Ci piace ringraziare Anand Swaroop per aver fornito topi Nrl-GFP, Jochen Haas per il supporto tecnico e Sindy Böhme ed Emely Lessmann per l'allevamento.	
	Questo lavoro è stato supportato dalla Deutsche Forschungsgemeinschaft (DFG): FZT 111 - Center of Regenerative Therapies Dresden, dal CRTD Seed Grant Program, dall'SFB 655 e dal ProRetina e.V. fondazione, il DIGS-BB Graduate Program Dresden e il Fundação para a Ciência e Tecnologia (SFRH/BD/60787/2009)

Uso etico e cura degli animali dichiarazione:
Tutti gli esperimenti sugli animali sono stati effettuati in stretta conformità con le leggi dell'Unione Europea e della Germania (Tierschutzgesetz) e hanno aderito alla dichiarazione ARVO per l'uso degli animali nella ricerca oftalmica e della visione. Tutti gli esperimenti sugli animali sono stati approvati dal comitato etico animale della TU Dresda e della Landesdirektion Dresden (numero di approvazione: 24D-9168.11-1/2008-33).
1. Prima di iniziare la dissociazione cellulare e l'ordinamento cellulare
<ol><li>Etichettare tre tubi di reazione da 15 ml con: Lavaggio (W), Frazione positiva (+) e Frazione negativa (-).</li>
</ol>2. Dissociazione della retina
<ol><li>Decapitare i cuccioli PN 4 usando una forbice.</li>
	<li>Risciacquare la testa dei cuccioli poco dopo nel 70% di etanolo seguito dal lavaggio in PBS.</li>
	<li>Trasferire la testa in HBSS freddo ed enucleare l'occhio (ripetere questo passaggio per tutte le teste). Per enucleare, aprire le palpebre e fissare l'occhio con forcep curve nella regione del nervo ottico. Quindi tira l'occhio con attenzione fuori dall'orbita.</li>
	<li>Dopo aver enucleato tutti gli occhi, isolare la retina: introdurre una forbice chiusa nel nervo ottico e aprire le lame. Sbirciare l'RPE/chorid out. Rimuovere le lenti e i vasi sanguigni utilizzando forcep curve.</li>
	<li>Trasferire le retine isolate in un tubo di reazione da 1,5 ml contenente la soluzione di papaina (fornita dal kit di dissociazione della papaina).</li>
	<li>Incubare le retine nella soluzione di papaina per 30-60 minuti in un bagno d'acqua o shaker (400 giri/min) a 37 °C.<ol>
<li>Mentre le retine incubano nella soluzione di papaina, pipetta 1 ml di soluzione ovomucoide in un tubo di reazione da 15 ml (etichetta "1").</li>
			<li>Aggiungere 60 μl di DNasi I (10 mg/ml) + 60 μl di soluzione ovomucoide (fornita dal kit) + 520 μl di EBSS in un tubo di reazione da 15 ml (etichetta "2").</li>
		</ol>
</li>
	<li>Dopo l'incubazione della papaina, trasferire la soluzione di papaina contenente le retine parzialmente digerite nel tubo di reazione "2".</li>
	<li>Utilizzando una pipetta lucida antincendio, eseguire la dissociazione meccanica (10 volte su e giù).</li>
	<li>Pipettare la sospensione a cella singola al tubo di reazione "1". Prova a generare 2 strati strati stratificando delicatamente la sospensione cellulare sopra la soluzione ovomucoide.</li>
	<li>Centrifuga per 5 min a 300 x g (circa 1.300 giri/min).</li>
	<li>Scartare il supernatante e rimescolare le cellule in 500 μl di tampone MACS.</li>
</ol>3. Ordinamento delle celle mediante MACS (Magnetic Associated Cell Sorting)
<ol><li>Aggiungere anticorpo anti-CD73 del ratto X μl a 500 μl per ottenere una concentrazione finale di 10 μg/ml.</li>
	<li>Incubare 5 minuti sul ghiaccio.</li>
	<li>Riempire il tubo di reazione da 15 ml a 10 ml con tampone MACS e centrifugarlo per 5 minuti ad una velocità di 300 x g.</li>
	<li>Rimuovere il supernatante.</li>
	<li>Rimpiorsi il pellet in tampone MACS da 480 μl e aggiungere 120 μl di microperline IgG di capra anti-ratto.</li>
	<li>Incubare 15 minuti sul ghiaccio; non agitarlo, scuoterlo o mescolarlo.</li>
	<li>Riempire il tubo di reazione da 15 ml a 5 ml con tampone MACS e centrifugarlo per 5 minuti a 300 x g.</li>
	<li>Poiché il tubo di reazione è stato centrifugato:<ol>
<li>Regolare una colonna LS nel supporto magnetico.</li>
			<li>Mettere il filtro di preseparazione sopra la colonna LS.</li>
