Gostamos de agradecer a Anand Swaroop por fornecer ratos NRL-GFP, Jochen Haas para suporte técnico, e Sindy Böhme e Emely Lessmann pela criação animal.	
	Este trabalho foi apoiado pela Deutsche Forschungsgemeinschaft (DFG): FZT 111 - Centro de Terapias Regenerativas Dresden, o Programa de Concessão de Sementes crtd, o SFB 655, e o ProRetina e.V. fundação, o Programa de Pós-Graduação DIGS-BB Dresden, e a Fundação para a Ciência e Tecnologia (SFRH/BD/60787/2009)

Uso ético e cuidado com a declaração dos animais:
Todos os experimentos em animais foram realizados em estrita conformidade com as leis da União Europeia e da Alemanha (Tierschutzgesetz) e aderiram à Declaração ARVO para o Uso de Animais em Pesquisa Oftalmômica e De Visão. Todos os experimentos em animais foram aprovados pelo comitê de ética animal do TU Dresden e do Landesdirektion Dresden (número de aprovação: 24D-9168.11-1/2008-33).
1. Antes de iniciar a dissociação celular e a classificação celular
<ol><li>Rotule três tubos de reação de 15 ml com: Lavagem (W), Fração positiva (+) e fração negativa (-).</li>
</ol>2. Dissociação da retina
<ol><li>Decapitar os filhotes PN 4 usando uma tesoura.</li>
	<li>Enxágüe a cabeça dos filhotes em pouco tempo em 70% de etanol seguido de lavagem na PBS.</li>
	<li>Transfira a cabeça para o HBSS frio e enuclear o olho (repita este passo para todas as cabeças). Para enuclear, abra as pálpebras e fixe o olho com fórceps curvos na região do nervo óptico. Em seguida, puxe o olho cuidadosamente para fora da órbita.</li>
	<li>Depois de enuclear todos os olhos, isole a retina: introduza uma tesoura fechada no nervo óptico e abra as lâminas. Peal o RPE/chorid para fora. Remova as lentes e vasos sanguíneos usando fórceps curvos.</li>
	<li>Transfira as retinas isoladas para um tubo de reação de 1,5 ml contendo a solução papain (fornecida pelo kit de dissociação papain).</li>
	<li>Incubar as retinas na solução papan por 30-60 min em um banho de água ou shaker (400 rpm) a 37 °C.<ol>
<li>Enquanto as retinas incubam na solução papain, pipeta 1 ml de solução ovomucóide para um tubo de reação de 15 ml (rótulo "1").</li>
			<li>Adicionar 60 μl de DNase I (10 mg/ml) + 60 μl de solução ovomucoid (fornecida pelo kit) + 520 μl de EBSS a um tubo de reação de 15 ml (rótulo "2").</li>
		</ol>
</li>
	<li>Após a incubação do papain, transfira a solução de papain contendo as retinas parcialmente digeridas para o tubo de reação "2".</li>
	<li>Usando uma pipeta polida de fogo, realize a dissociação mecânica (10x para cima e para baixo).</li>
	<li>Pipeta a suspensão de célula única ao tubo de reação "1". Tente gerar 2 camadas colocando suavemente a suspensão da célula em cima da solução ovomucoid.</li>
	<li>Centrífuga por 5 min a 300 x g (aproximadamente 1.300 rpm).</li>
	<li>Descarte o supernatante e resuspenque as células em 500 μl de tampão MACS.</li>
</ol>3. Classificação celular usando classificação celular associada magnética (MACS)
<ol><li>Adicione o anticorpo anti-CD73 de rato X μl aos 500 μl, a fim de alcançar uma concentração final de 10 μg/ml.</li>
	<li>Incubar 5 minutos no gelo.</li>
	<li>Encha o tubo de reação de 15 ml a 10 ml com tampão MACS e centrífugue por 5 minutos a uma velocidade de 300 x g.</li>
	<li>Remova o supernatante.</li>
	<li>Resuspenque a pelota em 480 μl de tampão MACS e adicione 120 μl de microesferas IgG anti-rato de cabra.</li>
	<li>Incubar 15 min no gelo; não agitar, agitar ou misturá-lo.</li>
	<li>Encha o tubo de reação de 15 ml a 5 ml com tampão MACS e centrífugue por 5 min a 300 x g.</li>
	<li>Como o tubo de reação é centrifuado:<ol>
<li>Ajuste uma coluna LS no suporte magnético.</li>
