Este trabalho foi financiado pela Fundação Carraresi e pelo MiND Foods Hub ID: 1176436. Todos os autores são membros do COST Action CA16119 In vitro 3D total cell guidance and fitness (CellFit).

Todos os estudos foram revisados e aprovados pelo Comitê de Ética da Universidade de Milão. Todos os experimentos em animais foram realizados de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório, publicado pelo Instituto Nacional de Saúde dos EUA (NIH). O isolamento de células humanas de indivíduos adultos saudáveis foi aprovado pelo Comitê de Ética do Ospedale Maggiore Policlinico, Milano. Todos os métodos do nosso estudo foram realizados de acordo com as diretrizes aprovadas.
1. Isolamento do fibroblasto de pele
NOTA: Todos os procedimentos descritos abaixo podem ser aplicados a fibroblastos isolados de diferentes espécies de mamíferos, incluindo camundongos, suínos e humanos. As células murinas foram isoladas de camundongos machos de 7 semanas de idade e tecido da pele suína foram coletados no matadouro local. As células humanas foram isoladas de pacientes adultos, após consentimento por escrito.
<ol><li>Prepare 0,1% solução de gelatina suína.<ol>
<li>Pesar 0,1 g de gelatina suína e dissolvê-la em 100 mL de água. Esterilizar a solução de gelatina por autoclaving antes de usar.</li>
		<li>Cubra placa de Petri de 35 mm com gelatina suína de 0,1%, adicionando 1,5 mL da solução preparada. Incubar por 2h em temperatura ambiente.</li>
	</ol>
</li>
	<li>Corte biópsias de pele de mamíferos (camundongos, suínos e humanos) de aproximadamente 2-5 cm de comprimento e coloque-os na linha salina tamponada fosfato (PBS) de Dulbecco contendo 2% de solução antimíctica antimíctica antimíctica. Armazenar a + 4 °C até usar. NOTA: As coletas de biópsia devem ser realizadas de acordo e após a aprovação do Comitê de Ética, de acordo com as diretrizes estabelecidas.</li>
	<li>Lave extensivamente as biópsias coletadas três vezes em PBS fresco estéril contendo 2% de solução antimicomolética antibiótico.</li>
	<li>Coletar biópsias da última lavagem e colocá-las em uma placa de Petri estéril de 100 mm. Use um bisturi estéril para cortá-los em pedaços de aproximadamente 2 mmde tamanho 3 .</li>
	<li>Ao final da incubação de 2 h, remova o excesso de solução de gelatina da placa de Petri de 35 mm (descrita na etapa 1.1.2) e, usando uma pinça cirúrgica estéril, coloque imediatamente 5-6 fragmentos de pele em cada prato de cultura pré-revestido.</li>
	<li>Molhe os fragmentos adicionando 100 gotículas μL de meio de isolamento do fibroblasto (Tabela 1) sobre cada uma delas. Cultura a 37 °C em 5% incubadora de CO2 . NOTA: Para evitar que o meio se evase, coloque a placa de Petri de 35 mm dentro de uma placa de Petri de 100 mm ou maior contendo água estéril. Certifique-se de tampar ambas as placas de Petri.</li>
	<li>Após 24h de cultura, verifique a quantidade do meio na placa de Petri cultura de 35 mm. Se necessário, adicione 500 μL de meio de isolamento do fibroblasto para manter molhado os fragmentos.</li>
	<li>Remova cuidadosamente o meio e atualize-o pelo menos a cada 2 dias de cultura usando uma pipeta.</li>
	<li>Quando os fibroblastos começam a crescer fora dos fragmentos de pele colocados na placa de Petri de 35 mm (descrita na etapa 1.5.) e começam a formar uma monocamada celular (geralmente 6 dias). Remova as peças da pele usando uma pinça cirúrgica estéril e cultura em 2 mL de meio de isolamento do fibroblasto.</li>
	<li>Continue a cultivar a monocamada celular a 37 °C em 5% de incubadora de CO2 até 80% de confluência e atualizar o meio a cada dois dias.</li>
</ol>2. Cultura da linha celular primária do fibroblasto
