이 작품은 리처드 토마스 Leukaemia 기금, 레이디 타타 기념관 신탁, Northwick 공원 병원 Leukaemia 연구 신탁 기금 및 건강 연구의 국립 연구소 (NIHR), 영국에 의해 재정 지원되었다.

1. 공사장 공중 발판의 발판 제조 및 바이오 작용화 <ol><li> 배양 접시 디스크의 형태로 우레탄 공사장 공중 발판 (기공 크기 1백~2백50밀리미터, 다공성 90~95%)를 조작하려면 다음 폴리머 솔루션 (Dioxan에서 5wt %)를 준비하여 열 유도된 상 분리에게 13 프로세스를 사용하여 동결 이후 용매 승화 (그림 1A). </li><li> ECM 단백질 (그림 1B)와 코팅 이전 0.5 X 0.5 X 0.5 mm의 조각으로 발판 디스크를 자르고. </li><li> 다음 1 분 및 인산으로 전송을 위해 에탄올 (70 %)에서 그들을 immersing하여 사전 서부 유럽 표준시 공사장 공중 발판은 20 분 동안 (PBS) 염분 버퍼. 그렇다면 3,630 XG에 10 분 동안 원심 분리기와 단백질 솔루션을 추가합니다. </li><li> 20 분 대한 1,420 XG에서 단백질 용액에 공사장 공중 발판을 원심 분리기. </li><li> 표면 모공 차단을 해제하고, 발판에 깊이 침투하는 놓는 세포를 허용하기 위해서는 10 분 동안 910 XG에 한 번만 더 공사장 공중 발판 원심 분리기PBS 인치 </li><li> 에탄올 2 H (70 %)에 대한 침수와 230 V, 50 Hz에서, 0.14, 자외선 램프,시 8 분 노출의 조합을 사용하여 공사장 공중 발판을 소독. 그 후, 30 % 태아 소 혈청 (FBS) 및 1퍼센트 페니실린과 스트렙토 마이신 (P / S)와 함께 보충 Iscove의 수정일 Dulbecco 중간 (IMDM)를 추가하기 전에 PBS로 두 번 공사장 공중 발판을 씻는다. ° C에서 5 % CO 2는 이전에 사용하기 37 3 일간 humidified 인큐베이터에서 공사장 공중 발판을 놓으십시오. 멸균 공사장 공중 발판은 24 잘 조직 배양 플레이트 (물론 당 하나 비계)에 배치됩니다. </li></ol> 2. Mononuclear 세포 분리 및 비계를 심는 <ol><li> Ficoll-Paque 밀도 기울기 원심 분리 (1500 rpm으로 35 분) (그림 1C)를 사용하여, 코드 혈액 또는 leukemic 골수 샘플로부터 인간 MNC의 압축을 풉니다. </li><li> IMDM의 Resuspend MNC 30 % FBS와 1 % P / S.를 포함하는 </li><li> 마이크로 피펫을 사용하여 공사장 공중 발판쪽으로 MNC 서스펜션의 종자 100μl (2 × 10 6 세포 / 비계). </li><li> 공사장 공중 발판으로 정착하는 세포에 대해 10 분 동안 5 % CO 2 하에서 37 ° C에서 씨앗을 품고 공사장 공중 발판을 품어. </li><li> 37시 인큐베이터 30 % FBS와 1 % P / S와 장소도 접시를 포함 IMDM 1.5 ML 각 잘 더더 이동 ° C와 실험 기간 동안 5퍼센트 CO 2. </li><li> 씨앗을 품고 공사장 공중 발판은 모든 미디어 일일 변경될 받고있다. </li></ol> 3 원위치 세포 증식과 형태론의 경우 :. MTS, SEM 및 Cytospins <ol><li> MTS 분석 : 문화 한 해제 번시드인하고 깨끗한 24 개의 새 항목 잘 플레이트에서 두 씨앗을 품고 공사장 공중 발판과 장소에서 제거합니다. 미디어 1 ML 37에서 MTS 솔루션 및 3 시에위한 품어 200 μl를 추가 ° C에서 5 % CO 2. <ol><li> microplate 판독기를 사용하여 490 nm의시 96 - 웰 플레이트 및 측정 흡광도에서 개최하며 뜨는에서 8 X 100 μl 샘플을 가져가라. </li></ol></li><li> 일 중 다른 시간 시점 : 전자 현미경 (SEM)을 스캔전자 문화, 미디어에서 MNC로 놓는 공사장 공중 발판을 제거하고 4에 40 분 2.5 % PBS 버퍼 글루 타 알데히드 용액 ° C로 고정 후 PBS로 두 번 씻는다. <ol><li> 탈수 4 시간 동안 무균 환경에서 10 분 건조를위한 에탄올의 등급 시리즈 (25, 50, 70, 80, 90, 95 및 100 %), 각각의 공사장 공중 발판. </li><li> 섹션 표본 후 사전 SEM 평가 2 분 아르곤 분위기 속에서 금을 스퍼터 코팅 그들은 20 kV의 가속 전압을 사용합니다. </li></ol></li><li> Cytospins : 문화에서 다른 시간 시점에서 발판에서 세포를 aspirating하여 2.0 X 10 4 세포 / 슬라이드를 수집합니다. <ol><li> 1,500 RPM에서 3 분 유리 슬라이드에 세포를 원심 분리기. </li><li> 라이트-Giemsa 수정된 청바지와 광학 현미경을 사용하여 관찰과 함께 슬라이드를 청바지. 소프트 이미징 시스템은 사진을 찍는하는 데 사용할 수있는 F-봅니다. </li><li> 현미경 관찰 이전 슬라이드를 씻으십시오. </li></ol></li><li> M<html> ultiphoton 분석 : 자세한 분석 시까지 -20시 에탄올 증기 (70 %) 숙박 후 건조하고 매장 ° C와 다른 시간 지점에서 픽스 공사장 공중 발판. 이전 multiphoton 현미경의 30 μm의 두께의 슬라이드로 섹션 비계 및 PBS와 젖은와 시각화합니다. <ol><li> 상온에서 30 분 10 % 태아 소 혈청으로 PBS에 씨앗을 품고 공사장 공중 발판을 차단합니다. </li><li> 마우스 안티 인간 CD71를 : ° C ~ 어둠 속에서 기본 단클론 항체의 1시 50분 희석과 4에서 공사장 공중 발판이 하룻밤 사이에 알을 품다. </li><li> PBS에서 샘플을 세 번 씻고 이차 항체에 얼룩 : 어둠의 실온에서 1 H를위한 안티 마우스 알렉사 형석 488. </li><li> PBS로 샘플을 세 번 씻고 물 방출 x60 목표 렌즈를 사용 공촛점 현미경으로 관찰합니다. </li><li> 형광단 알렉사 형석 488은 타악기 레이저로 488 nm의에서 흥분한다. Volocity 5.3.2 소프트웨어는 이후의 이미지 분석에 사용될 수 있습니다. </li></ol></li></ol>세포 인구 &quot;&gt; 4 jove_title. 