私たちは、Nrl-GFPマウス、技術サポートのためのヨッヘンハース、動物の畜産のためのシンディベーメとエメリーレスマンを提供してくれたアナンド・スワロップに感謝したいと思います。	
	この研究は、ドイツ・フォルシュングスゲミンシャフト(DFG):FZT 111 - 再生療法ドレスデンのセンター、CRTD種子補助金プログラム、SFB 655、およびProRetina e.Vによってサポートされました。 財団、DIGS-BB大学院プログラムドレスデン、およびフンダサンパラ・ア・シエンシア・エ・テクノロジア(SFRH/BD/60787/2009)

動物の倫理的使用とケア声明:
すべての動物実験は、欧州連合(EU)およびドイツの法律(Tierschutzgesetz)に厳密に従って行われ、眼科および視力研究における動物の使用に関するARVO声明に従った。すべての動物実験は、TUドレスデンとランデスディレクションドレスデンの動物倫理委員会によって承認されました(承認番号:24D-9168.11-1/2008-33)。
1. 細胞解離と細胞ソートを開始する前に
<ol><li>3つの15 ml反応チューブにラベルを付けます:ウォッシュ(W)、正の分数(+)および負の分数(-)。</li>
</ol>2. レチナ解離
<ol><li>はさみを使用してPN 4の子犬の首を切る。</li>
	<li>70%エタノールですぐに子犬の頭をすすいで、続いてPBSで洗浄します。</li>
	<li>ヘッドを冷たいHBSSに移し、目を核にします(すべてのヘッドに対してこのステップを繰り返します)。核を得るために、眼瞼を開き、視神経領域で湾曲した鉗子で目を固定する。その後、慎重に軌道から目を引きます。</li>
	<li>すべての目を核にした後、レティナを分離する:視神経に閉じたはさみを導入し、ブレードを開きます。ピールRPE/チョリドアウト。湾曲した鉗子を使用してレンズおよび血管を取り除く。</li>
	<li>単離したレチナをパパイン溶液を含む1.5ml反応チューブ(パパイン解離キットによって提供される)に移します。</li>
	<li>37°Cの水浴またはシェーカー(400rpm)で30〜60分間、パパイン溶液中のレチナをインキュベートします。<ol>
<li>レチナがパパイン溶液中でインキュベートする間、ピペット1mlのオボムコード溶液を15ml反応管に(ラベル「1」)にする。</li>
			<li>60 μlのDNase I(10 mg/ml)+60 μlのオボムコイド溶液(キット提供)+520 μlのEBSSを15ml反応チューブ(ラベル"2")に加えます。</li>
		</ol>
</li>
	<li>パパインインキュベーション後、部分的に消化されたレティナを含むパパイン溶液を反応管「2」に移す。</li>
	<li>火磨きピペットを使用して、機械的解離(10倍上下)を行います。</li>
	<li>単一細胞懸濁液を反応管「1」にピペットする。細胞懸濁液をオボムコイド溶液の上に優しく重ねて2層を生成するようにしてください。</li>
	<li>遠心分離機は300xg(約1,300rpm)で5分間を行った。</li>
	<li>上清を捨て、500 μlのMACSバッファで細胞を再中断します。</li>
</ol>3. 磁気関連セルソート(MACS)を用いたセルソート
<ol><li>X μlラット抗CD73抗体を500 μlに加えて、10 μg/mlの最終濃度を達成します。</li>
	<li>氷の上に5分をインキュベートします。</li>
	<li>15 ml の反応チューブに MACS バッファーを 10 ml 充填し、300 x g の速度で 5 分間遠心分離します。</li>
	<li>上清を取り除く。</li>
	<li>480 μl MACS バッファーにペレットを再懸濁し、120 μlのヤギアンチラット IgG マイクロビーズを追加します。</li>
	<li>氷の上に15分をインキュベート;攪拌、振ったり、混ぜたりしないでください。</li>
	<li>15 mlの反応管をMACSバッファーで5 mlに充填し、300 x gで5分間遠心分離します。</li>
	<li>反応管が遠心分離されているように:<ol>
<li>LSカラムを磁気スタンドに合わせて調整します。</li>
			<li>LS カラムの上に事前分離フィルタを配置します。</li>
			<li>3 ml MACS バッファで事前分離フィルターと LS カラムをハイドレートし、MACS バッファを洗浄(W)チューブに集めます。</li>
		</ol>
</li>
	<li>上清を取り除く。</li>
	<li>ペレットを500 μl MACSバッファに再懸濁します。</li>
	<li>500 μl のセル懸濁液をフィルターにロードし、フィルターに 1 ml MACS バッファーを追加し、負の分数を負の分数 (-) 反応管に集める。</li>
	<li>次に、3 x 3 ml の MACS バッファを列に追加して、列にバインドされていないセルを洗い流します。</li>
	<li>これらの9 ml がLSカラムを通り抜けると、磁気スタンドからカラムを取り外し、正の分数(+)反応管の上に取り付けます。</li>
	<li>5 ml の MACS バッファを LS カラムに素早くロードし、LS カラムにプランジャを入れ、バッファ全体がカラムを通るまで押します。</li>
	<li>300xgで5分間の正分を含む反応管を遠心分離する。</li>
	<li>上清を取り外し、MACSバッファの500 μlで再中断し、4°Cに保ちます。</li>
	<li>共通のセルカウントデバイスを使用してセルの合計量をカウントします。</li>
	<li>MACSバッファー内の2 x 105 セル/μlを含む正のソートされた分画から細胞懸濁液を調製し、4°Cに保つ</li>
</ol>4. マウスのレチナへのMACソートされた感光体前駆細胞の移植
<ol><li>1 mlの滅菌PBSまたはHBSS、10 μl DNAse I、1-2 ml無菌脱イオン化H2O、4°Cの細胞懸濁液分画のアリコートを用意してください。</li>
	<li>メデトミジン塩酸塩(0.01 mg/10 g体重)の腹腔内注射(0.01mg/10 g体重)、ケタミン(0.75mg/10 g体重)で成人マウス(すなわち2~4ヶ月)を麻酔する。その後、痛みの軽減のためにブプレノルフィン(0.05 mg /kg体重)の皮下注射を行います。</li>
	<li>フェニレフリンの低下で瞳孔を拡張 2.5%-トロピックアミド 0.5% .</li>
	<li>マウスヘッドホルダーにマウスを固定し、ステレオ顕微鏡の下に置きます。</li>
	<li>目の乾燥を防ぐために、Visidicゲルの滴を適用します。</li>
	<li>針に30 G 1/2の無菌を使用して、スクレアラと角膜(それぞれ 、オレ)の境界に小さな穴を作ります。</li>
	<li>ダイヤモンドペンを使用して一般的なカバースライドを小さな(約5mm x 5mm)部分にカットし、これらの片の1つを角膜の上に置き、レティナを直接視覚化できるようにします。</li>
	<li>殺菌したマイクロリットルのシリンジを脱イオン水で数回洗い流します。</li>
	<li>マイクロリットルシリンジに1 μlの細胞懸濁液を搭載し、結膜および結膜を通して針を直通し、点眼の半分の鼻の目視管理下に置きます。</li>
	<li>レチナル空間に到達するまで、静かにレチナに穴を開け、細胞懸濁液を皮下腔に注入する。</li>
	<li>出血することなく網膜空間の約1/4を覆い、均一であるべき網膜の雄牛剥離を観察する。</li>
	<li>注射器を優しく引き出し、レチナルホールが自動的にシールします。</li>
	<li>マイクロリットルシリンジを脱イオン水で数回洗い流します。</li>
	<li>ヘッドホルダーからマウスを離します。</li>
	<li>メデトミジン塩酸塩効果の逆転のためにアティパモゾール塩酸塩を注入することによってマウスを目覚めさせる。</li>
	<li>暖かい暗い部屋(およそ25°C)で目覚めの時間のためのマウスを置く。</li>
	<li>移植後2日後、ブプレノルフィン(体重0.05mg/kg)は、痛みの軽減のために、朝と午後に皮下注射によって投与されるべきである。</li>
</ol>