			<li>Coloque o filtro de pré-configuração em cima da coluna LS.</li>
			<li>Hidrate o filtro de pré-esparação e a coluna LS com tampão MACS de 3 ml e colete o tampão MACS no tubo de lavagem (W).</li>
		</ol>
</li>
	<li>Remova o supernatante.</li>
	<li>Resuspenda a pelota em 500 μl tampão MACS.</li>
	<li>Carregue a suspensão celular de 500 μl no filtro e adicione 1 ml de tampão MACS ao filtro e colete a fração negativa no tubo de reação de fração negativa (-).</li>
	<li>Em seguida, adicione 3 x 3 ml de tampão MACS à coluna para lavar as células que não estão ligadas à coluna</li>
	<li>Uma vez que estes 9 ml estejam através da Coluna LS, remova a coluna do suporte magnético e instale-a em cima do tubo de reação de fração positiva (+).</li>
	<li>Carregue rapidamente 5 ml de tampão MACS na coluna LS e coloque o êmbolo na Coluna LS e pressione-o até que todo o buffer esteja através da coluna.</li>
	<li>Centrifugar o tubo de reação contendo a fração positiva por 5 min a 300 x g.</li>
	<li>Remova o supernatante, resuspenda em 500 μl de tampão MACS e mantenha a 4 °C.</li>
	<li>Conte a quantidade total de células usando um dispositivo comum de contagem de células.</li>
	<li>Prepare uma suspensão celular a partir da fração classificada positiva contendo 2 x 105 células/μl no buffer MACS e mantenha a 4 °C</li>
</ol>4. Transplante de células precursoras do fotoreceptor classificados pelo MAC na Retina do Rato
<ol><li>Certifique-se de ter todas as seguintes soluções prontas: 1 ml de PBS ou HBSS estéreis, 10 μl DNAse I, 1-2 ml estéril desionizado H2O, alíquotas das frações de suspensão celular a 4 °C.</li>
	<li>Anestesiar o camundongo adulto (ou seja, 2-4 meses de idade) com uma injeção intraperitoneal de cloridrato de medetomidina (0,01 mg/10 g de peso corporal), cetamina (0,75 mg/10 g de peso corporal). Em seguida, faça uma injeção subcutânea de Buprenorfina (0,05 mg/kg de peso corporal) para alívio da dor.</li>
	<li>Dilatar pupilas com queda de Fenilefrino 2,5%-Tropicamid 0,5%.</li>
	<li>Fixar o mouse no suporte da cabeça do mouse e colocar sob microscópio estéreo.</li>
	<li>Aplique uma gota de gel vísico para evitar a secagem do olho.</li>
	<li>Faça um pequeno buraco na borda entre esclera e córnea (respectivamente ora serrata) usando uma agulha estéril de 30 G 1/2.</li>
	<li>Corte uma lâmina de cobertura comum em peças pequenas (aproximadamente 5 mm x 5 mm) usando uma caneta de diamante, coloque uma dessas peças em cima da córnea, permitindo visualização direta da retina.</li>
	<li>Lave a seringa microliter presterilizada várias vezes com água deionizada.</li>
	<li>Carregue a seringa microliter com 1 μl de suspensão celular, direcione a agulha tangencialmente através da conjuntiva e esclera e coloque sob controle visual na metade nasal da retina.</li>
	<li>Faça um furo suavemente na retina até chegar ao espaço subretinal, injete a suspensão da célula no espaço subretinal.</li>
	<li>Observe o descolamento bolhoso da retina, que deve ser uniforme, cobrindo aproximadamente 1/4 do espaço da retina sem qualquer sangramento.</li>
	<li>Retire a seringa suavemente, o orifício da retina sela automaticamente.</li>
	<li>Lave a seringa microlitera várias vezes com água deionizada.</li>
	<li>Solte o mouse do suporte da cabeça.</li>
	<li>Acorde o rato injetando cloridrato de atipamozole para reversão do efeito de cloridrato de medetomidina.</li>
	<li>Coloque o mouse para acordar na câmara escura quente (aproximadamente 25 °C).</li>
	<li>Nos próximos 2 dias após o transplante, a Buprenorfina (0,05 mg/kg de peso corporal) deve ser administrada por injeção subcutânea pela manhã e à tarde para alívio da dor.</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>

