Este trabalho foi financiado pelo Fundo Thomas Richard leucemia, a Senhora Tata Memorial Trust, do Northwick Park Hospital Fundo Fiduciário Leucemia Research e do Instituto Nacional de Pesquisa em Saúde (NIHR), Reino Unido.

1. Andaime Fabricação e Bio-funcionalização de Andaimes <ol><li> Para fabricar PU andaimes (tamanho de poro 100-250 mm, porosidade de 90-95%) sob a forma de discos de Petri, utilizar a separação de fases induzida termicamente 13 processo através da preparação de uma solução de polímero (5wt% em dioxano), seguido de congelamento e subsequente sublimação do solvente (Figura 1A). </li><li> Cortar o disco de andaime em cubos de 0,5 x 0,5 x 0,5 mm antes do revestimento com ECM proteínas (Figura 1B). </li><li> Pré-molhadas os suportes por imersão em etanol (70%) durante 1 min e depois transferir em salina tamponada com fosfato (PBS) durante 20 min. Em seguida, centrifugar durante 10 min a 3630 xg e adicionar a solução de proteína. </li><li> Centrifugar os suportes na solução de proteína a 1420 xg durante 20 min. </li><li> A fim de desbloquear os poros da superfície, e permitir que as células semeadas a penetrar mais profundamente na andaime, centrifugar o scaffolds mais uma vez em 910 xg durante 10 minem PBS. </li><li> Esterilizar os andaimes usando uma combinação de 8 min de exposição a 230 V, 50 Hz, 0,14 Uma lâmpada de UV, com a imersão durante 2 h em etanol (70%). Depois disso, lavar os suportes duas vezes em PBS antes da adição Modificado por Dulbecco Médio de Iscove (IMDM), suplementado com 30% de soro fetal bovino (FBS) e 1% de penicilina e estreptomicina (P / S). Colocar os suportes em um incubador humidificado durante 3 dias a 37 ° C e 5% de CO 2 antes de usar. Andaimes estéreis são colocados num placas de 24 poços de tecido-cultura (um andaime por poço). </li></ol> 2. Isolamento de células mononucleares e Plantio de andaime <ol><li> Extrair humano MNC do sangue do cordão ou amostras de medula óssea leucémicas, usando Ficoll-Paque centrifugação em gradiente de densidade (35 min a 1500 rpm) (Figura 1C). </li><li> Ressuspender o MNC em IMDM contendo 30% de FBS e 1% P / S. </li><li> 100 ul de semente de suspensão MNC (2 × 10 6 células / andaime) para a andaimes usando uma pipeta de micro-. </li><li> Incubar a andaimes semeadas a 37 ° C sob 5% de CO 2 durante 10 minutos para as células para se adaptar à andaimes. </li><li> Encher cada poço com 1,5 ml de IMDM contendo 30% de FBS e 1% P / S e as placas assim lugar na incubadora a 37 ° C e 5% de CO2 durante a duração da experiência. </li><li> Os arcabouços semeados sofrem diariamente mudam de todos os meios de comunicação. </li></ol> 3 na proliferação celular in situ e Morfologia:. MTS, SEM e cytospins <ol><li> Ensaio MTS: Retire uma cultura não-sementes e dois andaimes sem sementes e coloque em um 24 novo e limpo bem-chapa. Adicionar 1 ml de meio e 200 uL da solução de MTS e incubar durante 3 horas a 37 ° C e 5% de CO 2. <ol><li> Exame de 8 x 100 uL de amostras a partir do sobrenadante; lugar em uma placa de 96 poços e absorvância medida a 490 nm usando um leitor de microplacas. </li></ol></li><li> Microscopia eletrônica de varredura (MEV): em diferentes momentos do diacultura e, remover os andaimes semeadas com MNC a partir dos meios de comunicação, e fixar com 2,5% de PBS-tamponada solução de glutaraldeído por 40 min a 4 ° C, em seguida, lavar duas vezes com PBS. <ol><li> Desidratado os suportes em uma série graduada de etanol (25, 50, 70, 80, 90, 95 e 100%), cada um durante 10 min e seco num ambiente asséptico durante 4 horas. </li><li> Espécimes de secção e, em seguida, por pulverização catódica-revestimento com ouro numa atmosfera de árgon durante 2 min antes da avaliação SEM, utilizar uma voltagem de aceleração de 20 kV. </li></ol></li><li> Cytospins: Recolha 2,0 x 10 4 células / lâmina por aspiração das células do andaime em pontos de tempo diferentes na cultura. <ol><li> Centrifugar as células numa lâmina de vidro durante 3 min a 1500 rpm. </li><li> Coloração das lâminas com Wright-Giemsa modificado e observar por meio de um microscópio óptico. F-visualizar Imaging System Soft pode ser usado para tirar fotos. </li><li> Lave as lâminas antes da observação ao microscópio. </li></ol></li><li> M<html> análise ultiphoton: andaimes fixos em pontos de tempo diferentes, com vapor de etanol (70%) durante a noite e, em seguida, seco e armazenar a -20 ° C até análise posterior. Antes de visualização com a seção de microscópio multifotônica o andaime em 30 mm de espessura, slides e molhado com PBS. <ol><li> Bloquear a andaimes seeded em PBS com 10% de soro fetal de bovino durante 30 min à temperatura ambiente. </li><li> Incubar as andaimes durante a noite a 4 ° C na escuridão com uma diluição de 1:50 de anticorpo monoclonal primário: ratinho anti CD71 humano. </li><li> Lavar as amostras três vezes em PBS e coradas com o anticorpo secundário: anti-ratinho Alexa Fluor 488 durante 1 h à temperatura ambiente na escuridão. </li><li> Lavar as amostras três vezes com PBS e observar com um microscópio confocal usando uma água de emissão lente objectiva x60. </li><li> Fluoróforo Alexa Fluor 488 é excitado a 488 nm pelos lasers pulsáteis. O software Volocity 5.3.2 pode ser utilizado para análise de imagem subsequente. </li></ol></li></ol>jove_title &quot;&gt; 4. citometria de fluxo e da população celular <ol><li> Antes da análise celular no citómetro de fluxo (FC), rótulo as células de interesse com os anticorpos correspondentes de imunofluorescência para a detecção. Um certo número de amostras são preparadas de acordo com os anticorpos seleccionados, para