<ol>
	<li>Bartsch, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+cells+integrate+into+the+outer+nuclear+layer+and+differentiate+into+mature+photoreceptors+after+subretinal+transplantation+into+adult+mice.">Retinal cells integrate into the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice.</a> Exp. Eye Res. 86, 691-700 (2008).</li><li>MacLaren, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+repair+by+transplantation+of+photoreceptor+precursors.">Retinal repair by transplantation of photoreceptor precursors.</a> Nature. 444, 203-207 (2006).</li><li>Eberle, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+segment+formation+of+transplanted+photoreceptor+precursor+cells.">Outer segment formation of transplanted photoreceptor precursor cells.</a> PLoS One. 7, (2012).</li><li>Lakowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+photoreceptor+transplantation+in+models+of+the+childhood+retinopathy+Leber+congenital+amaurosis+using+flow-sorted+Crx-positive+donor+cells.">Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells.</a> Hum. Mol. Genet. 19, 4545-4559 (2010).</li><li>Barber, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+the+degenerate+retina+by+photoreceptor+transplantation.">Repair of the degenerate retina by photoreceptor transplantation.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 354-359 (2013).</li><li>Pearson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+vision+after+transplantation+of+photoreceptors.">Restoration of vision after transplantation of photoreceptors.</a> Nature. , (2012).</li><li>Singh, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+end-stage+retinal+degeneration+and+restoration+of+visual+function+by+photoreceptor+transplantation.">Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation.</a> Proc. Natl. Acad. Sci. U.S.A. 110, (2013).</li><li>Eberle, D., Schubert, S., Postel, K., Corbeil, D., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+integration+of+transplanted+CD73-positive+photoreceptor+precursors+into+adult+mouse+retina.">Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina.</a> Invest. Ophthalmol. Vis. Sci. 52, 6462-6471 (2011).</li><li>Koso, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD73,+a+novel+cell+surface+antigen+that+characterizes+retinal+photoreceptor+precursor+cells.">CD73, a novel cell surface antigen that characterizes retinal photoreceptor precursor cells.</a> Invest. Ophthalmol. Vis. Sci. 50, 5411-5418 (2009).</li><li>Lakowski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+transplantation+of+photoreceptor+precursor+cells+selected+via+cell+surface+antigen+expression.">Effective transplantation of photoreceptor precursor cells selected via cell surface antigen expression.</a> Stem Cells. 29, 1391-1404 (2011).</li><li>Akimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+GFP+to+newborn+rods+by+Nrl+promoter+and+temporal+expression+profiling+of+flow-sorted+photoreceptors.">Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 3890-3895 (2006).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Nakano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10, 771-785 (2012).</li><li>Osakada, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nat. Biotechnol. 26, 215-224 (2008).</li><li>Lee, M. Y., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+&amp;#34;Three-step+MACS&amp;#34;:+a+novel+strategy+for+isolating+rare+cell+populations+in+the+absence+of+known+cell+surface+markers+from+complex+animal+tissue.">Development of the &amp;#34;Three-step MACS&amp;#34;: a novel strategy for isolating rare cell populations in the absence of known cell surface markers from complex animal tissue.</a> J. Biomol. Tech. 23, 69-77 (2012).</li></ol>

