Os autores gostariam de agradecer biolitec Research GmbH (Jena, Alemanha) para gentilmente nos fornecer a formulação lipossomal mTHPC, com um agradecimento especial a Susanna Gräfe e Arno Wiehe pela sua ajuda e conhecimento técnico. Também gostaria de agradecer a Kerstin Reidy, que gerou uma grande parte dos resultados e foi co-responsável por sua publicação 11. Este trabalho foi financiado por doações do Schweizerischer Verein Balgrist, da Universidade de Zurique, o Krebsliga Zurique, bem como por uma concessão do Walter L. e Johanna Lobo Foundation, Zurich, na Suíça e uma bolsa da EuroNanoMed ERA-NET / SNF suíço National Science Foundation 31NM30-131004/1. Este trabalho também foi apoiado pelo programa (altamente especializada Medicine) para Musculoskeletal Oncology da Kanton Zurich HSM.

1. Comparação da absorção de mTHPC no Baixo respectiva e altamente metastático HOS e 143B OS linhas celulares <ol><li> Prepare a meio de cultura celular contendo DMEM, F12 de Ham, e inactivado por calor soro fetal de vitelo numa 4.5:4.5:1 proporção. Nota: A formulação lipossomal do PS mTHPC foi dissolvido em água a uma concentração final de 1,5 mg / ml mTHPC. </li><li> Placa de 0,2 x 10 6 e as células HOS 143B / poço em placas de 6 poços (triplica para cada condição) e deixar que as células aderir durante a noite. Nota: o número não tem de ser ajustada para as linhas de células individuais. A confluência de 80-90% é o mais adequado para o tempo da experiência. </li><li> No dia seguinte, incubar as células com uma concentração fixa de mTHPC para diferentes períodos de tempo (tempo de absorção dependente), ou com concentrações crescentes de mTHPC durante um período de tempo fixo (dose de absorção dependentes). </li><li> Incubar as células com mTHPC por tempo específico pontos ou concentrações de mTHPC. Nota: Neste estudo, ambas as linhas de células HOS e 143B foram incubadas com 0,6 ug / ml mTHPC para 0, 2,5, 5, ou 10 horas, ou com 0, 0,6, 2,5, e 10 ug / ml mTHPC durante 5 horas. Os períodos de tempo aplicados e as dosagens podem variar dependendo da linha de células que é utilizado. </li><li> Lembre-se de trabalhar sempre no escuro ou em condições de iluminação se apagaram para evitar a exposição à luz direta das células. </li><li> Após incubação das células com as condições indicadas, aspirar o meio e lava-se as células três vezes com PBS. Em seguida, retire as células com 0,5 ml de tripsina / EDTA e contar as células. </li><li> Depois de descolamento, centrifugar as células a 400 xg durante 5 min, aspirar o meio e ressuspender as células em PBS para uma densidade final de 200.000 células / ml. </li><li> Pipete 100 pi da suspensão de células obtida (ou seja, 20.000 células) em uma placa de 96 poços e medir a fluorescência destas células em um espectrofotómetro de fluorescência. A definiçãos para medições de fluorescência mTHPC são: 417 nm para a excitação e 652 nm para a emissão. </li><li> A fim de calcular a quantidade de mTHPC interiorizada por célula, a unidade de normalizar a fluorescência relativa (RFU) para o número de células e volume de células como se segue: </li><li> Prepara-se uma curva padrão, utilizando diferentes concentrações de mTHPC em PBS 0-4 ug / ml (medidas em triplicado) utilização. Pipetar 100 l de cada diluição numa placa de 96 poços e medir a fluorescência dos poços, utilizando as definições descritas no passo 1.6. </li><li> Dividir a RFU (obtido por 20000 células) pela inclinação da curva de calibração linear de mTHPC, que dá a concentração de mTHPC (em ug / ml) no poço. </li><li> Dividir esta concentração mTHPC por 10 para obter a quantidade