본 연구는 생물 의학 재료 (BMM) 연구소의 연구 프로그램에서 수행되었다. BMM은 경제 업무, 농업 혁신의 네덜란드 교육부에 의해 cofunded 있습니다. Nederlandse의 Hartstichting의 재정적 기여 기꺼이 인정 받고 있습니다.

1. 인간 베나 Saphena 세포의 배양 <ol><li> SCHNELL 등으로 프로토콜에 따라 보조 자료를 사용하는 지침에 따라 기증자로부터 얻은 베나 saphena 마그나, 9.에서 세포를 분리하고 액체 질소에 이러한 저장합니다. 여섯 잘 플레이트 문화에 2 × 2 ㎜의 한 기증자 컷 조각의 대정 saphena 마그나의 부분에서. 잘 당 2 개를 사용합니다. 일반적으로 충분한 세포는 액체 질소 0.25 X 10 6 세포와 약 3 병을 채우기 위해 얻을 수 있습니다. HVSCs는 vimentin에, 최근 desmin의 어떤 표현 α 평활근 액틴 10을 표현하는 모집단의 표현을 표시하여, 면역 조직으로 특징입니다. 다음 세포의 수를 증가 액체 질소에서 세포를 해동 프로토콜을 시작합니다. </li><li> T75 조직 문화 플라스크에 한 병에서 세포를 배치하고 고급 DMEM 50 ML 태아 소 혈청 (FBS), 5 ML 페니실린 / 미팅 성장 매체 (GM)를 추가스트렙토 마이신 5 ML L-glutamax. 2~3일 모든 매체를 변경합니다. </li><li> 세포는 약 14 일 후에 합류 성장. 스토어 0.5 액체 질소에 한 병에서 x 10 6 세포는 통로 1이라고합니다. </li></ol> 참고 : 세포의 냉동 HVSCs을 수확 할 때 필요하지 않습니다,하지만 전적으로 저장하는 데 사용됩니다. <ol start="4"><li> T175 조직 배양 플라스크에 한 병에서 HVSCs를 놓고 GM을 추가합니다. 2~3일 모든 매체를 변경합니다. 세포는 약 7 일 이후에 합류 성장. 상점 3 액체 질소에 한 병에서 x 10 6 세포는 통과 2라고도합니다. </li><li> rollerbottle에 한 병에서 세포를 배치하고 GM을 추가합니다. 2~3일 모든 매체를 변경합니다. 세포는 대략 8 일 후에 합류 성장. 하나 rollerbottle은 약 20 ~ 30 × 10 6 세포를 포함하고 있습니다. 통과 6 문화 세포집니다. 세포의 표현형이 변경 될 수 있기 때문에, 통과 9보다 높은 통로의 셀을 사용하지 마십시오. </li></oL&gt; 2. 섬유소 기반 조직 구문 공학 <ol><li> 경화제 (10시 1분)와 탄성 중합체를 혼합하여 실리콘 접착제를 준비합니다. 벨크로의 7 개의 x 3mm 직사각형 세그먼트를 잘라. 십자가를 형성하고, 벨크로 스트립 사이에 3mm의 제곱 공간을 남겨 유연한 막과 6 잘 문화 판에 접착제 벨크로. </li></ol> 주 : 만 벨크로의 부드러운면을 사용하고이 쪽을 위쪽으로 직면하고 있습니다. 때 접착 벨크로 만 실리콘 접착제 벨크로 커버, 잘 걸쳐 접착제를 전파하지 않는다. 문화 플레이트 실리콘 막 바닥을 가지고 있기 때문에, 플레이트에 쉽게 접착을 보장하기 위해 유연한 세포막을 보강 웰 플레이트 아래에 뭔가를 사용합니다. <ol start="2"><li> 접착제의 경화을 보장하기 위해 60 ° C의 오븐에서 하룻밤 실리콘 접착제를 건조. 30 분 동안 우물과 부화에 70 % 에탄올을 첨가하여 살균. PBS로 3 배 씻어 아에서 모든 PBS를 제거LS와 벨크로. 30 분 동안 UV 밑에두고 사용할 때까지 멸균 유지합니다. </li><li> 조직 공학 GM으로 구성된 중간 (TM) 및 L-아스 코르 빈산 2 - 인산의 130 밀리그램을 준비합니다. </li><li> TM에 1 밀리그램 / ML ε-아미노 카프로 산 (ACA)를 추가합니다. ACA는 굴욕 (11)로부터 섬유소를 방지하기 위해 문화의 첫번째 7 일에 사용됩니다. 또한, aprotinin을 사용할 수 있습니다. </li><li> 덩어리 형성을 방지하기 위해, 피브리노겐은 오픈 전에 실내 온도에 도달 할 수 있습니다. 