The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

참고 : 태반이 용어 선택 과목 제왕 섹션의 설정에, 싱글 건강한 임신에서 수집해야합니다. 작성된 동의서들은 태반의 수집을 위해 주어져야한다. 귀하의 관련 인간의 연구 윤리위원회는 승인 모든 수집 및 인체 조직의 사용을해야한다. 양막 상피 세포의 1. 분리 <ol style="margin-left: 40px;"><li> 클래스 II 생물 안전 캐비닛 내에서 멸균 표면에 태반을 놓습니다. </li><li> 멸균에게 행크스 평형 염 용액 (HBSS)을 사용하여 태반 표면 및 막으로부터 가능한 한 많은 혈액을 세척한다. </li><li> 탯줄이 위를 향하도록 배치 태반으로, 기계적으로 양막과 융모막 세포막의 바깥 쪽 가장자리를 구분합니다. </li><li> 분리되면, 수동으로 탯줄, 멤브레인의 에지 주변에서 작업, 태반의 융모막 막에서 양막 멤브레인 스트립. 참고 : caref 될UL은 양막에서 융모막 막 오염의 조각을 제거합니다. </li><li> 멸균 가위를 사용하여, 250 ml의 HBSS를 포함하는 용기를 밀봉 한 500㎖의 탯줄과 장소의 기지에서 약 2cm를 양막 막 잘라. </li><li> 양막 멤브레인을 포함하는 밀봉 된 병을 흔들어 깨끗이 씻으십시오. </li><li> 15cm 페트리 접시에 수집 멸균 집게를 사용하여 컨테이너, 장소에서 양막 막을 제거합니다. </li><li> 피 묻은거나 찢어 조각을 폐기, (집게 수직으로 개최 할 때) 약 5cm 긴 조각으로 양막 멤브레인을 잘라. </li><li> 500 ml의에있는 장소는 용기를 밀봉하고 250 ml의 HBSS로 약 3 ~ 5 번 씻거나 양막 막 더 오염 혈액 반투명 될 때까지. 참고 : 모든 혈액에 존재하는 솔루션을 소화 효소의 효율을 감소시킬 수있다. </li><li> 주의하면서 소화 용액을 50 ml의 효소를 함유하는 용기에 양막 막 조각을 모두 전송HBSS의 이월을 최소화 할 수 있습니다. </li><li> 남아있는 혈액을 제거하기 위해 교반과 함께 15 분 동안 37 ° C에서 물 목욕이나 따뜻한 방에서 솔루션을 소화 효소에 양막 조각을 품어. </li><li> 새로운 멸균 용기에 솔루션과 장소를 소화 효소의 양막 조각을 제거합니다. </li><li> 신선한 효소 100ml를 양막 멤브레인 용액​​을 소화하고 수욕 또는 부드러운 진탕 (제 1 분해) 따뜻한 방에서 60 분 동안 37 ℃에서 추가 배양한다. </li><li> 양막 막 조각을 제거하고 효소가 세포막의 각 부분에서 배출 솔루션을 소화 할 수 있도록 새로운 컨테이너 돌보는에 배치합니다. 주 : 이것은 용기 내부 림을 따라 양막 조각을 드래그함으로써 달성 될 수있다. </li><li> 신선한 효소의 100 ml의 양막 막 조각에 솔루션을 소화하고 부드러운 흔들림 (초 소화) 37 ° C에서 물 목욕이나 따뜻한 방에서 60 분을 품어 추가. </li><li> 셀 suspensi 전달10 분 동안 1,000 XG에 70 μm의 필터와 원심 분리기를 통해 단계 1.14에서에. </li><li> 제거하고 상층 액을 제거하고, 1 ㎎ / ㎖ 대두 계 효소 저해제를 함유하는 4 ml의 HBSS에서 세포 펠렛을 재현 탁. 부드럽게 단일 세포 현탁액을 수득 1ml를 피펫으로 반복해서 피펫. 주 : 제 다이제스트로부터 세포가 동일한 처리 단계가 될 때까지이 단계에서 세포를 얼음에 남아있을 수있다. </li><li> 양막 막 조각을 제거하고 효소가 세포막의 각 부분에서 배출 솔루션을 소화 할 수 있도록 새로운 컨테이너 돌보는에 배치합니다. 