total de mTHPC num poço (em ug, representando 100 ul de suspensão de células) e dividir esse valor, o volume de 20000 células. </li><li> Calcular o volume com a fórmula para uma esfera: (4/3 x π) xr 3. Obter o raio r medindo o diâmetro de 20 células tratadas com tripsina sob um microscópio, e dividindo esse valor por 2. </li><li> Finalmente, normalizar todos os valores para os obtidos para as células HOS após 10 horas de incubação (dose de absorção dependente) ou tratadas com 10 ug / ml mTHPC (tempo de absorção dependente), que foram definidos como 100%. </li></ol> 2. Medição da fototoxicidade do PS In Vitro <ol><li> Semente de 3.000 células / poço numa placa de 96 cavidades (triplicados para cada concentração de mTHPC utilizado) e deixá-los aderir durante a noite. Similarmente preparar uma placa de 96 poços adicional e tratar as células, tal como descrito abaixo, mas, em seguida, colocá-los de lado para a imagem de contraste de fase. Nota: o número tem que ser ajustada para cada linha celular individual. A confluência de 50-60% no tempo da experiência é ideal. <ol><li> Tenha em mente para adicionar o correspondente controlo de toxicidade no escuro de células para cada uma das concentrações mTHPC. Ose, são as células que irão ser incubadas na ausência ou na presença de concentrações seleccionadas de mTHPC, mas não irá ser iluminada. </li></ol></li><li> No dia seguinte, incubar as células com diferentes concentrações de mTHPC (0, 0,0001, 0,001, 0,01, 0,03, 0,075, 0,15, 0,3, 0,6, 1,25, 2,5, 5, e 10 ug / ml), dependendo das linhas de células e mantê-los no escuro por 5 horas. <ol><li> Consulte a Seção 1.3.2 Nota: O tempo de incubação antes da iluminação depende da linha celular e do PS usado, por conseguinte, é aconselhável para testar diferentes concentrações e em diferentes pontos de tempo em experiências separadas. </li></ol></li><li> Após a incubação para o período de tempo indicado, lavar as células duas vezes com PBS e adicionar novo meio fresco sem mTHPC. </li><li> Iluminar as células com um diodo laser de 652 nm, específico para o espectro de absorção mTHPC (ver Figura 1). <ol><li> Defina a laser para 21,88 mW / cm 2 poder a uma altura de 13,5 centímetros distance a partir das células, a fim de obter uma dose de energia de 5 J / cm 2. Iluminar as células para 230 seg. Não iluminar as células controle toxicidade escuras (com e sem mTHPC). Nota: Use sempre óculos para proteger os olhos da luz do laser usado para a excitação do PS. </li></ol></li><li> Depois de iluminação, manter as células no escuro a 37 º C por 24 horas. Posteriormente, adicione água de tetrazólio solúvel (WST-1) reagente (10 μl/100 mL de meio). </li><li> Três horas após a adição do reagente WST-1, mede-se a absorvância dos poços individuais da placa de 96 poços a 415 nm. </li><li> A fim de calcular a percentagem de células sobreviventes, definir a média dos valores obtidos para as células não tratadas mTHPC para cada condição de 100% (ou seja iluminado, sem mTHPC e nonilluminated, sem mTHPC). </li></ol> 3. Estimativa de número de celular por contagem celular <ol><li> Semente 20.000 células / nósll em uma placa de 24 poços e deixá-los a aderir durante a noite. </li><li> Incubar as células durante 5 horas com mTHPC (0,001, 0,003, 0,15, 0,6 e 1,25 ug / ml) e iluminá-los, tal como descrito acima. </li><li> Recolhe-se o meio, lavar as células uma vez com PBS, trypsinize los e recolhê-las por centrifugação a 400 xg durante 5 min. </li><li> Após a centrifugação, aspirar o meio e ressuspender as células em 200 ul de meio fresco. </li><li> Contar as células em uma câmara de Neubauer. </li></ol>

