우리는 편집 도움 제니퍼 Petrosino 다윗 DiSilvestro에게 감사의 말씀을 전합니다. 이 연구는 미국의 계란 이사회와 노보 노 디스크 제약에서 상 번호 10040042에서뿐만 아니라 OSU에서 식품 혁신 센터, 사무실 국제 문제에 대한 고급 기능 식품 연구 센터 및 기업가 정신에 의해뿐만 아니라 같은 상 번호 20020728에 의해 지원되었다 국립 과학 재단 (National Science Foundation)은 EEC-0914790 (LJL)을 부여합니다. 의료 연구에 대한 NIH 로드맵에 의해 건강 (OD)의 이사, 국립 연구소의 사무실에 의해 투자 및 지원 연구 자원을위한 국가 센터에서 수상 번호 R21OD017244 (OZ)과 UL1RR025755 (OSUCCC)에 의해 지원되었다 기술 프로젝트, NCI P30CA16058. 내용은 전적으로 저자의 책임이며 반드시 연구 자원을위한 국가 센터 또는 국립 보건원의 공식 견해를 대변하지 않습니다.

연구 프로토콜은 오하이오 주립 대학 윤리위원회에 의해 승인되었다. 동물 실험은 IACUC 프로토콜에 의해 승인되었다. 모든 절차는 층류와 레벨 2 바이오 안전성 캐비닛에서 수행 하였다. 우리는 모든 표준 안전 요구 사항 및 절차를 따랐다. 마이크로 캡슐의 제조를위한 마이크로 캡슐화 기술은, (18) (17) 기재된 바와 같이 수행되었다. 재료 1. 준비 <ol><li> 멸균 생리 식염수 (물 중 0.9 % 염화나트륨)에 2 %의 알긴산 나트륨 용액 10 ㎖를 준비한다. 생리 식염수 0.05 % PLL 솔루션을 준비합니다. 전날 이러한 솔루션을 준비하고 밤새 교반. 사용하기 전에 0.22 μm의 필터를 사용하여 솔루션을 필터링합니다. </li><li> 50 mM의 시트르산 나트륨 0.9 % 염화나트륨 용액에 100 mM의 CaCl2를 준비하고이를 오토 클레이브. </li><li> 모든 솔루션, 바늘, 전극 및 비커를 압력솥. 막힘 방지하기 와이어 철저히 바늘. </li><li> (102 마이크로 캡슐이 아니라 모든 CM이 필요한 캡슐 (18) 당 500 세포가 약가된다) 캡슐에 사용할 세포의 적절한 양을 결정합니다. 이백만 세포 당 알긴산 나트륨 1 ㎖를 사용합니다. </li><li> 높은 혈당에 &#39;섬유 아세포 성장 보통&#39;포함 10 % 혈청, 100 U / ml의 페니실린 / 스트렙토 마이신을 준비 (4,500 ㎎ / ℓ의 포도당) 둘 베코의 수정 이글의 중간 (DMEM). <ol><li> DMEM에서 10 % 소 태아 혈청, 10 μg의 / ㎖ 인슐린, 1 μM 덱사메타손 (dexamethasone), 0.5 mM의 3- 이소 부틸 -1- 메틸 크 산틴, 100 U / ml의 페니실린 / 스트렙토 마이신을 함유하는 &#39;분화 중간 I&#39;를 준비한다. </li><li> DMEM에서 &#39;분화 중간 II&#39;함유 10 % 소 태아 혈청, 10 μg의 / ㎖ 인슐린, 100 U / ml의 페니실린 / 스트렙토 마이신을 준비한다. </li></ol></li><li> (RI를 10 ml의 라​​디오 - 면역 침전 분석 완충액 당 프로테아제 억제제 정제를 함유하는 용해 버퍼를 준비PA 버퍼). </li></ol> 2. 알긴산 마이크로 비드 준비 (그림 1A) <ol><li> 세포 배양 플라스크에서 오래된 매체를 제거합니다. PBS의 10 ㎖로 세포를 씻어. 0.25 % 트립신 - EDTA (일 합류 T175 플라스크 당 2 ml)로 서스펜션에 세포를 가져와. </li><li> 혈구를 사용하여 세포 현탁액에서 세포를 계산합니다. 세포 현탁액에서 10 ml의 나누어지는을 사용하여 제조업체의 지침에 따라 세포를 계산합니다. 5 분 동안 실온에서 480 XG에서 원심 분리 배지에 남아있는 세포를 원심 분리기. </li><li> 단계 1.4에 기재된 바와 같이 알긴산 나트륨의 세포 펠렛을 일시. 5 mL의 주사기에 알긴산 나트륨 용액 세포 이동. </li><li> 23 게이지 바늘을 추가, 용액의 기포를 제거하고 공기의 1 ml의 포켓을 만들 주사기를 반전. </li><li> 캡슐의 바늘 주둥이에서 100 mM의 염화칼슘 2 용액 144 ml의를 포함하는 작은 비커 (180 ml)에 놓습니다. 