Agradecemos Hisayoshi Nozaki (Universidade de Tóquio), Takashi Nakada (Keio University), Yuka Marukawa Hashimoto (Universidade feminina do Japão) e Hiroyuki Sekimoto (Universidade feminina do Japão). Gostaríamos de agradecer os revisores para suas observações. Este estudo foi suportado por um subsídio para investigação científica (n. º 26440223 e 15H 04413) da sociedade para a promoção da ciência, Japão, Japão e por uma concessão da Fundação de desenvolvimento de nova tecnologia para Yuki Tsuchikane.

1. a recolha de uma amostra de água contendo algas
Nota: Conjugatophytes unicelulares crescer em áreas de água doce estagnadas, como Lagos, pântanos e arrozais. Uma amostra de água contendo algas é recolhida a partir de um desses ambientes para produzir uma tensão de cultura. Os seguintes itens são necessários antes de iniciar o processo: uma rede de plâncton, uma pipeta descartável e uma garrafa ou um tubo de 50 mL para a coleta de amostras de água.
<ol><li>Use uma rede em um ambiente de Lago de plâncton para coletar as algas da água. Use uma rede apropriada, dependendo da finalidade. Nota: A parte superior da rede é permeável à água. As algas pego pela net concentram-se no recipiente inferior da rede de plâncton. Uma malha grossa permite que pequenas algas a passagem. Uma malha fina entupir facilmente.</li>
	<li>Transferi a água (cerca de 50 mL) para o tubo de 50 mL ou uma garrafa de plástico de 100 mL. Levar a amostra de água concentrada para o laboratório.</li>
	<li>Conjugatophytes unicelulares também crescem em pântanos ou arrozais. Nestas áreas de águas rasas, a amostra de água contendo algas pode ser degustada diretamente com um conta-gotas. Em seguida, transferi a água (30-50 mL) para o tubo de 50 mL ou uma garrafa de plástico de 50 mL. Nota: A água cuidadosamente colhidas amostras acima da superfície do solo para que a amostra não contém qualquer tipo de solo.</li>
</ol>2. a confirmação das algas
Nota: Os seguintes itens são necessários: uma lupa ou microscópio portátil e um 60 mm prato para observação.
<ol><li>Para confirmar a presença de algas na amostra enquanto na pipeta descartável, use a lupa para observar a amostra diretamente. Nota: Células maiores de aproximadamente 400-1.000 µm de comprimento (por exemplo, Closterium ehrenbergii) pode ser facilmente observado usando esse método.</li>
	<li>Para confirmar as algas sob o microscópio portátil, despeje a amostra de água em um prato de plástico do laboratório.<ol>
<li>Colocar a amostra (cerca de 3 mL) na parte interna da parte superior (tampa) do prato de Petri 60 milímetros, em seguida, coloque a metade inferior, com a parte inferior virada para a parte interna da tampa, na parte superior. Nota: Esse método impede que a amostra se movendo e facilita a observação.</li>
	</ol>
</li>
</ol>3. preparação de uma pipeta capilar de vidro de uma pipeta Pasteur para a isolação
Nota: Antes de iniciar o processo, os seguintes itens são necessários: esterilizar pipetas Pasteur, um rack para as pipetas Pasteur, um bulbo de borracha, luvas, uma lâmpada de espírito, metanol para a lâmpada do espírito, fórceps, um isqueiro, uma lata de lixo para o vidro e uma sala limpa ou laminar capa de fluxo (se necessário).
<ol><li>Acenda uma lamparina.<ol>
<li>Para produzir uma pipeta capilar de vidro fino para o isolamento, certifique-se que a chama não está piscando. Aguçar a chama e derrete a pipeta Pasteur na sua ponta.</li>
	</ol>
</li>
	<li>Um vidro estéril pipeta Pasteur na chama, apoiada na parte lateral do bulbo de borracha com a mão e no lado de ponta usando fórceps de calor.</li>
	<li>Levar a pipeta Pasteur fora a chama e esticá-lo.<ol>
<li>Quando o vidro está a derreter, retire a pipeta Pasteur rapidamente a chama e puxe. Nota: Neste experimento, a pipeta Pasteur foi prorrogada por 15-20 cm. fazer claro a pipeta é desligado a chama durante o puxando.</li>
	</ol>
</li>
	<li>Quebre a ponta da pipeta vidro capilar para o comprimento correcto. Porque a parte da curvando-se é o melhor, é ideal para a ponta. Nota: Neste estudo, foi preparada uma pipeta capilar de vidro com uma ponta de 5 a 10 cm.<ol>
<li>Certifique-se de descartar o capilar depois derrete-lo, e confirmar que a chama é posta para fora.</li>
	</ol>
</li>
	<li>As bolhas libertadas a pipeta capilar de vidro indicam o tamanho da ponta da abertura. Selecione o tamanho apropriado para a célula a ser isolada. Cerca de 1 mm de bolhas é adequada para o isolamento de espécies Closterium , com larguras de célula de 10-20 µm.</li>
</ol>4. célula única isolamento por uma pipeta capilar de vidro
Nota: Os seguintes itens são necessários antes de começar: um microscópio invertido, vidros de relógio, um prato de plástico e estéril rosca tubos de ensaio contendo meio de CA (tabela 1) ou condicionado CA média7.
<ol><li>Prepare alguns vidros de relógio (22 mm de diâmetro e 1 mm de profundidade), cada contendo 1 mL de meio estéril de CA.</li>
	<li>Isole uma célula-alvo da amostra de água coletada no campo.<ol>
<li>Despeje a amostra de água (cerca de 30-40 mL) que contém as células de destino para o prato de plástico.</li>
		<li>Enquanto observa a amostra no prato plástico sob um microscópio invertido, pega células únicas usando a pipeta capilar de vidro.</li>
		<li>Trazer a pipeta capilar de vidro perto da célula para facilitar a aspiração da célula por ação capilar.</li>
	</ol>
</li>
	<li>Transferi as células em um vidro de relógio contendo 1 mL de meio de CA estéril (Figura 1a).</li>
	<li>Pegar as células novamente usando uma pipeta capilar de vidro novo do meio estéril de CA e transferi-lo para um segundo vidro de relógio contendo meio estéril de CA (Figura 1b). Nota: Este processo é repetido até que uma única célula é isolada na placa local (Figura 1C).</li>
	<li>Pegar a única célula usando uma pipeta capilar de vidro novo e finalmente, transferi-lo para um tubo de ensaio contendo 14 mL de meio de CA ou condicionados CA médio (Figura 1 d). Incubar a amostra a 23 ° C, sob uma luz: 8 16-h-h escuro ciclo por 2 semanas. Nota: O factor desconhecido para a proliferação celular, que é secretadas do crescimento de células para o meio ambiente, está incluídos no meio condicionado para facilitar a divisão celular4. Se for estabelecida uma cultura clonal, prepare o suporte de CA condicionado do meio de cultura4. A cultura vai ficar verde, se for bem sucedida.</li>
</ol>

