Agradecemos os esforços de colaboração do Dr. Dang Thao e membros de seu laboratório para o uso de seus equipamentos de imagem e microscópio. Agradecemos também a biorepository e Tissue Research Facility (BTRF) núcleo, Jeff Harper, e os moradores da Universidade de Virginia por nos fornecer o tecido uterino. Agradecemos Karol Szlachta pela ajuda com esquemática Overview.

Espécimes de histerectomia utilizadas neste manuscrito foram coletadas em concordância com um protocolo de ética da Universidade IRB-aprovado numeradas IRB-HSR # 14424. 1. Aquisição de exemplo de origem clínica <ol><li> Obter governo e diretrizes éticas baseadas em instituições e documentação de aprovação antes de começar. </li><li> Realizar todas as etapas condições estéreis. </li><li> Preservar tecido derivado do paciente em meio (RPMI ou DMEM / Glucose alta) em um tubo de 50 ml a 4 ° C, se a amostra não pode ser processada em cultura imediatamente. As amostras podem ser armazenadas neste estado por um período máximo de 24 horas. </li><li> Lavar amostra de tecido, três vezes com 1X tampão fosfato salino estéril (PBS) e descartar a solução entre as lavagens. </li></ol> 2. Preparação de linhas de células primárias, utilizando o Método de raspagem <ol><li> Adicionar 4-10 ml de meio de crescimento (RPMI ou DMEM / alto teor de glucose suplementado com 10% de soro fetal bovino e 1% de Penicilina-streptomycin) de tecido e adiciona Fungizona (0,25 ug / ml de concentração final) durante 30 min. </li><li> Descartar o meio de cultura. </li><li> Lavar o tecido duas vezes com 1X PBS. </li><li> Colocar o tecido de uma placa de cultura de 6 centímetros de células para distinguir entre miométrio e endométrio camadas (ver as Figuras 2A-2C para uma descrição mais detalhada). Separa-se o endométrio. </li><li> Usando uma lâmina de bisturi ou navalha, transecto o tecido em pedaços pequenos, enquanto arranhando sobre o prato de cultura de seis centímetros celular. Estes riscos facilitar a fixação das células endometriais primárias emergentes. Em comparação com o método de tripsina, é necessário mais do tecido (ver Figura 2D para uma aproximação). </li><li> Gentilmente, adicionar 2 ml de meio de cultura para prato riscado (fragmentos de tecido será visível e idealmente imobilizado a partir do movimento coçar). </li><li> Transferir a placa (s) para uma incubadora de cultura de células designada (37 ° C e 5% CO 2). Designar um lugar para as linhas de células primárias, longe de other placas de cultura de células. As culturas primárias são mais sensíveis para a infecção e contaminação. </li><li> Examine as células sob um microscópio de luz por dia. Pequenas populações de células que surgem de dois dias ou de três em torno dos tecidos cortadas (ver Figura 3B). </li><li> Para manter as culturas, lavar cuidadosamente com PBS 1X e adicionar o meio de crescimento fresco (2 mL) a cada três dias. Quando aumenta a taxa de proliferação, o meio de crescimento adicional (3-4 ml) pode ser adicionado à placa de cultura de 6 centímetros (s). Nota: Durante a lavagem e mídia alterações, pedaços de tecido provavelmente será aspirado. Isso não vai afetar o crescimento de células aderentes. Pedaços de tecido deve ser aspirado pelo passo subsequente (2,10) como novas colónias é improvável que emergem após uma semana. </li><li> Quando a placa de 6 cm é cerca de 75 - 80% confluentes (ver Figura 3B), a passagem utilizando 0,05% de tripsina. Pilhas não podem ser passadas por tempo indeterminado - congelar baixo uma placa de células assim que duas ou três placas são maintained. Se forem necessárias mais passagens, siga um protocolo de imortalização (revisado em Ref 3). </li></ol> 3. Preparação de linhas celulares primárias utilizando o Método de tripsina <ol><li> Coloque um pequeno pedaço de tecido endometrial (ver Figura 2D para uma aproximação) em 2 ml de tripsina a 0,25% suplementado com canamicina (0,03 mg / ml de concentração final). </li><li> Incubar tecido a 37 ° C na plataforma rotativa durante 30 min. </li><li> Resumidamente