<ol><li>Quando os fibroblastos atingirem 80% de confluência, remova cuidadosamente as células de isolamento do fibroblasto e lave as células três vezes com 3 mL de PBS contendo 1% de solução antimicocótica antibiótico.</li>
	<li>Para desapego celular, adicione 600 μL de solução trypsin-EDTA de 0,25% no prato de cultura e incubar a 37 °C por 3-5 min.</li>
	<li>Adicione 5,4 mL de cultura fibroblasto para neutralizar a trippsina quando as células começarem a se desprender do prato de cultura (Tabela 1).</li>
	<li>Desalojar células por tubulação repetida e suave. Células de placas em novos pratos de cultura (sem gelatina), mantendo a razão de passagem entre 1:2 e 1:4 (dependendo da taxa de crescimento). NOTA: A centrifugação não é necessária.</li>
	<li>Manter as células na cultura e mudar de meio a cada 2 dias, até que elas tenham atingido 80% de confluência e passá-las. NOTA: Propagar fibroblastos duas vezes por semana para manter o crescimento vigoroso.</li>
</ol>3. Exposição do fibroblasto ao 5-aza-CR
<ol><li>Prepare a solução fresca de estoque 1 mM 5-aza-CR.<ol>
<li>Pese 2,44 mg de 5-aza-CR e dissolva-o em 10 mL de DMEM de alta glicose. Resuspend o pó por vórtice. Esterilize a solução com filtro de 0,22 μm. NOTA: A solução de estoque de 5 aza-CR deve ser preparada imediatamente antes do uso.</li>
		<li>Prepare a solução de trabalho de 5 aza-CR diluindo 1 μL de solução de estoque de 5 aza-CR (3.1.1.) em 1 mL de meio de cultura fibroblasto. NOTA: A concentração da solução de trabalho de 5 aza-CR é de 1 μM 1,2,3,8,9.</li>
	</ol>
</li>
	<li>Células de trypsinize como descrito anteriormente (2.1.-2.3.) e desalojam células repetidamente e suavemente.</li>
	<li>Pegue a suspensão da célula e transfira-a para um tubo cônico.</li>
	<li>Conte células usando uma câmara de contagem sob um microscópio óptico à temperatura ambiente. Calcule o volume de células médias necessárias para re suspendê-los para obter 4 x 104 células em 30 μL de cultura fibroblasto complementada com 1 μM 5-aza-CR (ver passo 3.1.2.). NOTA: A fórmula a ser utilizada depende do tipo específico de câmara. <img alt="Equation 1" src="/files/ftp_upload/61655/61655equ01v3.jpg">
</li>
	<li>Centrifugar a suspensão celular a 150 x g por 5 min a temperatura ambiente. Remova a pelota supernascedora e resuspend com o meio de cultura fibroblasto suplementado com 1 μM 5-aza-CR (ver passo 3.1.2.). Para que o volume do meio de cultura do fibroblasto seja utilizado, consulte o passo 3.4. NOTA: Como controle negativo, as células resuspend ad a mesma concentração no meio de cultura fibroblasto sem 5-aza-CR e proceder com encapsulamento celular em pó PTFE (passo 4.1.-4.13.).</li>
</ol>4. Encapsulamento de fibroblasto em micro-bioreatores PTFE
<ol><li>Encha uma placa de Petri de 35 mm com pó de politetrafluoroetileno (PTFE) para produzir uma cama (Figura 1A). NOTA: Use placas de Petri de bacteriologia de 35 mm para evitar a adesão ao mármore líquido. Para obter uma fina concha hidrofóbica e porosa, use um pó PTFE com um tamanho médio de partícula de 1 μm e produzido com uma moagem máxima de 2,0 NPIRI. Isso permite a criação de mármores líquidos permeáveis a gás. Além disso, o revestimento translúcido facilita a observação de processos de agregação celular em tempo real Tamanho de partícula maior leva a alta polidispersidade que pode causar evaporação elevada, deformidade e perda da forma esférica, e a dissolução prematura dos micro-bioreatores.</li>
	<li>Dispense 30 μL de gotícula única contendo 4 x 104 células (ver passos 3.4.- 3,5.) no leito de pó (Figura 1B).</li>
	<li>Gire suavemente a placa de Petri de 35 mm em um movimento circular para garantir que o pó PFTE cubra inteiramente a superfície da gota líquida para formar um micro-bioreator de mármore líquido (Figura 1C).</li>
