamda 90 dakika boyunca silika gofretlere adsorbe edilmesine izin verin.</li>
	<li>Matrisleri pH 7.2'de 0.1 M sodyum kakodilat tamponunda %2.5 glutaraldehit içinde sabitleyin. NOT: Gluteraldehit tehlikelidir ve çeker ocakta eldiven ve göz koruması ile kullanılmalı ve tehlikeli atık olarak atılmalıdır.</li>
	<li>pH 7.2'de 0.1 M sodyum kakodilat tamponunda %1 osmiyum tetroksit içinde sonek matrisleri. NOT: Osmiyum tetroksit tehlikelidir ve çeker ocakta eldiven ve göz koruması ile kullanılmalı ve uygun şekilde atılmalıdır. Kakodilat tehlikelidir ve çeker ocakta eldiven ve göz koruması ile kullanılmalı ve uygun şekilde atılmalıdır.</li>
	<li>pH 7.2'de sodyum kakodilat tamponunda 3 kez yıkayın.</li>
	<li>Artan etanol konsantrasyonları ile dehidrat.</li>
	<li>İnce bir altın tabakası ile matrisleri püskürtün.</li>
	<li>Taramalı elektron mikroskobu kullanılarak görüntü.</li>
</ol>

<ol>
	<li>Carey, S. P., Kraning-Rush, C. M., Williams, R. M., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+control+of+invasive+tumor+cell+behavior+by+extracellular+matrix+microarchitecture.">Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture.</a> Biomaterials. 33 (16), 4157-4165 (2012).</li><li>Wang, K., 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=Stiffening+and+unfolding+of+early+deposited-fibronectin+increase+proangiogenic+factor+secretion+by+breast+cancer-associated+stromal+cells.">Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells.</a> Biomaterials. 54, 63-71 (2015).</li><li>Bruekers, S. M. C., 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=Fibrin-fiber+architecture+influences+cell+spreading+and+differentiation.">Fibrin-fiber architecture influences cell spreading and differentiation.</a> Cell Adhesion &amp; Migration. 10 (5), 495-504 (2016).</li><li>Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+Activities+of+Laminins+that+Drive+Basement+Membrane+Assembly+and+Function.">Integrating Activities of Laminins that Drive Basement Membrane Assembly and Function.</a> Curr Top Membr. 76, 1-30 (2015).</li><li>Colognato, H., Winkelmann, D. A., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+Polymerization+Induces+a+Receptor-Cytoskeleton+Network.">Laminin Polymerization Induces a Receptor-Cytoskeleton Network.</a> The Journal of Cell Biology. 145 (3), 619-631 (1999).</li><li>Powell, S. K., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+laminins+and+their+cellular+receptors.">Neuronal laminins and their cellular receptors.</a> International Journal of Biochemistry &amp; Cell Biology. 29 (3), 401-414 (1997).</li><li>Li, M. L., 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=Influence+of+a+reconstituted+basement+membrane+and+its+components+on+casein+gene+expression+and+secretion+in+mouse+mammary+epithelial+cells.">Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (1), 136-140 (1987).</li><li>Fiore, A., 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=Laminin-111+and+the+Level+of+Nuclear+Actin+Regulate+Epithelial+Quiescence+via+Exportin-6.">Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6.</a> Cell Rep. 19 (10), 2102-2115 (2017).</li><li>Inman, J. L., Robertson, C., Mott, J. D., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development:+cell+fate+specification,+stem+cells+and+the+microenvironment.">Mammary gland development: cell fate specification, stem cells and the microenvironment.</a> Development. 142 (6), 1028-1042 (2015).</li><li>Weaver, V. 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=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> Journal of Cell Biology. 137 (1), 231-245 (1997).</li><li>Furuta, S., Ren, G., Mao, J. -. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+signals+initiate+the+reciprocal+loop+that+informs+breast-specific+gene+expression+and+homeostasis+by+activating+NO,+p53+and+microRNAs.">Laminin signals initiate the reciprocal loop that informs breast-specific gene expression and homeostasis by activating NO, p53 and microRNAs.</a> eLife. 7, 26148 (2018).</li><li>Brown, S. C., 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=Dystrophic+phenotype+induced+in+vitro+by+antibody+blockade+of+muscle+alpha-dystroglycan-laminin+interaction.">Dystrophic phenotype induced in vitro by antibody blockade of muscle alpha-dystroglycan-laminin interaction.