vortex do tecido. </li><li> Centrifugar a amostra a 200-400 x g durante 2 min e desprezar o sobrenadante. </li><li> Adicionar tripsina fresco e Kanamycin. </li><li> Incubar o tecido a 37 ° C durante a rotação durante uma hora. </li><li> Vortex o tecido durante 5-10 seg. </li><li> Adicionar 2 ml de meio de crescimento (suplementado com 10% de soro fetal bovino e 1% de penicilina-estreptomicina) para desactivar a tripsina. </li><li> Células centrifugar a 200-400 g durante 2-3 min. </li><li> Desprezar o sobrenadante e adicionar 2 ml de crescerª mídia para as células peletizadas. </li><li> Placa para um prato de cultura de 6 centímetros de células. Raspar a placa como no Passo 2.5 de Preparação Lines célula primária utilizando o Método raspagem não é necessário, mas melhora a fixação e crescimento de culturas primárias. </li><li> Monitorar as células em um microscópio de luz por dia. As células são geralmente visíveis ao microscópio de luz após 24-48 horas (Figura 3C). </li><li> Para manter as culturas, monitorar as células e meios de mudar a cada 2-3 dias, como no passo 2.9. </li><li> Para passagem, as células, utilizar 0,05% ou 0,25% de tripsina, quando as células atingem 75-80% de confluência (ver Figura 3C). </li></ol> 4. Salvando tecido extra para Análise (pressão congelamento e formalina Fixation) <ol><li> Lavar as amostras duas vezes com 1X PBS. </li><li> Aspirar o máximo de líquido possível. </li><li> Por pressão de congelamento, coloque amostra de tecido endometrial em um tubo de centrífuga de 1,7 (ver Figuras 2F e 2G). Coloque a 1,7tubo de centrífuga, em azoto líquido durante 10 segundos ou até que ele pode ser visto que o tecido foi congelado para baixo. Nota: Tenha cuidado para não tocar diretamente nitrogênio líquido durante a preparação de amostras. As amostras podem ser armazenadas a -80 ° C até processamento adicional ou análise. </li><li> Para a fixação em formalina, corte um pequeno pedaço de tecido endometrial (ver Figura 2H), e submergir em 10% de formalina tamponada a zinco. Após 24 horas, descartar formalina e adicionar 70% de etanol para as amostras de tecido, até processamento posterior. </li></ol> 5. Imunofluorescência (IF) <ol><li> Fixação celular <ol><li> Prepare as células em um slide cultura ou prato. </li><li> Delicadamente, lave as células com PBS 1X. </li><li> Adicionar pré-arrefecida (4 ° C), metanol e acetona (misturadas a uma razão de 1:1) durante 5 min. </li><li> O ar seco do slide antes de prosseguir. Slide pode ser armazenado a 4 ° C durante 1-2 semanas, se necessário. </li></ol></li><li> Processamento IF <ol><li> Rehidratar células com 1X PBS durante 10 min. Para o following Passos 1-6, realizar todas as lavagens e incubações em uma plataforma giratória. </li><li> Bloco utilizando 1X PBS suplementado com 1% de Albumina de Soro Bovino (BSA) e 1% de espécies de soro específico (fonte de anticorpo 2 nd) durante 30 min à temperatura ambiente (RT). </li><li> Incubar no anticorpo primário (ver recomendações do fabricante para diluições de anticorpos). Consulte Materiais para detecção de anticorpos específicos. Dilui-se o anticorpo primário em tampão de bloqueio fresco como no Passo 5.2.2. Esta incubação pode ser realizada por pelo menos duas horas à temperatura ambiente ou durante a noite a 4 ° C. </li><li> Lavar 3 vezes com PBS 1X durante 5 minutos cada. </li><li> Incubar no anticorpo secundário (ver recomendações do fabricante para diluições de anticorpos). Dilui-se o anticorpo secundário em tampão de bloqueio fresco como no Passo 5.2.2. Para evitar foto-branqueamento, é importante para a realização desta e os passos subsequentes na ausência de luz. </li><li> Lavar 3 vezes com PBS 1X durante 5 minutos cada. </li><li> Thoroughly secar os slides. </li><li> Adicionar mídia de montagem e solução contracoloração DAPI. </li><li> Aplicar lamela e selar as bordas com selante (s). As lâminas podem ser armazenadas a 4 ° C no escuro, durante cerca de 2 semanas. </li></ol></li></ol> 6. Extração de RNA <ol><li> Granulado de 1 x 10 7 células por centrifugação. Todos os materiais e reagentes neste protocolo deve ser livre de RNase. </li><li> Lisar as células em 1 ml de reagente TRIZOL, e incuba-se as amostras homogeneizadas durante 10 min à temperatura ambiente para completar a dissociação de complexos nucleoproteicos. </li><li> Adicionar 200 ul de clorofórmio por cada 1 ml de TRIZOL. Agitar vigorosamente à mão por 15 segundos e incubar por 2-3 min à temperatura ambiente. </li><li> Centrifugar a uma velocidade máxima (15000 xg) durante 15 minutos a 4 ° C. Três camadas vai resultar. </li><li> Transferir a fase aquosa superior para um tubo de microcentrifugação fresco. Adicionar 1 mL de glicogénio para aumentar o rendimento de ARN; no entanto, esta etapa só é necessária quando uma pequenaquantidade de ARN é antecipado. </li><li> Adicionar 0,5 ml de álcool isopropílico para a camada aquosa, e inverter as amostras 3-5 vezes. Incubar as amostras a temperatura ambiente durante 10 min. Para aumentar o rendimento, as amostras podem ser colocadas em -20 ° C ou -80 ° C durante 30 min. </li><li> Centrifugar a uma velocidade máxima durante 10 minutos a 4 ° C. </li><li> Neste ponto, uma pequena bola de ARN deve ser visível. Desprezar o sobrenadante. </li><li> Lava-se a ARN com 1 ml de etanol a 75%. </li><li> Centrifugar a uma velocidade máxima durante 5 minutos e descarta-se o sobrenadante. As amostras podem ser lavadas mais para eliminar contaminantes inorgânicos, uma vez, mas este passo não é necessário. </li><li> Resumidamente, secar o sedimento de ARN ARN até pelota é seca (normalmente demora 7-10 min). Nota: Um passo adicional pode ser usada para diminuir a matéria inorgânica e diminuir o tempo de secagem. Após o descarte do sobrenadante da lavagem de etanol, as amostras de rotação à velocidade máxima por mais um minuto e aspirado líquido residual. Tome cuidado para não aspirar pelota. </li><li> Dissolve-se o ARN em água livre de RNase, dependendo do tamanho do sedimento de ARN. Volumes variam tipicamente 10-50 ul. </li><li> Incubar as amostras a 55 ° C durante 10 min, e medir a concentração de ARN. </li><li> Armazenar as amostras a -20 ° C até posterior processamento. </li></ol> 7. Transcrição Reversa <ol><li> Traga o ARN da amostra (1-3 mg) com um volume de 9 mL com H 2 O. </li><li> ARN de calor a 70 ° C durante 10 min. </li><li> Para gerar uma mistura principal de AMV, combinar os reagentes seguintes: 5 ul de dNTPs (concentração de trabalho de 50 uM), 5 ul de oligonucleótidos de ADN de N6 (concentração de trabalho de 80 uM), 5 ul de tampão 5X AMV, e 1 ul de AMV . Esta solução master mix foi concebido por uma amostra. </li><li> Adicionar 16 ul da mistura de base para a ARN aquecida. O volume de reacção final de 25 ul de reacção. </li><li> Use as seguintes condições de PCR para a transcrição reversa: (fase 1), 42 ° C por 90 min, (STAge 2) 95 ° C durante 5 min, e (Fase 3) 4 ° C até posterior processamento. </li></ol> 8. PCR em Tempo Real <ol><li> Realizar as reacções de PCR utilizando os iniciadores, a temperatura de recozimento, números de ciclos, e os reagentes presentes no Quadro 1. Nota: É ideal para acompanhar as instruções do fabricante ao utilizar reagentes de PCR (consulte os números da empresa e catálogo de materiais). </li></ol> 9. In vitro decidualização Protocol (Derivado Ref 4 e 5) <ol><li> Placa células endometriais. </li><li> Crescer até uma confluência de 75-85%. </li><li> Após as células se tornam confluentes, lavar uma vez com 1X PBS. </li><li> Rapidamente, adicionar mídia sem hormônio (fenol livre RPMI suplementado com 5% de carvão tira FBS e 1% de penicilina-estreptomicina). </li><li> Após 24 horas, adicionar mídia da hormona livre suplementados com acetato de medroxiprogesterona (MPA), a uma concentração final de 1 uM e 8-bromoadenosine 3 &#39;, 5&#39;Monofosfato cíclico (cAMP), a uma concentração final de 0,5 mM. </li><li> Depois de pelo menos 48 horas, parar a reacção e salvar a chapa (s) para posterior processamento. </li></ol>