수풀 피브리노겐없이 더 쉽게 용해됩니다. ACA와 보충 10 ㎎ 실제 단백질 / ㎖ TM 7의 농도 섬유소를 용해. 살균 필터 피브리노겐 용액은 여러 개의 필터가 필요할 수 있습니다. 사용할 때까지 얼음에 저장합니다. </li></ol> 참고 : 너무 많은 거품 형성을 방지하기 위해 부드럽게 피브리노겐 혼합 용해. <ol start="6"><li> ACA와 보충 10 IU / ㎖ TM 7의 농도에 트롬빈을 녹입니다. 사용할 때까지 얼음에 저장합니다. </li> &lt;/ OL&gt; 참고 : 얼음 저장은 트롬빈과 섬유소의 조기 겔화를 방지하기 위해 필요합니다. <ol start="7"><li> GM과 개수에 resuspend을 세포를 Trypsinize. </li><li> 섬유소 기반의 조직 구조 (조직 공학 심장 판막 7 방법에 따라 농도) 시드 15 X 10 6 세포 / ML을 사용합니다. 단일 젤 100 μL GOF 엘, 따라서 1.5 X 10 6 세포로 구성되어 있습니다. 될 것입니다 젤의 수만큼 원심 분리기 튜브의 결과로, 하나의 원심 분리 관에 1.5 X 10 6 세포를 넣습니다. </li><li> 7 분 350 XG에서 세포를 원심 분리하고 상층 액을 버린다. 50 μl의 트롬빈에있는 세포를 resuspend을. 이 현탁액에 파란색 형광 폴리스티렌 마이크로 4 μl를 추가합니다. 유리 병에 피브리노겐의 50 μl를 넣습니다. 세포를 50 μL 트롬빈을 차지하기 위해 피펫을 사용합니다. 100 μL에 피펫의 볼륨을 높입니다. 트롬빈의 50 μL 솔루션을 피펫피브리노겐을 유리 병에 세포는 트롬빈과 섬유소를 혼합하고, 100 ㎕의 혼합물을 차지합니다. </li></ol> 참고 : 형광 폴리스티렌 마이크로는 이미지 분석을위한 내부 기준 마커로 사용됩니다. 혼합 트롬빈과 피브리노겐은 신중 혼합물을 pipetting하여 기포의 형성을 방지합니다. 기포는 섬유소 젤에 구멍 발생합니다. <ol start="10"><li> 에와 벨크로 스트립 사이에 젤 혼합물을 피펫. 섬유소 젤의 일반적인 겔화 시간은 한 구성 요소가 혼합되어, 20 초 순서입니다. </li></ol> 주 : 혼합물이 잘 판에 피펫되기 전에 겔화를 방지하기 위해 가능한 한 빨리이 작업을 수행합니다. 세포와 구슬을 사용하기 전에 연습합니다. <ol start="11"><li> ACA와 문화의 TM을 추가하기 전에 겔화 할 수 있도록 가습 95 % 공기 / 5 % CO 2 배양기에서 37 ° C에서 30 분 동안 현탁액을 품어. </li><li> 최초 7 일 ACA 2-3일 모든과 TM을 대체합니다. 일주가 ACA없이 배양 할 수 후 젤은 충분히 안정적이다. 첫 주 후 2-3 일마다 TM을 대체합니다. 잘 당 TM 6 ML을 추가합니다. 하루에 12 세포는 시각화를위한 충분한 콜라겐을 생산했다. </li></ol> 3. 스트레인 및 제약 조건의 적용 변형 유도를 변경 <ol><li> 정적 변형은 세포 견인 및 압축 12에 의한 직접 세포에 의해 적용됩니다. 콜라겐 조직 개편을 유도하기 위해, 조직이 한 방향으로 두 개의 벨크로 스트립에서 느슨한 구성 잘라. 단방향 제약 (그림 1A)이 발생합니다. 구조는 다음 안정 충분하고 콜라겐이 입금 된 이후, 12 일에이 문제를 수행합니다. </li><li> FlexCell의 FX4000T 시스템의 사용과 유연한 막과 문화 접시의 바닥에 진공을 적용하여주기적인 변형을 적용합니다. 로드 포스트 위에 (이는 일의 한 부분에베이스 플레이트에 플레이트를 놓고전자 주기적 로딩 장치). 펌프는 멤브레인 위에 진공을 적용하여 아래 누워 사각형 포스트에 막 가져옵니다. 조직의 십자가 모양의 첨부 파일로 인해 막 (벨크로 통해) 생성하고 직사각형 게시물 적용주기 변형 (그림 1B) 단축됩니다. </li><li> 처음에는 순환 변형 응용 프로그램이 6 일째에 시작하기 전에, 초기 기계적인 무결성을 달성하기 위하여 5 일 동안 정적 문화를 사용합니다. 진공 펌프 컨트롤러에 프로그램은 주기적 얼룩 프로토콜입니다. 