This research was supported by the S-Innovation project of the Japan Science and technology Agency (JST), the Graduate Program for Leaders in Life Innovation (GPLLI) of the University of Tokyo, and the Research Fellowship for Young Scientists of Japan Society for the Promotion of Science. We thank nac Image Technology Inc. for taking movies using a hi-speed camera.

1. Materiais Preparando <ol><li> Preparar tampão HEPES 10 mM. Ajustar o pH para 7,0 à TA e adicionar NaCl a 0,9%. </li><li> Preparar a solução de alginato e 5% de solução de EDTA a 10 mM através da mistura de solução salina tamponada com HEPES preparado em 1.1. Ajustar o pH para 7,0 à temperatura ambiente. </li><li> Autoclave os reagentes (1.1, 1.2) durante 20 min a 121 ° C. </li><li> Preparar 60 milímetros pratos revestidos com gelatina em camadas com 1,2-2,0 x 10 6 de fibroblastos de rato embrionário (MEF) células, as quais são tratadas como células de alimentação por incubação dentro de DMEM contendo 10% de FBS ES-qualificados e 10 ug / ml de mitomicina C para 90 - 120 min. </li><li> Manter rato induzida estaminais pluripotentes (IPS) sobre o prato de células com as células de alimentação na presença de 5 ml de DMEM de alto teor de glucose contendo 20% de FBS ES-qualificado, 50 uM de 2-mercaptoetanol, aminoácidos não-essenciais, e antibióticos ( 100 unidades / ml de penicilina G sódio, 100 ug / ml de sulfato de estreptomicina, e 250 ng / ml de amphotericin B). </li><li> Semente 4 × 10 5 células iPS sobre uma placa de 60 mm revestidas com gelatina e incubar a 37 ° C e 5% de CO 2. Alterar o meio de cultura todos os dias durante a incubação. Após 3-4 dias de cultura, as células trypsinize durante 1 min com 500 ul de tripsina a 0,05% contendo EDTA a 0,02% a 37 ° C e 5% de CO 2. Depois disso, retire as células tocando no prato de cultura dez vezes. </li><li> Adicionar 2,5 ml de DMEM com 10% de FBS e antibióticos ES-qualificado (seguindo as células iPS dissociação do rato em células individuais por pipetagem com 1.000 mL micropipeta, três vezes) suspensão de células centrifugar a 160 × g por 3 min e remover o sobrenadante e contar as células . Seguindo a contagem das células, propagar novamente as células recolhidas num prato revestido com gelatina e incubar durante 30 min para isolar as células iPS a partir das células MEF. Após a contagem das células (geralmente 1-3 × 10 6 células / prato pode ser coletado), reseed 4 × 10 5 células iPS emo prato. </li></ol> 2. celular encapsulação em cápsulas de Hidrogel de Alginato <ol><li> Recolher as células por o mesmo procedimento descrito em 1.5 e 1.6. Após a centrifugação a 160 x g durante 3 minutos, lava-se a pelete de células iPS com solução salina tamponada com HEPES três vezes. Depois de re-suspender as células em solução salina tamponada com HEPES, filtrar a suspensão de células através de um filtro de 40 um da célula, a fim de remover grandes agregados, o que poderia de outro modo entupir o bocal. </li><li> Misturar 2 ml de suspensão de células numa solução salina tamponada com HEPES e 3 ml de solução a 5% Alg-Na. Finalmente 5 ml de suspensão de células numa solução de 3% Alg-Na é obtido. A densidade celular é desejavelmente superior a 10 6 células / ml. </li><li> Depois de recolher a suspensão de células para uma seringa de 5 ml, instalar um bocal co-axial (Figura 1A, B) na seringa e colocá-las sobre a bomba de seringa. Emit N2 fluxo de gás (inferior a 1 L / min) através da agulha exterior do bico de co-axial eexpelir a suspensão de células através do bocal interno. </li><li> Recolhe gotas em 250 ml de 0,5% de solução de CaCl2 e esperar 10-20 min para a gelificação enquanto se agitava a 60-90 rpm. </li></ol> 3. Tratamento de cápsulas de alginato (opcional) <ol><li> Incubar cápsulas Alg-Ca em 0,05% de poli-L-lisina (PLL) solução durante 5 min a 37 ° C e 5% (w / v) de CO 2. </li><li> Após lavagem das cápsulas com solução salina tamponada com HEPES, incubar em DMEM contendo FBS a 10% a fim de neutralizar a carga eléctrica na sua superfície. Utilizá-los na forma de cápsulas revestidas (Figura 1C). </li><li> Incubam-se as cápsulas em solução salina tamponada com HEPES contendo 10 mM de EDTA durante 5 minutos. As cápsulas tratado com EDTA são utilizados na forma de cápsulas ocas (Figura 1C). </li></ol> 4. Cultura e coletar células em alginato Cápsulas <ol><li> Incubar as células encapsuladas a 75 rpm com agitação a 37 ° C e 5% de CO 2durante 10 dias. </li><li> Recolha e incubar as cápsulas em EDTA 10 mM a 37 ° C e 5% de CO 2 durante 10 min. Recolher as células a partir das cápsulas de alginato PLL sem tratamento por centrifugação a 1000 × g durante 3 min. </li><li> Se as cápsulas de alginato são tratadas com PLL, romper a membrana Alg-PLL, pipetando para cima e para baixo em torno de dez vezes com uma agulha ligada a uma seringa e centrifuga-se que em 1000 - 5000 × g durante 5 min. Diâmetro da agulha deve ser menor do que o tamanho da cápsula e 25 g (260 ^ m) de agulhas são utilizadas nesta experiência (600 um) </li><li> Após a centrifugação, coletar sedimento celular estritamente a partir de membranas Alg-PDD quebrados se você quiser coletar amostras de mRNA a partir de células. Restantes membranas Alg-PLL possivelmente prevenir a purificação de ARNm. </li></ol>

