우리는 Nrl-GFP 마우스를 제공 아난드 스와롭, 기술 지원을위한 요헨 하스, 동물 축산에 대한 신디 뵈메와 에멜리 레스만을 제공 감사드립니다.	
	이 작품은 도이치 포충스게마인샤프트(DFG): FZT 111 - 재생 치료 드레스덴 센터, CRTD 종자 보조금 프로그램, SFB 655 및 프로레티나 e.V. 재단, DIGS-BB 대학원 프로그램 드레스덴, 그리고 Fundação 파라 시엔시아 e Tecnologia (SFRH/BD/60787/2009)

동물의 윤리적 사용 및 관리 성명서:
모든 동물 실험은 유럽 연합 및 독일 법률 (Tierschutzgesetz)에 따라 엄격하게 수행되었으며 안과 및 시력 연구에서 동물의 사용에 대한 ARVO 성명서를 준수했습니다. 모든 동물 실험은 TU 드레스덴과 Landesdirektion 드레스덴의 동물 윤리위원회에 의해 승인되었다 (승인 번호: 24D-9168.11-1/2008-33).
1. 세포 절개 및 셀 정렬을 시작하기 전에
<ol><li>세 개의 15ml 반응 튜브라벨: 세척(W), 포지티브 분수(+) 및 음수분(-).</li>
</ol>2. 망막 해리
<ol><li>가위를 사용하여 PN 4 새끼를 참수하십시오.</li>
	<li>강아지의 머리를 70 % 에탄올로 헹구고 PBS에서 세척합니다.</li>
	<li>머리를 차가운 HBSS로 옮기고 눈을 방출합니다 (모든 머리에 대해이 단계를 반복하십시오). 신경을 방출하려면 눈꺼풀을 열고 시신경 부위의 곡면으로 눈을 고정시합니다. 그런 다음 시야를 조심스럽게 궤도밖으로 끌어냅니다.</li>
	<li>모든 눈을 방출 한 후 망막을 분리하십시오 : 닫힌 가위를 시신경에 도입하고 블레이드를 엽니 다. RPE /chorid 밖으로 peal. 구부러진 집게를 사용하여 렌즈와 혈관을 제거합니다.</li>
	<li>분리된 망막을 파팡 용액을 함유하는 1.5 ml 반응 튜브로 옮김(파팡 해리 키트에 의해 제공).</li>
	<li>37°C에서 수조 또는 셰이커(400rpm)에서 30-60분 동안 파팡 용액의 망막을 배양한다.<ol>
<li>망막은 파팡 용액에 배양하는 동안, 15ml 반응 튜브 (라벨 "1")에 ovomucoid 용액의 파이펫 1 ml.</li>
			<li>DNase I (10 mg/ml) + ovomucoid 용액 60 μl (키트제공) + EBSS 520 μl을 15ml 반응 튜브 (라벨 "2")에 추가합니다.</li>
		</ol>
</li>
	<li>파파인 인큐베이션 후, 부분적으로 소화된 망막을 포함하는 파팡 용액을 반응 튜브 "2"로 전달한다.</li>
	<li>불을 연마한 파이펫을 사용하여 기계적 해리(10배 상하)를 수행합니다.</li>
	<li>파이펫 반응 튜브 "1"에 단일 셀 서스펜션. 난소용액 위에 셀 서스펜션을 부드럽게 겹쳐서 2층을 생성해 보십시오.</li>
	<li>300 x g (약 1,300 rpm)에서 5 분 동안 원심 분리기.</li>
	<li>상체를 버리고 MACS 버퍼의 500 μl에서 셀을 다시 일시 중지합니다.</li>
</ol>3. 자기 연관된 세포 분류(MACS)를 사용하여 셀 정렬
<ol><li>10 μg/ml의 최종 농도를 달성하기 위해 X μl 쥐 항 CD73 항체를 500 μl에 첨가하십시오.</li>
	<li>얼음에 5 분 인큐베이션.</li>
	<li>MACS 버퍼로 15ml 반응 튜브를 10ml로 채우고 원심분리기는 300 x g의 속도로 5 분 동안 작성합니다.</li>
	<li>상부체를 제거합니다.</li>
	<li>480 μl MACS 버퍼에서 펠릿을 다시 중단하고 염소 안티 쥐 IgG 마이크로 비드의 120 μl을 추가합니다.</li>
	<li>얼음에 15 분 인큐베이션; 동요하지 마십시오, 흔들어 또는 혼합.</li>
	<li>MACS 버퍼로 15ml 반응 튜브를 5ml로 채우고 원심분리기는 300 x g에서 5 분 동안 작성하십시오.</li>
	<li>반응 튜브가 원심 분리됨에 따라:<ol>