흐름 Cytometric 분석 <ol><li> 흐름 cytometer (FC)의 세포 분석하기 전에 검출에 해당 immunofluorescence 항체와 관심 세포에 라벨을 달고. 샘플의 수를 검출을위한 선택된 항체에 따라 준비가되어 있습니다. MNC 검출 항체의 경우 다음과 같은 조합이 사용됩니다 : CD45-FITC/CD71-PE/CD235a-PE-Cy5. </li><li> 다른 시간 지점과 원심에 있던 발판에서 세포를 기음. 100 μl FC 완충액 (PBS + 0.1 % 나트륨 azide)에서 6 1x10 주변 세포의 세포 펠렛를 해산하고 각 항체 형광 색소의 10 μL를 추가합니다. 4에 30 분 ° C에 대해 세포를 품어, PBS로 두 번 씻어 마침내 FC 버퍼에 다시 일시 중지합니다. </li><li> 항체 표현의 &quot;부정적&quot;를 설정하고 흐름 cytometer 컨트롤과 함께 검출 채널을 교정 isotype 제어 흐름 cytometer의 스테인드로 샘플을로드합니다. </li><li> 샘플 스테인드 w 읽기<html> 세 가지 다른 항체 ith 그리고 마지막 WinList 소프트웨어를 사용하는 데이터를 나타냅니다. </li></ol> 5. 대표 결과 외인성 성장 요인 이외없이 조혈 세포 성장 속도론의 예제는 그림 2에 표시됩니다. 조혈의 이질적인 성격, 서로 다른 두 가지 세포로 인해 : 정상과 비정상 조혈 세포는 그림하고 있습니다. 그림 2A에서 인간 CBMNC의 세포 증식은 문화가 28 일 후에 분명하다. 그림 2B는 인간의 기본 leukemic 세포를 사용하여 흉내의 성장 속도론을 보여줍니다. 세포의 증식은 흡광도와 관련하여 세포 신진 대사 활동을 측정하는 MTS 분석을 사용하여 평가된다. 두 경우 모두, 세포가 확산하고 모델에서 설립. 성장 속도론의 차이가 관찰되며, 문화를 구축 정상 조혈 세포를 빠르게 leukemic 세포보다. 공사장 공중 발판의 _content &quot;&gt; 외인성 성장 요인 부재의 문화의 28 일 정상 조혈 세포 (그림 3A)와 leukemic 세포 (그림 3B)의 전형이었다 후에도 수확 세포의 형태. 중앙 부분은 후 SEM으로 분석되었다 공사장 공중 발판은 문화에서 제거하고 클러스터에와 &quot;틈새 같은&quot;구조 (그림 3A &#39;&amp; B&#39;)에서 자신을 설립, 발판에 걸쳐 씨앗을 품고 세포의 확산을 보여주되었다. 그림 C는 보여준다 unseeded의 기공 크기와 분포 비계는 컨트롤로 사용됩니다. 28일 후 Multiphoton 현미경은 원래의 장소에 3D 발판 내에서 세포의 분포를 강조하기 위해 사용하고 마커의 표현에 의해 (그림 4) 비계의 중앙 섹션에 erythroid 섬의 존재를 보여주되었다 erythroblasts에 긍정적이다. CD71 이것은 성숙하고 성숙 세포 뒤의 중요성을 증명링 적혈구 조혈. 인간 CBMNCs 및 그림 5B 비정상 조혈 세포 설명 : 기본 leukemic 세포를 마지막으로, 세포 사전 시딩의 유동세포계측법 그래프는 그림 5A가 정상 조혈 세포를 대표하는 조혈 세포의 표현형의 차이를 보여줍니다. CD235a의 수준 + 및 CD45 + 각각 leukemias의 hemoblastic 성격을 강조 leukemic보다 일반적인 샘플에 높은 있습니다 적혈구 및 leukocytes에 대응. <img alt="그림 1" src="/files/ftp_upload/3654/3654fig1.jpg" /> 그림 1. 우레탄 발판 제조 및 바이오 작용화에 관련된 프로세스의 삽화. ) 우레탄은 Dioxan (5wt %)에 녹아 있으며, 열 유도된 상 분리 공정 및 후속 용매 승화하여 발판 Safinia 외 13에서 설명한 바와 같이, 생산하고 있습니다. B) 비계 디스크는 다음 INT 절단되어O 장치의 큐브 0.5 X 0.5 엑스트라 셀룰러 매트릭스 (ECM) 단백질과 원심 분리에 의해 코팅 X 0.5 mm 후. C) MNC는 인간의 탯줄 CB이나 micropipette으로 밀도 구배 원심 분리 및 우레탄 공사장 공중 발판에 씨앗을 (2x10 6 셀 / 비계)를 사용 BM 흡출됨으로써 압축이 풀립니다. <img alt="그림 2" src="/files/ftp_upload/3654/3654fig2.jpg" /> . 그림 2 세포 증식은 MTS 분석을 사용하여 측정 :) 인간의 탯줄 혈액 MNCs 사용하는 인간의 기본 leukemic 세포를 사용하여) B를. 외인성 크린 시토킨의 추가없이 우레탄 공사장 공중 발판에 놓는 때 열은 시간에 따른 세포의 성장을 보여줍니다. <img alt="그림 3" src="/files/ftp_upload/3654/3654fig3.jpg" /> 그림 3 : 공사장 공중 발판 주위 세포 형태학 및 유통이 scanni를 cytospins를 사용하여NG 전자 micrographs. (AB) 문화의 28 일 후에 우레탄 공사장 공중 발판에서 수집한) 코드 혈액 mononuclear 세포의 대표 라이트-Giemsa 스테인드 cytospins 및 B) 뼈 aspirated leukemic 세포 문화 날로부터 14 일 후 우레탄 공사장 공중 발판에서 수집했습니다. 두 실험 시토킨 자유로운 상태로 진행​​되었다. (A&#39;-B &#39;)) 씨앗을 품고 코드 혈액 MNCs와 B&#39;) 28 일 동안 배양해 거쳐 leukemic 세포없이 세포와 C) 제어 비계의 우레탄 발판 SEM의 micrographs 대표 중앙 섹션 &#39;. <img alt="그림 4" src="/files/ftp_upload/3654/3654fig4.jpg" /> 그림 4. erythroid 마커 CD71과 문화와 스테인드 28 D 후 탯줄 혈액 MNCs와 우레탄 발판 씨앗의 Multiphoton의 현미경. <img alt="그림 5" src="/files/ftp_upload/3654/3654fig5.jpg" /> 그림 5. 유동세포계측법 3D 도트 플롯은 CD45, CD71 및 CD235a 표면 발현 마커에 대해 묻다. 얻은 isotype 컨트롤도 상대 형광 강도의 비교를 위해 제공됩니다. 패널은 위의 마커에 대한 긍정적인 인간 코드 혈액 mononuclear 세포를 보여줍니다, 패널 B는 인간의 기본 leukemic 세포를 보여줍니다.