팁 approxim로 캡슐에 전극을 부착100 mM의 CaCl2를 용액의 표면 위 ately 2.5 cm. </li><li> 단단히 주사기 펌프에 알긴산 나트륨을 세포를 포함하는 용액을 주사기 놓는다. 주사기의 구멍에 고무 튜브를 연결합니다. 알긴산 나트륨 셀 솔루션은 튜브를 통해 중간에 들어갈 때까지 플런저를 밀어 넣습니다. 5.4 kV의에 전압을 조정합니다. 주사기 펌프에 12.06 mm 직경을 설정합니다. 3 ㎖ / 시간에 속도를 조정합니다. 펌프를 시작합니다. 캡슐의 전원을 켜고 5.4 kV로의 전압을 유지한다. </li><li> 후드의 창을 닫고 알긴산 마이크로 비드의 형성 끝날 때까지 불필요한 진동을 피하십시오. 모든 용액이 바늘을 통과 한 후, PLL로 코팅하기 전에 추가로 20 분 동안 100 mM의 CaCl2를 용액에 알긴산 공 모양의 마이크로 비드를 고화. </li></ol> PLL 3. 코팅 마이크로 비드 (그림 1B) <ol><li> 알긴산 나트륨 셀 볼 형상 마이크로 비드를 함유하는 비이커를 제거하고 이들 microb 전송할50 ㎖ 원심 분리 튜브에 EADS. 마이크로 비드 펠렛에서 염화칼슘 2 용액을 제거합니다. </li><li> 0.9 %의 NaCl 30 ㎖를 첨가하여 알긴산 세포 공 모양의 마이크로 비드를 씻는다. 부드럽게 손으로 튜브를 흔들어. 마이크로 비드가 중력에 의해 침전 한 후 25 mL를 피펫으로 0.9 %의 NaCl을 제거합니다. 3 세척 총 두 번 더 반복합니다. </li><li> 알긴산 나트륨 수용액의 각 1 ml를 0.05 %의 PLL 용액 10 ㎖을 사용한다. 10 분 동안 분당 1,000 회전에서 0.05 % PLL과 소용돌이를 추가합니다. 주 : 일반적으로, 10 분 PLL 코팅 충분하다. </li><li> PLL 코팅이 형성된 후에, PLL 용액을 제거하고, 3.2에 기재된 바와 같이 캡슐을 3 회 세척 하였다. </li></ol> 알긴산 코어 4. 제거 (그림 1C) <ol><li> 50 mM 시트르산 나트륨 용액 30 ㎖를 추가한다. 5 분 또는 모든 알긴산 나트륨이 용해 될 때까지 기다립니다. 단계 3.1.1에 기재된 바와 같이 세척 세 번 캡슐. </li><li> , 0.9 % NaCl을 제거 50 ML 튜브에 배지 20 ㎖를 추가및 세포 배양 용 플라스크에 세포를 포함하는 모든 캡슐을 전송. 표준 세포 배양 조건 18 세 미만의 캡슐화 된 세포를 처리합니다. </li></ol> 체외 응용 프로그램 5. 세포 사이의 이종 이식 및 숙주 세포의 상호 작용이나 대사 물질 유입의 속도론 / 유출을 연구하기 위해 (그림 2) <ol><li> 공동 문화에 대한 합류 할 때까지 24 웰 플레이트에 문화 숙주 세포. 배양 preadipocytes의 &#39;섬유 아세포 성장 매체&#39;를 사용합니다. </li><li> 24 웰 플레이트 함유 합류 숙주 세포 내로 전송 마이크로 캡슐. 단층을 달성하기 위해 마이크로 캡슐 (102 마이크로 캡슐 / 웰의 CM 2)를 추가합니다. </li><li> 육일에 대한 차별화 중간 II에 전 지방 세포의 분화 중간 I.와 차별화마다 48 시간, 변화 미디어를 유도한다. RIPA 완충액으로 세포를 Lyse. 웨스턴 블롯을 사용하여 단백질 발현을 분석 조건 당 50 μg의 단백질을 사용합니다. </li></ol> Treatm에 대한 생체 응용 프로그램 6.비만의 ENT (그림 3) <ol><li> 마취의 유지 보수를 위해 산소와 마취 2 % 이소 플루 란의 유도를위한 산소와 4 % 이소 플루 란을 섞는다. 발가락 핀치에 의한 적절한 마취 깊이를 확인합니다. <ol><li> 건조로부터 각막을 보호하기 위해 눈에 안티 가려움 연고를 적용합니다. </li></ol></li><li> 이러한 녹색 형광 단백질 (GFP)와 같은 영구적 인공 형광 단백질로 표시되어 캡슐화 된 세포를 사용합니다. </li><li> 3 ML의 주사기 및 캡슐화 된 세포를 주입하는 20 게이지 바늘을 사용합니다. <ol><li> 0.5 × 10 6 세포가도 3에 도시 된 바와 같이, 생식선 및 신장 사이 복강 지역에있는 각 IAB 지방 저장소 내로 PBS 0.2 ㎖에 현탁 분사.의 평균 중량 생쥐이 PBS의 부피와 세포 수를 사용 40g. 다른 중량 용량 의존적 효과의 측정을 위해이 생쥐의 세포 수와 볼륨을 조정합니다. </li></ol></li></ol>