			<li>Idratare il filtro di preseparazione e la colonna LS con tampone MACS da 3 ml e raccogliere il tampone MACS nel tubo di lavaggio (W).</li>
		</ol>
</li>
	<li>Rimuovere il supernatante.</li>
	<li>Rimosi il pellet in tampone MACS da 500 μl.</li>
	<li>Caricare la sospensione cellulare da 500 μl sul filtro, quindi aggiungere 1 ml di tampone MACS al filtro e raccogliere la frazione negativa nel tubo di reazione della frazione negativa (-).</li>
	<li>Aggiungere quindi 3 x 3 ml di buffer MACS alla colonna per lavare via le celle non associate alla colonna</li>
	<li>Una volta che questi 9 ml sono attraverso la colonna LS, rimuovere la colonna dal supporto magnetico e installarla sopra il tubo di reazione della frazione positiva (+).</li>
	<li>Caricare rapidamente 5 ml di buffer MACS nella colonna LS e mettere lo stantuffo nella colonna LS e premerlo verso il basso fino a quando l'intero buffer non passa attraverso la colonna.</li>
	<li>Centrifugare il tubo di reazione contenente la frazione positiva per 5 min a 300 x g.</li>
	<li>Rimuovere il supernatante, resuspend in 500 μl di tampone MACS e mantenere a 4 °C.</li>
	<li>Contare la quantità totale di celle utilizzando un dispositivo comune di conteggio delle celle.</li>
	<li>Preparare una sospensione cellulare dalla frazione ordinata positiva contenente 2 x 105 celle/μl nel buffer MACS e mantenerla a 4 °C</li>
</ol>4. Trapianto di cellule precursori fotorecettore ordinate da MAC nella retina del topo
<ol><li>Assicurarsi di avere tutte le seguenti soluzioni pronte: 1 ml di PBS sterile o HBSS, 10 μl DNAse I, 1-2 ml sterile deionizzato H2O, aliquote delle frazioni di sospensione cellulare a 4 °C.</li>
	<li>Anestetizzare il topo adulto (cioè 2-4 mesi)con un'iniezione intraperitoneale di cloridrato di medetomidina (0,01 mg/10 g di peso corporeo), chetamina (0,75 mg/10 g di peso corporeo). Quindi fare un'iniezione sottocutanea di Buprenorfina (0,05 mg / kg di peso corporeo) per alleviare il dolore.</li>
	<li>Dilatare gli alunni con un calo di Fenilefrina 2.5%-Tropicamid 0.5%.</li>
	<li>Fissare il mouse nel supporto della testa del mouse e posizionarlo al microscopio stereo.</li>
	<li>Applicare una goccia di gel visidico per evitare l'asciugatura dell'occhio.</li>
	<li>Fare un piccolo foro al confine tra sclera e cornea (rispettivamente ora serrata)usando uno sterile 30 G 1/2 in ago.</li>
	<li>Tagliare un coperchio comune scivolare in piccoli pezzi (circa 5 mm x 5 mm) utilizzando una penna diamanta, posizionare uno di questi pezzi sopra la cornea, consentendo la visualizzazione diretta della retina.</li>
	<li>Sciacquare più volte la siringa microliter presterilizzata con acqua deionizzata.</li>
	<li>Caricare la siringa microliter con 1 μl di sospensione cellulare, dirigere l'ago tangenzialmente attraverso la congiuntiva e la sclera e posizionare sotto controllo visivo nella metà nasale della retina.</li>
	<li>Punzonare delicatamente un foro nella retina fino a raggiungere lo spazio subretinale, iniettare la sospensione cellulare nello spazio subretinale.</li>
	<li>Osservare il distacco bolloso della retina, che dovrebbe essere uniforme, coprendo circa 1/4 dello spazio retinico senza alcun sanguinamento.</li>
	<li>Ritirare delicatamente la siringa, il foro retinica si sigilla automaticamente.</li>
	<li>Sciacquare più volte la siringa del microlitro con acqua deionizzata.</li>
	<li>Rilasciare il mouse dal supporto della testa.</li>
	<li>Svegliare il topo iniettando cloridrato di atipamozolo per invertire l'effetto cloridrato di medetomidina.</li>
	<li>Posizionare il mouse per il tempo di risveglio in una camera calda e scura (circa 25 °C).</li>
	<li>I successivi 2 giorni dopo il trapianto, la buprenorfina (0,05 mg / kg di peso corporeo) deve essere somministrata per iniezione sottocutanea al mattino e al pomeriggio per alleviare il dolore.</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>