주 : 이것은 용기 내부 림을 따라 양막 조각을 드래그함으로써 달성 될 수있다. </li><li> 10 분 동안 1,000 XG에 70 μm의 필터와 원심 분리기를 통해 단계 1.18에서 세포 현탁액을 전달합니다. </li><li> 제거하고 상층 액을 제거하고, 1 ㎎ / ㎖ 대두 계 효소 저해제를 함유하는 4 ml의 HBSS에서 세포 펠렛을 재현 탁. 부드럽게 1 ML의 페이지 반복적으로 피펫단일 세포 현탁액을 수득 ipette. </li><li> 두 다이제스트 또는 대안에서 풀 세포는 세포 수 때까지 분리 된 세포 다이제스트을 유지합니다. 참고 :이 높은 생존 세포에 잠재적으로 낮은 생존 세포의 혼합을 방지 할 수 있습니다. </li><li> 10 ~ 20 ml의 최종 부피에 HBSS를 추가하고 생존 능력을 측정하기 위해 트리 판 블루를 사용하여 세포 수를 수행합니다. </li><li> 유동 세포 계측법에 의한 상피 세포 표현형을 확인합니다. 안티는 EpCAM-PE 사용하여 적절한 항체 칵테일을 확인 (1 : 2 희석) 항 CD90-PeCy5 (1 : 250) 및 안티 CD105-APC (1 : 100) HBSS에. </li><li> 25 × 106 세포 / ml의 농도로 하나의 세포 또는 항체 칵테일의 두 다이제스트의 서브 세트를 재현 탁. </li><li> 유동 세포 계측법, 얼룩 이소 컨트롤을 포함하는 항체 칵테일 1 × 10 6 세포의 경우 (예. IgG의 1 이소 제어-PE + 안티 CD90-PeCy5 + 안티 CD105-APC). 설정 사이토 흐름에 대한 흠 ~ 1 × 10 6 세포를 보관하십시오. </li><li> 항체 세포를 품어60 분 동안 4 ° C에서 칵테일. </li><li> 워시 세포는 유동 세포 계측법 1 ㎖ 당 5-10000000 세포에서 HBSS 및 재현 탁에 3 배. </li><li> 유동 세포 계측법 및 참고 결과를 수행합니다. 셀 분리 (는 EpCAM 양성 및 CD90 / CD105 음성 세포의 백분율)의 순도 및 각각 90-95% &lt;1 %가 될 것으로 예상된다. </li><li> 최종 응용 프로그램에 의해 결정되는 적절한 품질 관리 또는 방출 기준에 대한 테스트를 위해 따로 세포의 적절한 수를 설정합니다. 참고 :이 질환 / 바이러스 차단, 또는 다른 안전 또는 생물학적 기준을 포함​​ 할 수 있습니다. </li><li> 나머지 세포의 경우, 냉동 보존 프로토콜에 따라, 또는 대안 (문화 단계 3.6 직접) 문화에 직접 배치합니다. </li></ol> hAECs 2. 냉동 보존 <ol style="margin-left: 40px;"><li> 데이터 또는 트리 판 블루 배제와 수동 계산 유동 세포 계측법을 사용하여 세포 수와 생존을 결정합니다. </li><li> 5 분 동안 700 XG에 원심 분리기. </li><li> 재현 탁 셀동물 제품 무료 냉​​동 보존 매체에 1 ㎖ 당 1-10000000 세포 사이의. </li><li> O 고리 동결 튜브에 세포의 적절한 볼륨을 피펫 및 냉각이 약 발생하는 위치 제어 속도 냉동 장치에 배치 1 ° C / 분까지 -80 C이 달성 °. </li><li> 장기간 저장을 위해 액체 질소로 전송 튜브. </li></ol> Cyropreserved hAECs 3. 해동 문화 <ol style="margin-left: 40px;"><li> 액체 질소에서 냉동 보존 튜브를 제거하고 얼음에 배치합니다. 