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Part I: evaluation of different techniques for the encapsulation of pamidronate di-sodium salt.</a> Journal of microencapsulation. 20, 509-524 (2003).</li><li>Smidsrod, O., Skjak-Braek, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+as+immobilization+matrix+for+cells.">Alginate as immobilization matrix for cells.</a> Trends in biotechnology. 8, 71-78 (1990).</li><li>Lewinska, D., Rosinski, S., Werynski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+process+conditions+during+impulsed+electrostatic+droplet+formation+on+size+distribution+of+hydrogel+beads.">Influence of process conditions during impulsed electrostatic droplet formation on size distribution of hydrogel beads.</a> Artificial cells, blood substitutes, and immobilization biotechnology. 32, 41-53 (2004).</li><li>Chan, E. 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V., Yasmeen, R., Ziouzenkova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+vitamin+A+supplementation+in+obese+mice+regulates+tissue+factor+and+cytokine+production+in+a+sex-specific+manner.">Moderate vitamin A supplementation in obese mice regulates tissue factor and cytokine production in a sex-specific manner.</a> Archives of biochemistry and biophysics. 539, 239-247 (2013).</li><li>Ziouzenkova, O., 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=Retinaldehyde+represses+adipogenesis+and+diet-induced+obesity.">Retinaldehyde represses adipogenesis and diet-induced obesity.</a> Nature. 13, 695-702 (2007).</li><li>Yasmeen, R., Jeyakumar, S. 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No conflicts of interest declared.

다양한 건조 방법, 압출, 및 에멀젼을 포함하여 19 세포를 캡슐화하는데 사용되어왔다. 이 방법에서는, 알기 네이트 비드는 PLL로 코팅 및 알지네이트 코어 캡슐화를 완료 용해되고, 바늘 통해 압출된다. 이 방법이 수년 동안 사용되었지만, 원하는 크기와 형상으로 구형 비드의 형성은 여전히​​ 도전. 캡슐의 크기는 알긴산 나트륨 용액, 압출기는 직경 바늘 끝과 CaCl2를 용액 (2...