</a> Journal of Cell Science. 112 (2), 209 (1999).</li><li>Calof, A. L., Campanero, M. R., O'Rear, J. J., Yurchenco, P. D., Landert, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain-specific+activation+of+neuronal+migration+and+neurite+outgrowth-promoting+activities+of+laminin.">Domain-specific activation of neuronal migration and neurite outgrowth-promoting activities of laminin.</a> Neuron. 13 (1), 117-130 (1994).</li><li>Li, 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=Laminin-sulfatide+binding+initiates+basement+membrane+assembly+and+enables+receptor+signaling+in+Schwann+cells+and+fibroblasts.">Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.</a> The Journal of Cell Biology. 169 (1), 179-189 (2005).</li><li>Sonnenberg, A., 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=Integrin+recognition+of+different+cell-binding+fragments+of+laminin+(P1,+E3,+E8)+and+evidence+that+alpha+6+beta+1+but+not+alpha+6+beta+4+functions+as+a+major+receptor+for+fragment+E8.">Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.</a> The Journal of Cell Biology. 110 (6), 2145-2155 (1990).</li><li>Sorokin, L., Sonnenberg, A., Aumailley, M., Timpl, R., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+the+laminin+E8+cell-binding+site+by+an+integrin+possessing+the+alpha+6+subunit+is+essential+for+epithelial+polarization+in+developing+kidney+tubules.">Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.</a> The Journal of Cell Biology. 111 (3), 1265-1273 (1990).</li><li>Horejs, C. -. 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=Biologically-active+laminin-111+fragment+that+modulates+the+epithelial-to-mesenchymal+transition+in+embryonic+stem+cells.">Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 111 (16), 5908-5913 (2014).</li><li>Hochman-Mendez, C., Cantini, M., Moratal, D., Salmeron-Sanchez, M., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fractal+Nature+for+Polymerized+Laminin.">A Fractal Nature for Polymerized Laminin.</a> PLOS ONE. 9 (10), 109388 (2014).</li><li>Kent, A. 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=The+microstructure+of+laminin-111+compensates+for+dystroglycan+loss+in+mammary+epithelial+cells+in+downstream+expression+of+milk+proteins.">The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins.</a> Biomaterials. 218, 119337 (2019).</li><li>Weir, M. L., 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=Dystroglycan+loss+disrupts+polarity+and+beta-casein+induction+in+mammary+epithelial+cells+by+perturbing+laminin+anchoring.">Dystroglycan loss disrupts polarity and beta-casein induction in mammary epithelial cells by perturbing laminin anchoring.</a> Journal of Cell Science. 119, 4047-4058 (2006).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Palmero, C. Y., 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+follicular+thyroid+cell+line+PCCL3+responds+differently+to+laminin+and+to+polylaminin,+a+polymer+of+laminin+assembled+in+acidic+pH.">The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.</a> Molecular and Cellular Endocrinology. 376 (1), 12-22 (2013).</li><li>Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylaminin+recognition+by+retinal+cells.">Polylaminin recognition by retinal cells.</a> Journal of Neuroscience Research. 92 (1), 24-34 (2014).</li><li>Leal-Lopes, C., Grazioli, G., Mares-Guia, T. R., Coelho-Sampaio, T., Sogayar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+laminin+incorporation+into+alginate-based+microcapsules+reduces+pericapsular+overgrowth+and+inflammation.">Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (10), 1912-1922 (2019).</li><li>Paccola Mesquita, F. C., Hochman-Mendez, C., Morrissey, J., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+as+a+Potent+Substrate+for+Large-Scale+Expansion+of+Human+Induced+Pluripotent+Stem+Cells+in+a+Closed+Cell+Expansion+System.">Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.</a> Stem Cells International. 2019, 9 (2019).</li><li>Walker, J. M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , 61-67 (2002).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