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The authors have nothing to disclose.

<html> A encapsulação pode ser cultura em comparação com as culturas em suspensão directos. Cultura em suspensão é um método mais simples para obter grandes quantidades de células estaminais pluripotentes que os métodos de encapsulamento. No entanto, controlando a agregação de células numa cultura em suspensão é ainda difícil. No método de encapsulamento, agregação celular é limitado em cápsulas e podem, portanto, ser bem controlada. A publicação anterior mostrou que células encapsuladas formado a...

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient1. However, conventional dish culture is only able to obtain 1 &#215; 105 cells/cm2, thus requiring 1 m2 of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier2 and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells3. Indeed, suspended cells are exposed to shear stress, which causes cell damage3 or differentiation4.
Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose5, PEG6, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg&#8722;Ca hydrogel is formed immediately after dropping alginate solution into a CaCl2 solution and is also readily digested by enzymes or chelating reagents. 
Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N2 gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N2 and concentration of both CaCl2 and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mouse embryo fibroblast</td>
			<td>Cell Biolabs</td>
			<td>SNL 76/7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse induced pluripotent stem cell</td>
			<td>RIKEN Bio resorce centre</td>
			<td>iPS-MEF-Ng-20D-17</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high-glucose</td>
			<td>GIBCO</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>ES qualified&#160; FBS</td>
			<td>GIBCO</td>
			<td>16141079</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibacterial Antibiotics</td>
			<td>GIBCO</td>
			<td>15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acid</td>
			<td>GIBCO</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>GIBCO</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor</td>
			<td>Merck Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>GIBCO</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>26G/16G needle</td>
			<td>Hoshiseido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>TERUMO</td>
			<td>SS-10ESZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium&#160; Chloride</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Alginate</td>
			<td>Wako</td>
			<td>194-09955</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Wako</td>
			<td>039-00475</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (Mw=15,000-30,000)</td>
			<td>SIGMA</td>
			<td>P7890</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>DOJINDO</td>
			<td>345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylinge pump</td>
			<td>AS ONE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IRB</td>
			<td></td>
		</tr>
		<tr>
			<td>Hispeed camera</td>
			<td>nac image technology</td>
			<td>Memrecam HX-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

alginate encapsulation of pluripotent stem cells using a co-axial nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

No protocolo de 2,5, a solução de alginato expelido forma uma forma esférica imediatamente após a expulsão (Figura 2A - H). Se a suspensão é expulso com uma taxa de fluxo de N2 inferior a 1 L / min, o tamanho das gotículas é uniforme (Figura 2I). No entanto, se o fluxo de N2 é superior a 1 L / min, a gotícula de quebra (Figura 2G, de setas brancas) e o tamanho das gotas se torna heterogéneo (Figura 2J). Sendo assim, é ...

Watch this Scientific Journal Video about Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle at JoVE.com

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Encapsulation alginato de pluripotentes células estaminais utilizando um bocal Co-axial

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

alginate-encapsulation-pluripotent-stem-cells-using-co-axial

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Developmental Biology

12.5K visualizações.  The University of Tokyo. We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.