detecção. Para a detecção MNC a seguinte combinação de anticorpos são utilizados: CD45-FITC/CD71-PE/CD235a-PE-Cy5. </li><li> Aspirar as células do andaime em pontos de tempo diferentes e centrifugar. Dissolve-se uma pelete de células de cerca de 1x10 6 células em 100 uL de tampão FC (PBS azida de sódio + 0,1%) e adicionar 10 uL de cada corante de fluorescência de anticorpos. Incubar as células durante 30 min a 4 ° C, lavar duas vezes com PBS e finalmente re-suspender em tampão de FC. </li><li> Carregar a amostra para o manchado citómetro de fluxo com o controlo de isotipo para definir o &quot;negatividade&quot; da expressão do anticorpo e calibrar os canais de detecção com o controle citómetro de fluxo. </li><li> Leia a amostra manchado w<html> om os três anticorpos diferentes e, finalmente, representam os dados no uso do software WinList. </li></ol> 5. Os resultados representativos Um exemplo de hematopoiéticas cinética de crescimento celular, sem a adição de factores de crescimento exógenos é mostrado na Figura 2. Devido à natureza heterogénea de hematopoiese, duas células diferentes: normais e anormais células hematopoiéticas são ilustrados. Na Figura 2A, a proliferação celular de CBMNC humana é evidente após 28 dias em cultura. Figura 2B mostra a cinética de crescimento no mimetismo usando células humanas leucémicas primárias. Proliferação celular é avaliada utilizando o ensaio de MTS que mede a actividade metabólica celular em relação à absorvância. Em ambos os casos, as células proliferaram e estabeleceu no modelo. As diferenças na cinética de crescimento são observados; células hematopoiéticas normais estabelecer a cultura mais rápido do que as células leucémicas. _content &quot;&gt; A morfologia das células colhidas, mesmo após 28 dias de cultura na ausência de factores de crescimento exógenos era típica de células hematopoiéticas normais (Figura 3A) e células leucémicas (Figura 3B). secções centrais dos suportes foram analisados ​​por SEM após os andaimes foram retirados da cultura e mostrou a propagação das células semeadas em todo o andaime, estabelecendo-se em agrupamentos e em &quot;nicho-estruturas semelhantes (Figura 3A &#39;&amp; B&#39;). Figura C mostra o tamanho dos poros e distribuição em um unseeded andaime usado como um controlo. microscopia multifotônica após 28 dias foi utilizado para destacar a distribuição das células no interior da andaime 3D in situ e que mostrou a presença de eritróide ilhas nas secções centrais do andaime (Figura 4) pela expressão do marcador CD71, que é positiva em eritroblastos. Isso prova a importância da du madura e células vencimentoeritropoiese anel. Finalmente, os gráficos de citometria de fluxo da semeadura células antes mostra a diferença no fenótipo de células hematopoiéticas, em que A Figura 5A representa células hematopoiéticas normais: CBMNCs humanos e Figura 5B ilustra anormais células hematopoiéticas: células leucémicas primárias. Níveis de CD235a + e CD45 + correspondente a eritrócitos e leucócitos, respectivamente são superiores na amostra normal do que no leucémicas destacando a natureza hemoblastic das leucemias. <img alt="A Figura 1" src="/files/ftp_upload/3654/3654fig1.jpg" /> Figura 1. Ilustração dos processos envolvidos na fabricação e PU andaime bio-funcionalização. A) PU é dissolvido em dioxano (5wt%) e pelo processo termicamente induzida fase de separação e sublimação solvente subsequente do andaime é produzido, como descrito por Safinia et al 13. B) O disco de andaime é então cortada intO cubos de 0,5 x 0,5 x 0,5 mm e, então, revestido por centrifugação com matriz extra-celular (ECM) proteínas. C) MNC são extraídos umbilical humano CB ou a partir de aspiração BM utilizando centrifugação em gradiente de densidade e semeado (2x10 6 células / andaime) na andaimes PU com uma micropipeta. <img alt="A Figura 2" src="/files/ftp_upload/3654/3654fig2.jpg" /> . Figura 2 proliferação celular medido utilizando o ensaio de MTS: A) usando sangue do cordão umbilical humano EMNs; B), utilizando células humanas leucémicas primárias. As colunas mostram o crescimento de células ao longo do tempo, quando semeadas no andaimes PU, sem a adição de citocinas exógenas. <img alt="A Figura 3" src="/files/ftp_upload/3654/3654fig3.jpg" /> Figura 3: A morfologia e distribuição em torno dos andaimes utilizando cytospins, scannimicrografias ng elétrons. (AB) Representante Wright-Giemsa cytospins manchadas de A) células mononucleares do sangue do cordão coletadas em PU andaimes após 28 dias de cultura, e B) osso aspirados células leucêmicas coletadas de PU andaimes após 14 dias de cultura. Ambos os experimentos foram realizados em condições de citocinas livre. (A&#39;-B &#39;) Representante seções centrais das micrografias de MEV de PU andaime A&#39;) cabo semeado sangue multinacionais e B &#39;) células leucêmicas após serem cultivadas durante 28 dias, e C) controle andaime sem células. <img alt="A Figura 4" src="/files/ftp_upload/3654/3654fig4.jpg" /> Figura 4. Multifotônica micrografia de um andaime PU semeado com sangue do cordão umbilical multinacionais após 28 d na cultura e corados com o marcador CD71 eritróide. <img alt="A Figura 5" src="/files/ftp_upload/3654/3654fig5.jpg" /> Figura 5. Citometria de Fluxo 3D Dot-plots coradas para CD45, CD71 e marcadores de expressão CD235a superfície. O controlo de isotipo obtido é também apresentados para comparação da intensidade relativa da fluorescência. O painel A mostra células do cordão humanos de sangue mononucleares positivos para os marcadores acima; B Painel mostra células leucémicas humanas primárias.