Os autores declaram não haver interesses financeiros concorrentes.

O transplante subretinal de células precursoras fotorreceptoras representa uma ferramenta confiável para alcançar a integração dessas células sensíveis à luz em retinas hospedeiras em números significativos1,2. Isso pode permitir o estabelecimento de uma terapia celular para o tratamento de doenças degenerativas da retina no futuro6. A população de doadores de células, atualmente isoladas das retinas PN 4, é uma mistura de diferentes tipos de células, das quais apenas as células precursoras do fotorecept...

A visão é um dos principais sentidos dos humanos. O comprometimento desse sentido e da cegueira são uma das principais razões para a incapacidade nos países industrializados. A causa predominante para comprometimento da visão ou cegueira é a degeneração da retina, caracterizada pela perda celular fotorreceptora, pois pode ser observada na degeneração macular, retinite pigmentosa, distrofia cone-rod e outras condições. Até o momento, uma terapia eficaz para restaurar a visão perdida não está disponível. Em 2006 e 2008, dois laboratórios diferentes relataram, independentes um do outro, um transplante bem sucedido de células precursoras de fotorreceptor de varas em retinas de camundongos adultos do tipo selvagem1,2. Assim, decorrente da possibilidade de transplante de células precursoras fotorreceptores também em uma retina degenerada, para substituir fotorreceptores degenerados e restaurar a visão. De fato, foi demonstrado recentemente que tais células precursoras fotorreceptoras transplantadas provocam critérios morfológicos de fotorreceptores maduros do tipo selvagem, como segmentos externos devidamente desenvolvidos3, terminais sinápticos próximos a células bipolares endógenas e um corpo de células redondas localizados na camada nuclear externa2-4,bem como a capacidade de integrar-se funcionalmente ao circuito neural hospedeiro5-7. Um dos principais princípios dessa estratégia é o uso do pós-natal dia 4 (PN 4, PN0 é definido como dia de nascimento) retinas de camundongos jovens, resultando em uma mistura de diferentes tipos de células para transplante. No pano de fundo de uma futura aplicação terapêutica, essa mistura deve ser purificada para células precursoras fotorreceptoras. CD73 foi descrito como o primeiro marcador de superfície celular específico para fotorreceptores jovens na retina8-10. Aqui, demonstramos um método de purificação celular precursora fotoreceptor baseado neste marcador de superfície celular e com o uso da técnica de classificação celular associada a magnético (MACS). O MACS pode ter vantagens em comparação com as técnicas de classificação celular ativadas por fluorescentes, devido aos tempos de triagem rápidos e ao ajuste mais fácil às condições de GMP. Poderíamos demonstrar um enriquecimento de ~90% e uma taxa de integração até 3 vezes maior ao transplantar a população enriquecida para o espaço subretinal em retinas adultas do tipo selvagem. Assim, o enriquecimento celular precursor do fotoreceptor baseado em MACS e o transplante subretinal são técnicas confiáveis e promissoras para o desenvolvimento de uma estratégia terapêutica regenerativa para o tratamento da degeneração da retina.