<ol>
	<li>Wickett, N. 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=Phylotranscriptomic+analysis+of+the+origin+and+early+diversification+of+land+plants.">Phylotranscriptomic analysis of the origin and early diversification of land plants.</a> Proceedings of the National Academy of Sciences. 111, E4859-E4868 (2014).</li><li>Delwiche, C. F., Cooper, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+origin+of+a+terrestrial+flora.">The evolutionary origin of a terrestrial flora.</a> Current Biology. 25, R899-R910 (2015).</li><li>Daste, P., Neuville, D., Victor-Baptiste, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+obtaining+clonal+isolation+of+diatoms.">A simple procedure for obtaining clonal isolation of diatoms.</a> British Journal of Psychology. 18, 1-3 (1983).</li><li>Pringsheim, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pure Cultures of Algae: Their Preparation and Maintenance. , 119 (1946).</li><li>Sieracki, M., Poulton, N., Crosbie, N., Andersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+isolation+techniques+for+microalgae.">Automated isolation techniques for microalgae.</a> Algal Culturing Techniques: Automated isolation techniques for microalgae. , 101-116 (2005).</li><li>Andersen, R. A., Kawachi, M., Andersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+microalgae+isolation+techniques.">Traditional microalgae isolation techniques.</a> Algal Culturing Techniques: Traditional micro algae isolation techniques. , 83-100 (2005).</li><li>Abe, 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=Stable+nuclear+transformation+of+the+Closterium+peracerosum-strigosum-littorale+complex.">Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex.</a> Plant and Cell physiology. 52, 1676-1685 (2011).</li><li>Tsuchikane, Y., Tsuchiya, M., Kokubun, Y., Abe, J., Sekimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+processes+of+Penium+margaritaceum+(Zygnemophyceae,+Charophyta).">Conjugation processes of Penium margaritaceum (Zygnemophyceae, Charophyta).</a> Phycological Research. 59, 74-82 (2011).</li><li>Kanda, 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=CRISPR/Cas9-based+knockouts+reveal+that+CpRLP1+is+a+negative+regulator+of+the+sex+pheromone+PR-IP+in+the+Closterium+peracerosum-strigosum-littorale+complex.">CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.</a> Scientific Reports. 7, 17873 (2017).</li><li>Ichimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+types+and+reproductive+isolation+in+Closterium-ehrenbergii+Meneghini.">Mating types and reproductive isolation in Closterium-ehrenbergii Meneghini.</a> Botanical Magazine-Tokyo. 94, 325-334 (1981).</li><li>Sekimoto, H., Satoh, S., Fujii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+physiological+properties+of+a+protein+inducing+protoplast+release+during+conjugation+in+the+Closterium+peracerosum-strigosum-littorale+complex.">Biochemical and physiological properties of a protein inducing protoplast release during conjugation in the Closterium peracerosum-strigosum-littorale complex.</a> Planta. 182, 348-354 (1990).</li><li>Ichimura, T., Watanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Closterium+calosporum+complex+from+the+Ryukyu+Islands-Variation+and+taxonomical+problems.">The Closterium calosporum complex from the Ryukyu Islands-Variation and taxonomical problems.</a> Memoirs of the National Science Museum. 7, (1974).</li><li>Provasoli, L., Pintner, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+media+for+fresh-water+algae:+problems+and+suggestions+.">Artificial media for fresh-water algae: problems and suggestions .</a> The Ecology of Algae, a symposium held at the Pymatuning Laboratory of Field Biology on June 18 and 19, 1959. , 84-96 (1960).</li><li>Provasoli, L., Tryon, C. A., Hartmann, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media++&amp;+prospects+for+the+cultivation+of+marine+algae.">Media &amp; prospects for the cultivation of marine algae.</a> Proceedings of U. S.-Japan Conference in Hakone, Japan. , 63-75 (1968).</li><li>Guillard, R. R. L., Andersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+methods+for+microalgae.">Purification methods for microalgae.</a> Algal Culturing Techniques: Purification methods for microalgae. , 117-132 (2005).</li></ol>