Bu çalışma, ABD Enerji Bakanlığı'nın himayesinde, Lawrence Livermore Ulusal Laboratuvarı tarafından DE-AC52-07NA27344 Sözleşmesi kapsamında gerçekleştirilmiştir. IM sürümü LLNL-JRNL-799443.

Lamininler, epitel, kas ve nöral organ sistemlerinde ECM'nin önemli bir unsurunu temsil eder ve in vitro hücrelerin fonksiyonel farklılaşmasını düzenlemede önemli roller oynadığı gösterilmiştir. Artan kanıtlar, hücrelere gösterilen laminin yapısının, sinyalizasyonunu ve sonuçta ortaya çıkan hücre fenotipini15,16 düzenlediğini göstermektedir, bu nedenle Ln1 mikroyapısını kontrol etme yöntemleri, bu alanlarda çalışan temel biyologl...

Büyüme faktörleri veya sitokinlerin aksine, hücre dışı matriks (ECM) proteinleri yapısal ağlarda birleşebilir. ECM disfonksiyonu birçok hastalık durumunun önemli bir unsuru olduğundan, hücrelerin ECM bileşiminden, mikro yapısından ve biyomekaniğinden gelen sinyalleri algıladığı ve ilettiği mekanizmalar terapötik gelişim için çekici hedeflerdir. Artan kanıtlar, bu ağların mikro yapısının, bu proteinlerin hücrelere nasıl sinyal verdiği konusunda önemli bir rol oynadığını göstermektedir. Bugüne kadar, ECM mikroyapısı ve hücre fonksiyonu arasındaki bu bağlantı, kollajen I1, fibronektin2 ve fibrin3 için gösterilmiştir.
Laminin-111 (Ln1), epitel hücreleri4, kas hücreleri5 ve sinir hücreleri6 ile temas eden hücre dışı matrisin önemli bir yapısal bileşeni olan trimerik, çapraz şekilli bir proteindir. Meme bezinde, Ln1, meme bezi epitel hücrelerinin süt üreten hücrelere 7,8,9 fonksiyonel farklılaşması için gereklidir ve Ln1, tümör geri dönüşünün indüksiyonu için çok önemlidir10,11. Ln1 ve diğer lamininler, kasta kortikal aktin ağlarının ve sarkolemma organizasyonunungelişimi için gereklidir 12,13 ve nöral hücre göçü ve nörit büyümesi13 için gereklidir. Bu nedenle, laminin sinyallerinin hücrelere aktarıldığı mekanizmaları anlamak aktif bir araştırma alanıdır.
Ln1, integrinler, syndekanlar, distroglikan, LBP-110 ve LamR dahil olmak üzere çok çeşitli reseptörler için çoklu yapışkan alanlar içerir (Şekil 1A)4,14. Laminin α1'in c-terminal küresel alanlarını içeren E8 fragmanı, distroglikan ve integrinler α6β1 ve α3β115'in bağlanması için gereklidir ve epitel hücrelerinin fonksiyonel farklılaşması için gereklidir15,16. Buna karşılık, kısa kollar farklı biyolojik aktivitegösterir 17, bu da ağ oluşumundan sonra bu farklı Ln1 alanının tanınmasının hücre davranışını değiştirebileceği anlamına gelir.
Yakın zamanda, polimerik bir ağ halinde düzenlenmiş Ln1'in fraktal özellikler (yani, çoklu uzunluk ölçeklerinde kendine benzer bir yapı) sergilediğini gösterdik.18. Fraktal ağlar, bu düzenlemeden yoksun olan Ln1'e kıyasla epitel hücrelerine farklı sinyal verir19. Bu fraktal ağ, uzun kolun tercihen18 hücrelerine maruz kaldığı belirli bir montaj yapısını gösterir. Bu farklı uzun kol görüntüsünün bir sonucu olarak, epitel hücreleri, iyi organize edilmiş sıkı bağlantılara sahip süt üreten hücrelere fonksiyonel farklılaşmaya ve aktin stres liflerinin baskılanmasına uğrar19.
Ln1 uzun kolunun yüksek bir görüntüsünü gösteren kısa kol-kısa kol bağlanma etkileşimlerinden Ln1 ağları oluşturmak için 3 yöntem açıklıyoruz: 1) asidik tamponlarla hücresiz montaj yoluyla, 2) glikoproteinlerle birlikte inkübasyon yoluyla veya 3) hücre yüzeyi distroglikanı ile etkileşim yoluyla. Bu üç yöntem, deneysel tasarımda esnekliğe izin veren üç farklı mekanizma aracılığıyla benzer laminin mikro yapıları üretir. Önceki çalışmalar, bu üç yöntemin biyolojik sağlamlığı temsil edebileceğini düşündürmektedir: meme bezi epitelinde, hücre yüzeyi distroglikan, glikoproteinler veya yapay olarak yapılandırılmış laminin, fonksiyonel farklılaşmayı indükleyebilir19. Bu nedenle, araştırmacılar çalışma konusuna göre bu yöntemler arasında seçim yapabilirler: distroglikan eksprese eden hücreler, hücre zarındaki distroglikan bir fraktal ağa doğru polimerizasyonu indüklediğinden, Ln-1 mikro yapısına duyarlılık göstermez19,20. Lamininler ve glikoproteinlerin(21) bir karışımı olan Matrigel, Ln-1'in en ekonomik kaynağını temsil eder ve20'yi ayırt etmek için distroglikan nakavt meme hücrelerini indüklemek için gerekli mikro yapıyı sunar, ancak partiden partiye değişkenliğe sahip karmaşık bir protein karışımı içerir. PolyLM, bu işlevi sağlayan en temiz, en indirgemeci sistemi temsil eder, ancak Matrigel19'a kıyasla meme hücresi farklılaşmasını indüklemek için daha yüksek maliyet ve daha düşük etkinlikle.
Bu üç yöntem, difüzyon sınırlı agregasyonun18,19 karakteristik fraktal ağ yapısına sahiptir ve çoklu deney sistemlerinde 22,23,24,25 agrega laminine kıyasla değişmiş biyolojik aktivite gösterir. Bu yöntemleri kullanan gelecekteki çalışmalar, epitelyal farklılaşma için gerekli reseptörleri ve sinyalleri tanımlayabilir ve tümörler gibi anormal mikro ortamlardaki20 hücrelerde normal hücre-matris etkileşimlerini geri yüklemeyi belirleyebilir.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
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		<col />
		<col />
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% formalin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">HT501128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin primary antibody</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Rabbit Alexafluor 488 secondary antibody</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A32731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C4901 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">Heracell 150 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge for microcentrifuge tubes</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:161px;">5418 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal Microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">LSM 710 or equivalent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">25CIR-1 or similar</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">11330107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF (Epidermal Growth Factor)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">11376454001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescently Labeled Laminin</td>
			<td style="width:121px;">Cytoskeleton Inc</td>
			<td style="width:161px;">LMN02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">VWRV0304-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">320331 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortisone</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">H0888-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">16634-50mg</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MepG Dystroglycan Knockout Cells</td>
			<td style="width:121px;">LBNL</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S8045 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normocin</td>
			<td style="width:121px;">Invivogen</td>
			<td style="width:161px;">ant-nr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium Tetroxide</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">75633</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovine Prolactin</td>
			<td style="width:121px;">Los Angeles Biomedical Research Institute</td>
			<td style="width:161px;">Ovine Pituitary Prolactin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phalloidin</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A22287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">10010023 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purified Laminin-111</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Recombinant human Nidogen-1, carrier free</td>
			<td style="width:121px;">R&#38;D systems</td>
			<td style="width:161px;">2570-ND-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Acetate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S2889 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cacodylate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile filters</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">SLGP033RS or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">252859 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-low protein binding tubes</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">76322-516, 76322-522 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure water</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">15230253 or similar</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a fractal microstructure of laminin-111 to signal to cells