만성 질환 1의 증가 빈도는 치료 세포 인구 2의 이식에 대한 연구를 자극했다. Syngenic 동종 또는 줄기 세포는 이러한 애플리케이션 2에 대한 가장 일반적으로 사용되는 세포 유형이다. 그러나 이러한 치료는 이식 후 분화 줄기 세포의 이동의 제어를 허용하지 않는 효율적인 비용이되지 않습니다. 유익한 기능을 가진 유전자 변형 세포의 이식은 많은 질병의 치료 개선을 기대하고있다. 그러나, 유전자 세포 변형이 호스트의 면역 시스템에 의해 인식되고, 따라서, 이러한 치료는 면역 3이 필요합니다. 인슐린 생성 세포의 캡슐화 박사 창 (4)에 의해 개발되었다. 기술은 염화칼슘 수용액에 침지 알긴산 방울 세포의 캡슐화에 기초한다. 알긴산 분자 (G) (M) 만누 및 guluronic 산 구성 및 CA로 연결 될 수 2+. 겔화 후, 비드를 폴리 -L- 라이신 (PLL) 용액에 현탁된다. 이 단계에서, PLL은 캡슐의 멤브레인을 설정 알지네이트 분자에 G와 M에 결합한다. 캡슐의 막의 다공성은 M 및 PLL 농도, 인큐베이션 시간, 온도 변화에 의해 조절 될 수있다. PLL의 결합도 유형과 알긴산의 농도에 따라 달라집니다. 칼슘 이온과 가교 알긴산 행렬, 생리 학적 환경이나 인산과 구연산 이온의 농도가 높은 일반적인 버퍼 솔루션의 불안정하다. 이 버퍼는 알긴산에서 칼슘을 추출하고 핵심을 녹일 수 있습니다. 알지네이트 코어의 액화는 세포 이동과 성장을위한 캡슐 내부에 공간을 제공합니다. 폴리 양이온 폴리 -L- 라이신 (APL)와 음이온 알지네이트에 캡슐화 된 세포는 면역 글로불린에 대한 침투하지만 독소의 영양소의 유입과 유출이 있습니다. 이 APL의 특성은 장기 SU 가능유 전적으로 서로 다른 호스트로 이식 후 캡슐화 된 세포의 rvival. 엘리엇 등 알. 구년 주입 후 5 인간의 환자에서 캡슐 돼지의 췌장 세포 기능의 생존을보고했다. 캡슐화 기술은 마이크로 캡슐화 (3-800 μm의) 및 macroencapsulation (1000 μm의보다 큰)로 분류 될 수있다. 마이크로 캡슐은 macrocapsules 6보다 더 내구성. 박사 장과 1964 년 동료의 발견 이후, 마이크로 캡슐화 널리 인슐린을 생산하는 근육 세포, 다른 호르몬 및 생리 활성 분자 (7)의 캡슐화를 위해 사용되어왔다. 이러한 치료는 섬유화 및 면역 반응 (8)를 포함하는 호스트 조직의 여러 도전에 직면했다. 우선, 생체 고분자의 품질과 관련된 부작용이 해결되었다. 그러나, 근육 세포의 이식은 여전히​​ 호르몬 overpr의 결과로서, 예컨대 섬유증과 같은 부작용을 개시전문 동맥의 외부 PREPARATION 기초 조작 ADVANCED. 최근 수십 년간, 비만과 제 2 형 당뇨병은 전염병 비율 9에 도달했습니다. 성인 사람들의 30 % 이상은 전 세계적으로 과체중과 비만 (10)이다. 증가 된 복강 (IAB) 지방 형성은 만성 염증의 발생 빈도를 증가시키고, 제 2 형 당뇨병, 심혈관 질환, 특정 암 및 기타 합병증 11-13을 촉진합니다. 증거의 여러 라인은 IAB 지방과 관련된 기전이 특정 지방 세포에 의해 피할 수 있음을 제안했다. 최근의 연구는 신진 대사를 개선하고 생체 (14) 설치류에서 비만과 인슐린 저항을 줄일 수 IAB 영역으로 피하 지방 세포의 이식을 보여 주었다. 비만과 인슐린 저항성의 효과적인 감소는 열 (15, 16)의 형태로 에너지를 방출 할 수있는 열 발생 지방 세포와 연결되었습니다. 지방 세포의 열 발생은 안정한 형질 변형함으로써 달성 될 수있다이러한 언 커플 링 단백질 1 (UCP1)로서 또는 다른 열 발생 및 UCP1 유전자 15,16의 발현을 조절하는 유전자의 미토콘드리아의 언 커플 링 프로톤, 참여 유전자. 우리의 최근의 연구는 알데히드 탈수소 효소 (1) A1 (Aldh1a1)의 결핍이 쥐 17, 18에서 비만과 인슐린 저항을 감소 IAB 지방의 열 발생 리모델링에 이르게 것으로 나타났다. 특히, 열 발생 Aldh1a1 결핍의 캡슐화 (Aldh1a1는 - / -) preadipocytes는 IAB 지방 (18)의 치료를위한 새로운 치료 기회를 제안, 비만 야생형 마우스에 IAB 지방에 같은 치료 효과를 매개한다. 실험 설정에서 캡슐화 된 세포는 비용 효과적인 방법으로 19에서 특정 세포 집단의 효과를 연구하기 위해 연구자 수 있습니다. 여기에서는 비만의 마우스 모델에서 열 발생 이화 세포주의 캡슐화 및 실험실 및 치료 적용 방법을 논의한다. 이 프로토콜은 T를 설명마이크로 캡슐의 제조 (도 1)에 대한하는 hree 단계 : 알지네이트 마이크로 비드 (도 1a), 폴리 양이온 성 폴리 -L- 라이신 (PLL) 마이크로 비드의 표면 (도 1b)에 멤브레인의 형성 및 제거의 형성 알지네이트 코어 (그림 1C).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Encapsulation device (VAR V1)</td>
			<td>Nisco</td>
			<td>LIN-0042</td>
			<td>None</td>
		</tr>
		<tr>
			<td>KD scientific syringe pump</td>
			<td>KD scientific</td>
			<td>780100Y</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Olympus microscope&#160;</td>
			<td>Olympus Optical</td>
			<td>IX70-S8F2</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Sigma</td>
			<td>MKBP8122V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Poly-l-lysine hydrobromide (PLL)</td>
			<td>Sigma</td>
			<td>020M5006V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>SLBJ2662V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma</td>
			<td>030M0200</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>SLBD2595V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Gels</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td>None</td>
		</tr>
		<tr>
			<td>ATGL primary antibody (from rabbit)</td>
			<td>Cell Signaling</td>
			<td>2138S</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Secondary anti body (anti rabbit)</td>
			<td>LI-COR</td>
			<td>926-68071</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Radio-Immunoprecipitation Assay (RIPA) buffer</td>
			<td>Boston BioProducts</td>
			<td>D25Y6Z</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>RNBD2893</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Cortizone 10 anti-itch ointment</td>
			<td>Cortizone 10</td>
			<td>C4029138</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Newborn calf serum (CS)</td>
			<td>Sigma</td>
			<td>N4762</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td>None</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I0516</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Insulin (bovine)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