<li>LS 컬럼을 마그네틱 스탠드에 조정합니다.</li>
			<li>LS 열 위에 사전 분리 필터를 놓습니다.</li>
			<li>3ml MACS 버퍼로 프리분리 필터와 LS 컬럼을 수분화하고 MACS 버퍼를 세척(W) 튜브로 수집합니다.</li>
		</ol>
</li>
	<li>상부체를 제거합니다.</li>
	<li>500 μl MACS 버퍼에서 펠릿을 다시 중단합니다.</li>
	<li>500 μl 셀 서스펜션을 필터에 로드한 다음 필터에 1ml MACS 버퍼를 추가하고 음극분획(-) 반응 튜브에 음극기를 수집합니다.</li>
	<li>그런 다음 컬럼에 3 x 3 ml의 MACS 버퍼를 추가하여 열에 바인딩되지 않은 셀을 씻어 내다</li>
	<li>이 9ml가 LS 컬럼을 통과하면 자기 스탠드에서 컬럼을 제거하고 양극분수(+) 반응 튜브 위에 설치합니다.</li>
	<li>LS 열에 5 ml의 MACS 버퍼를 빠르게 로드하고 LS 열에 플런저를 넣고 전체 버퍼가 열을 통과할 때까지 아래로 누릅니다.</li>
	<li>300 x g에서 5 분 동안 양성 분획을 포함하는 반응 튜브를 원심 분리합니다.</li>
	<li>상체를 제거하고 MACS 버퍼의 500 μl에서 다시 중단하고 4 °C에서 유지하십시오.</li>
	<li>공통 셀 계수 장치를 사용하여 셀의 총 양을 계산합니다.</li>
	<li>MACS 버퍼에서 2 x 105 셀/μl을 포함하는 양극 분수에서 셀 서스펜션을 준비하고 4°C에서 보관하십시오.</li>
</ol>4. 마우스 망막에 MAC 정렬 광수용체 전구체 세포의 이식
<ol><li>다음 의 모든 솔루션을 준비해야 합니다: 1ml 멸균 PBS 또는 HBSS, 10 μl DNAse I, 1-2 ml 멸균 제H2O, 4°C에서 셀 서스펜션 분획의 알리쿼트.</li>
	<li>메데토미딘 염산염(0.01 mg/10g 체중), 케타민(0.75 mg/10g 체중)의 인트라피오네 주사로 성인마우스(즉, 2-4개월)를 마취한다. 그런 다음 통증 완화를 위해 Buprenorphine (0.05 mg/kg 체중)의 피하 주사를 수행하십시오.</li>
	<li>페닐레프린 2.5%-트로피카미드 0.5% 하락으로 동공을 넓히게 한다.</li>
	<li>마우스 헤드 홀더에 마우스를 고정하고 스테레오 현미경 아래에 배치합니다.</li>
	<li>눈의 건조를 방지하기 위해 Visidic 젤 한 방울을 바르십시오.</li>
	<li>바늘에 멸균 30 G 1/2를 사용하여 clera와 각막 (각각 오라 세라타)사이의 경계에 작은 구멍을 확인합니다.</li>
	<li>다이아몬드 펜을 사용하여 일반적인 커버 슬라이드를 작은 (약 5mm x 5mm) 조각으로 자르고 각막 위에 이 조각 중 하나를 배치하여 망막의 직접 시각화를 허용합니다.</li>
	<li>미리 살균 된 마이크로 리터 주사기를 탈화 된 물로 여러 번 플러시하십시오.</li>
	<li>마이크로리터 주사기를 셀 서스펜션 1μl로 적재하고, 결막과 클era를 통해 직접 바늘을 접선으로 적재하고 망막의 비강 반의 비강에서 시각적 제어 하에 놓습니다.</li>
	<li>망막에 구멍을 부드럽게 뚫고 침전 공간에 도달할 때까지 셀 서스펜션을 적재 공간에 주입합니다.</li>
	<li>결막의 황소 분리를 관찰, 어떤 출혈없이 망막 공간의 약 1/4를 커버, 균일해야한다.</li>
	<li>주사기를 부드럽게 인출하면 망막 구멍 씰이 자동으로 밀봉됩니다.</li>
	<li>마이크로리터 주사기를 탈온화물로 여러 번 플러시합니다.</li>
	<li>헤드 홀더에서 마우스를 놓습니다.</li>
	<li>메데토미딘 염산염 효과의 반전을 위해 아티파노졸 염산염을 주입하여 마우스를 깨우는 다.</li>
	<li>따뜻한 어두운 챔버 (약 25 °C)에서 일어나 시간을 위해 마우스를 놓습니다.</li>
	<li>이식 후 다음 2일, 부프레노르핀(0.05 mg/kg의 체중)은 통증 완화를 위해 아침과 오후에 피하 주사에 의해 투여되어야 한다.</li>
</ol>