Usando o método presente, cepas de 9 cultura clonal de SP. Closterium foram estabelecidas de 15 células isoladas da amostra de água (Inba1), que representa uma taxa de sucesso de 60%. Identificação de espécies realizarão no futuro por observação morfológica, bem como análises de ADN, tais como análise filogenética molecular8.
No presente método, o frescor da amostra de água é importante para a taxa de sucesso. É melhor isolar as células a partir das amostras de água logo que possível após a coleta de campo. Embora as células da espécie Closterium (por exemplo, c. ehrenbergii, c. peracerosum-strigosum-littorale complexo) podem ser mantidas na amostra de água para cerca de 7 d armazenando-a 4-8 ° C, o isolamento das células é desejável no dia da coleção ou no dia seguinte. Também é importante remover todos os contaminadores que não sejam as células alvo, aumentando assim o número de repetições de lavagem (i.e., > 3 x) podem impedir a contaminação das culturas por microorganismos no solo e células.
Uma limitação dessa técnica é que os meios de cultura utilizados neste protocolo (CA ou condicionados CA médio) nem sempre são adequados para todas as algas de conjugação. Em alguns casos, pode ser necessário considerar o uso de outro meio, bem como a luz diferente e a condições de temperatura. Além disso, como é difícil provar se uma cultura cresceu de uma única célula, é necessário buscar com precisão apenas 1 célula ao transferir células para um tubo de ensaio. Portanto, a transferência deve ser feito com uma nova pipeta capilar de vidro cada vez.
Estabelecer uma cultura clonal com este método de lavagem de pipeta é um método clássico/padrão e é o método mais confiável para usar para isolar as células desejadas para um estudo. Estabeleceram-se as culturas clonais de conjugação algas usadas em muitos estudos8,9,10,11 usando o método actual. É difícil analisar a conjugação algas sem uma cultura clonal. Além disso, nos últimos anos, sequências do genoma inteiro de novo tornou-se fácil de obter (por exemplo, usando o MinION nanopore sequencer), e estabelecer culturas clonais facilitará ainda mais o sequenciamento de novo . Em outras palavras, esse método é o primeiro passo no futuro estudar destas algas para compreender melhor a sua biodiversidade.
A fim de estabelecer uma tensão estável de cultura, é necessário realizar determinadas etapas críticas com precisão dentro do protocolo. O passo mais importante é a preparação de uma pipeta capilar de vidro com uma abertura ideal para permitir a passagem de apenas uma única célula. A abertura da pipeta capilar de vidro deve ser ligeiramente mais do que o comprimento do eixo menor da célula de interesse. A pipeta capilar de vidro ideal pode aspirar a uma única célula por ação capilar. No entanto, se o diâmetro da abertura é muito grande, o capilar também desenhará em materiais de contaminantes não-vivos ou mesmo (um) outro microrganismo, além da célula-alvo.
Para obter uma pipeta capilar de vidro com um ideal de abertura, os seguintes pontos são importantes para observação. Primeiro, o pilar de fogo deve ser estreito ao aquecer a pipeta capilar de vidro. Em seguida, quando puxar a pipeta Pasteur, tem que ser removido da chama; caso contrário, a abertura da pipeta Pasteur entrará em colapso.
Os equipamentos e recipientes mostrados neste protocolo são básicos e podem ser modificados. Por exemplo, usando placas multi bem em vez de vidros de relógio com um poço, a lavagem da célula pode ser feita mais eficiente. Além disso, os recipientes podem ser substituídos por outros semelhantes. Através da transferência de uma única célula para o meio de cultura para a última etapa, uma cultura clonal pode ser estabelecida.
Preparar um recipiente esterilizado e trabalhando em um capuz estabelecerá axénica cepas (não contaminadas). Para evitar a contaminação, é necessário repetir a etapa de lavagem com alíquotas frescas mais de 3x. Às vezes, as bactérias também são esterilizadas pela adição de antibióticos15.
Esse método focado em gênero (Zygnematales) isolamento, mas alterando o tamanho da abertura da pipeta capilar de vidro, pode ser aplicado para o isolamento de diferente tamanho plâncton.