<ol>
	<li>Orkin, S., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis:+an+evolving+paradigm+for+stem+cell+biology.">Hematopoiesis: an evolving paradigm for stem cell biology.</a> Cell. 132, 631-644 (2008).</li><li>Spradling, A., Drummond-Barbosa, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Panoskaltsis, N., Mantalaris, A., Wu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+mimicry+of+bone+marrow+tissue+ex+vivo.">Engineering a mimicry of bone marrow tissue ex vivo.</a> J Biosci. Bioeng. 100, 28-35 (2005).</li><li>Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Mantalaris, A., Bourne, P., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+osteoclasts+in+a+three-dimensional+bone+marrow+culture+system.">Production of human osteoclasts in a three-dimensional bone marrow culture system.</a> Biochem. Eng. J. 20, 189-196 (2004).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+bioprocessing:+fundamentals+and+principles.">Stem cell bioprocessing: fundamentals and principles.</a> J. R. Soc. Interface. 6, 209-232 (2009).</li><li>Dexter, T., Testa, N., Prescott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 14, 387-405 (1976).</li><li>Piacibello, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+growth+factor+requirement+of+primitive+cord+blood+hematopoietic+stem+cell+for+self-renewal+and+amplification+vs+proliferation+and+differentiation.">Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.</a> Leukemia. 12, 718-727 (1998).</li><li>Yoshida, T., Takagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+processing+engineering+for+ex+vivo+expansion+of+hematopoietic+cells:+a+review.">Cell processing engineering for ex vivo expansion of hematopoietic cells: a review.</a> Biochemical Engineering Journal. 20, 99-106 (2004).</li><li>Lim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intelligent+bioprocessing+for+haemotopoietic+cell+cultures+using+monitoring+and+design+of+experiments.">Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.</a> Biotechnol. Adv. 25, 353-368 (2007).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+three-dimensional+scaffold+for+ex+vivo+biomimicry+of+human+acute+myeloid+leukaemia.">The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.</a> Biomaterials. 31, 2243-2251 (2010).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Aqel, N., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cytokine-free+expansion+of+cord+blood+mononuclear+cells+in+three-dimensional+scaffolds.">Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds.</a> Biomaterials. 32, 9263-9270 (2011).</li><li>Safinia, L., Datan, N., Hohse, M., Mantalaris, A., Bismarck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+methodology+for+the+effective+surface+modification+of+porous+polymer+scaffolds.">Towards a methodology for the effective surface modification of porous polymer scaffolds.</a> Biomaterials. 26, 7537-7547 (2005).</li></ol>