	<li>Pegue o micro-bioreator de mármore líquido usando uma ponta de pipeta de 1.000 μL, cortada na borda, para acomodar o diâmetro do mármore (Figura 1D,E). Coloque o micro-bioreator de mármore líquido em uma placa de Petri bacteriologia limpa para estabilizá-la (Figura 1F). NOTA: Para criar um atrito para segurar o mármore dentro da ponta, corte as pontas da pipeta com um diâmetro aproximadamente ligeiramente menor do que o diâmetro do mármore líquido.</li>
	<li>Transfira o micro-bioreator de mármore líquido da placa de Petri para uma placa de 96 poços (um mármore/bem) (Figura 1G).</li>
	<li>Adicione lentamente 100 μL de mídia da margem do poço. O micro-bioreator começa a flutuar em cima da mídia (Figura 1H). NOTA: O micro-bioreator quebra em contato líquido direto, devido ao rompimento da hidrofobiidade ptfe. Como abordagem alternativa, os micro-bioreatores de mármore líquido podem ser colocados individualmente em um prato de cultura bacteriologia de 35 mm. Neste caso, para evitar a evaporação líquida do mármore, a placa de Petri de 35 mm contendo o micro-bioreator deve ser inserida em uma placa de Petri de 100 mm, anteriormente aliquoed com água estéril</li>
	<li>Incubar micro-bioreator de mármore líquido por 18 h a 37 °C em incubadora de CO2 de 5% 1,2,3,8,9. NOTA: O tamanho da partícula PTFE de 1 μm pode garantir uma troca de gás ideal entre o líquido interior e o ambiente circundante.</li>
	<li>Após a incubação de 5 aza-CR por 18 h, colete o micro-bioreator de mármore líquido usando um corte de ponta de pipeta de 1.000 μL na borda (ver passo 4.5).</li>
	<li>Coloque o micro-bioreator em uma nova placa petriologia de 35 mm (Figura 1D-F).</li>
	<li>Use uma agulha para perfurar o mármore líquido e quebrá-lo.</li>
	<li>Recuperar esferoides formados com uma ponta de pipeta de 200 μL, cortada na borda, sob um estereomicroscope (Figura 1I,J). NOTA: Células epigeneticamente apagadas encapsuladas em PTFE formam uma estrutura esférica 3D (um agregado em cada mármore líquido).</li>
	<li>Para avaliar a aquisição de estado pluripotente em resposta ao 5-aza-CR, verifique o início da expressão genética relacionada à pluripotência, OCT4, NANOG, REX1 e SOX2, por PCR qualitativo (Tabela 2).</li>
	<li>Prossiga com a segunda etapa do protocolo, conforme descrito abaixo.</li>
</ol>5. Cultura em micro-bioreatores ptfe de células epigeneticamente apagadas
<ol><li>Prepare o meio de cultura ESC fresco (Tabela 1).</li>
	<li>Transfira organoides em uma placa de Petri contendo meio ESC para lavagem de resíduos de 5 aza-CR (ver etapas 5.1.-5.2.).</li>
	<li>Prepare uma nova placa petriologia de 35 mm contendo cama de pó PTFE (veja também o passo 4.1.).</li>
	<li>Dispense um organoide único em uma gotícula de 30 μL ESC meio de cultura no leito de pó usando uma ponta de pipeta de 200 μL, cortada na borda (ver passos 4.9.; 5.3.).</li>
	<li>Gire suavemente a placa de Petri de 35 mm em um movimento circular para formar um novo micro-bioreator de mármore líquido, pegue-a usando uma ponta de pipeta de 1.000 μL, corte na borda e coloque o micro-bioreator recém-formado em um poço de placa de 96 poços (um mármore/bem) (ver passos 4.3.-4.6.).</li>
	<li>Para flutuar os micro-bioreatores, adicione 100 μL de mídia da margem do poço para banhar lentamente o mármore (ver nota 4.7.).</li>
	<li>Micro-bioreatores de mármore líquido de cultura a 37 °C em incubadora de CO2 de 5% pelo tempo necessário. Troque de meio a cada dois dias, seguindo o procedimento descrito em 5.3.-5.7. NOTA: No presente manuscrito, são fornecidos resultados obtidos com cultura organoides durante 28 dias. No entanto, se necessário, um período de cultura mais longo pode ser realizado.</li>
</ol>