Conjugação de algas (da ordem Zygnematales), também conhecido como conjugatophytes, ocupam uma posição-chave filogenética como os parentes vivos mais próximos dos antepassados imediatos da terra plantas1,2. Estabelecidas culturas clonais de algas têm levado a uma grande variedade de estudos taxonômicos, fisiológicos e genéticos ao longo dos últimos anos. As técnicas de isolamento de microorganismos de um campo natural têm uma longa tradição de3,4. Nos últimos anos, isolamento automatizado técnicas usando citometria de fluxo foram também estabelecidos5. O método mais comum usado para obter uma única célula para o estabelecimento de uma cultura clonal é isolamento de célula única usando uma pipeta capilar de vidro6. Esse método tradicional requer habilidade e um protocolo experimental preciso.
Neste artigo, demonstramos a amostragem de algas de amostras de campo e o estabelecimento de culturas clonais de algas unicelulares de conjugação, tais como a espécie Closterium , usando uma pipeta capilar de vidro. Uma amostra de água contendo as células vegetativas é coletada de um site de campo (por exemplo, um lago, lagoa ou arrozal). As células são isoladas da amostra de água usando uma pipeta capilar de vidro e em seguida lavadas. As células são cultivadas em um tubo de ensaio contendo meio de nitrogênio-completada (médio CA) a 24 ° C, sob uma luz: 8 16-h-h regime de escuro. Esse método também é apropriado para isolar outras microalgas para gerar culturas clonais.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sampling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plankton net</td>
			<td>RIGO, Japan</td>
			<td>N-NO380T</td>
			<td>Mesh size: 32 &#181;m</td>
		</tr>
		<tr>
			<td>Portable microscope (e.g., Nature Scope FABRE)</td>
			<td>Nikon, Japan</td>
			<td>JAN: 4960759 206725</td>
			<td>Magnification: 20&#215;</td>
		</tr>
		<tr>
			<td>Loupe (e.g., Peak 1985 Steinheil System Magnifier)</td>
			<td>Tohkai Sangyo Co. Ltd., Japan</td>
			<td>1985-20</td>
			<td>Magnification: 20&#215;</td>
		</tr>
		<tr>
			<td>Spuit</td>
			<td>EIKEN CHEMICAL CO. LTD. Japan</td>
			<td>Sterile spuit No.4</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Tube (50 mL centrifuge tubes with screw caps)</td>
			<td>Labcon, USA</td>
			<td>3181-345-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle (100 mL Polycarbonate bottle)</td>
			<td>AS ONE Corporation, Japan</td>
			<td>1-7403-01</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm dish</td>
			<td>Asahi glass Co. Ltd., Japan</td>
			<td>1010-060</td>
			<td>&#160;For observation of algae.&#160; 60 mm/non-treated dish</td>
		</tr>
		<tr>
			<td>Preparation of a micropipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (cotton plugged 9&#8221; Pasteur Pipettes)</td>
			<td>Fisher Scientific, USA</td>
			<td>13-678-8B</td>
			<td>7 &#215; 225 mm</td>
		</tr>
		<tr>
			<td>Rubber bulb (SPOID SILICONE, 1 cc)</td>
			<td>AS ONE Corporation, Japan</td>
			<td>5-5669-01</td>
			<td>10 &#215; 40 mm</td>
		</tr>
		<tr>
			<td>Stainless forceps</td>
			<td>AS ONE Corporation, Japan</td>
			<td>PT-09</td>
			<td>110 mm</td>
		</tr>
		<tr>
			<td>Single-cell isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass (Blood reaction board)</td>
			<td>Sekiya Rika Co., Ltd, Japan</td>
			<td>F14-155-030</td>
			<td>to rinse cells during isolation. 22 mm diameter and 1 mm depth</td>
		</tr>
		<tr>
			<td>Threaded test tubes&#160;</td>
			<td>Fujimotorika, Co., Ltd, Japan</td>
			<td>XX142</td>
			<td>18 &#216; &#215; 170 mm</td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>CKX41N</td>
			<td>for isolation of algae.</td>
		</tr>
		<tr>
			<td>Plastic dish</td>
			<td>Asahi glass Co. Ltd., Japan</td>
			<td>SH90-15E&#160;</td>
			<td>&#160;90 &#215; 15 mm</td>
		</tr>
	</tbody>
</table>

