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. Materiales Preparación <ol><li> Preparar 10 mM de tampón HEPES. Ajustar el pH a 7,0 a temperatura ambiente y añadir NaCl al 0,9%. </li><li> Preparar la solución de alginato de 5% y solución de EDTA 10 mM mediante la mezcla de solución salina tamponada con HEPES preparado en 1.1. Ajustar el pH a 7,0 a temperatura ambiente. </li><li> Autoclave los reactivos (1.1, 1.2) durante 20 min a 121 ° C. </li><li> Preparar platos 60 mm recubiertos de gelatina en capas con 1,2 a 2,0 × 10 6 de fibroblastos embrionarios de ratón (MEF) las células, que se tratan como células alimentadoras por incubación en DMEM que contiene 10% de SFB ES-calificados y 10 g / ml de mitomicina C para 90 - 120 min. </li><li> Mantener inducida ratón madre pluripotentes (iPS) células en el plato con las células alimentadoras en presencia de 5 ml de DMEM de alta glucosa que contiene 20% de ES-calificado FBS, 50 mM de 2-mercaptoetanol, aminoácido no esencial, y antibióticos ( 100 unidades / ml de penicilina G sódica, 100 g / ml de sulfato de estreptomicina y 250 ng / ml de amphotericin B). </li><li> Semilla de 4 × 10 5 células iPS en un 60 mm plato de gelatina recubiertos y incubarlos a 37 ° C y 5% de CO 2. Cambie el medio de cultivo cada día durante la incubación. Después de 3 - 4 días de cultivo, trypsinize células durante 1 min con 500 l de tripsina al 0,05% que contenía 0,02% de EDTA a 37 ° C y 5% de CO 2. Después de eso, separar las células tocando la placa de cultivo diez veces. </li><li> Añadir 2,5 ml de DMEM con 10% de (tres veces siguientes iPS disociación de ratón células en células individuales por pipeteo con 1000 l micropipeta) FBS y antibióticos ES-calificado suspensión de células se centrifuga a 160 × g durante 3 min y retirar el sobrenadante y contar las células . Tras el recuento de células, resembrar las células recogidas en un plato de gelatina con recubrimiento y se incuba durante 30 min para aislar las células iPS a partir de células MEF. Después de contar de las células (generalmente 1-3 × 10 6 células / placa puede ser recogida), reseed 4 × 10 5 células iPS enel plato. </li></ol> 2. encapsulación de células en alginato de hidrogel Cápsulas <ol><li> Recoger las células por el mismo procedimiento descrito en 1.5 y 1.6. Después de centrifugación a 160 xg durante 3 min, lavar el sedimento de células iPS con solución salina tamponada con HEPES tres veces. Después de volver a suspender las células en solución salina tamponada con HEPES, filtrar la suspensión celular a través de un filtro de 40 micras de células con el fin de eliminar grandes agregados, que de otro modo podrían obstruir la boquilla. </li><li> Mezclar 2 ml de suspensión de células dentro de solución salina tamponada con HEPES y 3 ml de solución de Na Alg-5%. Finalmente se obtuvieron 5 ml de suspensión de células dentro de solución Alg-Na 3%. La densidad celular es deseablemente más de 10 6 células / ml. </li><li> Después de recoger la suspensión de células en una jeringuilla de 5 ml, instalar una boquilla co-axial (Figura 1A, B) en la jeringa y los puso en la bomba de jeringa. Emit N 2 de flujo de gas (inferior a 1 litro / min) a través de la aguja exterior de la boquilla co-axial yexpulsar la suspensión celular a través de la boquilla interior. </li><li> Recoger gotitas en 250 ml de solución de 0,5% CaCl 2 y esperar 10-20 min para la gelificación mientras se agita a 60-90 rpm. </li></ol> 3. Tratamiento de alginato Cápsulas (opcional) <ol><li> Incubar cápsulas Alg-Ca en 0,05% (w / v) de poli-L-lisina (PLL) solución durante 5 min a 37 ° C y 5% de CO 2. </li><li> Después de lavar las cápsulas con solución salina tamponada con HEPES, incubar en DMEM que contenía FBS al 10% para neutralizar la carga eléctrica en su superficie. Utilizarlos como cápsulas recubiertas (Figura 1C). </li><li> Incubar las cápsulas en solución salina tamponada con HEPES que contiene EDTA 10 mM durante 5 min. Las cápsulas tratados con EDTA se utilizan como cápsulas huecas (Figura 1C). </li></ol> 4. Cultura y recoger las células en cápsulas de alginato <ol><li> Se incuban las células encapsuladas en 75 rpm con agitación a 37 ° C y 5% de CO 2durante 10 días. </li><li> Recoger e incubar las cápsulas en EDTA 10 mM a 37 ° C y CO 2 al 5% durante 10 min. Recoger las células de las cápsulas de alginato sin tratamiento PLL por centrifugación a 1000 × g durante 3 min. </li><li> Si cápsulas de alginato son tratados con PLL, romper la membrana Alg-PLL pipeteando arriba y hacia abajo alrededor de diez veces con una aguja unida a una jeringa y se centrífuga a 1.000 - 5.000 × g durante 5 min. Diámetro de la aguja debe ser menor que el tamaño de cápsula y 25 G (260 m) se utilizan agujas en este experimento (600 micras) </li><li> Después de la centrifugación, recoger sedimento celular estrictamente de membranas Alg-PLL rotos si quieres recoger muestras de ARNm a partir de células. El resto de las membranas Alg-PLL posiblemente prevenir purificación de ARNm. </li></ol>

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The authors have nothing to disclose.

<html> Cultura encapsulación puede ser comparado con cultivos en suspensión directos. Cultivo en suspensión es un método más simple para obtener grandes cantidades de células madre pluripotentes que los métodos de encapsulación. Sin embargo, el control de la agregación de células en cultivo en suspensión es todavía difícil. En el método de encapsulación, la agregación celular está limitado en cápsulas y puede por lo tanto ser bien controlada. Una publicación anterior mostró que las células encapsulad...

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.

En protocolo 2.5, la solución de alginato expulsado forma una forma esférica inmediatamente después de la expulsión (Figura 2A - H). Si la suspensión se expulsa con una velocidad de flujo de N 2 inferior a 1L / min, el tamaño de las gotas es uniforme (Figura 2I). Sin embargo, si el flujo de N 2 es superior a 1L / min, la gotita se rompe (Figura 2G, de flecha blanca) y el tamaño de las gotitas se vuelve heterogénea (Figura 2J).</stro...

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.

La encapsulación de alginato de células madre pluripotentes utilizando una boquilla-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 vistas.  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.