Gli autori non dichiarano interessi finanziari concorrenti.

Il trapianto subretinale di cellule precursori del fotorecettore rappresenta uno strumento affidabile per ottenere l'integrazione di queste cellule sensibili alla luce nelle retine ospiti innumero significativo 1,2. Ciò potrebbe consentire l'istituzione di una terapia cellulare per il trattamento delle malattie degenerative retiniche in futuro6. La popolazione donatrice di cellule, attualmente isolata dalle retine PN 4, è una miscela di diversi tipi di cellule, da cui solo le cellule precursori del fotorecettore si in...

La visione è uno dei sensi primi degli umani. La compromissione di questo senso e della cecità sono una delle principali ragioni della disabilità nei paesi industrializzati. La causa predominante di compromissione della vista o cecità è la degenerazione retinica, caratterizzata da perdita cellulare fotorecettore, in quanto può essere osservata nella degenerazione maculare, retinite pigmentosa, distrofia conica-asta e altre condizioni. Ad oggi, non è disponibile una terapia efficace per ripristinare la visione persa. Nel 2006 e nel 2008 due diversi laboratori hanno riportato, indipendenti l'uno dall'altro, un trapianto riuscito di cellule precursori del fotorecettore di bastone in retine di topi adulti di tiposelvatico 1,2. Così, derivante dalla possibilità di trapianto di cellule precursori del fotorecettore anche in una retina degenerata, per sostituire i fotorecettori degenerati e ripristinare la visione. Infatti, è stato dimostrato di recente che tali cellule precursori fotorecettori trapiantate suscitano criteri morfologici di fotorecettori maturi di tipo selvatico, come i segmentiesterni 3adeguatamente sviluppati, terminali sinaptici in prossimità di cellule bipolari endogene e un corpo cellulare rotondo situato nello stratonucleare esterno 2-4,nonché la capacità di integrarsi funzionalmente nel circuito neurale ospite5-7. Uno dei principi principali di questa strategia è l'uso della retina dei giovani topi postnatale giorno 4 (PN 4, PN0 è definita come giorno di nascita), con conseguente miscela di diversi tipi di cellule per il trapianto. Sullo sfondo di una futura applicazione terapeutica, questa miscela deve essere purificata per le cellule precursori del fotorecettore. CD73 è stato descritto come il primo marcatore di superficie cellulare specifico per i giovani fotorecettori nella retina8-10. Qui, dimostriamo un metodo di purificazione delle cellule precursori del fotorecettore basato su questo marcatore di superficie cellulare e con l'uso della tecnica macs (magnetic-associated cell sorting). MACS potrebbe avere vantaggi rispetto alle tecniche di smistamento delle celle attivate fluorescentmente, a causa dei tempi di smistamento rapidi e della più facile regolazione delle condizioni GMP. Potremmo dimostrare un arricchimento di ~ 90% e un tasso di integrazione fino a 3 volte superiore quando trapiantiamo la popolazione arricchita nello spazio subretinale nelle retine adulte di tipo selvatico. Pertanto, l'arricchimento cellulare precursore del fotorecettore basato su MACS e il trapianto subretinale sono tecniche affidabili e promettenti per lo sviluppo di una strategia terapeutica rigenerativa per il trattamento della degenerazione retinica.