<ol>
	<li>Orkin, S., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis:+an+evolving+paradigm+for+stem+cell+biology.">Hematopoiesis: an evolving paradigm for stem cell biology.</a> Cell. 132, 631-644 (2008).</li><li>Spradling, A., Drummond-Barbosa, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Panoskaltsis, N., Mantalaris, A., Wu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+mimicry+of+bone+marrow+tissue+ex+vivo.">Engineering a mimicry of bone marrow tissue ex vivo.</a> J Biosci. Bioeng. 100, 28-35 (2005).</li><li>Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Mantalaris, A., Bourne, P., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+osteoclasts+in+a+three-dimensional+bone+marrow+culture+system.">Production of human osteoclasts in a three-dimensional bone marrow culture system.</a> Biochem. Eng. J. 20, 189-196 (2004).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+bioprocessing:+fundamentals+and+principles.">Stem cell bioprocessing: fundamentals and principles.</a> J. R. Soc. Interface. 6, 209-232 (2009).</li><li>Dexter, T., Testa, N., Prescott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 14, 387-405 (1976).</li><li>Piacibello, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+growth+factor+requirement+of+primitive+cord+blood+hematopoietic+stem+cell+for+self-renewal+and+amplification+vs+proliferation+and+differentiation.">Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.</a> Leukemia. 12, 718-727 (1998).</li><li>Yoshida, T., Takagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+processing+engineering+for+ex+vivo+expansion+of+hematopoietic+cells:+a+review.">Cell processing engineering for ex vivo expansion of hematopoietic cells: a review.</a> Biochemical Engineering Journal. 20, 99-106 (2004).</li><li>Lim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intelligent+bioprocessing+for+haemotopoietic+cell+cultures+using+monitoring+and+design+of+experiments.">Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.</a> Biotechnol. Adv. 25, 353-368 (2007).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+three-dimensional+scaffold+for+ex+vivo+biomimicry+of+human+acute+myeloid+leukaemia.">The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.</a> Biomaterials. 31, 2243-2251 (2010).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Aqel, N., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cytokine-free+expansion+of+cord+blood+mononuclear+cells+in+three-dimensional+scaffolds.">Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds.</a> Biomaterials. 32, 9263-9270 (2011).</li><li>Safinia, L., Datan, N., Hohse, M., Mantalaris, A., Bismarck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+methodology+for+the+effective+surface+modification+of+porous+polymer+scaffolds.">Towards a methodology for the effective surface modification of porous polymer scaffolds.</a> Biomaterials. 26, 7537-7547 (2005).</li></ol>