著者らは、競合する財政的利益を宣言しない。

感光体前駆細胞の低レチナル移植は、これらの光感受性細胞を有意な数1,2の宿主のレチナに統合することを達成するための信頼できるツールを表す。これにより、将来6のレチン性変性疾患の治療のための細胞療法の確立が可能になる可能性があります。現在PN4のレティナから単離されている細胞のドナー集団は、異なる細胞タイプの混合物であり、そこから、感光体前駆細胞のみが、皮膜?...

ビジョンは人間の主要な感覚の一つです。この感覚と失明の障害は、先進国における障害の主な理由の一つです。視力障害または失明の主な原因は、黄斑変性症、網膜色素変性症、円錐棒ジストロフィー、およびその他の条件で観察することができるように、感光体細胞喪失を特徴とする網膜変性である。現在までに、失われた視力を回復するための効果的な治療法は利用できません。2006年および2008年には、2つの異なるラボが報告され、互いに独立して、ロッド感光体前駆体細胞を成体の野生型マウスのレチナ1,2に移植することに成功した。このように、感光体前駆細胞移植の可能性も生じて退化したretinaに、退化した感光体を置き換え、視力を回復させる。実際、最近、このような移植された感光体前駆体細胞は、適切に発達した外層3、内因性双極細胞に近接したシナプス末端、および外核層2-4に位置する丸い細胞体などの成熟した野生型感光受容体の形態学的基準を引き出し、また宿主神経回路5-7に機能的に統合する能力を有することが最近実証されている。この戦略の主な原則の1つは、出生後4日目(PN4、PN0は出生日として定義される)若いマウスのレティナの使用であり、移植のための異なる細胞タイプの混合物をもたらす。将来の治療アプリケーションの背景に、この混合物は、感光体前駆細胞のために精製されなければならない。CD73は、若い感光体に特異的な第1の細胞表面マーカーとして、レティナ8-10に記載されている。ここでは、この細胞表面マーカーに基づく光受容体前駆細胞精製法と、磁気関連細胞選別法(MACS)法を用いた方法を示す。MACSは、高速なソート時間とGMP条件の調整が容易であるため、蛍光活性化細胞選別技術と比較して利点があります。成人型の野生型レチナの皮下空間に濃縮された集団を移植する際に、〜90%の濃縮率と最大3倍の高い統合率を実証することができた。従って、MACSベースの感光体前駆細胞の濃縮および皮膜下移植は、レチナル変性の治療のための再生治療戦略の開発に対する信頼できる有望な技術である。