<ol>
	<li>Juarranz, A., Jaen, P., Sanz-Rodriguez, F., Cuevas, J., Gonzalez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+cancer.+Basic+principles+and+applications.">Photodynamic therapy of cancer. Basic principles and applications.</a> Clin. Transl. Oncol. 10, 148-154 (2008).</li><li>Agostinis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+cancer:+an+update.">Photodynamic therapy of cancer: an update.</a> CA Cancer. 61, 250-281 (2011).</li><li>D'Adamo, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appraising+the+current+role+of+chemotherapy+for+the+treatment+of+sarcoma.">Appraising the current role of chemotherapy for the treatment of sarcoma.</a> Semin. Oncol. 38 Suppl 3, 19-29 (2011).</li><li>Allison, D. 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=A+meta-analysis+of+osteosarcoma+outcomes+in+the+modern+medical+era.">A meta-analysis of osteosarcoma outcomes in the modern medical era.</a> Sarcoma. , (2012).</li><li>Anninga, J. 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=Chemotherapeutic+adjuvant+treatment+for+osteosarcoma:+where+do+we+stand.">Chemotherapeutic adjuvant treatment for osteosarcoma: where do we stand.</a> Eur. J. Cancer. 47, 2431-2445 (2011).</li><li>Bacci, G., 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=Pattern+of+relapse+in+patients+with+osteosarcoma+of+the+extremities+treated+with+neoadjuvant+chemotherapy.">Pattern of relapse in patients with osteosarcoma of the extremities treated with neoadjuvant chemotherapy.</a> Eur. J. Cancer. 37, 32-38 (2001).</li><li>Briccoli, 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=Resection+of+recurrent+pulmonary+metastases+in+patients+with+osteosarcoma.">Resection of recurrent pulmonary metastases in patients with osteosarcoma.</a> Cancer. 104, 1721-1725 (2005).</li><li>Milla Sanabria, 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=Direct+and+indirect+photodynamic+therapy+effects+on+the+cellular+and+molecular+components+of+the+tumor+microenvironment.">Direct and indirect photodynamic therapy effects on the cellular and molecular components of the tumor microenvironment.</a> Biochim. Biophys. Acta. 1835, 36-45 (2013).</li><li>Mitra, S., Foster, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photophysical+parameters,+photosensitizer+retention+and+tissue+optical+properties+completely+account+for+the+higher+photodynamic+efficacy+of+meso-tetra-hydroxyphenyl-chlorin+vs+Photofrin.">Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin.</a> Photochem. Photobiol. 81, 849-859 (2005).</li><li>Ma, L., Moan, J., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+new+photosensitizer,+meso-tetra-hydroxyphenyl-chlorin,+for+use+in+photodynamic+therapy:+a+comparison+of+its+photobiological+properties+with+those+of+two+other+photosensitizers.">Evaluation of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin, for use in photodynamic therapy: a comparison of its photobiological properties with those of two other photosensitizers.</a> Int. J. 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Os autores declaram não competindo interesses financeiros ou conflitos de interesses.

Para alcançar citotoxicidade ideal em resposta a PDT, é fundamental para escolher as configurações de luz de laser direita e tempos de incubação. Os procedimentos aqui descritos são consistentes e eficiente para determinar a absorção de PS e para quantificar a citotoxicidade induzida por PDT, in vitro. Usando os comprimentos de onda de absorção específicas do PS mTHPC, a absorção PS celular pode ser determinada de um modo directo, e o PS pode ser activada para gerar espécies reactivas de oxigéni...

