기금은 보건 국립 연구소 (EB 001013), 자연 과학 및 캐나다의 공학 연구 협의회 및 건강 연구 캐나다 연구소에 의해 제공되었다. 우리는 그녀의 전문 박사 AP 맥기건 감사와 모듈의 개발에 도움이됩니다.

관 1) 준비 <ol><li> 폴리에틸렌 튜브 2 ~ 3 미터 길이 (0.76 mm ID를 X 1.22 mm OD)를 자르고. </li><li> 튜브의 한쪽 끝을로 20 G1 바늘에 실을 꿰다. </li><li> 변환기 자체 밀봉 주머니 (7 1 / 2 &quot;X 13&quot;)에 코일 튜빙 장소. 튜빙의 두 끝을은 가방의 unsealed 끝에 위치 및 가스 살균 테이프와 장소에서 녹화한다. </li><li> 인감 가방 및 가스 소독. </li></ol> 콜라겐 2) 중화 참고 : 콜라겐 1 ML는 폴리에틸렌 튜빙의 약 2 미터 채워집니다. <ol><li> 15 ML 원뿔 관에 튜브를 원하는 길이에 필요한 콜라겐의 필요한 양을 놓고 얼음 계속. </li><li> 128 UL 추가 10X α - 멤 당 ML 튜브와 반복 솔루션을 pipetting으로 혼합하는 콜라겐니다. 참고 : (1) 솔루션은 산성의 콜라겐에 α - 멤 같은 노란색이 혼합된 것입니다 설정해야하며 (2) 혼합시 거품을 최소화하려고합니다. </li><li> 콜라겐은 7.4의 산도를 가지고 때까지 0.8 M 나트륨 중탄산염을 추가하여 콜라겐을 중화. 산도의 추가 나트륨 중탄산염에 대한 일반적인 추정을위한 노란색 콜라겐 솔루션은 밝은 핑크색 색상을 발견되기 전까진. 참고 : 일반적으로 콜라겐의 각 ML 당 30-60 μL 걸립니다. </li><li> 무력화 콜라겐으로 사용할 준비까지 겔 4 보관하면되지 않습니다 얼음에 무력화 콜라겐 계속 ° C. </li></ol> 셀 3) 삽입 (옵션) 참고 : 세포 종류에 따라 세포가 ML 당 20,000,000 세포 ML 당 백만 세포 사이의 농도에서 임베디드 수 있습니다. 당신이 포함하고자하는 세포에 필요한 매체를 사용하십시오. <ol><li> 세포에서 미디어를 제거하고 PBS로 5 ML 씻으십시오. </li><li> PBS를 제거 및 3 ML 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)를 추가합니다. </li><li> 37 5 분 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)에있는 세포를 품어 ° C. </li><li> 7 ML 매체에 세포를 Resuspend. </li><li> 세포를 세어보세요. </li><li> 15 ML 원뿔 관을 삽입에 대한 세포의 필요한 번호를 전송합니다. </li><li> 5 분 300 XG에 펠렛은 세포. </li><li> 세포 펠렛에서 미디어를 대기음. </li><li> 무력화 콜라겐의 세포를 Resuspend 얼음 계속. </li></ol> 콜라겐 4) 겔링 <ol><li> 생물 학적 안전 캐비넷에서 폴리에틸렌 튜브가 들어있는 가방의 밀봉 끝을 잘라. </li><li> 멸균 핀셋을 사용하여 튜브의 한쪽 끝을에서 20G 바늘로 3 ML의 주사기를 연결합니다. 가방에 튜빙을 유지. </li><li> 끝이 살균 핀셋을 사용하여 원뿔 관의 하단에까지 무력화 콜라겐에 튜브의 다른 쪽 끝을 삽입합니다. 원뿔 튜브 얼음에 보관하십시오. </li><li> retracting 천천히 주사기의 플런저에 의해 폴리에틸렌 튜브에 콜라겐을 당겨. 참고 : 콜라겐이 빠르게 다음 거품이 콜라겐의 형성에 튜브에 뽑아 경우. </li><li> 튜빙을 통해 콜라겐을 당기는 후, 튜브 밖으로 20G 바늘을 끌어와 가방에 다시 양쪽을 넣어. 테이프 가방 종료. </li><li> ° C 60 분 37 가방을 품다. 콜라겐은 세포가 포함되어있는 경우 다음 각 15 분 이상의 가방을 플립. 그래서 세포는 모듈의 한쪽으로 해결하지 않습니다. </li></ol> 5) 튜브의 절단은 Gelled 콜라겐을 포함 <ol><li> 절단 장치와 튜브를 잡아 트레이에 대한 압력솥 헤더와 블레이드. </li><li> 절단 장치를 조립. </li><li> 플레이스 장치를 절단 앞에 살균 트레이에 튜브 및 절단 장치에 튜빙을로드합니다. </li><li> 컷 모듈을 수집하는 절단 장치의 콘센트에서 미디어의 25 ML을 포함한 50 ML 원뿔 튜브를 설정합니다. 