<ol>
	<li>Pennarossa, 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=Brief+demethylation+step+allows+the+conversion+of+adult+human+skin+fibroblasts+into+insulin-secreting+cells.">Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 8948-8953 (2013).</li><li>Pennarossa, 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=Reprogramming+of+pig+dermal+fibroblast+into+insulin+secreting+cells+by+a+brief+exposure+to+5-aza-cytidine.">Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine.</a> Stem Cell Reviews and Reports. 10 (1), 31-43 (2014).</li><li>Brevini, T. A. 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=Morphological+and+molecular+changes+of+human+granulosa+cells+exposed+to+5-azacytidine+and+addressed+toward+muscular+differentiation.">Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation.</a> Stem Cell Reviews and Reports. 10 (5), 633-642 (2014).</li><li>Mirakhori, F., Zeynali, B., Kiani, S., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+azacytidine+step+allows+the+conversion+of+suspension+human+fibroblasts+into+neural+progenitor-like+cells.">Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells.</a> Cell Journal. 17 (1), 153-158 (2015).</li><li>Tan, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+tissue+engineering+and+regeneration+through+epigenetic+reprogramming+and+scaffold+manipulation.">Muscle tissue engineering and regeneration through epigenetic reprogramming and scaffold manipulation.</a> Scientific Reports. 5, 16333 (2015).</li><li>Brevini, T. A. L., Pennarossa, G., Acocella, F., Brizzola, S., Zenobi, A., Gandolfi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+conversion+of+adult+dog+skin+fibroblasts+into+insulin-secreting+cells.">Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells.</a> Veterinary Journal. 211, 52-56 (2016).</li><li>Chandrakanthan, V., 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=PDGF-AB+and+5-Azacytidine+induce+conversion+of+somatic+cells+into+tissue-regenerative+multipotent+stem+cells.">PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (16), 2306-2315 (2016).</li><li>Manzoni, E. F. 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=5-azacytidine+affects+TET2+and+histone+transcription+and+reshapes+morphology+of+human+skin+fibroblasts.">5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.</a> Scientific Reports. 6, 37017 (2016).</li><li>Pennarossa, 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=Epigenetic+erasing+and+pancreatic+differentiation+of+dermal+fibroblasts+into+insulin-producing+cells+are+boosted+by+the+use+of+low-stiffness+substrate.">Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate.</a> Stem Cell reviews and reports. 14 (3), 398-411 (2018).</li><li>Pennarossa, 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=Use+of+a+PTFE+micro-bioreactor+to+promote+3D+cell+rearrangement+and+maintain+high+plasticity+in+epigenetically+erased+fibroblasts.">Use of a PTFE micro-bioreactor to promote 3D cell rearrangement and maintain high plasticity in epigenetically erased fibroblasts.</a> Stem Cell Reviews and Reports. 15 (1), 82-92 (2019).</li><li>Constantinides, P. G., Jones, P. A., Gevers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+striated+muscle+cells+from+non-myoblast+precursors+following+5-azacytidine+treatment.">Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment.</a> Nature. 267 (5609), 364-366 (1977).</li><li>Taylor, S. M., Jones, 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=Multiple+new+phenotypes+induced+in+10T1/2+and+3T3+cells+treated+with+5-azacytidine.">Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.</a> Cell. 17 (4), 771-779 (1979).</li><li>Taylor, S. M., Constantinides, P. A., Jones, 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=5-Azacytidine,+DNA+methylation,+and+differentiation.">5-Azacytidine, DNA methylation, and differentiation.</a> Current Topics in Microbiology and Immunology. 108, 115-127 (1984).</li><li>Jones, 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=Effects+of+5-azacytidine+and+its+2'-deoxyderivative+on+cell+differentiation+and+DNA+methylation.">Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation.</a> Pharmacology &amp; Therapeutics. 28 (1), 17-27 (1985).</li><li>Palii, S. S., Van Emburgh, B. O., Sankpal, U. T., Brown, K. D., Robertson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+inhibitor+5-Aza-2'-deoxycytidine+induces+reversible+genome-wide+DNA+damage+that+is+distinctly+influenced+by+DNA+methyltransferases+1+and+3B.">DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B.