<ol>
	<li>Heller, D., Heller, 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> in The endometrium: a clinicopathologic approach. , 56-75 (1994).</li><li>Emera, D., Romero, R., Wagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+menstruation:+a+new+model+for+genetic+assimilation:+explaining+molecular+origins+of+maternal+responses+to+fetal+invasiveness.">The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.</a> Bioessays. 34, 26-35 (2011).</li><li>Gudjonsson, T., Villadsen, R., Ronnov-Jessen, L., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+protocols+used+in+cell+culture+models+of+human+breast+morphogenesis.">Immortalization protocols used in cell culture models of human breast morphogenesis.</a> Cell Mol Life Sci. 61, 2523-2534 (2004).</li><li>Brosens, J. J., Hayashi, N., White, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+regulates+decidual+prolactin+expression+in+differentiating+human+endometrial+stromal+cells.">Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.</a> Endocrinology. 140, 4809-4820 (1999).</li><li>Jones, 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=Regulation+of+the+SUMO+pathway+sensitizes+differentiating+human+endometrial+stromal+cells+to+progesterone.">Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone.</a> Proc Natl Acad Sci U S A. 103, 16272-16277 (2006).</li><li>Salamonsen, L. 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=Proteomics+of+the+human+endometrium+and+uterine+fluid:+a+pathway+to+biomarker+discovery.">Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery.</a> Fertil Steril. 99, 1086-1092 (2013).</li><li>Haouzi, D., Dechaud, H., Assou, S., De Vos, J., Hamamah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+human+endometrial+receptivity+from+transcriptomic+and+proteomic+data.">Insights into human endometrial receptivity from transcriptomic and proteomic data.</a> Reprod Biomed Online. 24, 23-34 (2012).</li><li>Chen, J. I., 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=Proteomic+characterization+of+midproliferative+and+midsecretory+human+endometrium.">Proteomic characterization of midproliferative and midsecretory human endometrium.</a> J Proteome Res. 8, 2032-2044 (2009).</li><li>Talbi, 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=Molecular+phenotyping+of+human+endometrium+distinguishes+menstrual+cycle+phases+and+underlying+biological+processes+in+normo-ovulatory+women.">Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.</a> Endocrinology. 147, 1097-1121 (2006).</li><li>Punyadeera, 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=Oestrogen-modulated+gene+expression+in+the+human+endometrium.">Oestrogen-modulated gene expression in the human endometrium.</a> Cell Mol Life Sci. 62, 239-250 (2005).</li><li>Ponnampalam, A. P., Weston, G. C., Susil, B., Rogers, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+human+endometrium+during+the+menstrual+cycle.">Molecular profiling of human endometrium during the menstrual cycle.</a> Aust N Z J Obstet Gynaecol. 46, 154-158 (2006).</li><li>Zhang, L., Rees, M. C., Bicknell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+long-term+culture+of+normal+human+endometrial+epithelium+and+stroma.+Expression+of+mRNAs+for+angiogenic+polypeptides+basally+and+on+oestrogen+and+progesterone+challenges.">The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges.</a> J Cell Sci. 108 (1), 323-331 (1995).