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. Matériaux Préparation <ol><li> Préparer tampon HEPES 10 mM. Ajuster le pH à 7,0 à température ambiante et ajouter du NaCl à 0,9%. </li><li> Préparer la solution d&#39;alginate à 5% et une solution 10 mM d&#39;EDTA en mélangeant une solution saline tamponnée au HEPES préparé en 1.1. Ajuster le pH à 7,0 à température ambiante. </li><li> Autoclave les réactifs (1.1, 1.2) pendant 20 min à 121 ° C. </li><li> Préparer des plats 60 mm revêtues de gélatine en couches avec de 1,2 à 2,0 × 10 6 souris fibroblaste embryonnaire (MEF) des cellules, qui sont traités comme cellules nourricières par incubation à l&#39;intérieur du DMEM contenant 10% de FBS ES-qualifiés, et 10 ug / ml de mitomycine C pendant 90 - 120 min. </li><li> Maintenir la souris induit souches pluripotentes (iPS) cellules sur le plat avec les cellules nourricières en présence de 5 ml de DMEM riche en glucose contenant 20% d&#39;ES qualifié FBS, 50 M de 2-mercaptoéthanol, acide aminé non essentiel, et des antibiotiques ( 100 unités / ml de pénicilline G sodique, 100 ug / ml de sulfate de streptomycine et 250 ng / ml de amphotericin B). </li><li> Seed 4 × 10 5 cellules iPS sur une boîte de 60 mm revêtu de gélatine et les incuber à 37 ° C et 5% de CO 2. Changer le milieu de culture tous les jours pendant l&#39;incubation. Après 3-4 jours de culture, les cellules trypsiniser pendant 1 min avec 500 pi de trypsine à 0,05% contenant 0,02% d&#39;EDTA à 37 ° C et 5% de CO 2. Après cela, détacher les cellules en tapant sur la boîte de culture dix fois. </li><li> Ajouter 2,5 ml de DMEM avec 10% de FBS et des antibiotiques ES-qualifié (après iPS dissociation de souris des cellules dans des cellules individuelles par pipetage avec 1000 ul micropipette trois fois) suspension de cellules de centrifugation à 160 g pendant 3 min et retirez surnageant et compter les cellules . Après le comptage des cellules, réensemencer les cellules recueillies sur un plat revêtu de gélatine et incuber pendant 30 min à isoler les cellules iPS à partir des cellules MEF. Après comptage des cellules (généralement de 1 - 3 × 10 6 cellules / boîte peut être recueillie), réensemencer 4 × 10 5 cellules iPS surle plat. </li></ol> 2. Encapsulation cellulaire dans Alginate hydrogel Capsules <ol><li> Recueillir les cellules par le même mode opératoire décrit dans 1.5 et 1.6. Après centrifugation à 160 x g pendant 3 min, laver le culot de cellules iPS avec une solution saline tamponnée au HEPES trois fois. Après remise en suspension des cellules dans une solution saline tamponnée de HEPES, filtrer la suspension de cellules à travers un tamis de 40 um de la cellule afin d&#39;éliminer les gros agrégats, ce qui pourrait autrement obstruer la buse. </li><li> Mélanger 2 ml de suspension de cellules au sein de la solution saline tamponnée de HEPES et 3 ml de solution à 5% Alg-Na. Enfin 5 ml de suspension cellulaire à moins de 3% de solution de Na-Alg est obtenu. La densité cellulaire est de préférence plus de 10 6 cellules / ml. </li><li> Après avoir recueilli la suspension de cellules dans une seringue de 5 ml, installer une buse de co-axial (figure 1A, B) sur la seringue et les mettre sur la pompe à seringue. N 2 Emit écoulement de gaz (inférieure à 1 L / min) à travers l&#39;aiguille extérieure de la buse de co-axial etexpulser la suspension de cellules à travers la buse intérieure. </li><li> Recueillir gouttelettes dans 250 ml de solution à 0,5% de CaCl2 et attendez 10 - 20 min pour la gélification tout en agitant à 60-90 rpm. </li></ol> 3. Traitement des capsules d&#39;alginate (facultatif) <ol><li> Incuber capsules Alg-Ca à 0,05% de poly-L-lysine (PLL) solution pendant 5 minutes à 37 ° C et 5% (p / v) de CO 2. </li><li> Après lavage des capsules avec une solution saline tamponnée de HEPES, incuber dans du DMEM contenant 10% de FBS dans le but de neutraliser la charge électrique sur leur surface. Utiliser les capsules enrobées comme (figure 1c). </li><li> Incuber les capsules dans une solution saline tamponnée au HEPES contenant 10 mM d&#39;EDTA pendant 5 min. Les capsules traitées EDTA sont utilisés sous forme de capsules creuses (Figure 1C). </li></ol> 4. Culture et recueillir des cellules dans de l&#39;alginate Capsules <ol><li> Incuber les cellules encapsulées à 75 tours par minute sous agitation à 37 ° C et 5% de CO 2pendant 10 jours. </li><li> Recueillir et incuber les capsules dans 10 mM d&#39;EDTA à 37 ° C et 5% de CO2 pendant 10 min. Recueillir les cellules des capsules d&#39;alginate sans traitement PLL par centrifugation à 1 000 g pendant 3 min. </li><li> Si les capsules d&#39;alginate sont traités avec PLL, briser la membrane Alg-PLL par pipetage de haut en bas autour de dix fois avec une aiguille fixée à une seringue et centrifuger à 1,000 - 5,000 g pendant 5 min. diamètre de l&#39;aiguille doit être inférieure à la taille de la capsule et G 25 (260 um) d&#39;aiguilles sont utilisées dans cette expérience (600 um) </li><li> Après centrifugation, recueillir culot cellulaire strictement cassés membranes Alg-PLL si vous voulez prélever des échantillons d&#39;ARNm à partir de cellules. Restants membranes Alg-PLL éventuellement, de prévenir la purification de l&#39;ARNm. </li></ol>

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The authors have nothing to disclose.

<html> culture d&#39;encapsulation peut être comparé à des cultures en suspension directe. Suspension de culture est un procédé plus simple d&#39;obtenir de grandes quantités de cellules souches pluripotentes que les méthodes d&#39;encapsulation. Toutefois, le contrôle de l&#39;agrégation des cellules en culture en suspension est encore difficile. Dans le procédé d&#39;encapsulation, l&#39;agrégation cellulaire est limitée dans des capsules et peuvent donc être bien contrôlée. Une publication antérieure ...

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.

Au protocole 2.5, la solution d&#39;alginate expulsé forme une forme sphérique immédiatement après l&#39;expulsion (Figure 2A - H). Si la suspension est expulsé avec une vitesse d&#39;écoulement inférieure à 2 N 1L / min, la taille des gouttelettes est uniforme (figure 2I). Cependant, si le débit de N 2 est supérieur à 1 L / min, la gouttelette tombe en panne (figure 2G, repéré par une flèche blanche) et la taille des gouttelettes dev...

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 alginate de cellules souches pluripotentes en utilisant une buse de co-axiale

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

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12.5K vues.  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.