</a> Molecular and Cellular Biology. 28 (2), 752-771 (2008).</li><li>Vining, K. H., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+forces+direct+stem+cell+behaviour+in+development+and+regeneration.">Mechanical forces direct stem cell behaviour in development and regeneration.</a> Nature Reviews. Molecular Cell Biology. 18 (12), 728 (2017).</li><li>Matamoro-Vidal, A., Levayer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+influences+of+mechanical+forces+on+cell+competition.">Multiple influences of mechanical forces on cell competition.</a> Current Biology. 29 (15), 762-774 (2019).</li><li>Yim, E. K., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-dependent+cell+signaling+in+stem+cell+differentiation.">Force-dependent cell signaling in stem cell differentiation.</a> Stem Cell Research &amp; Therapy. 3 (5), 41 (2012).</li><li>Kumar, A., Placone, J. K., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+extracellular+forces+that+determine+cell+fate+and+maintenance.">Understanding the extracellular forces that determine cell fate and maintenance.</a> Development. 144 (23), 4261-4270 (2017).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Current Opinion in Cell Biology. 15 (6), 753-762 (2003).</li><li>Streuli, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+mediates+tissue-specific+gene+expression+in+mammary+epithelia.">Laminin mediates tissue-specific gene expression in mammary epithelia.</a> The Journal of Cell Biology. 129 (3), 591-603 (1995).</li><li>Christman, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Azacytidine+and+5-aza-2[prime]-deoxycytidine+as+inhibitors+of+DNA+methylation:+mechanistic+studies+and+their+implications+for+cancer+therapy.">5-Azacytidine and 5-aza-2[prime]-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.</a> Oncogene. 21, 5483-5495 (2002).</li><li>Stresemann, C., Lyko, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+action+of+the+DNA+methyltransferase+inhibitors+azacytidine+and+decitabine.">Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine.</a> International Journal of Cancer. 123 (1), 8-13 (2008).</li><li>Ledda, S., Idda, A., Kelly, J., Ariu, F., Bogliolo, L., Bebbere, 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+novel+technique+for+in+vitro+maturation+of+sheep+oocytes+in+a+liquid+marble+microbioreactor.">A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.</a> Journal of Assisted Reproduction and Genetics. 33 (4), 513-518 (2016).</li><li>Vadivelu, R. 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=Generation+of+three-dimensional+multiple+spheroid+model+of+olfactory+ensheathing+cells+using+floating+liquid+marbles.">Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles.</a> Scientific Reports. 5, 15083 (2015).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marble+as+bioreactor+for+engineering+three-dimensional+toroid+tissues.">Liquid Marble as bioreactor for engineering three-dimensional toroid tissues.</a> Scientific Reports. 7 (1), 12388 (2017).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marbles+as+bioreactors+for+the+study+of+three-dimensional+cell+interactions.">Liquid Marbles as bioreactors for the study of three-dimensional cell interactions.</a> Biomedical Microdevices. 19 (2), 31 (2017).</li><li>Varelas, X., 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=TAZ+controls+Smad+nucleocytoplasmic+shuttling+and+regulates+human+embryonic+stem-cell+self-renewal.">TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal.</a> Nature Cell Biology. 10 (7), 837-848 (2008).</li><li>Panciera, T., 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+Expandable+Tissue-Specific+Stem/Progenitor+Cells+through+Transient+Expression+of+YAP/TAZ.">Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.</a> Cell Stem Cell. 19 (6), 725-737 (2016).</li><li>Pennarossa, G., Paffoni, A., Ragni, G., Gandolfi, F., Brevini, T. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+signaling-directed+YAP/TAZ+regulation+encourages+3D+spheroid+colony+formation+and+boosts+plasticity+of+parthenogenetic+stem+cells.">Rho signaling-directed YAP/TAZ regulation encourages 3D spheroid colony formation and boosts plasticity of parthenogenetic stem cells.</a> Advances in Experimental Medicine and Biology. 1237, 49-60 (2020).</li></ol>