우리는 공개 할게 없다.

여기서 제시 전직 생체내 3D 문화 시스템은 우리가 조혈의 3D biomimicry를 확립 수 있도록 그 recapitulates 원래 BM 건축과 외인성 크린 시토킨의 독립 세포 표현형. 3D 모델은 구조와 정상 및 비정상 조혈 세포가 생체내에 발생한 것과 유사한 조건에서 분아 따위에 의해 중식 수 microenvironment를 제공합니다. 고분자 비계 재료의 선택 biomimicry 디자인에 중요한 단계를 나타...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue No</td>
		</tr>
		<tr>
			<td>Dioxan</td>
			<td>Invitrogen</td>
			<td>D20,186-3</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>MTS</td>
			<td>Promega</td>
			<td>G3580</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka Biochemika</td>
			<td>49624</td>
		</tr>
		<tr>
			<td>Wright-Giemsa</td>
			<td>Sigma-Aldrich</td>
			<td>WG32</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10108-165</td>
		</tr>
		<tr>
			<td>CD71</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32272</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>BD Pharmigen</td>
			<td>74895</td>
		</tr>
		<tr>
			<td>CD71-PE</td>
			<td>BD Pharmigen</td>
			<td>555537</td>
		</tr>
		<tr>
			<td>CD235a-PE-Cy5</td>
			<td>BD Pharmigen</td>
			<td>555570</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
		</tr>
	</tbody>
</table>