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Papain Dissociation System</td>
			<td style="width:111px;">Worthington&#160; Biochemical Corporation</td>
			<td style="width:103px;">LK003150</td>
			<td style="width:176px;">supplied DNase I is not used in the method</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">purified rat anti-mouse CD73, clone TY/23</td>
			<td style="width:111px;">BD Pharmingen</td>
			<td style="width:103px;">550738</td>
			<td style="width:176px;">Stock concentration 0.5mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Goat Anti-Rat IgG MicroBeads</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-048-501</td>
			<td style="width:176px;">Total volume of 2ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">PBS</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">10010-015</td>
			<td style="width:176px;">Used to count the total number of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">DNase I</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">D5025-150KU</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">HBSS</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">14025050</td>
			<td style="width:176px;">Used for dissociation of the retinas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Trypan blue</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">Fluka93595</td>
			<td style="width:176px;">Used to count the total number of cells</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:195px;">Vidisic</td>
			<td style="width:111px;">Dr. Mann Pharma / Andreae-Noris Zahn AG</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Domitor</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:103px;">76579</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Ketamin 10%</td>
			<td style="width:111px;">Ratiopharm</td>
			<td style="width:103px;">7538843</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Antisedan</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:103px;">76590</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:195px;">Phenylephrin 2.5%-Tropicamid 0.5%</td>
			<td style="width:111px;">University clinics Dresden pharmacy</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Name of Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:176px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pre-Separation Filters</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-041-407</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">LS Columns</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-042-401</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">MACS MultiStand</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-042-303</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">QuadroMACS Separator</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-090-976</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">fire polish glass pasteur pipette</td>
			<td style="width:111px;">Brand</td>
			<td style="width:103px;">74777 20</td>
			<td style="width:176px;">The pipette&#8217;s tips need to be fire-polished and autoclaved.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">MACS 15ml tube rack</td>
			<td style="width:111px;">Miltenyi</td>
			<td style="width:103px;">130-091-052</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cell count chamber</td>
			<td style="width:111px;">Carl Roth</td>
			<td style="width:103px;">T728.1</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Sterile 15ml tubes</td>
			<td style="width:111px;">Greiner Bio-one</td>
			<td style="width:103px;">188271</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Leica M651 MSD</td>
			<td style="width:111px;">Leica</td>
			<td style="width:103px;">M651 MSD</td>
			<td style="width:176px;">can be used instead of Olympus SZX10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Olympus SZX10</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:103px;">SZX10</td>
			<td style="width:176px;">can be used instead of Leica M651 MSD</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Olympus inverted stereo microscope CKX41</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:103px;">CKX41</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Cell culture hood Thermo Scientific MSC-Advance</td>
			<td style="width:111px;">Thermo scientific</td>
			<td style="width:103px;">51025411</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">1.5ml reaction tube</td>
			<td style="width:111px;">Sarstedt</td>
			<td style="width:103px;">727706400</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">2ml reaction tube</td>
			<td style="width:111px;">Sarstedt</td>
			<td style="width:103px;">72695</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Eppendorf Centrifuge 5702</td>
			<td style="width:111px;">VWR (Eppendorf)</td>
			<td style="width:103px;">521-0733</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Mouse head holder</td>
			<td style="width:111px;">myNeurolab</td>
			<td style="width:103px;">471030</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">BD Microlance 3 30G 1/2&#8221;</td>
			<td style="width:111px;">BD Pharmingen</td>
			<td style="width:103px;">304000</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Hamilton microliter syringe 5&#181;l, 75RN</td>
			<td style="width:111px;">Hamilton</td>
			<td style="width:103px;">065-7634-01</td>
			<td style="width:176px;">delivered without needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Hamilton RN special needle GA34</td>
			<td style="width:111px;">Hamilton</td>
			<td style="width:103px;">065-207434</td>
			<td style="width:176px;">Blunt, 12mm length</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Vannas-T&#252;bingen Spring Scissors - 5mm Blades Straight</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">15003-08</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Dumont #7 Forceps - Titanium Biologie</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11272-40</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Diamond pen</td>
			<td style="width:111px;">Tools-tech</td>
			<td style="width:103px;">-</td>
			<td style="width:176px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">15x15mm Cover slips</td>
			<td style="width:111px;">Sparks</td>
			<td style="width:103px;">MIC3366</td>
			<td style="width:176px;">-</td>
		</tr>
	</tbody>
</table>