참고 : 모듈이 언론에 FBS없이 튜브에서 분리하지 않는 콜라겐 전용 모듈도 FBS를 포함하는 사용 미디어. 모듈이 포함되어 있으면 세포가 포함된 세포에 필요한 미디어를 사용합니다. 있다면 모듈에 포함된 세포가 내피 세포에 대한 미디어를 사용하지 않습니다. </li><li> 튜빙의 2mm 길이를 잘라 튜브를 잘라 절단 장치를 설정합니다. </li><li> 37 모듈을 잘라 품어 ° 50 ML 원뿔 튜브 60 분 C. </li></ol> 6) 튜브에서 콜라겐 모듈 제거 <ol><li> 10 초에 대한 튜브의 모듈을 포함하는 소용돌이 50 ML 원뿔 관. 참고 : 파손을 피하기 위해 높은 세포 밀도와 임베디드 모듈을 분리 때 부드럽게한다. </li><li> 모듈 튜브의 바닥에 정착하자. 약 5 분 정도 소요됩니다. </li><li> 넓은 입 10 ML serological 피펫을 사용하면 15 ML 원뿔 관에 정착한 모듈을 전송합니다. </li><li> 단계를 6.1-6.3 두 번 반복합니다. </li><li> 아닌 조직 문화 처리 강판에 내피 세포 또는 전송 모듈과 코트 모듈 7 단계로 이동합니다. </li></ol> 내피 세포와 함께 7) 코팅 모듈을 <ol><li> 일 미디어의 5 ML과 15 ML 원뿔 튜브의 바닥에 모듈을 정착E는 세포를 내장. </li><li> 내피 세포에서 미디어를 제거하고 PBS로 5 ML 씻으십시오. </li><li> PBS를 제거 및 3 ML 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)를 추가합니다. </li><li> 37 5 분 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)에있는 세포를 품어 ° C. </li><li> 7 ML 매체에 세포를 Resuspend. </li><li> 세포를 세어보세요. 원뿔 관의 하단에있는 포장 모듈 ML 당 5x10 6 내피 세포가 필요 해요. </li><li> 5 ML 내피 세포 미디어에서 5 분 및 resuspend 300 XG에 펠렛은 세포. </li><li> 모듈에 내피 세포가 들어있는 미디어의 5 ML을 추가합니다. 참고 : 이것은 우리가 두 세포 유형의 생존과 기능에 대한 작업 찾은 두 미디어 유형의 50:50 비율을 제공합니다. 사용 그것을 사용하기 전에 두 세포 유형의 phenotypes의 적절한 유지 보수를 위해 공식화 공동 문화 매체를 테스트합니다. </li><li> 모두 정적 및 동적으로 내피 세포와 모듈을 품어. </li><li> 정적 배양 믹스 역전의 내피 세포와 모듈과 37 수직 튜브를 설정할 ° 15 분 C하십시오. 반복합니다. </li><li> 부드럽게 37 튜브를 락을하여 동적으로 씨앗이 내피 세포 ° 60 분 C. </li><li> 두 정적 부화를 반복합니다. </li><li> 아닌 조직 문화가 처리 강판의 모듈을 놓고 37 밤새 품어 ° C. </li><li> 새로운 미디어와 새로운 비 조직 문화 처리 강판의 다음날 장소의 모듈 (50:50 혼합하는 경우 사용하는 공동 문화 시스템) 무소속의 내피 세포를 제거하려면 다음과 같이하십시오. </li><li> 그들은 모듈의 표면의 100 %를 커버까지 내피 세포와 입혀져 있습니다 모듈을 품어. 이것은 일반적으로 약 7 일이 소요됩니다. </li></ol> 8) 대표 검색 결과 : 그들이 처음 튜브에서 제거하는 경우 모듈은 원통 모양 것입니다. 모듈이 포함된 셀 및 /를 포함 또는 내피 세포로 코팅하는 경우 그들은 볼륨 50 %까지 계약하고 타원형 모양 (그림 1)을 개발할 수 있습니다. 모듈은 또한 가벼운 현미경 (그림 1)에서 볼 때 밀도가 더 불투명하게 될 것입니다. 또한 내피 세포가 모듈의 표면에 합류하는 경우 VE - Cadherin의 immunofluorescence 얼룩 (그림 2)에서보실 수 있습니다 꽉 분기점의 형성이 있습니다. 대량 전송 분석 모듈 (대형 직경의 예 : 모듈, D = 1.4 대규모 조직에서 흔히 문제는 불충 분한 산소 수송에 의한 괴사성 핵심을 개발하지 않고 높은 세포 밀도를 (8x10 7 세포 / cm 3) 지원 할 수있다 증명했다 mm) (그림 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="그림 1" /> 그림 1 : 모듈 제조 및 수축. 