참고 : 전송의 경우 - 다음 단계로 빠르게 진행합니다. </li><li> 얼음이 남아의 작은 조각 (~ 5mm X 5mm) 때까지 37 ℃에서 급속 냉동 보관 유리 병을 녹여. 주 : 세포 용액은 동결 배지에서 DMSO의 독성을 감소시키는 가온하지 않는 것이 중요하다. </li><li> 감기 (~ 4 ° C) 혈청이없는 매체와 냉동 보존 매체 10 배 희석 점적 적당한 크기에 추가D 튜브 또는 용기. </li><li> RT에서 5 분 700 XG에 원심 분리기. </li><li> 1 mL 당 1 백만 세포의 농도로 근사, 무 혈청 배지에 재현 탁. 트리 판 블루 배제로 자동 또는 수동 계산을 사용하여 세포 수와 생존을 결정합니다. 예상 생존 후 해동 미리 냉동 보존 생존보다 5 ~ 10 % 적습니다. </li><li> 플레이트 표준 세포 배양 처리 된 플라스틱 제품에 세포, 또는 선택적으로 사용 유형 I 코트 문화 표면에 콜라겐. </li><li> I 형 콜라겐 코팅는 아래의 프로토콜을 따라 <ol><li> 인간의 태반 (25 ml의 탈 이온수 50 μL 빙초산 15 mg의 콜라겐)에서 산 가용성 I 형 콜라겐을 준비합니다. </li><li> 파라 필름으로 비이커를 커버하고 콜라겐 가닥이 용해 될 때까지 37 ° C에서 적당히 저어. </li><li> 0.22 μm의 필터를 사용하여 멸균 여과 콜라겐 용액. </li><li> 탈 이온수로 필터링 된 콜라겐 스톡 1:10 희석. 이 희석 주식은 작업 conce입니다코팅 플라스틱 및 막 표면에 대한 ntration 솔루션입니다. </li><li> 콜라겐 코트 플라스틱, 37 ° C의 실온에서 2 시간 동안 유리와 막 표면, 또는 O / N 2-8 ° C에서. </li><li> 코팅 된 표면에서 초과 액체를 제거하고 O / N을 건조 할 수 있습니다. </li><li> 세포와 매체를 도입하기 전에 무균 조직 배양 등급의 물 또는 HBSS로 씻어. </li></ol></li><li> 플레이트 혈청 배지에서 25,000 세포 / ㎠의 밀도로 hAECs, 그리고 첨부 72-96 시간 동안 발생할 수있다. </li><li> 첨부 파일에 따라주의 깊게 흡입 및 무 혈청 배지의 적절한 볼륨 2-3 일마다로 대체하여 미디어를 변경합니다. </li><li> 아래의 프로토콜을 이용하여 세포 통로 <ol><li> 기음 세포 배양 미디어 및 무균 HBSS의 1 배 부피로 세척 </li><li> 세포는 세포 배양 물 표면으로부터 분리 될 때까지 관찰되는 효소의 양을 추가 1X 용액을 소화하고 5-15 분 동안 인큐베이션하거나. </li><li> 용액은 세포를 포함하는 소화 효소 CE 보장 모아서LLS 반복 세포 배양면에 용액을 소화 효소를 피펫 팅하여 세포 배양 물 표면으로부터 분리되고 </li><li> 5 분 동안 700 XG에 원심 분리기. </li><li> 1 ㎎ / ㎖ 대두 계 효소 저해제를 함유하는 HBSS의 적당한 부피에서 세포 펠렛을 재현 탁. 부드럽게 단일 세포 현탁액을 수득하여 1ml 피펫으로 반복해서 피펫. </li><li> 1 백만 세포 / ml의 대략적인 농도 무 혈청 배지를 추가합니다. 트리 판 블루 배제로 자동 또는 수동 계산을 사용하여 세포 수와 생존을 결정합니다. </li><li> 플레이트 혈청 배지에서 25,000 세포 / ㎠의 밀도로 hAECs하고 표준 방법으로 배양을 계속. </li></ol></li></ol>