subretinal transplantation of macs purified photoreceptor precursor cells into the adult mouse retina

視覚障害と、レチナの光感知細胞、すなわち感光体の喪失による失明 は 、先進国における障害の主な理由を表している。細胞移植による退化光受容体の置換は、将来の臨床応用における可能な治療選択肢を表す。実際、最近の前臨床試験では、出生後4日目に新生児マウスのレチナから分離された未熟な感光体が、副腎移植後に成人マウスの残毛に溶け込む可能性があることを実証した。ドナー細胞は、内外セグメント、外核層に位置する丸い細胞体、内因性双極細胞に近接したシナプス末端を含む成熟した感光体形態を生成した。実際、最近の報告では、ドナー感光体が機能的に宿主マウスの神経回路に統合することを示した。このような細胞置換アプローチの将来の臨床適用のために、選択した細胞の精製懸濁液を生成し、眼に適切に統合するために正しい位置に配置する必要があります。感光体前駆体の濃縮のために、並べ替えは、ドナー細胞の遺伝子レポーター修飾を避けるために、特異的な細胞表面抗原に基づくべきである。ここでは、磁気関連細胞選別(MACS)−細胞表面マーカーCD73に基づく感光体特異的レポーターマウスの新生児性レチナから分離された移植可能なロッド感光体前駆体の濃縮を示す。抗CD73抗体と共にマイクロビーズ共役二次抗体を用いたインキュベーションにより、MACSによるロッド感光体前駆体の濃縮が約90%となった。フローサイトメトリーと比較して、MACSはGMP規格に適用しやすく、また、大量の細胞を相対的に短期間にソートできるという利点があります。成人野生型マウスの皮下腔への濃縮細胞懸濁液の注入は、未ソートの細胞懸濁液と比較して3倍高い集積率をもたらした。

マウスのレチナに統合するロッド感光体の能力を評価するために、マウスレポーターラインを使用し、GFPが神経性retinaロイシンジッパー(Nrl、Nrl-GFP)プロモーター11によって駆動される。Nrlは、成人期を通じてE12.5でその発現を開始するロッド感光体の最も初期のマーカーであり、ドナーロッド感光体細胞の特定の標識を可能にする。
PN 4 Nrl-GFPの子犬は首を切られ?...

Watch this Scientific Journal Video about Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina at JoVE.com

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

細胞移植は、感光体喪失を特徴とするレチン系変性の治療のための戦略を表す。ここでは、移植可能な感光体の濃縮方法と、その副レチング移植を成体マウスに説明する。

MACSの精製された感光体前駆体細胞の成人マウス・レチナへの副腎移植

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

subretinal-transplantation-macs-purified-photoreceptor-precursor

Research

JoVE Journal

Medicine

10.6K ビュー.  Technische Universität Dresden. 細胞移植は、感光体喪失を特徴とするレチン系変性の治療のための戦略を表す。ここでは、移植可能な感光体の濃縮方法と、その副レチング移植を成体マウスに説明する。

ビデオ: Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

マウスの眼の中に遺伝子治療ベクターと幹細胞の網膜下注入

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

医学

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

ヒアルロン酸ナトリウムの網膜下注入によるげっ歯類における網膜剥離モデル

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

異心臓移植のマウスモデルを用いた肺動脈弁の移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

マウス腎臓移植：同種移植片拒絶のモデル

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

懸濁液中の網膜色素上皮細胞を送達するげっ歯類における網膜下注射の実行

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

移植のためのカプセル化サーモ前脂肪細胞の脂肪組織デポへ

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

マウスで同所後肢移植

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

ウサギにおける網膜下手術のためのステッププロトコルによってステップ

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

マウスの卵巣脂肪パッドに移植

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部マウス心臓移植モデルにおける血液回路再建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...