establishment of a clonal culture of unicellular conjugating algae

É essencial para estabelecer culturas clonais de microalgas para uso em estudos de vários temas, tais como fisiologia, genética, taxonomia e Microbiologia. Assim, é extremamente importante desenvolver técnicas para estabelecer culturas clonais. Neste artigo, vamos demonstrar o estabelecimento de culturas clonais de uma alga de conjugação. São coletadas amostras de água do campo. Posteriormente, as células são isoladas, usando uma pipeta capilar de vidro, colocado na mídia e cresceu em condições adequadas para a geração de uma cultura clonal.

Uma amostra de água (Inba1) de 50 mL foi coletada de um ponto de amostragem no lago Inba-numa (pH 8.1, 24,7 ° C; 35 ° 44′30. 2″N 140 ° 12′08. 2″E) no Sakura-shi, Chiba, Japão, em 25 de junho de 2016. A amostra de água era armazenada em 4-8 ° C. No dia seguinte após a coleta, uma amostra de Closterium SP. foi isolado a partir da amostra de água contendo as células vegetativas. As células vegetativas quinze de morfologia idêntica (Inba1-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12,-13, -14 e -15) foram isoladas a partir da amostra e em seguida, lavou usando a pipeta método de lavagem. Quinze células únicas isoladas foram cultivadas em tubos de ensaio independentes, contendo 15 mL de meio de CA condicionado. Depois de 2 semanas de incubação, observou-se proliferação celular em 9 tubos de ensaio [Inba1-1, -3, -4, -5 (Figura 2a), -6, -9, -10, -12 (Figura 2b) e-13; Tabela 2]. Estabeleceram-se nove culturas clonais dos Inba1 isolados de amostra (Figura 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57761/57761fig1.jpg"> Figura 1 : O fluxo de isolamento de célula única. (a) transferência a célula algal único alvo a amostra de água original para o estéril médio no primeiro vidro de relógio. (b, c) Transferi o celular novamente para o vidro de relógio próximo para lavar. Este procedimento deve ser repetido até que não haja nenhuma outras células/contaminantes do que o alvo a médio; três vidros de relógio são mostrados no diagrama aqui como exemplo. (d) finalmente, transferir a célula lavada para um tubo de ensaio do meio adequado para o crescimento. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57761/57761fig2v2.jpg"> Figura 2 : Fotografias de células vegetativas de Closterium SP. (um) Inba1-5) e (b) Inba1-12 são mostrados aqui. Barra de escala = 50 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig2v2large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57761/57761fig3.jpg"> Figura 3 : Estabelecimento de uma cultura clonal da amostra Inba1. Os tubos de ensaio, cultivados por 2 semanas foram fotografados do fundo. Um asterisco indica a proliferação celular. Barra de escala = 2 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig3large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Nome da mídia</td>
			<td>Referência</td>
			<td colspan="2">Comentários</td>
		</tr>
<tr>
<td rowspan="13">CA</td>
			<td rowspan="13">Ichimura e Watanabe12
</td>
			<td>O CA (não3)2·4H2</td>
			<td>2 mg</td>
		</tr>
<tr>
<td>KNO3
</td>
			<td>10 mg</td>
		</tr>
<tr>
<td>NH4não3
</td>
			<td>5 mg</td>
		</tr>
