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.準備材料<ol><li> 10mMのHEPES緩衝液を準備します。 RTでpHを7.0に調整し、0.9％に塩化ナトリウムを加えます。 </li><li> 1.1で調製したHEPES緩衝生理食塩水を混合することにより、5％アルギン酸塩溶液および10mM EDTA溶液を準備します。 RTでpHを7.0に調整します。 </li><li> 121℃で20分間、試薬（1.1、1.2）をオートクレーブ。 </li><li> 90、10％ES-修飾FBSおよびマイトマイシンC10μg/ mlのを含むDMEM内でインキュベートすることにより、フィーダー細胞として処理され、2.0×10 6マウス胚性線維芽細胞（MEF）細胞、 - 1.2層状ゼラチンコーティングされた60ミリメートルの料理を準備します - 120分。 </li><li>マウス人工多能性幹（iPS）皿上の細胞を5mlの存在ES修飾FBS、2-メルカプトエタノール、非必須アミノ酸、50μM、および抗生物質（20％を含むDMEM高グルコース中のフィーダー細胞を維持しますペニシリンGナトリウム、硫酸ストレプトマイシンを100μg/ mlと100単位/ ml、およびamphoteriの250 ng / mlでCINのB）。 </li><li>シード4×10 5ゼラチンコーティングされた60ミリメートル皿にiPS細胞と37℃、5％CO 2でそれらをインキュベートします。インキュベーション中に、毎日培地を変更します。 3の後- 4日間培養し、37℃で0.02％EDTA、5％CO 2を含む0.05％トリプシンの500μlの1分間細胞をトリプシン処理。その後、培養皿を10回タップすることで細胞を剥離。 </li><li> （3回千μlをマイクロピペットでピペッティングにより単一細胞に解離マウスiPS細胞を以下）ES-修飾FBSおよび抗生物質の10％を含むDMEM 2.5 mlを加え3分間160×gで遠心分離細胞懸濁液、上清を除去し、細胞をカウント。細胞計数の後、ゼラチンコートディッシュに回収した細胞を再シードし、MEF細胞からiPS細胞を単離するために30分間インキュベートします。細胞の計数した後（通常は1 - 3×10 6細胞/皿を収集することができます）、上の4×10 5 iPS細胞を再シード皿。 </li></ol>アルギン酸ハイドロゲルカプセルに2細胞のカプセル化<ol><li> 1.5および1.6に記載の方法と同様の方法により細胞を収集します。 3分間160×gで遠心分離した後、三回HEPES緩衝生理食塩水でのiPS細胞ペレットを洗浄します。 HEPES緩衝化生理食塩水で細胞を再懸濁した後、それ以外の場合はノズルを詰まらせる可能性が大きな凝集体を除去するために40μmのセルストレーナーを通して細胞懸濁液を濾過します。 </li><li> HEPES緩衝生理食塩水内の細胞懸濁液2mlを、5％ALG-ナトリウム溶液3mlを混合します。最後に、3％のALG-Na溶液中で細胞懸濁液5mlを得られます。細胞密度は、望ましくは10以上6細胞/ mlです。 </li><li> 5ミリリットルシリンジに細胞懸濁液を収集した後、注射器に同軸ノズル（ 図1A、B）をインストールし、シリンジポンプに設定してください。同軸ノズルの外針を通してN 2ガ ​​ス流量（リットル/分より低い）を放出し、内側ノズルを通して細胞懸濁液を追い出します。 </li><li> 0.5％のCaCl 2溶液250ml中に液滴を収集し、10を待つ- 60〜90 rpmで攪拌しながら、ゲル化のために20分。 </li></ol>アルギン酸カプセルの3処理（オプション） <ol><li> 0.05％、37℃で5分間、5％CO 2（w / v）のポリ-L-リジン（PLL）溶液をALG-カルシウムカプセルをインキュベートします。 </li><li> HEPES緩衝生理食塩水でカプセルを洗浄した後、それらの表面上の電荷を中和するために10％FBSを含むDMEM中でそれらを培養します。コーティングされたカプセル（ 図1C）として、それらを利用しています。 </li><li> 5分間、10mMのEDTAを含むHEPES緩衝生理食塩水でカプセルをインキュベートします。 EDTA処理したカプセルは、中空カプセル（ 図1C）として利用されます。 </li></ol> 4.文化アルギン酸カプセルの細胞を収集<ol><li> 37℃および5％CO 2で振とうしながら75 rpmでカプセル化された細胞をインキュベート10日間。 </li><li>収集し、10分間CO 2を 37℃で、10mMのEDTAでカプセルをインキュベートし、5％。 3分間1,000×gで遠心分離することにより、PLL処理なしのアルギン酸カプセルから細胞を収集します。 </li><li> 5分間5,000×gで - アルギン酸塩カプセルはPLLで処理している場合は、シリンジに取り付けられた1000でそれを遠心針で上下にピペッティング10回の周りによってALG-PLL膜を破ります。針の直径は、カプセルサイズ25 G（260μm）の針が、この実験で使用されているよりも低くなければならない（600ミクロン） </li><li>あなたは、細胞からのmRNAのサンプルを収集したい場合は、遠心分離した後、壊れたALG-PLL膜から厳密に細胞ペレットを収集します。残りのALG-PLL膜は、おそらくmRNA精製を防ぎます。 </li></ol>

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The authors have nothing to disclose.

<html>カプセル化培養物を直接懸濁培養物と比較することができます。浮遊培養は、カプセル化方法よりも多能性幹細胞を大量に得るための簡単​​な方法です。しかし、懸濁培養中の細胞の凝集を制御することは依然として困難です。カプセル化方法において、細胞凝集は、カプセル内に限定されるので、十分に制御することができます。以前の刊行物は、大きな細胞塊が自由懸濁培養7<...

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.

プロトコル2.5で、排出されたアルギン酸塩溶液は直ちに放出（ - H図2A）の後に球状をなします。懸濁液を1L / minより低いN 2流量で排出された場合、液滴の大きさが均一（ 図2I）です。 N 2流量は、1L /分よりも大きい場合には、液滴（ 図2G、白矢印）を分解し、液滴の大きさは、不均一（ 図2J）となります。これを考えると、こ...

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.

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

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 ビュー.  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.