Não temos nada a divulgar.

O ex vivo sistema de cultura 3D apresentado aqui permite-nos estabelecer uma biomimicry 3D da hematopoiese que recapitula a arquitetura original BM e fenótipo celular independente de citocinas exógenas. O modelo 3D fornece a estrutura e do microambiente que permite normais e anormais células hematopoiéticas para proliferar em condições similares àquelas encontradas in vivo. A seleção do material polimérico andaime representou um passo crítico no projeto biomimicr...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue No</td>
		</tr>
		<tr>
			<td>Dioxan</td>
			<td>Invitrogen</td>
			<td>D20,186-3</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>MTS</td>
			<td>Promega</td>
			<td>G3580</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka Biochemika</td>
			<td>49624</td>
		</tr>
		<tr>
			<td>Wright-Giemsa</td>
			<td>Sigma-Aldrich</td>
			<td>WG32</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10108-165</td>
		</tr>
		<tr>
			<td>CD71</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32272</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>BD Pharmigen</td>
			<td>74895</td>
		</tr>
		<tr>
			<td>CD71-PE</td>
			<td>BD Pharmigen</td>
			<td>555537</td>
		</tr>
		<tr>
			<td>CD235a-PE-Cy5</td>
			<td>BD Pharmigen</td>
			<td>555570</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
		</tr>
	</tbody>
</table>