Os autores não têm nada a revelar.

Durante as últimas décadas, diversos estudos se concentraram no desenvolvimento de estratégias para reverter uma célula terminalmente diferenciada para um estado permissivo menos comprometido e maior. O protocolo aqui descrito permite a geração e manutenção a longo prazo de células pluripotentes a partir de células adultas maduras terminais diferenciadas. O método combina duas etapas independentes que envolvem a indução de um estado permissivo elevado que é alcançado através de apagamento epigenético qu?...

Durante as últimas décadas, o conceito amplamente aceito de progressão unidirecional em direção ao comprometimento e diferenciação celular foi completamente revisto. Foi demonstrado que a especificação celular pode ser revertida, e uma célula terminantemente diferenciada pode ser empurrada para um estado permissivo menos comprometido e mais alto, usando diferentes métodos.
Entre os vários métodos propostos, um dos métodos mais promissores envolve o uso de compostos químicos para induzir células em uma chamada pluripotência quimicamente induzida. As pequenas moléculas utilizadas nesta abordagem são capazes de interagir e modificar a assinatura epigenética de uma célula adulta madura, evitando a necessidade de qualquer vetor transgênico e/ou viral 1,2,3,4,5,6,7,8,9,10 . Inúmeros estudos têm mostrado recentemente que é possível mudar as células de um fenótipo para outro, fornecendo estímulos bioquímicos e biológicos específicos que induzem a reativação de genes hipermetilados 11,12,13,14,15. Esses eventos de desmetilação permitem a conversão de células terminais diferenciadas em um progenitor primitivo, um multipotente ou uma célula de alta plasticidade/pluripotente 1,2,3,4,5,6,7,8,9,10.
Paralelamente, muitos estudos têm se concentrado recentemente na compreensão de pistas relacionadas à mecanosensagem e, mais especificamente, na possibilidade de usar forças mecânicas para influenciar diretamente a plasticidade celular e/ou diferenciação 16,17,18,19. De fato, foi claramente demonstrado que a matriz extracelular (ECM) desempenha um papel fundamental no controle do destino celular. Em particular, os sinais biomecânicos e biofísicos produzidos pelo ECM regulam diretamente mecanismos moleculares e vias de sinalização, influenciando o comportamento celular e funções20,21. Esses dados recentes abriram caminho para o desenvolvimento de novos sistemas de cultura 3D que imitam mais de perto o microambiente de células in vivo, replicando estímulos mecânicos e físicos impulsionando o comportamento celular.
Descrevemos aqui um protocolo de duas etapas que combina o uso de apagamento epigenético químico com pistas relacionadas à mecanosensagem, para gerar células pluripotentes de mamíferos. No primeiro passo, as células são incubadas com a molécula desmetilante 5-aza-citidina (5-aza-CR). Este agente é capaz de induzir uma significativa desmetilação global de DNA através de um efeito combinado da translocação direta 2 (TET2)-mediada ação 8,10 e a inibição indireta das metiltransferases de DNA (DNMT)22,23. Esta etapa induz a remoção dos blocos epigenéticos com uma posterior reativação da expressão genética relacionada à pluripotency e, portanto, a geração de células de alta plasticidade 1,2,3,8,10, a partir de agora referidas como "células apagadas epigenéticas". Na segunda etapa, as células são encapsuladas em um sistema de cultura 3D. Para isso, o composto sintético hidrofóbico não reativo politetrafluoroetileno (PTFE; com tamanho de partícula de 1 μm) é usado como micro-bioreator, que permite a criação de um microambiente celular inalcançável através do uso de sistemas tradicionais de cultura 2D10. As partículas de pó PTFE aderem à superfície da gota líquida na qual as células são re-suspensas e isolam o núcleo líquido da superfície de suporte, permitindo a troca de gás entre o líquido interior e o ambiente circundante24. O "micro-bioreator PTFE" obtido, também conhecido como "Mármore Líquido", incentiva as células a interagir livremente entre si, promovendo o rearranjo de células 3D 25,26,27, e estende e mantém de forma estável o estado de alta plasticidade adquirido, embora a sinalização relacionada à bio-mecanosensing 10.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7522</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>5-Azacytidine</td>