ex vivo mimicry of normal and abnormal human hematopoiesis

골수 (BM)은 spatially 조직 세포 microenvironments에 의해 규제되는 복잡한 입체 (3D) 조직이 어떤 점에서의 조혈입니다 틈새에게 2-4 되나, 조혈 줄기 세포는 혈액 세포 형성 1을 유지하기 위해 독특한 microenvironment를 필요로합니다. BM 틈새의 조직은 정상 또는 악성 BM 5의 기능 또는 기능 장애에 중요합니다. 따라서 전직 생체내의 흉내를 사용하여 생체내 microenvironment에의 이해 우리 leukemogenesis 6, 분자 세포 및 microenvironmental determinants을 명료하게하다 도움이 될 것이다. 현재 조혈 세포 중 allogenic 또는 이종 개체의 stromal 세포와 공동의 문화 또는 외인성 크린 시토킨 8의 추가 요구 (2D) 2 차원의 조직 문화 flasks / 잘 접시 7에서 체외에서 배양해 있습니다. 이러한 조건 인공 있으며 생체내에 따라 다를 </eM&gt;은 3D 휴대폰 틈새 시장이 부족하고 pluripotency 9,10의 분화 및 손실을 초래할 수있는 비정상적으로 높은 시토킨 농도로 세포를 노출 점에서 microenvironment. 여기, 우리는 생체내 3D 성장 환경에서을 시뮬레이션 및 외인성 성장 요인 부재에 multilineage의 조혈을 지원하는 새로운 3D 골수 배양 시스템을 제시한다. 폴리 우레탄 (PU)으로 만들어진이 시스템에 사용되는 높은 다공성 발판은 2D 11에서 기존의 단일층 문화보다 높은 특정 표면 영역에 걸쳐 고밀도 세포 성장을 용이하게합니다. 우리의 연구는이 모델은 인간의 탯줄 혈액 (CB) mononuclear 세포 (MNC) 외인성 크린 시토킨의 부재에서 12 일차 leukemic 세포의 성장을 지원하는 것을 시사하고있다. 이 소설 차원 흉내는 조혈을 연구하고 백혈병에 대한 새로운 치료법을 탐구하는 인간의 실험적인 모델의 개발을위한 실행 가능한 플랫폼을 제공합니다.

Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

조혈을위한 3D 문화 시스템은 인간 코드 혈액과 leukemic 골수 세포를 사용하여 설명합니다. 방법은 세포외 기질 단백질로 코팅 다공성 합성 폴리 우레탄 발판의​​ 사용을 기반으로합니다. 이 발판은 세포의 광범위한 수용하는 적응이다.

정상과 비정상적인 인간 조혈의 전직 생체내 흉내

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

ex-vivo-mimicry-of-normal-and-abnormal-human-hematopoiesis

Research

JoVE Journal

Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

15.4K 조회수.  Imperial College London. 조혈을위한 3D 문화 시스템은 인간 코드 혈액과 leukemic 골수 세포를 사용하여 설명합니다. 방법은 세포외 기질 단백질로 코팅 다공성 합성 폴리 우레탄 발판의​​ 사용을 기반으로합니다. 이 발판은 세포의 광범위한 수용하는 적응이다.

비디오: Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

고해상도 3D 영상의 전 VIVO</em마이크로 중부 표준시로> 생물 학적 샘플

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

연구

생체공학

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

3 차원의 인간 피부 모델을 재구성 : 도구는 일반 피부와 흑색증의 진행을 공부하기

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

에 대한 기계적 적응 고분자 나노 복합 재료의 환경 제어 Microtensile 테스트<em&gt; 생체</em&gt; 특성

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

변성 콜라겐 물가 이미징<em&gt; 생체 내</em&gt;와<em&gt; 예 생체 내</em&gt; 리테이너 타입 콜라겐 미메시스 펩티드의 사진 트리거 하이브리드를 통해

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

내 시경 Submucosal 주입 재료 성능 평가 대 한 새로운 Ex Vivo 모델

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

광학 확산 상관 분광법을 사용하여 인간의 뇌의 대뇌 혈류 기반 휴식 상태 기능 적 연결

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

동시 브라이트필드, 형광, 및 수축 심장 Trabeculae Ex Vivo의 광학 일관성 지형 이미징

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

다기능 시스템을 사용하여 인간 암 및 정상 세포에 있는 예 관련 단백질의 모든 광학 Mechanobiology 심문

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...