ex vivo mimicry of normal and abnormal human hematopoiesis

Células-tronco hematopoéticas requer um microambiente único, a fim de sustentar a formação do sangue célula 1, a medula óssea (BM) é um complexo tridimensional (3D) tecido hematopoiese em que é regulada por espacialmente organizadas microambientes celulares denominados nichos 2-4. A organização dos nichos BM é crítico para a função ou disfunção de BM normal ou maligno 5. Por conseguinte, uma melhor compreensão do microambiente em in vivo utilizando um mimetismo ex vivo nos ajudaria elucidar os determinantes moleculares, celulares e microambiental de leucemogênese 6. Actualmente, as células hematopoiéticas são cultivadas in vitro em frascos de tecidos bidimensionais (2D) de cultura / poço de placas 7 que requerem ou de co-cultura com alogénico ou xenogénica células estromais ou adição de citocinas exógenas 8. Estas condições são artificiais e diferem do in vivo </em&gt; microambiente na medida em que faltam os nichos 3D celulares e expor as células a concentrações anormalmente elevados de citocinas que podem resultar em diferenciação e perda de 9,10 pluripotência. Aqui, apresentamos um romance 3D osso sistema de cultura de medula que simula o ambiente de crescimento in vivo em 3D e suporta hematopoiese multilinhagens na ausência de fatores de crescimento exógenos. O andaime altamente poroso utilizado neste sistema de poliuretano (PU), facilita a alta densidade crescimento celular através de uma maior área superficial específica do que a cultura em monocamada em 2D convencional 11. O nosso trabalho indicou que este modelo de suporte o crescimento de sangue do cordão umbilical humano (CB), as células mononucleares (MNC) 12 e primárias de células leucémicas, na ausência de citocinas exógenas. Este mimetismo 3D romance proporciona uma plataforma viável para o desenvolvimento de um modelo humano experimental para estudar a hematopoiese e para explorar novos tratamentos para a leucemia.

Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Um sistema de cultura de 3D para a hematopoiese é descrito usando sangue do cordão umbilical humano e células de medula óssea leucémicas. O método baseia-se na utilização de um poliuretano poroso sintético andaime revestido com proteínas da matriz extracelular. Este andaime é adaptável para acomodar uma grande variedade de células.

Ex vivo Mimetismo de normal e anormal Hematopoese Humanos

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

ex-vivo-mimicry-of-normal-and-abnormal-human-hematopoiesis

Research

JoVE Journal

Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

15.4K visualizações.  Imperial College London. Um sistema de cultura de 3D para a hematopoiese é descrito usando sangue do cordão umbilical humano e células de medula óssea leucémicas. O método baseia-se na utilização de um poliuretano poroso sintético andaime revestido com proteínas da matriz extracelular. Este andaime é adaptável para acomodar uma grande variedade de células.

Vídeo: Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

A Pele Tridimensional Humanos Reconstruir Modelo: uma Ferramenta para o Estudo da Pele Normal e Progressão Melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengenharia

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.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

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

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Teste de microtração ambientalmente controlado mecanicamente adaptativas nanocompósitos poliméricos para<em&gt; Ex vivo</em&gt; Caracterização

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinação da taxa de transporte de xenobióticos e nanomateriais através da placenta usando o<em&gt; Ex vivo</em&gt; Modelo de perfusão da placenta humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imagiologia desnaturadas colágeno Vertentes<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via foto desencadeada Hibridização do Caged Colágeno mimético Peptídeos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

Conectividade funcional do estado de repouso do cérebro humano por meio de espectroscopia de correlação difusa óptica

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

Brightfield simultâneo, Fluorescência e Imagem Tomográfica de Coerência Óptica de Contrair Trabeculae Cardíaca Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

Interrogatório de Mecanobiologia totalmente óptica de proteína associada a sim em câncer humano e células normais usando um sistema multifuncional

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...

Um novo biorreator impresso em 3D para cultura ex vivo de vasos sanguíneos em perfusão

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Autor em destaque: EasyFlow - Um biorreator de perfusão econômico e adaptável para cultura de grandes vasos sanguíneos 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo Cultura de Perfusão de Grandes Vasos Sanguíneos em Biorreator Impresso em 3D

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...