<ol>
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저자는 경쟁 적인 재정적 이익을 선언하지 않습니다.

광수용체 전구체 세포의 침전체 이식은 이러한 빛에 민감한 세포의 통합을 달성하기 위한 신뢰할 수 있는 도구를 나타내며, 이는상당수1,2에서숙주 망막으로 통합된다. 이것은 미래에 망막 퇴행성 질병의 처리를 위한 세포 치료의 설치를 허용할 지도 모릅니다6. 현재 PN 4 망막으로부터 분리된 세포의 기증자 인구는 다른 세포 유형의 혼합물이며, 이 세포는 감전 주사 후 광수용체 전구체 세포만 ?...

시력은 인간의 주요 감각 중 하나입니다. 이러한 감각과 실명의 장애는 선진국의 장애의 주된 이유 중 하나입니다. 시력 장애 또는 실명에 대한 주된 원인은 황반 변성, 망막 색소, 콘로드 이영양증 및 기타 조건에서 관찰될 수 있으므로 광수용체 세포 손실을 특징으로 하는 망막 변성입니다. 현재까지 잃어버린 시력을 복원하는 효과적인 치료법은 제공되지 않습니다. 2006년과 2008년에 는 서로 독립적으로, 영성 야생형 마우스망막1,2로막대 광수용체 전구체 세포를 성공적으로 이식하는 두 개의 상이한 실험실을 보고하였다. 따라서, 광수용체 전구체 세포 이식의 가능성을 발생시키고 또한 퇴화 된 망막으로, 퇴화 광수용체를 대체하고 시력을 회복시킨다. 실제로, 최근에는 이식된 광수용체 전구체세포가 제대로 발달된 외부 세그먼트3,시냅스 말단과 외부 핵층2-4에위치한 둥근 세포 체와 근접한 성숙한 야생형 광수용체의 형태학적 기준을 유도하고 있으며, 뿐만 아니라, 소각 회로 신경에 기능적으로 통합할 수 있는 능력도 입증되었다. 이 전략의 주요 원리 중 하나는 산후 4일 (PN 4, PN0은 출생의 날로 정의) 젊은 마우스 망막의 사용으로, 이식을위한 다른 세포 유형의 혼합물의 결과. 미래의 치료 응용 프로그램의 배경에, 이 혼합물은 광수용체 전구체 세포에 대해 정제되어야한다. CD73은 망막8-10에서젊은 광수용체에 대한 제1 세포 표면 마커로 기재되었다. 여기서, 우리는 이러한 세포 표면 마커에 기초하여 광수용체 전구체 세포 정화 방법을 시연하고 자기 관련 세포 선별(MACS) 기술을 사용한다. MACS는 빠른 정렬 시간과 GMP 조건에 대한 쉽게 조정으로 인해 형광 활성화 셀 정렬 기술에 비해 장점이 있을 수 있습니다. 우리는 성인 야생 형 망막에서 하수 공간으로 농축 된 인구를 이식 할 때 ~ 90 % 농축 및 최대 3 배 높은 통합 률을 입증 할 수 있습니다. 따라서 MACS 기반 광수용체 전구체 세포 농축 및 피망 이식은 망막 퇴성의 치료를 위한 재생 치료 전략의 개발을 위한 신뢰할 수 있고 유망한 기술이다.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
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	<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