</li><li>Richards, R. G., Brar, A. K., Frank, G. R., Hartman, S. M., Jikihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+cells+from+term+human+decidua+closely+resemble+endometrial+stromal+cells:+induction+of+prolactin+and+insulin-like+growth+factor+binding+protein-1+expression).">Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression).</a> Biol Reprod. 52, 609-615 (1995).</li><li>Gellersen, B., Brosens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+and+progesterone+receptor+cross-talk+in+human+endometrium:+a+decidualizing+affair.">Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair.</a> J Endocrinol. 178, 357-372 (2003).</li><li>Marsh, M. M., Hampton, A. L., Riley, S. C., Findlay, J. K., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+endothelin+released+by+human+endometrial+epithelial+cells+in+culture.">Production and characterization of endothelin released by human endometrial epithelial cells in culture.</a> J Clin Endocrinol Metab. 79, 1625-1631 (1994).</li><li>Siegfried, J. M., Nelson, K. G., Martin, J. L., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+identification+of+cultured+cells+from+human+endometrium.">Histochemical identification of cultured cells from human endometrium.</a> In Vitro. 20, 25-32 (1984).</li><li>Rawdanowicz, T. J., Hampton, A. L., Nagase, H., Woolley, D. E., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+production+by+cultured+human+endometrial+stromal+cells:+identification+of+interstitial+collagenase,+gelatinase-A,+gelatinase-B,+and+stromelysin-1+and+their+differential+regulation+by+interleukin-1+alpha+and+tumor+necrosis+factor-alpha.">Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.</a> J Clin Endocrinol Metab. 79, 530-536 (1994).</li><li>Dimitriadis, E., Robb, L., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+11+advances+progesterone-induced+decidualization+of+human+endometrial+stromal+cells.">Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 8, 636-643 (2002).</li><li>Sakai, N., 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=Involvement+of+histone+acetylation+in+ovarian+steroid-induced+decidualization+of+human+endometrial+stromal+cells.">Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells.</a> J Biol Chem. 278, 16675-16682 (2003).</li><li>Logan, P. C., Ponnampalam, A. P., Steiner, M., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyclic+AMP+and+estrogen/progesterone+on+the+transcription+of+DNA+methyltransferases+during+the+decidualization+of+human+endometrial+stromal+cells.">Effect of cyclic AMP and estrogen/progesterone on the transcription of DNA methyltransferases during the decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 19, 302-312 (2013).</li><li>Tamura, I., 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=Induction+of+IGFBP-1+expression+by+cAMP+is+associated+with+histone+acetylation+status+of+the+promoter+region+in+human+endometrial+stromal+cells.">Induction of IGFBP-1 expression by cAMP is associated with histone acetylation status of the promoter region in human endometrial stromal cells.</a> Endocrinology. 153, 5612-5621 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Society for Reproductive Medicine: Quick facts about infertility. , (2013).</li><li>Salker, 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=Natural+selection+of+human+embryos:+impaired+decidualization+of+endometrium+disables+embryo-maternal+interactions+and+causes+recurrent+pregnancy+loss.">Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.</a> PLoS One. 5, (2010).</li></ol>