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The authors have nothing to disclose.

이 방법의 성공에 중요한 영향을 미칠 수있는 몇 가지 중요한 매개 변수가 있습니다. hAECs의 분리하기 전에 최대 3 시간 동안 태반이나 양막의 저장 그러나 그것은 조직은 가능한 한 빨리 처리하는 것이 좋습니다, 물류 또는 스케줄링을 위해 바람직 할 수있다. 티슈가 저장 될 경우에는 저장 양막 막의 박리 및 세정 다음 수행하는 것이 권장된다. 양막은 39 ° C에서 멸균 항생제를 함유하는 HBSS에 저?...

이러한 태반과 같은 주 산기 소스, 태반 막, 탯줄과 양수에서 파생 된 세포는 재생 의학 1,2 세포의 잠재적 인 소스로 연구자와 임상의의 주목을 받고있다. 이 관심의 이유는이 세포 유형 모두 소성 및 면역 기능 3, 그들의 잠재적 인 치료 응용 프로그램에 대한 기본적인 특성을 어느 정도 가지고 있다는 것입니다. hAECs 회생 세포 물질의 풍부한 전위 소스를 제공, 용어 또는 용어 사전 양막 멤브레인 (4)로부터 유도 될 수 상피 이종 집단이다. 세포 치료 매력적 hAECs을 속성은 자신의 다 능성, 낮은 면역 원성 및 항 염증 기능을 포함한다. hAECs은 (높은 시험 관내 및 생체 내 둘 다 능성 중배엽 계통으로 분화 할 수 cardiom를 밝혀졌다yocytes, 근육 세포, 골 세포, 지방 세포), 내배엽 계통 (췌장 세포, 간 세포, 폐 세포)와 외배엽 계통 (헤어, 피부, 신경 세포와 성상 세포) 5-10. 안심, 자신의 다 능성의 hAECs 어느 형태의 종양에 표시 또는 생체 내에서 종양 발생을 촉진하지 않습니다에도 불구하고. 또한, hAECs 또한 클래스 II 인간 백혈구 항원 (HLAs) 8의 낮은 수준을 표현, 특권 면역 있습니다. 이 속성은 가능성이 면역 능력 원숭이, 토끼, 기니피그, 쥐, 돼지 11 ~ 13을 사용하여 연구에서 입증 된 바와 같이, 동종 및 이종 이식 후 면역 거부를 회피 할 수있는 능력의 기초가. hAECs는 강력한 면역 조절 및 면역 속성을 표시함으로써자가 면역 질환 치료에 잠재적 인 임상 응용 프로그램에 대한 중요한 실용적인 장점을 제공한다. hAECs는 모두 선천성 및 후천성 면역 시스템에서 면역 기능을 발휘하는 것으로 추정된다. 에면역의 분비가 (14) 요인을 통해 제안 된 메커니즘의 전자입니다. 전임상 동물 질병 모델에서 hAECs 현재 애플리케이션은 뇌졸중, 다발성 경화증, 간 질환, 당뇨병 및 만성 폐 질환의 치료를 포함한다. 연구자들은 그들의 고유 한 속성에 뇌졸중 후 뇌 염증을 치료하는 hAECs를 사용에 관심을 보여 주었다. hAECs, 그들은 생착 할 수있는 혈액 뇌 장벽을 통과 최대 60 일 동안 생존, 뉴런으로 분화, 염증을 감소시키고 신경 질환 (15)의 동물 모델에서 손상된 중추 신경계 조직의 재생을 촉진 할 수 있다는 증거가있다. hAECs 타겟팅 및 다발성 경화증의 발생 및 진행에 기여하는 여러 병리학 적 경로를 취소하는 기능을 제공한다. 예를 들면, 전임상 동물 실험의 결과 hAECs 강하게 면역 것을 제안하고잠재적으로 말초 면역 관용을 유도하고 지속적인 염증 반응을 취소 할 수 있습니다. hAECs는 또한 생체 내에서 신경 세포로 분화 및 신경성의 방대한 16 인자 분비를 통해 내인성 신경 재생을 향상시키는 능력을 갖는 것을 나타내고있다. 설치류 및 인간 양막 상피 세포는 이미 동물 모델에서 간 질환의 치료를위한 그들의 치료 효능을 증명하고있다. 감소 된 간세포 자멸사 수반 간 질환, 간에서의 생착 가능한 hAECs HAEC에 이식 리드의 사염화탄소 유도 손상 모델에서 간 염증 및 섬유증 및 17의 감소. hAECs는 인슐린과 포도당 수송을 포함하여 표현 췌장 요인으로 자극 할 수있다. 몇몇 연구는 당뇨병 마우스 (18)에서 혈당 수준을 복원 hAECs 가능성을 조사 하였다. 마우스에서 수신hAECs는 모두 동물 체중 및 혈당 수준은 감염 후 세포의 정상 수준으로 감소 하였다. 이들 연구는 당뇨병의 치료를위한 hAECs의 사용을위한 강력한 바이다. hAECs는 성인과 신생아 모델 19 두의 예방 및 실험 급성 및 만성 폐 손상의 수리 검증 역할을한다. 이러한 연구는 hAECs은 낭포 성 섬유증 막 횡단 전도력 레귤레이터 (CFTR), 낭포 성 섬유증 (20) 환자에서 돌연변이 이온 채널 등 다양한 폐 관련 단백질을 발현하는 기능 폐 상피 세포로 체외에서 분화 것을 발견했다. hAECs가 부상당한 성인과 신생아 폐에 전달 될 ​​때 또한, 그들은 호중구, 대 식세포와 림프구 21-23을 포함한 폐 백혈구 모집을 감소, 호스트 면역 세포의 변조를 통해 자신의 수복 효과를 발휘한다. 자신의 풍요 로움을 감안할 때,안전 기록, 여러 질병에 대한 입증 된 임상 적용, hAECs를 사용하여 임상 시험은 불가피하다. 임상 시험에 HAEC 치료의 번역을 촉진 목표로, 우리는 현재 좋은 제조 기준 (cGMP의) 지침에 따라 동물 제품 무료 시약을 사용하여, 임상 시험에 적합한 방식으로 문화 hAECs을 분리 cryopreserve과하는 방법을 개발 . 우리는이 프로토콜에게 우리가 동물 유래 시약 6을 사용 hAECs을 분리 성공적으로 사용하고 이전에 출판 된 프로토콜을 기반으로. 우리는 동물 제품 무료 시약 동물 유래 제품을 대체 할 수있는 원래의 프로토콜을 변경, 이후의 최적화는 세포 수율, 생존 및 순도를 최적화하기 위해 실시 하였다. 우리의 목표는 인간의 임상 시험에 대한 전지 제조에 대한 규제 기준을 준수 할 프로토콜을 개발하는 것이 었습니다.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
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			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:119px;">13-361B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>S17329&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:220px;">Scissors - Sharp/Blunt Straight</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>NC0562592&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:220px;">Protective Apparel (Gown)</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protective Apparel</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolation gowns</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">General purpose face mask</td>
			<td style="width:119px;">Cardinal Health</td>
			<td style="width:119px;">AT7511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bonnets</td>
			<td style="width:119px;">Medline</td>
			<td style="width:119px;">CRI1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shoe covers</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-7873W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Media and Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T3449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130 - 091-253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