망막의 빛 감지 세포, 즉 광수용체의 손실로 인한 시력 장애 및 실명은 선진국에서 장애의 주된 이유를 나타냅니다. 세포 이식에 의한 퇴화 광수용체의 교체는 향후 임상 적용에서 가능한 치료 옵션을 나타낸다. 실제로, 최근 전임상 연구는 산후 4일째에 신생아 마우스 망막에서 분리된 미성숙광수용체가 침전 이식 후 성인 마우스 망막에 통합될 가능성이 있음을 입증했습니다. 기증자 세포는 내과성 양극성 세포에 근접하여 내부 및 외부 세그먼트, 외부 핵 층에 위치한 둥근 세포 체, 시냅스 말기를 포함한 성숙한 광수용체 형태를 생성하였다. 실제로, 최근 보고서는 기증자 광수용체가 기능적으로 숙주 마우스의 신경 회로에 통합한다는 것을 보여주었습니다. 이러한 세포 교체 접근법의 향후 임상 적용을 위해, 선택의 세포의 정제 된 현탁액을 생성하고 눈에 적절한 통합을위한 올바른 위치에 배치해야합니다. 광수용체 전구체의 농축을 위해, 선별은 기증자 세포의 유전 기자 수정을 피하기 위하여 특정 세포 표면 항원에 기초해야 합니다. 여기서 우리는 자기 관련 세포 선별(MACS) -세포 표면 마커 CD73에 기초하여 광수용체 특이기자 마우스의 신생아 망막으로부터 분리된 이식 가능한 막대 광수용체 전구체의 농축을 보여준다. 항 CD73 항체를 함유한 배양은 마이크로 비드 컨쥬게이드 이차 항체에 이어 MACS에 의한 로드 광수용체 전구체의 농축을 약 90%까지 허용하였다. 흐름 세포측정에 비해 MACS는 GMP 표준에 더 쉽게 적용할 수 있으며 많은 양의 세포를 상대적인 짧은 기간에 정렬할 수 있다는 장점이 있습니다. 성인 야생형 마우스의 하수 공간에 농축된 세포 현탁액을 주입하면 분류되지 않은 세포 현탁액에 비해 3배 더 높은 통합률을 보였습니다.

마우스 망막에 통합하는 막대 광수용체의 능력을 평가하기 위해 마우스 리포터 라인이 사용되었으며, GFP는 신경망막 류신 지퍼(Nrl, Nrl-GFP)프로모터(11)에의해 구동된다. Nrl은 항봉 광수용체세포의 특정 라벨링을 허용하는 성인기 전반에 걸쳐 E12.5에서 발현을 시작하는 로드 광수용체의 초기 마커이다.
PN 4 Nrl-GFP 새끼는 참수되었고 눈이 에클레아트되었습니다. 망막은 ?...

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

세포 이식은 광수용체 손실을 특징으로 하는 망막 변성의 치료를 위한 전략을 나타낸다. 여기에서 우리는 이식 가능한 광수용체의 농축 및 성인 마우스로그들의 감하 이식을 위한 방법을 기술합니다.

성인 마우스 망막으로 MACS 정제 광수용체 전구체 세포의 감하 이식

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 조회수.  Technische Universität Dresden. 세포 이식은 광수용체 손실을 특징으로 하는 망막 변성의 치료를 위한 전략을 나타낸다. 여기에서 우리는 이식 가능한 광수용체의 농축 및 성인 마우스로그들의 감하 이식을 위한 방법을 기술합니다.

비디오: 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.

마우스 눈에 유전자 치료 벡터와 줄기 세포의 Subretinal 주입

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 ...

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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.

나트륨 히알루 론산의 망막 주사 설치류에있는 망막 박리 모델

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.

이소성 심장 이식 마우스 모델을 사용하여 폐동맥 판막 이식

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.

마우스 신장 이식 : 동종 이식 거부의 모델

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.

서스펜션에 망막 색소 상피 세포를 제공하기 위해 설치류에서 망막 주사를 수행

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.

지방 조직의 종점에 이식 캡슐화 열 발생 Preadipocytes

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).

마우스에서 소성을 뒷다리 이식

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.

토끼의 망막 수술에 대한 단계 프로토콜에 의해 단계

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.

마우스 난소 지방 패드 속으로 이식

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.

복부 마우스 심혼 이식 모형에 있는 혈액 회로 재건

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