Estado da arte do tratamento de osteossarcoma (OS) de hoje, um tumor ósseo primário, abrange uma combinação de quimioterapia adjuvante e neo cirurgia. Este regime de tratamento revelou um aumento na taxa de sobrevivência de pacientes com doença localizada a partir de cerca de 20% antes do uso de quimioterapia, a atualmente entre 60-70% 3, 4. No entanto, nas últimas duas décadas, a sobrevida global dos pacientes com doença do sistema operacional local tem plateaued 4, 5. Além disso, 30-40% dos pacientes com recaída no prazo de 3 anos após o diagnóstico e os pacientes com doença metastática continuam a ter uma fraca sobrevivência de 20-30% 4, 6, 7. Para melhorar o resultado de esses pacientes, novas estratégias terapêuticas têm de ser desenvolvidas. Terapia Fotodinâmica (PDT), uma terapia anticâncer em vez novela, usa a luz de um comprimento de onda específico para a excitação de um photosensiTizer (PS), que se acumula nas células tumorais após a sua injecção na corrente sanguínea. A luz do laser de excitação do PS gera radicais de oxigénio, na presença de oxigénio, os quais induzem a reacção citotóxica em células tumorais e a morte celular. Além deste mecanismo primário, dois PDT adicional evocado processos biológicos contribuir para o crescimento do tumor reduzido: TFD causa a vasoconstrição e a formação de trombos da microcirculação do tumor e, consequentemente, a hipóxia e anóxia locais no interior do tumor, levando a enfarte do tumor. Finalmente, PDT feridos e moribundos células tumorais desencadear uma resposta imune local, uma característica bastante singular do PDT. Isto envolve o sistema do complemento e a activação das células apresentadoras de antigénios dendríticas 8. Assim, são criadas condições para a apresentação de antígenos tumorais com subsequente ativação de células linfóides, levando a imunidade específica do tumor. Até agora, a terapia fotodinâmica tem sido utilizada para tratar diversos tipos de tumores de tecidos moles e hyperplásia de, como a ceratose actínica, esôfago de Barrett, tumores endobrônquica, câncer de bexiga, carcinomas basocelulares e tratamento paliativo de câncer de cabeça e pescoço 2. O tratamento é conhecido por induzir a necrose de grande escala local com apenas pequenos efeitos secundários, e, portanto, tem o potencial para eliminar selectivamente o tecido do tumor. Apesar destas vantagens, a aplicação de PDT permanece tecnicamente mais exigente do que a administração de fármacos quimioterapêuticos. A fim de atingir a eficácia máxima, a concentração de PS, o tempo de exposição à luz e de transferência total de energia de luz têm de ser optimizados. Isto pode ser feito em experiências in vivo, mas, por causa do grande número relativo de parâmetros que têm de ser optimizados, é mais eficiente para determinar inicialmente a condições óptimas in vitro. Nas experiências descritas em seguida, testou-se a eficácia in vitro de PDT utilizando o PS 5,10,15,20-tetraquis-(meta-hidroxifenil) chlorin, abreviado mTHPC (Figura 1A). mTHPC é a substância activa do medicamento Foscan, que é usado atualmente na clínica para o tratamento paliativo de câncer de cabeça e pescoço. É um dos PS mais potente, induzindo enormes danos celulares já em baixas concentrações, e demonstrou-se ser superior a outras PS em termos de penetração do tecido 9, 10. O seu espectro de absorção de luz (Figura 1B) mostra dois picos proeminentes, uma em 417 nm e um segundo de 652 nm, que são utilizados para a localização de tecido de acumulação de PS e para a indução de PDT, respectivamente. Atualmente, uma formulação lipossomal para mTHPC está em desenvolvimento. Aqui, descrevemos os procedimentos para quantificar a absorção desta formulação lipossomal, e para realizar PDT em duas linhas de células do sistema operacional humanos, o baixo HOS metastático e as células metastáticas 143B altos. Alguns dos dados aqui apresentados foram relatados anteriormente 11. The abordagem aqui descrita permite-nos estudar o efeito de um fenótipo metastático em PDT eficácia. Células 143B, ortotopicamente injetadas nos membros posteriores de ratos SCID imunodeficientes causar tumores primários metástase intratibial, um modelo que imita de perto a doença metástase humana. Assim, as experiências in vitro propostos são perfeitamente adequados para avaliar os ajustes de PDT óptimas para ser usado mais tarde em experiências in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:251px;">PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-883</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HAM F12</td>
			<td>PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">heat-inactivated fetal calf serum</td>
			<td>GIBCO, Basel, Switzerland</td>
			<td style="width:127px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:291px;">mTHPC</td>
			<td style="width:251px;">biolitec research GmbH, Jena, Germany</td>
			<td style="width:127px;"></td>
			<td style="width:287px;">As this liposomal formulation is originating from R &#38; D 
			no catalogue number is available at the moment. Stock: 1.5mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &#62;99% purity)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectramax Gemini XS plate reader</td>
			<td style="width:251px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:127px;">ID# 861</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Microscope Zeiss Observer.Z1</td>
			<td style="width:251px;">Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ceralas PDT 652 nm Laser</td>
			<td style="width:251px;">Biolitec AG, Jena, Germany</td>
			<td style="width:127px;">LD652nm2W400u</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Water-soluble tetrazolium (WST) reagent</td>
			<td style="width:251px;">Roche Diagnostics AG, Rotkreuz, Switzerland</td>
			<td style="width:127px;">1644807</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

