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. Materiali Preparazione <ol><li> Preparare 10 tampone HEPES mM. Portare il pH a 7,0 a RT e aggiungere NaCl allo 0,9%. </li><li> Preparare la soluzione di alginato 5% e la soluzione di EDTA 10 mM mescolando salina tamponata HEPES preparato in 1.1. Portare il pH a 7,0 a RT. </li><li> Autoclave i reagenti (1.1, 1.2) per 20 minuti a 121 ° C. </li><li> Preparare 60 millimetri piatti di gelatina rivestite con strati 1,2-2,0 × 10 6 topo fibroblasti embrionali (MEF), le cellule, che sono trattati come cellule di alimentazione mediante incubazione all&#39;interno DMEM contenente 10% FBS ES-qualificati e 10 mg / ml di mitomicina C per 90 - 120 min. </li><li> Mantenere topo staminali pluripotenti indotte (iPS), le cellule sul piatto con le cellule di alimentazione in presenza di 5 ml di glucosio DMEM contenente il 20% di ES-qualificato FBS, 50 micron di 2-mercaptoetanolo, aminoacido non essenziale, e gli antibiotici ( 100 unità / ml di penicillina G sodio, 100 ug / ml di solfato di streptomicina, e 250 ng / ml di amphotericin B). </li><li> Seed 4 × 10 5 cellule ips su 60 mm piatto gelatina rivestite e incubare a 37 ° C e 5% CO 2. Modificare il terreno di coltura ogni giorno durante l&#39;incubazione. Dopo 3 - 4 giorni di coltura, trypsinize cellule per 1 min con 500 ml di 0,05% tripsina contenente 0,02% EDTA a 37 ° C e 5% CO 2. Dopo di che, staccare le cellule toccando il piatto cultura dieci volte. </li><li> Aggiungere 2,5 ml di DMEM con 10% di FBS e antibiotici ES-qualificato (seguenti cellule iPS topo dissociazione in cellule singole pipettando con 1.000 microlitri micropipetta tre volte) sospensione cellulare centrifugare a 160 g per 3 minuti e rimuovere il surnatante e contare le cellule . Dopo il conteggio delle cellule, reseed le cellule raccolte su un piatto di gelatina a polvere e incubare per 30 minuti per isolare le cellule iPS dalle cellule MEF. Dopo il conteggio delle cellule (di solito 1-3 × 10 6 cellule / piatto può essere raccolto), reseed 4 × 10 5 cellule iPS suil piatto. </li></ol> 2. Cella incapsulamento in alginato Hydrogel Capsule <ol><li> Raccogliere le cellule con la stessa procedura descritta in 1.5 e 1.6. Dopo la centrifugazione a 160 g per 3 minuti, lavare il pellet di cellule iPS con soluzione salina HEPES-buffered tre volte. Dopo ri-sospensione le cellule in soluzione salina tamponata HEPES, filtrare la sospensione cellulare attraverso un filtro 40 micron cellula per rimuovere grandi aggregati, che potrebbero intasare l&#39;ugello. </li><li> Mescolare 2 ml di sospensione cellulare all&#39;interno di soluzione salina tamponata HEPES e 3 ml di soluzione al 5% Alg-Na. Infine 5 ml di sospensione cellulare entro il 3% soluzione Alg-Na si ottiene. Densità cellulare è vantaggiosamente maggiore di 10 6 cellule / ml. </li><li> Dopo aver raccolto la sospensione cellulare in una siringa da 5 ml, installare un ugello coassiale (Figura 1A, B) sulla siringa e disporli su pompa a siringa. Emit N 2 flusso di gas (inferiore 1L / min) attraverso l&#39;ago esterna dell&#39;ugello coassiali eespellere la sospensione cellulare attraverso l&#39;ugello interno. </li><li> Raccogliere gocce in 250 ml di soluzione allo 0,5% di CaCl 2 ed attendere 10 - 20 min per gelificazione mentre agitazione a 60-90 rpm. </li></ol> 3. Trattamento di alginato Capsule (opzionale) <ol><li> Incubare capsule Alg-Ca a 0,05% (w / v) di poli-L-lisina (PLL) soluzione per 5 min a 37 ° C e 5% CO 2. </li><li> Dopo aver lavato le capsule con soluzione salina tamponata con HEPES-, incubare in DMEM contenente 10% FBS per neutralizzare la carica elettrica sulla loro superficie. Utilizzare loro sotto forma di capsule rivestite (Figura 1C). </li><li> Incubare le capsule in soluzione salina tamponata HEPES contenente EDTA 10 mM per 5 min. Le capsule trattato con EDTA sono utilizzati sotto forma di capsule cavi (Figura 1C). </li></ol> 4. Cultura e raccogliere le cellule in alginato Capsule <ol><li> Incubare le cellule incapsulate a 75 rpm con agitazione a 37 ° C e 5% CO 2per 10 giorni. </li><li> Raccogliere e incubare le capsule EDTA 10 mM a 37 ° C e 5% CO 2 per 10 min. Raccogliere le cellule dalle capsule alginato senza trattamento PLL per centrifugazione a 1000 g per 3 min. </li><li> Se capsule di alginato sono trattati con PLL, rompere la membrana Alg-PLL pipettando su e giù intorno dieci volte con un ago attaccato ad una siringa e centrifugare a 1000 - 5000 xg per 5 min. Diametro ago deve essere inferiore formato di capsule e 25 g (260 micron) aghi sono utilizzati in questo esperimento (600 micron) </li><li> Dopo la centrifugazione, raccogliere pellet cellulare rigorosamente da rotte membrane Alg-PLL, se si vuole raccogliere campioni di mRNA da cellule. Rimanenti membrane Alg-PLL eventualmente prevenire mRNA purificazione. </li></ol>

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The authors have nothing to disclose.

<html> Cultura incapsulamento può essere confrontato con le culture diretti sospensione. Coltura in sospensione è un metodo semplice per ottenere grandi quantità di cellule staminali pluripotenti rispetto ai metodi di incapsulamento. Tuttavia, controllando l&#39;aggregazione di cellule in coltura in sospensione è ancora impegnativo. Nel metodo di incapsulamento, aggregazione cellulare è limitata in capsule e può quindi essere ben controllata. Una pubblicazione precedente ha mostrato che le cellule incapsulate forma...

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.

Al protocollo 2.5, la soluzione di alginato espulso forma una forma sferica subito dopo l&#39;espulsione (Figura 2A - H). Se la sospensione viene espulso con una portata N 2 inferiore 1L / min, la dimensione delle goccioline è uniforme (figura 2I). Tuttavia, se il flusso di N 2 è superiore a 1L / min, la goccia si rompe (figura 2G, a freccia bianca) e la dimensione delle goccioline diventa eterogenea (Figura 2J). Dato questo, è ...

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.

Incapsulamento alginato di cellule staminali pluripotenti usando un ugello coassiale

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 Views.  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.