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Outros grupos descritos e adaptado metodologia para a preparação de culturas do estroma do endométrio, a maioria dos quais utilizam colagenase 4,12,13,15-18. Neste manuscrito, nós fornecemos metodologia e evidências para dois métodos de cultura de estroma endometriais simplificados primários, os quais são utilizados por nosso laboratório por razões económicas ea disponibilidade conveniente de tripsina e / ou uma lâmina de barbear. Ao comparar os nossos dois métodos, a...

O corpo do útero humano é composto por três camadas, a perimétrio (ou serosa), miométrio e endométrio. Distinguindo cada uma destas camadas é um passo importante para estabelecer linhas de células do endométrio. O perimétrio é a camada mais exterior do útero e do composto, as células serosas finas. O miométrio é a espessura da camada, meio do útero e composta por células de músculo liso. O endométrio é identificada como a camada interna do útero e inclui populações epiteliais e células estromais. O endométrio é subdividida na camada basal, cuja população de células-tronco é a hipótese de repovoar a camada functionalis aproximadamente a cada 28 dias 1. A camada functionalis do endométrio humano é submetido a alterações bioquímicas e morfológicas significativas na resposta a hormonas circulantes. Estas hormonas são derivados a partir da glândula pituitária e os ovários. Oprodução coordenada e liberação de hormônios resulta em um ciclo reprodutivo. O ciclo reprodutivo é projetado para preparar o endométrio para potenciais eventos de implantação do embrião. Nos seres humanos, o ciclo de reprodução é conhecido como &quot;ciclo menstrual&quot; e divide-se em três fases - proliferativa, secretora, e menstruais. A fase proliferativa envolve a proliferação do endométrio functionalis camada enquanto que a fase de secreção está marcada por functionalis maturação. Especificamente, as alterações extracelulares, secreções e diferenciação celular sinalizar uma implantação potencial. Se o implante não ocorrer antes do final da fase de secreção, a camada functionalis endométrio é eliminado durante a fase menstrual. A importância da menstruação e os eventos que provocam o desprendimento da camada functionalis ainda estão sendo debatidas. Nos seres humanos, tem-se colocado que a menstruação é o resultado de um evento específico mid-secretora diferenciação fase conhecidacomo &quot;decidualização espontânea&quot; 2. Neste manuscrito, nós fornecemos metodologia detalhada para ambos os métodos de isolamento de células do estroma endometrial, e usar uma combinação de imunofluorescência e imagens digitais para demonstrar a eficácia dessas abordagens. Além disso, foi aplicado um comumente utilizado em modelo in vitro de decidualização espontânea para confirmar o isolamento de células do estroma endometrial.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">0.25 Trypsin or 0.05% Trypsin&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH3023602 or SH30004202&#160;&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="27">
			<td dir="LTR" height="27" style="height:27px;width:235px;">1.7 micro Centrifuge Tube&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">22-272A&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">1&#181;l,20&#181;l, 200ml and 1000&#181;l Pipette&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">24-401,24-402, 24-412, 24-430&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">15ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339650</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">50ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339652</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">6cm Cell Culture Dish&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-556-002&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">8 well Chambers&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Acetate&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher scientific&#160;</td>
			<td dir="LTR" style="width:216px;">C4-100&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">AMV RT Enzyme/Buffer&#160;</td>
			<td dir="LTR" style="width:201px;">Bio Labs&#160;</td>
			<td dir="LTR" style="width:216px;">M077L&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Bovine Serum Albumin (BSA)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-1605-100&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Buffered Zinc Formalin&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">59201ZF&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Charcoal strip FBS&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher&#160;</td>
			<td dir="LTR" style="width:216px;">NC9019735&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Chloroform&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cover slip&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Brand&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-544D&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cyclic AMP (cAMP)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">B7880</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">DMEM/High Glucose&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH30243FS&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">dNTP&#160;</td>
			<td dir="LTR" style="width:201px;">Bioline&#160;</td>
			<td dir="LTR" style="width:216px;">BIO-39025&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Anti Goat -TRITC&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-3855&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Serum&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s lab&#160;</td>
			<td dir="LTR" style="width:216px;">&#160;017-000-002&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">E Cadherin Antibody&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">1702-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Ethanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">BP2818-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific</td>
			<td dir="LTR" style="width:216px;">03-600-511&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fungizone Amphotericin B&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">15290-018&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">GAPDH Probe&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">HS99999905&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Glycogen&#160;</td>
			<td dir="LTR" style="width:201px;">5Prime&#160;</td>
			<td dir="LTR" style="width:216px;">2301440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Goat Anti Mouse -FITC&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s Lab&#160;</td>
			<td dir="LTR" style="width:216px;">115-096-003&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Isopropanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP2618-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Kanamycin&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP906-5&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Medroxyprogesterone acetate (MPA)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">M1629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">MeOH (Methanol)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">A4-08-1&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Mounting Media (w/DAPI)&#160;</td>
			<td dir="LTR" style="width:201px;">Vector Labratories&#160;</td>
			<td dir="LTR" style="width:216px;">H-1500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">N6 DNA Oligos&#160;</td>
			<td dir="LTR" style="width:201px;">Invitrogen&#160;</td>
			<td dir="LTR" style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Number 15 Scraper&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">BD&#160;</td>
			<td dir="LTR" style="width:216px;">371615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Pan Cytokeratin&#160; Mouse mAB&#160;</td>
			<td dir="LTR" style="width:201px;">Cell Signaling&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">4545</td>
			<td></td>
		</tr>
		<tr height="29">
			<td dir="LTR" height="29" style="height:29px;width:235px;">PBS (phosphate buffered saline)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-399-4&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Penicillin-Streptomycin Glutamine Solution 100X&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">SV30082.01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">PML Anti Goat Anti body&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-9862&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Primer(s)&#160;</td>
			<td dir="LTR" style="width:201px;">Eurofins&#160;</td>
			<td dir="LTR" style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">RPMI&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">SH30027FS&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">RPMI (Phenol free)&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">11835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Sybr Green&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Taqman&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Trizol&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Vimentin Antibody&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">4211-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