Laminin-111 (Ln1) epitel, kas ve sinir sistemlerinde hücre dışı matriksin önemli bir parçasıdır. Daha önce, Ln1'in mikro yapısının, hücrelere sinyal verme şeklini değiştirdiğini, muhtemelen Ln1'in ağlara montajının farklı yapışkan alanları açığa çıkarması nedeniyle göstermiştik. Bu protokolde, polimerize Ln1 üretmek için üç yöntem açıklıyoruz.

Laminin-111, kısa kollarının ucunda kendi kendine birleşen üç alana sahip trimerik bir proteindir (Şekil 1A). Uygun şekilde işlendiğinde, kısa kollar, daha büyük uzamsal ölçeklerde difüzyonla sınırlı agrega fraktal özellikleri gösteren altıgen bir kafes (Şekil 1B,1C) halinde polimerize olabilir (Şekil 2). Kalsiyum içeren nötr-pH tamponlarında inkübe edilen laminin, bir anti-Ln1 antikoru i...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Polimerize edilmemiş Ln1'e kıyasla hücrelere farklı sinyal veren fraktal özelliklere sahip Ln1 polimerleri üretmek için üç yöntem açıklıyoruz.

Hücrelere sinyal vermek için laminin-111'in fraktal mikro yapısının oluşturulması

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

920 Görüntüleme.  Texas Heart Institute. Polimerize edilmemiş Ln1'e kıyasla hücrelere farklı sinyal veren fraktal özelliklere sahip Ln1 polimerleri üretmek için üç yöntem açıklıyoruz.

Video: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Biyomühendislik

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

Laminer Akış Kesme Stres altında endotel progenitör hücrelerin değerlendirin Paralel plakalı Akış Odası ve Sürekli Akış Devre

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A Tam Cilt Hata Modeli Biyomateryallerin vaskülarizasyonunu değerlendirin<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Hücre içi sinyal iletimi Upconversion nanopartikül aracılı kinaz Photoactivation tarafından uzaktan tetiklemek için entegre bir sistem

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: Hücresel enerji metabolizması araştırmak için bir güçlü, invaziv olmayan bir yaklaşım

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Stres birden çok paralel-plaka akışı Chambers kullanarak yamultmak için maruz endotel hücrelerinin gen ifade Analizi

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

Mikrobiyal Davranışı İncelemek Için Kontrollü, Dinamik Kimyasal Manzaralar Üretmek

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Yumuşak Litografi Değişimi Ile Kök Yüzey Mikroyapısının Biyomimetik Replikasyonu

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Sessiz İnsan Hematopoetik Kök Hücrelerini Korumak İçin Bir Kültür Yöntemi

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Hücrelere sinyal vermek için laminin-111'in fraktal mikro yapısının oluşturulması

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Devirli Basınç Biyoreaktör Tasarım Ex vivo Eğitim