Nos últimos anos, tem sido a dificuldade em encontrar terapias mais eficazes contra o câncer com menos efeitos colaterais sistêmicos. Portanto Terapia Fotodinâmica é uma nova abordagem para um tratamento selectivo mais tumor. Terapia Fotodinâmica (PDT) que faz uso de um fotossensibilizador não tóxico (PS), que, após a activação com luz de um comprimento de onda específico, na presença de oxigénio, gera radicais livres de oxigénio, que provocam uma resposta citotóxica 1. Apesar de sua aprovação há quase vinte anos pelo FDA, PDT hoje é apenas usado para tratar um número limitado de tipos de câncer (de pele, bexiga) e doenças nononcological (psoríase, queratose actínica) 2. A principal vantagem da utilização da TFD é a capacidade de executar um tratamento local, o que evita os efeitos secundários sistémicos. Além disso, permite o tratamento de tumores em locais delicados (por exemplo, em torno de nervos ou vasos sanguíneos). Aqui, um intraoperataplicação ive do PDT é considerada no osteossarcoma (OS), um tumor do osso, para atingir satélites tumor primário deixado para trás em torno do tecido tumoral após a ressecção cirúrgica do tumor. O tratamento tem como objetivo diminuir o número de recidivas e na redução do risco de (pós-operatório) metástase. No presente estudo, apresentamos in vitro procedimentos PDT para estabelecer as configurações PDT ideais para o tratamento eficaz de linhas de células do sistema operacional amplamente utilizados que são usados ​​para reproduzir a doença humana em modelos do rato OS intratibial bem estabelecidos. A captação do PS mTHPC foi examinada com um espectrofotómetro e fototoxicidade foi provocado com a luz laser de excitação de mTHPC em 652 nm para induzir a morte celular avaliada com um ensaio de WST-1 e pela contagem de células sobreviventes. As técnicas estabelecidas nos permitem definir as configurações PDT ideais para futuros estudos em modelos animais. Eles são uma ferramenta fácil e rápida para a avaliação da eficácia da TFD <em&gt; In vitro antes de uma aplicação in vivo.

Com as técnicas aqui relatadas, investigamos PDT-based mTHPC em células do sistema operacional humanos. Primeiro, o tempo e a dose de absorção dependente mTHPC foi investigada no baixo HOS metastático e nas linhas de células 143B OS altamente metastáticas. mTHPC absorção pode ser avaliada através da medição da fluorescência de mTHPC com um espectrofotómetro de fluorescência (Figura 2, reproduzida com a permissão do Reidy et al. 11). Figura 2A ilustra ...

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Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

O protocolo relatado aqui permite a avaliação da eficácia da terapia fotodinâmica (PDT) em linhas de células e a optimização das condições de PDT, antes de aplicar a terapia em modelos animais.

Citotóxicos Eficácia da Terapia Fotodinâmica no osteossarcoma Células<em&gt; In Vitro</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

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Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

12.7K visualizações.  Balgrist University Hospital, Zurich, Switzerland.  O protocolo relatado aqui permite a avaliação da eficácia da terapia fotodinâmica (PDT) em linhas de células e a optimização das condições de PDT, antes de aplicar a terapia em modelos animais. 

Vídeo: Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

Injeção de sub-retiniana de Vetores da terapia genética e células-tronco no olho do rato

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Medicina

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Em Enriquecimento vitro de câncer de ovário de iniciação Células Tumorais

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inchaço na Organóides intestinal induzida por forscolina: An<em&gt; In Vitro</em&gt; Ensaio para a Avaliação de Medicamentos Response na Fibrose Cística Os pacientes

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

preparação e<em&gt; In Vitro</em&gt; Caracterização de magnetizado miR-modificado Células Endoteliais

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

Um método para quantificar Desempenho Membro Superior na Vida Diária utilizem acelerómetros

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Uma abordagem In Vitro para terapia fotodinâmica

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Preparação de grânulos de Gushukang (GSK) para experimentos in vivo e in vitro

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.