예를 들어 일축 방향으로 13로, 이전에 설정된 간헐적으로 순환 변형 프로토콜을 사용합니다. 이 3 시간 휴식 기간과 교대로 3 시간의 기간 0 ~ 5 % 변형에 긴장 1 Hz에서와 사인파의 간헐적 인 긴장으로 구성되어 있습니다. 정렬 콜라겐 조직을 유도 7 일이주기적인 변형 프로토콜을 수행합니다. </li><li> 일반적으로 십이일 후 HVSCs은 정렬 된 콜라겐의 조직을 생산N. 정렬 콜라겐 조직에 도달하면, 원래의 변형 방향에 수직으로, 단축 주기적 변형 방향을 변경합니다. 90 ° 직사각형 게시물을 설정하여이 작업을 수행합니다. </li></ol> 4. 시각화 세포와 콜라겐 <ol><li> 세포 생존이나 콜라겐 형성을 방해하지 않는 프로브를 활성화 실시간 콜라겐 리모델링, 라벨 샘플을 시각화합니다. 찬란 세포의 세포질과 콜라겐 얼룩 프로브를 사용합니다. </li><li> 공 촛점 레이저 스캐닝 현미경을 사용하여 또는 차 고조파 세대 780 nm의 500-550 nm의 사이의 감지에 여기에자가 형광 14을 사용하여 콜라겐 구조를 시각화하는 데 사용할 수 있습니다. </li><li> 3 시간의 휴식 기간 동안 주기적으로 긴장 샘플, 설치 및 인큐베이터에서 배양 접시를 제거하고 세포와 콜라겐을 시각화하기 위해 공 촛점 레이저 스캐닝 현미경에 그 (것)들을 수송한다. </li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+Collagen+Organization+at+the+Micro-level+in+Fibrin-based+Engineered+Tissue+Constructs.">Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs.</a> Ann. Biomed. Eng. 41, 763-774 (2013).</li><li>Merryman, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+heart+valve+interstitial+cell+stiffness+and+transvalvular+pressure:+implications+for+collagen+synthesis.">Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen synthesis.</a> Am. J. Physiol. 290, (2006).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cellular+mechanotransduction+to+biologically+inspired+engineering:+2009+Pritzker+Award+Lecture,+BMES+Annual+Meeting+October+10,+2009.">From cellular mechanotransduction to biologically inspired engineering: 2009 Pritzker Award Lecture, BMES Annual Meeting October 10, 2009.</a> Ann. Biomed. Eng. 38, 1148-1161 (2009).</li><li>Sander, E. A., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39, 714-729 (2010).</li><li>van der Schaft, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Skeletal+Muscle+Tissues+from+Murine+Myoblast+Progenitor+Cells+and+Application+of+Electrical+Stimulation.">Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation.</a> J. Vis. Exp. (73), e4267 (2013).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