encapsulation thermogenic preadipocytes for transplantation into adipose tissue depots

세포 캡슐화 반투막 내에 생존 세포를 포획하기 위해 개발되었다. 접목 캡슐화 된 세포를 장기 생존율을 달성하기 위해 처리 된 숙주의 조직에 저 분자량 대사를 교환 할 수있다. 반투막 면역계에 의해 거부를 피하기 위하여 캡슐화 된 세포를 접목 허용한다. 캡슐화 과정은 인슐린과 같은 생체 활성 화합물, 기타 호르몬, 사이토 카인의 방출을 제어 할 수 있도록 설계되었다. 여기서 우리는 비만 마우스의 복강 지방 조직에 열 발생과 에너지 손실 (열 발생)에 대한 지질 소비 이화 세포 캡슐화하는 방법을 설명한다. 열 발생 이화 세포의 캡슐화는 비만의 예방 및 치료에 잠재적으로 적용 가능하고 2 형 당뇨병있다. 이화 셀들의 또 다른 잠재적 인 애플리케이션은 알코올 대사 또는 기타 독성 및 환경 오염 물질의 해독을 포함 할 수있다.

도 1은 마이크로 비드 제조의 모든 단계가 현미경으로 제어 될 수 있음을 나타낸다.도 2a는 공동 배양하는 방법을 지방 세포를 캡슐화 된 세포의 단층으로 도시한다.도 2b는 지방 세포 / 마이크로 공동 배양을 이용하여 정량적 연구의 대표적인 예이며 그 지방 세포는 웨스턴 블롯을 이용하여 분석 하였다 제 5 해물에 설명했다. 캡슐화 된 세포는 본 실험에서 분...

Watch this Scientific Journal Video about Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots at JoVE.com

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

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 ...

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Research

JoVE Journal

Medicine

9.2K 조회수.  The Ohio State University. 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.

비디오: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

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

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 in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

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 ...

연구

의학

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 ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

견갑골 갈색 지방 조직의 Cerenkov 발광 이미징

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

현지화, 식별 및 절제 쥣과 지방이 종점의

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

인간의 갈색 지방 조직의 종점은 자동으로 양전자 방출 단층 촬영 / 전산화 단층 촬영 분단과 자기 공명 이미지 등록

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT 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 ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

비 투 열 방법을 통해 복 부 피하 지방 조직 생 검에 대 한 기술

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

인간 지방 조직 Microvascular 함수 Videomicroscopy를 사용 하 여 평가

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

쥐 모델에 부분 Colectomy 후 누설을 줄이기 위해 지방 조직 유래 줄기 세포의 이식

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

지방 조직 유래 Stromal 혈관 분수 잠재적인 연골 재생을 위한 생산의 임상 프로토콜

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...