<tr>
<td>Β – at2glycerophosphate·5H2O</td>
			<td>3 mg</td>
		</tr>
<tr>
<td>O MgSO4·7H2</td>
			<td>2 mg</td>
		</tr>
<tr>
<td>Vitamina B12</td>
			<td>0,01 μg</td>
		</tr>
<tr>
<td>Biotina</td>
			<td>0,01 μg</td>
		</tr>
<tr>
<td>Tiamina HCl</td>
			<td>1 μg</td>
		</tr>
<tr>
<td>Metais PIV</td>
			<td>0,1 mL</td>
		</tr>
<tr>
<td>Fe (como EDTA; molar de 1:1)</td>
			<td>0,1 mL</td>
		</tr>
<tr>
<td>HEPES</td>
			<td>40 mg</td>
		</tr>
<tr>
<td colspan="2">Adicione água para completar 100 mL</td>
		</tr>
<tr>
<td colspan="2">ajustar o pH com NaOH a 7.2</td>
		</tr>
<tr>
<td rowspan="7">Metais P IV</td>
			<td rowspan="7">Provasoli e Pintner13
</td>
			<td>At2EDTA·2H2, O</td>
			<td>100 mg</td>
		</tr>
<tr>
<td>O FeCl3·6H2</td>
			<td>19,6 mg</td>
		</tr>
<tr>
<td>MnCl2· 4H2O</td>
			<td>3,6 mg</td>
		</tr>
<tr>
<td>ZnCl2*</td>
			<td>1,04 mg</td>
		</tr>
<tr>
<td>O CoCl2·6H2</td>
			<td>0,4 mg</td>
		</tr>
<tr>
<td>At2MoO4·2H2, O</td>
			<td>0,25 mg</td>
		</tr>
<tr>
<td colspan="2">Adicione água para completar 100 mL</td>
		</tr>
<tr>
<td rowspan="3">Fe (como EDTA; molar de 1:1)</td>
			<td rowspan="3">Provasoli14
</td>
			<td>Fe (NH4)2(então4)2·6H2O,</td>
			<td>70,2 mg</td>
		</tr>
<tr>
<td>Na2EDTA·2H2O,</td>
			<td>66 mg</td>
		</tr>
<tr>
<td colspan="2">Adicione água para completar 100 mL</td>
		</tr>
<tr>
<td>Médio de CA condicionado</td>
			<td>Abe et al.7
</td>
			<td colspan="2">Incube as células em meio fresco CA 14 – 20 dias. Coletar o médio cultivado por filtração utilizando papel de filtro qualitativo e esterilizar o meio filtrado em autoclave (121 ° C por 15 min).</td>
		</tr>
<tr>
<td colspan="4">* O NIES, 1,04 mg ZnCl2 redacção 2,2 mg ZnSO4·7H2O.</td>
		</tr>
</tbody></table>Tabela 1: Formulações de meios de comunicação utilizados neste estudo.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Amostra de água</td>
			<td>Nome da célula</td>
			<td>Estatuto após a cultura</td>
			<td>Nome de estirpe</td>
		</tr>
<tr>
<td rowspan="15">Inba1</td>
			<td>-1</td>
			<td>As células se proliferaram</td>
			<td>Inba1-1</td>
		</tr>
<tr>
<td>-2</td>
			<td>Não mudou</td>
			<td></td>
		</tr>
<tr>
<td>-3</td>
			<td>As células se proliferaram</td>
			<td>inba1-3</td>
		</tr>
<tr>
<td>-4</td>
			<td>As células se proliferaram</td>
			<td>inba1-4</td>
		</tr>
<tr>
<td>-5</td>
			<td>As células se proliferaram</td>
			<td>inba1-5</td>
		</tr>
<tr>
<td>-6</td>
			<td>As células se proliferaram</td>
			<td>inba1-6</td>
		</tr>
<tr>
<td>-7</td>
			<td>Não mudou</td>
			<td></td>
		</tr>
<tr>
<td>-8</td>
			<td>Não mudou</td>
			<td></td>
		</tr>
<tr>
<td>-9</td>
			<td>As células se proliferaram</td>
			<td>inba1-9</td>
		</tr>
<tr>
<td>-10</td>
			<td>As células se proliferaram</td>
			<td>inba1-10</td>
		</tr>
<tr>
<td>-11</td>
			<td>Não mudou</td>
			<td></td>
		</tr>
<tr>
<td>-12</td>
			<td>As células se proliferaram</td>
			<td>inba1-12</td>
		</tr>
<tr>
<td>-13</td>
			<td>As células se proliferaram</td>
			<td>inba1-13</td>
		</tr>
<tr>
<td>-14</td>
			<td>Não mudou</td>
			<td></td>
		</tr>
<tr>
<td>-15</td>
			<td>Não mudou</td>
			<td></td>
		</tr>
</tbody></table>Tabela 2: O isolamento julgamento sumário.