세포 인구 섬유소 구조의 설명 모델 시스템은 세포와 콜라겐의 연구에 큰 잠재력을 가지고 (재) 조직 (드 Jonge 등. 15), 조직 공학의 목적으로 사용될 예. 초기 세포 캐리어로 섬유소를 사용하여 섬유소 분해 한 후, 조직은 세포와 내생 매트릭스에서만 생성됩니다. 이러한 방법으로, 세포 경계 강성 12 감지, 또는 변형 방지를 표시 수축력에게 16, 17을 적용하여, ?...

심장 혈관 조직은 탁월한 하중 기능을 가지고 있습니다. 세포 외 기질의 콜라겐 섬유의 특정 내용과 조직의 하중 특성에 기여하고 전체 조직의 힘 1을 지배. 조직 공학 구조물의 기계적 조절이 사용됩니다 - 일반적으로 (주기적) 긴장 요법으로 구성 - 조직의 조직과 기계적 성질 2,3을 향상 할 수 있습니다. 미리 정의 된 콜라겐 구조를 가진 조직을 만드는 복잡한 조직 형상의 변형에 의한 콜라겐 조직의 완전한 이해는 아직 달성되었다 아닙니다. 이 개발 조직에 콜라겐 리모델링 우리의 제한된 지식에 주로 때문입니다. 기존 모델은 주로 정적 변형률 4-6의 사용과 콜라겐 리모델링의 최종 그물 결과에 대한 정보를 제공합니다. 여기에 우리가 영향을 받아 3D로, 실시간 방식으로 콜라겐 (재) 조직의 연구를 할 수있는 고도 조정 가능한 모델 시스템을 제공정적 또는 주기적 변형. 조직 구조는 구조의 모든 콜라겐 내생 것을 보장 섬유소 기반으로하고 있습니다. 구조의 셀 콜라겐 조직은 시각, 그리고 내부 참조 시스템은 우리가 시간 경과 분석을위한 세포와​​ 콜라겐 구조를 재배치 할 수 있습니다. 이러한 세포는 기초하여 향상된 여분의 세포 매트릭스 생산 및 엔지니어링 심장 혈관 조직 7 매트릭스와 우리의 설립을 사용 개조 할 수있는 능력에 대한 알려져 있기 때문에이 프로토콜에서 우리는, 인간 베나 Saphena 세포 (HVSCs)를위한 모델 시스템의 사용을 설명합니다 드 Jonge 등. 8 일에