모듈 수축이 시딩 상당히 작은 모듈 직경 및 길이 (P &lt;0.001) (A)를 결과 HUVEC - C (내피 세포 라인)을 다음과 같은 세 가지 일 동안 발생했습니다. 임베디드 HepG2 세포가 균일하게 제조시 모듈 내에 배포 및 높은 생존 능력 (B)를 유지했다. [세포가 녹색 라이브, 죽은 세포 레드] [코스토핀 2010에서 적응] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="그림 2" /> 그림 2 : 모듈의 표면에 코팅 십일 RAEC 후 EC 꽉 세포 분기점의 VE - Cadherin immunofluorescent 얼룩. 스케일 바 = 50 μm의. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="그림 3" /> 그림 3 : 메이슨의 전형적인 모듈의 trichrome 염색법 (0.76 mm 초기 직경) 및 대형 모듈 (1.4 mm 초기 직경)는 산소 확산 제한의 효과를 보여줍니다. 제조 아래 세븐 일, 죽은 세포의 큰 숫자가 가능한 세포의 단지 얇은 테두리를 (~ 200μm 두께) 떠나, 대형 모듈 (오른쪽 아래 패널)의 핵심 내에서 형성했다. 반대로 작은 모듈은 라이브 세포의 균일한 높은 분포를 유지. [모듈 HepG2 세포와 임베디드 및 내피 세포와 코팅.] [코스토핀 2010에서 가져온] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="그림 4" /> 그림 4 : poloxamine에 (A) HMEC - 1 씨앗 - 콜라겐 모듈. poloxamine - 콜라겐 모듈은 자신의 원통형 모양을 유지하고 제한된 세포 부착 속성 콜라겐에만 모듈에 비해있다 시딩의 1 일 후. 스케일 바 = 200 μm의. PLGA 기반 생분해성 microspheres를 포함하는 콜라겐 모듈의 (B) 라이트 현미경 이미지. 스케일 바 = 500 μm의. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="그림 5" /> 그림 5 : 시험 관내 assays에의 예. (A) Angiogenesis 분석 : 모세와 같은 형태 내피 세포와 씨앗 모듈에 쉽게 지방 - 유래 줄기 세포로 감지하고 배양 5 일 후에 계량하실 수 있습니다. (B)의 모듈에 대한 라이브 / 죽은 분석의 공촛점 현미경 이미지라이브 세포에 대한 taining 것은 녹색과 죽은 세포를 위해 빨간색입니다. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="그림 6" /> 그림 6 : 임베디드 mesenchymal 줄기 세포와 내피 세포의 표면 레이어를 포함하는 콜라겐 모듈. 모듈은 microfluidic 챔버에서 7 일 동안 전단 응력 (~ 0.64 dyn / cm 2) 노출 및 연락처 지점에서 융합을 표시하기 시작했다. 내피 세포 vWF (갈색)와 스테인드 및 mesenchymal 줄기 세포의 핵은 청색 (H &amp; E) 표시됩니다. 스케일 바 = 100μm. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="그림 7" /> 그림 7 : 지방 - 유래 줄기 세포 (ASC)와 인간 Microvascular 내피 세포와 코팅을 포함하는 콜라겐 모듈의 수백은 (HMEC) 지방의 재생 어플 리케이션을위한 생체내에 ASC / HMEC 상호 작용을 연구하기 위해 마우스 칩을 피부 밑에 심어입니다.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 서로 다른 세포 유형 1-11와 임베디드 모듈을 사용하여 여러 가지 마이크로 조직을 가공했습니다. 우리는 성공적으로 기본 cardiomyocytes, 아일 레츠, 지방 줄기 세포 및 mesenchymal stromal 세포뿐만 아니라, HepG2, NIH 3T3 및 복제 구 간 세포를 포함한 여러 세포 라인을 내장했습니다. 우리는 쥐 대동맥 내피 세포, 인간 제대 정맥 내피 세포 및 인간의 microvascular 내피 세포를 포함한 내피 세포?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of micro-tissues using modules of collagen gel containing cells