Watch this Scientific Journal Video about Establishment of a Clonal Culture of Unicellular Conjugating Algae at JoVE.com

Establishment of a Clonal Culture of Unicellular Conjugating Algae

Estabelecimento de uma cultura Clonal de algas unicelulares de conjugação

Neste artigo, vamos demonstrar o estabelecimento de culturas clonais de espécies de algas conjugação unicelulares coletadas de um site de campo natural.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

establishment-of-a-clonal-culture-of-unicellular-conjugating-algae

Nanyang Technological University

Research

JoVE Journal

Biology

13.1K visualizações.  Japan Women's University. Neste artigo, vamos demonstrar o estabelecimento de culturas clonais de espécies de algas conjugação unicelulares coletadas de um site de campo natural.

Vídeo: Establishment of a Clonal Culture of Unicellular Conjugating Algae

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biologia

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Isolamento de CD133 + células tronco do fígado de Expansão Clonal

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Scale Up de Cultura de células de mamíferos utilizando um Flask New Multicamadas

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Estabelecimento de Culturas Microbianas enriquecimento eucarióticas a partir de um quimicamente estratificada Antarctic Lake e Avaliação do Potencial de Fixação de Carbono

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

O método de cultura fatia para acompanhar o desenvolvimento de germes dentários Em Explant Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In Vitro Reconstituição de Complexos de Light-colheita de plantas e algas verdes

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Estabelecimento de um pH extracelular ácido sistema de cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

Estabelecimento e Manutenção de Baratas Gnotobióticas Americanas (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Cultura do tecido do córtex ovariano bovino

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.