Muitos esforços têm sido dedicados a estabelecer em sistemas de cultura de células in vitro. Estes sistemas são concebidos para modelar um vasto número de processos in vivo. Sistemas de cultura de células resultantes de amostras endometriais humanos não são excepção. As aplicações vão desde processos fisiológicos normais cíclicos de patologias endometriais, tais como cancros ginecológicos, doenças infecciosas, e deficiências na função reprodutora. Aqui, oferecemos dois métodos para o estabelecimento de células estromais endometriais primários de espécimes ressecados cirurgicamente histerectomia endometriais. O primeiro método é referido como &quot;o método de raspagem&quot; e incorpora raspagem mecânica usando lâminas cirúrgicas ou de barbear ao passo que o segundo método é denominado &quot;o método de tripsina.&quot; Este último método utiliza a actividade enzimática da tripsina para promover a separação de células e primário conseqüência celular. Nós ilustramos metodologia passo-a-passo através de imagens digitais e microscopia. Nós também provide exemplos para validar as linhas de células estromais do endométrio através de tempo real, as reacções em cadeia da polimerase quantitativa (qPCR) e imunofluorescência (IF).

Conforme enfatizado na seção de protocolo, não se esqueça de realizar todos os métodos sob governo, institucionais e diretrizes éticas ao manusear e preparar o tecido humano. Incluídos neste manuscrito é uma ilustração do fluxo de trabalho geral de &quot;o método de raspagem&quot; (Figura 1A) e &quot;o método de tripsina&quot; (Figura 1B) utilizados para estabelecer culturas primárias de endométrio. Estes métodos são descrito...

Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Estabelecimento de sistemas de cultura primária de células do estroma endometrial a partir de espécimes de histerectomia é uma técnica biológica valioso e um passo crucial antes de prosseguir uma vasta gama de pesquisa objetiva. Aqui, descrevemos dois métodos utilizados para estabelecer culturas de tecidos do estroma endometriais ressecados cirurgicamente de pacientes humanos.

Dois métodos para estabelecer células endometriais Humano estromais primários de espécimes de histerectomia

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

two-methods-for-establishing-primary-human-endometrial-stromal-cells

Research

JoVE Journal

Medicine

22.5K visualizações.  University of Virginia.  Estabelecimento de sistemas de cultura primária de células do estroma endometrial a partir de espécimes de histerectomia é uma técnica biológica valioso e um passo crucial antes de prosseguir uma vasta gama de pesquisa objetiva. Aqui, descrevemos dois métodos utilizados para estabelecer culturas de tecidos do estroma endometriais ressecados cirurgicamente de pacientes humanos. 

Vídeo: Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Geração Experimental de Carcinoma Associado fibroblastos (FAC) de Fibroblastos Humanos mamária

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Análise de Transcriptoma Humanos espécimes cirúrgicos retina utilizando Jornal

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para obtenção de células do cancro do ovário primárias de amostras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Enriquecimento para resistente à quimioterapia do cancro do ovário células-tronco de linhas de células humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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 ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Um método de isolamento do romance grau clínico para rim humano Perivascular de células do estroma

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento de células humanas primárias do Decidual de membranas fetais de termo placentas

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolamento de Adipócitos Viáveis e Fração Vascular Estromal do Tecido Adiposo Visceral Humano Adequado para Análise de RNA e Fenotipagem de Macrófagos

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...