<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

La compromissione della vista e la cecità dovute alla perdita delle cellule che percepiscono la luce della retina, cioè dei fotorecettori, rappresentano la ragione principale della disabilità nei paesi industrializzati. La sostituzione dei fotorecettori degenerati con il trapianto di cellule rappresenta una possibile opzione di trattamento nelle future applicazioni cliniche. Infatti, recenti studi preclinici hanno dimostrato che i fotorecettori immaturi, isolati dalla retina neonatale del topo al giorno 4 postnatale, hanno il potenziale per integrarsi nella retina del topo adulto dopo il trapianto subretinale. Le cellule donatrici hanno generato una morfologia fotorecettori matura che include segmenti interni ed esterni, un corpo cellulare rotondo situato nello strato nucleare esterno e terminali sinaptici in prossimità delle cellule bipolari endogene. In effetti, recenti rapporti hanno dimostrato che i fotorecettori dei donatori si integrano funzionalmente nei circuiti neurali dei topi ospiti. Per una futura applicazione clinica di tale approccio di sostituzione cellulare, le sospensioni purificate delle cellule scelte devono essere generate e posizionate nella posizione corretta per una corretta integrazione nell'occhio. Per l'arricchimento dei precursori del fotorecettore, lo smistamento dovrebbe basarsi su specifici antigeni di superficie cellulare per evitare la modifica genetica delle cellule donatrici da parte dei reporter. Qui mostriamo lo smistamento cellulare associato al magnetico (MACS) - arricchimento dei precursori del fotorecettore a bastone trapiantabile isolati dalla retina neonatale di topi reporter specifici del fotorecettore basati sul marcatore di superficie cellulare CD73. L'incubazione con anticorpi anti-CD73 seguita da anticorpi secondari coniugati con micro-perline ha permesso l'arricchimento dei precursori del fotorecettore dell'asta da parte del MACS a circa il 90%. Rispetto alla citometria a flusso, MACS ha il vantaggio che può essere più facile da applicare agli standard GMP e che elevate quantità di cellule possono essere ordinate in brevi periodi di tempo relativi. L'iniezione di sospensioni cellulari arricchite nello spazio subretinale dei topi adulti di tipo selvatico ha comportato un tasso di integrazione 3 volte superiore rispetto alle sospensioni cellulari nonsortite.

Al fine di valutare la capacità dei fotorecettori dell'asta di integrarsi nella retina del mouse, è stata utilizzata una linea di reporter del topo, in cui GFP è guidata dalla cerniera neurale retina leucina (Nrl, Nrl-GFP)promotrice 11. L'Nrl è il primo marcatore dei fotorecettori di aste che inizia la sua espressione all'E12.5 per tutta l'età adulta, consentendo una specifica etichettatura delle cellule fotorecettori dell'asta del donatore.
I cuccioli di PN 4 Nrl-GFP sono stat...

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

Il trapianto cellulare rappresenta una strategia per il trattamento della degenerazione retinica caratterizzata dalla perdita di fotorecettore. Qui descriviamo un metodo per l'arricchimento dei fotorecettori trapiantabili e il loro innesto subretinale in topi adulti.

Trapianto subretinale di cellule precursori del fotorecettore purificato MACS nella retina del topo 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 Views.  Technische Universität Dresden. Il trapianto cellulare rappresenta una strategia per il trattamento della degenerazione retinica caratterizzata dalla perdita di fotorecettore. Qui descriviamo un metodo per l'arricchimento dei fotorecettori trapiantabili e il loro innesto subretinale in topi adulti.

Video: 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.

Iniezione sottoretinico di vettori di terapia genica e cellule staminali nel Eye mouse

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 ...

Ricerca

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.

Distacco della retina Modello in Roditori da iniezione sottoretinico di ialuronato di sodio

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

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.

Mouse trapianto renale: Modelli di Allograft Rifiuto

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.

Esecuzione sottoretinico Iniezioni in roditori di agire cellule retiniche epitelio pigmentato in sospensione

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.

Incapsulamento termogenico preadipocytes al trapianto nel tessuto adiposo Depositi tessuti

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).

Ortotopico degli arti posteriori del trapianto di 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.

Un passo per passo protocollo per la chirurgia sottoretinico in Conigli

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.

Trapianto nel Mouse Pad ovarico Fat

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

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Intravitale Microscopia e Trombo induzione nel lobo di un mouse Hairless

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

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.

Ricostruzione del circuito sanguigno in un modello di trapianto di cuore di topo addominale

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

Methods Article

Trapianto subretinale di cellule precursori del fotorecettore purificato MACS nella retina del topo adulto