<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

A deficiência visual e a cegueira devido à perda das células de sensoriamento de luz da retina, ou seja, fotorreceptores, representam a principal razão para a incapacidade nos países industrializados. A substituição de fotorreceptores degenerados por transplante celular representa uma possível opção de tratamento em futuras aplicações clínicas. De fato, estudos pré-clínicos recentes demonstraram que fotorreceptores imaturos, isolados da retina do camundongo neonatal no pós-natal dia 4, têm o potencial de se integrar à retina do camundongo adulto após o transplante subretinal. As células doadoras geraram uma morfologia fotorreceptora madura, incluindo segmentos internos e externos, um corpo de células redondas localizado na camada nuclear externa, e terminais sinápticos próximos a células bipolares endógenas. De fato, relatórios recentes demonstraram que os fotorreceptores doadores se integram funcionalmente aos circuitos neurais dos camundongos hospedeiros. Para uma futura aplicação clínica de tal abordagem de substituição celular, suspensões purificadas das células de escolha devem ser geradas e colocadas na posição correta para a adequada integração ao olho. Para o enriquecimento dos precursores do fotorreceptor, a classificação deve ser baseada em antígenos de superfície celular específicos para evitar a modificação genética das células doadoras. Aqui mostramos a classificação celular associada a magnético (MACS) - enriquecimento de precursores fotorreceptor de haste transplantáveis isolados da retina neonatal de camundongos repórteres específicos do fotorreceptor com base no marcador de superfície celular CD73. A incubação com anticorpos anti-CD73 seguidos por anticorpos secundários conjugados por micro-contas permitiu o enriquecimento de precursores fotorreceptor de haste pelo MACS para aproximadamente 90%. Em comparação com a citometria de fluxo, o MACS tem a vantagem de que pode ser mais fácil aplicado aos padrões GMP e que altas quantidades de células podem ser classificadas em períodos relativos curtos de tempo. A injeção de suspensões celulares enriquecidas no espaço subretinal de camundongos adultos do tipo selvagem resultou em uma taxa de integração 3 vezes maior em comparação com suspensões celulares não sortidas.

Para avaliar a capacidade dos fotorreceptores de haste de integrar-se à retina do rato, foi utilizada uma linha de repórteres de camundongos, na qual o GFP é impulsionado pelo zíper de leucina de retina neural (Nrl, Nrl-GFP) promotor11. Nrl é o marcador mais antigo de fotorreceptores de vara iniciando sua expressão em E12.5 durante toda a idade adulta, permitindo uma rotulagem específica de células fotorreceptoras de vara de doador.
PN 4 Filhotes de NRL-GFP foram decapitados...

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

O transplante celular representa uma estratégia para o tratamento da degeneração da retina caracterizada pela perda de fotorreceptor. Aqui descrevemos um método de enriquecimento de fotorreceptores transplantáveis e seu enxerto subretinal em camundongos adultos.

Transplante subretinal de células precursoras do fotoreceptor purificado MACS na retina do rato adulto

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 visualizações.  Technische Universität Dresden. O transplante celular representa uma estratégia para o tratamento da degeneração da retina caracterizada pela perda de fotorreceptor. Aqui descrevemos um método de enriquecimento de fotorreceptores transplantáveis e seu enxerto subretinal em camundongos adultos.

Vídeo: 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.

Injeção de sub-retiniana de Vetores da terapia genética e células-tronco no olho do rato

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 ...

Medicina

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.

Descolamento de Retina modelo em roedores por injeção sub-retiniana de hialuronato de sódio

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.

O transplante de válvula pulmonar Usando um modelo do rato de Heterotópica Transplante de Coração

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.

Rato Transplante Renal: Modelos de rejeição de enxertos

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.

Realizando Subretinal Injeções em roedores para entregar epitélio pigmentar da retina células em suspensão

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.

Encapsulation termogênico pré-adipócitos para transplante em adiposo Depots Tissue

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).

Ortotópico Hind Limb Transplantation no mouse

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.

A Passo a Passo Protocolo de Cirurgia sub-retiniana em Coelhos

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.

Transplantação no Pad Mouse ovário Fat

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.

Reconstrução do circuito sanguíneo em um modelo de transplante de coração de rato abdominal

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