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

콜라겐 함량과 콜라겐 조직 개발 조직은 지방 조직의 긴장과 조직의 제약 조건에 의해 영향을받을 수있다. 조직 엔지니어는 미리 정의 된 콜라겐 구조와 조직을 만들려면 다음 원칙을 사용하는 것을 목표로하고 있습니다. 최종 조직 구조를 제어하는​​ 콜라겐 리모델링의 정확한 기본 프로세스의 완전한 이해는, 그러나, 부족이다. 특히, 약간은 조직의 기계적 부하 조건 변화에 따라 콜라겐 섬유의 (재) 방향에 대한 알려져있다. 우리는 더 콜라겐을 명료하게, 이축이 제한된 근섬유-시드 섬유소 구조로 구성된 체외 모델 시스템을 개발 I 님의 질문에 (재) 방향) 단축 정적 하중 조건 및 II에 이축 복귀) 이축 - 제약의주기 단축 하중은 FlexCell의 FX4000T 로딩 장치의 사용과, 방향로드의 변화 전후의 구조. 시간 경과 공 촛점 이미징은 vi를하는 데 사용됩니다비파괴 방식으로 콜라겐 (재) 방향을 sualize. 구조의 셀 콜라겐 조직은 실시간으로 시각화 할 수 있으며, 내부 참조 시스템은 우리가 시간 경과 분석을위한 세포와​​ 콜라겐 구조를 재배치 할 수 있습니다. 모델 시스템의 다양한 측면은 세포의 소스 또는 건강하고 병에 걸린 세포의 사용과 같이 조정할 수 있습니다. 첨가물 예를 MMPs를 추가 또는 인테그린을 ​​차단하는으로, 더 메커니즘을 기본 콜라겐 리모델링을 명료하게 할 수 있습니다. 구조의 모양과 크기는 쉽게 세포와 콜라겐 (재) 조직을 연구하는 고도 조정 가능한 모델 시스템의 결과로, 특정 요구에 적용 할 수 있습니다.

이 모델 시스템은 배양 근섬유 - 시드 섬유소 젤 수 있습니다. 그림 1A 정적 이축 제약 조건 첫 번째 배양 조직을 보여줍니다. 조직의 제약 단축 정적 제약 조건을 만들려면 두 가지 조건에서 섬유소 젤을 절단하여 발표 및 조직 압축 및 후 개조 (그림 1A)입니다. 주기적인 변형의 경우, 조직은뿐만 아니라 정적 이축 제약 조건 하에서 배양한다. 후에 5 일주기 단축 스트레인?...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

이 모델 시스템은 비파괴 방식으로 내생 콜라겐 (재) 조직 리얼 타임으로 공부하는 데 사용할 수있는 근섬유 채워진 섬유소 젤에서 시작됩니다. 모델 시스템은 서로 다른 세포 소스, 중간 첨가제와 함께 사용할 수있는, 매우 조정할 수 있으며, 특정 요구에 쉽게 적용 할 수 있습니다.

엔지니어링 섬유소 기반 조직의 면역 조직 및 제약 조건의 적용에서 생성하고 세포와 콜라겐 (재) 조직을 유도하는 스트레인

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K 조회수.  Eindhoven University of Technology.  이 모델 시스템은 비파괴 방식으로 내생 콜라겐 (재) 조직 리얼 타임으로 공부하는 데 사용할 수있는 근섬유 채워진 섬유소 젤에서 시작됩니다. 모델 시스템은 서로 다른 세포 소스, 중간 첨가제와 함께 사용할 수있는, 매우 조정할 수 있으며, 특정 요구에 쉽게 적용 할 수 있습니다. 

비디오: Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

자기 조립 셀룰러이 Biomechanical 분석 및 조직 공학을위한 셀 유래 조직 반지를 조작하는 감독

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

연구

생체공학

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

라만 분광학을 사용하여 세포와 세포외 기질의 비 접촉, 레이블이없는 모니터링

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

전기 자극의 손쥐 Myoblast의 전구 세포 및 응용에서 공학 골격근 조직도

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

운동 조건의 혈청을 이용한 인대 구조의 치료 : Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

재조합 콜라겐 나 세포 확장과 생물에 셀 납품 시스템으로 그들의 잠재적인 사용에 대 한 펩 티 드 Microcarriers 모델

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

큰 힘줄 결함을 복구하는 조직 엔지니어링 콜라겐 이식과 새로운 테노르래피 봉합사 기술

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

인쇄 3D 세포 -라덴 췌장 조직 구성을 인쇄를위한 췌장 조직 유래 세포 외 매트릭스 바이오 잉크

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...