			<td>Sigma-Aldrich</td>
			<td>A2385</td>
			<td>5-aza-CR, for fibroblast epigenetic erasing</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR</td>
			<td>Bio-Rad Laboratories</td>
			<td>NA</td>
			<td>Thermal cycler for quantitative PCR</td>
		</tr>
		<tr>
			<td>Cytidine</td>
			<td>Sigma-Aldrich</td>
			<td>C4654</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>41966052</td>
			<td>For fibroblast isolation and culture medium</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>31885023</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>PBS; for biopsy and cell wash and for solution preparation</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT Micro Purification Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>61021</td>
			<td>mRNA estraction</td>
		</tr>
		<tr>
			<td>ESGRO Recombinant Mouse LIF Protein</td>
			<td>Sigma-Aldrich</td>
			<td>ESG1106</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>FGF-Basic (AA10-155) Recombinant Human Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0024</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td>For dish coating</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 2700</td>
			<td>Applied Biosystems</td>
			<td>NA</td>
			<td>Thermal cycler for qualitative PCR</td>
		</tr>
		<tr>
			<td>Global DNA Methylation ELISA Kit</td>
			<td>CELL BIOLABS</td>
			<td>STA-380</td>
			<td>Methylation study</td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M7801</td>
			<td>Qualitative PCR</td>
		</tr>
		<tr>
			<td>Guanosine</td>
			<td>Sigma-Aldrich</td>
			<td>G6264</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-10 Nutrient Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>31550031</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>KOVA glasstic slide 10 with grids</td>
			<td>Hycor Biomedical</td>
			<td>87144</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Leica MZ APO Stereo Microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td>For organoid observation</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Millex-GS 0.22 &#181;m pore filters</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>For solution sterilization</td>
		</tr>
		<tr>
			<td>M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant</td>
			<td>Promega</td>
			<td>M3681</td>
			<td>mRNA reverse transcription</td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>51119000</td>
			<td>For ELISA plate reading</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE300 Inverted Phase Contrast Microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>For cell observation</td>
		</tr>
		<tr>
			<td>Perkin Elmer Thermal Cycler 480</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Thermal cycler for reverse transcription</td>
		</tr>
		<tr>
			<td>Poly(tetrafluoroethylene) 1 &#956;m particle size</td>
			<td>Sigma-Aldrich</td>
			<td>430935</td>
			<td>For generating micro-bioreactor</td>
		</tr>
		<tr>
			<td>PureLink Genomic DNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K182001</td>
			<td>Genomic DNA estraction</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Cells-to-CT Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1728</td>
			<td>Quantitative PCR</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 100X20 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833902</td>
			<td>For fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833900</td>
			<td>For Fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Suspension</td>
			<td>Sarstedt</td>
			<td>833900500</td>
			<td>Bacteriology petri dish for liquid marble culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 Well,Standard,F</td>
			<td>Sarstedt</td>
			<td>833924005</td>
			<td>For liquid marble culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>For fibroblast dissociation</td>
		</tr>
		<tr>
			<td>Tube 15ml, 120x17mm, PS</td>
			<td>Sarstedt</td>
			<td>62553041</td>
			<td>For cell suspension centrifugation</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
	</tbody>
</table>