이 프로토콜은 모듈이라는 마이크로 조직의 유형의 제조에 대해 설명합니다. 모듈 접근 방식은 유니폼, 확장성과 vascularized 조직을 생성합니다. 모듈은 콜라겐뿐만 아니라 다른 gelable 또는 crosslinkable 재료 만들 수 있습니다. 그들은 제조시 직경 길이 약 2mm와 0.7 mm 있지만 임베디드 세포 또는 경우 모듈이 내피 세포로 코팅 아르와 크기로 축소. 개별 모듈은 임베디드 세포가 산소와 다른 영양소의 확산 제한 내에 있지만 모듈 perfusable 아르 더 큰 조직을 형성하기 위해 함께 포장 수있는 정도로 작은 수 있습니다. 그들이 복잡한 조직을 형성하기 위해 함께 모여되기 전에 다른 셀 형식에 포함된 또는 모듈에 코팅된 수 있기 때문에 이러한 조직 건설에 모듈입니다. 모듈을 만들기 위해 세 가지 주요 단계가 있습니다 : (1) 콜라겐을 중화하고 그 안에 세포를 포함, 튜브 (2) 겔화 콜라겐과 내피 세포와 모듈과 (3) 코팅 모듈을 절단.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

임베디드 세포와 어떤 표면을 포함하는 모듈 불리는 원통형 콜라겐 젤을 사용하여 마이크로 조직의 생성은 내피 세포로 코팅됩니다.

콜라겐의 모듈을 사용하여 미세 조직의 제조가 포함 세포를 젤

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.7K 조회수.  University of Toronto. 임베디드 세포와 어떤 표면을 포함하는 모듈 불리는 원통형 콜라겐 젤을 사용하여 마이크로 조직의 생성은 내피 세포로 코팅됩니다.

비디오: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

동적 마스크 프로젝션 석판술를 사용하여 신경 문화 시스템의 Micropatterned Hydrogels의 제작

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

연구

생체공학

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 ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

금 나노 막대를 포함하는 세포 차량의 제조 및 광 음향 분석

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

젤 보조 재수를 사용하여 거대한 크기의 Polymersomes 형성

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

제조 및 Macrostructure를 포함 하는 광학 조직 팬텀의 특성화

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

준비, 정화, 및 지방산 포함 된 리의 사용

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Falcarindiol 인간 중간 엽 줄기 세포에 의해 포함 된 새로운 지질 코팅 나노 입자의

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...