이 절차가 제대로 따라하면, 1 억 2 천만 hAECs의 평균 수익률은 80-160000000 세포의 전형적인 범위, 예상해야한다. 이러한 수율에서 83 ± 4 %의 평균 생존 능력을 기대할 수있다. 임상 방법에 증가 된 평균 수익률과 약간 낮은 가능성으로 인해 혈청 단백질의 부족으로 아마 또한 동물성 제품보다 더 높은 트립신의 활동으로 인해, 그리고 수 있습니다. 격리 hAECs은 &lt;1 % CD90, CD105 간충직 마커 양성 세포와 ...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

분리, 냉동 보존 및 문화 임상 응용 프로그램에 대한 인간 양막 상피 세포

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K 조회수.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

비디오: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

연구

의학

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

임상 및 전임상 연구 응용 프로그램에 대한 종양 세포 (CTC) 분석 실험을 순환 반자동의 적응

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

종양 특이 내피 세포의 분리 및 문화 확장

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

인간의 뇌하수체 선종 세포의 3 차원 알긴산 비드 문화

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

개 동맥과 정맥에서 차 내피 세포의 분리 및 문화

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

인간의 신장 혈관 주위 Stromal 세포에 대 한 새로운 임상 등급 격리 방법

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

인간 비강 상피 오가노이드의 문화와 이미징

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

인간 지방 유래 줄기 세포의 분리 및 배양 (Isolation and culture of human adipose-derived stem cells with an innovative Xenogeneic-free method for human therapy)

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...