a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells

O fenótipo celular pode ser invertido ou modificado com diferentes métodos, com vantagens e limitações específicas para cada técnica. Aqui descrevemos uma nova estratégia que combina o uso de apagamento epigenético químico com pistas relacionadas à mecanosensagem, para gerar células pluripotentes de mamíferos. Dois passos principais são necessários. Na primeira etapa, as células adultas maduras (terminantemente diferenciadas) são expostas à borracha epigenética 5-aza-cytidina para levá-las a um estado pluripotente. Esta parte do protocolo foi desenvolvida, com base na compreensão crescente dos mecanismos epigenéticos que controlam o destino e a diferenciação celular, e envolve o uso do modificador epigenético para apagar o estado diferenciado celular e, em seguida, dirigir para uma janela transitória de alta plasticidade.
Na segunda etapa, as células apagadas são encapsuladas em micro-bioreatores de politetrafluoroetileno (PTFE), também conhecidos como Mármores Líquidos, para promover o rearranjo celular 3D para estender e manter a alta plasticidade adquirida. O PTFE é um composto sintético hidrofóbico não reativo e seu uso permite a criação de um microambiente celular, que não pode ser alcançado em sistemas tradicionais de cultura 2D. Este sistema incentiva e aumenta a manutenção da pluripotência, embora as pistas relacionadas à bio-mecanosensing.
Os procedimentos técnicos aqui descritos são estratégias simples para permitir a indução e manutenção de um estado de alta plasticidade em células somáticas adultas. O protocolo permitiu a derivação de células de alta plasticidade em todas as espécies de mamíferos testadas. Uma vez que não envolve o uso de transfecção genética e é livre de vetores virais, pode representar um notável avanço tecnológico para aplicações de medicina translacional. Além disso, o sistema micro-bioreator fornece um notável avanço na tecnologia organoide de células-tronco, recriando in vitro um microambixo específico que permite a cultura de longo prazo de células de alta plasticidade, ou seja, como ESCs, iPSCs, células epigeneticamente apagadas e MSCs.

O presente protocolo descreve todas as etapas a serem executadas para gerar e manter as células pluripotentes dos mamíferos a partir de células somáticas adultas. Este método tem sido bem sucedido com fibroblastos isolados de diferentes espécies de mamíferos, ou seja, camundongos, suínos e humanos. Os resultados representativos aqui relatados são obtidos de todas as linhas celulares, independentemente da espécie de origem.
Análises morfológicas mostram que, após a incubação de 1...

Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Apresentamos aqui um método que combina o uso de apagamento epigenético químico com pistas relacionadas à mecanosensagem para gerar eficientemente células pluripotentes de mamíferos, sem a necessidade de transfecção genética ou vetores retrovirais. Essa estratégia é, portanto, promissora para a medicina translacional e representa um notável avanço na tecnologia organoide de células-tronco.

Uma estratégia de duas etapas que combina modificação epigenética e sinais biomecânicos para gerar células pluripotentes mamíferas

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

a-two-step-strategy-that-combines-epigenetic-modification

Research

JoVE Journal

Biology

2.2K visualizações.  Università degli Studi di Milano. Apresentamos aqui um método que combina o uso de apagamento epigenético químico com pistas relacionadas à mecanosensagem para gerar eficientemente células pluripotentes de mamíferos, sem a necessidade de transfecção genética ou vetores retrovirais. Essa estratégia é, portanto, promissora para a medicina translacional e representa um notável avanço na tecnologia organoide de células-tronco.

Vídeo: A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing e Subcultura células de mamíferos

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Biologia

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Cromatina imunoprecipitação (chips) para a Modificação do ensaio dinâmico Histone na expressão dos genes ativados em células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualização de mRNAs retículo endoplasmático localizados em células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-Sua purificação: A Two-step Affinity Purification Protocolo Cedendo Full-length proteínas purificadas

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

Isolamento de hepatócitos humanos por duas etapas Collagenase Perfusão Procedimento

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Reverter levedura Sistema de dois híbridos para identificar mamíferos Receptor Nuclear Resíduos que interagem com ligantes e / ou antagonistas

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

Automatizando Experiments chip-seq para gerar perfis epigenéticos em 10.000 células HeLa

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...