저자는 배양 그림 2에 표시되는 조직을위한 Yabin 우 감사 할 사진이 바트 반 Overbeeke로 촬영되었다. 작품은 재정적 SenterNovem, 교부금 ISO 42022에 의해 지원되었다.

1. 손쥐 Myoblast의 전구 세포 또는 C2C12 세포의 문화 <ol><li> 처음 Shefer 및 동료 (10)에 의해 출판하고 나중에 콜린스 외. 11 Boonen 외. 12 적응 프로토콜에 따라 세포를 분리 및 액체 질소에 이것들을 저장합니다. 이 마우스, 예를 들어 C57Bl / 6이 필요합니다. 다른 방법은 예를 들어 리 Y 외하여 시각화 실험의 저널에 출판하는 방법, 다른 실험실에 사용됩니다. 13. 시약 및 장비를 들어 당신은 7 페이지와 8의 표라고합니다. 하나의 마우스에서 근육에서 일반적으로 액체 질소의 약 20 병을 cryopreserve하기에 충분한 세포를 얻을 수 있습니다. 다음, 우리는 액체 질소 저장에서 세포를 해동 할 수있는 프로토콜을 시작합니다. </li><li> 25cm 2 Matrigel (1 MG / ML) 코팅 조직 배양 플라스크에 한 유리 병에서 세포를 놓고 이루어진 성장 매체 (GM) (335 ML DMEM 고급, 100 ML 태아 bovin을 추가전자 혈청 (FBS), 50 ML HS, 5 ML 페니실린 / 스트렙토 마이신, 5 ML L-glutamin, 5 ML 닭 배아 추출물 (혼)). </li></ol> 참고 : 상온에서 2 시간에 코트 흡인하여 Matrigel을 제거합니다. <ol start="3"><li> 세포는 약 삼일마다 1시 3분 하위 문화. 이 말의 의미는 : 일 3 : 한 75cm 2 플라스크에서 두 150cm 2 플라스크 (코팅 플라스크에 preplating 30 분 포함)에 대한 통로 셀 : 일 6시 75cm 2 플라스크에 한 25cm 2의 항로 세포. 일 9 전송 한 150cm 2 병 세 150cm 2 플라스크 (필요한 경우 preplate가 다시 세포의 표현형에 따라)에 사용할 1 개의 트리플 150cm 2 병, 또는 경우에. 하루에 12에 세포가 근육 구조에 퍼 뜨리고위한 준비가되어 있습니다. </li></ol> 참고 사항 : - 트리플 150cm 2에 따라 세포의 수는 약 4.5 X 10 6 세포 될 것입니다. -로 사용다른 isolations의 재산이 시딩시 세포의 혼합 인구를 얻기 위해 그들을 혼합합니다. 참고 : 기본 셀 작동하지 않을 경우, C2C12 myoblasts는 좋은 대안입니다. 2. 공학 골격근 화장지에게 <ol><li> &quot;경화 대리인&quot;(10시 1분)와 &quot;탄성&quot;을 혼합하여 실리콘 접착제를 준비합니다. 한 삼각면 (; 그림 1 모양 집과 같은)에 벨크로의 5mm 평방 세그먼트 - + / 잘라. 사각형 사이의 약 12​​ mm의 공간에서 6 잘 문화 판의 우물에 접착제 벨크로. </li></ol> 참고 사항 : - 만 벨크로의 부드러운면을 사용하고 측면 위쪽을 향하고 있습니다. - 지붕이 서로 마주 있는지 확인하십시오. - 만 실리콘 접착제와 벨크로를 커버 잘 전역에 접착제를 전염되지 않습니다. - 나중에 전기 자극의 경우는 잘 PL 수직 방향으로 구조를 정렬하는 관련(긴 축을 따라) 먹었다. <ol start="2"><li> 진공 오븐에 하룻밤 건조하게 놔둬, 가열하지, 주로 기포를 제거합니다. 15 분의 우물과 부화 70 % EtOH를 추가하여 살균. PBS로 3 배 씻어 15 분에 UV 아래 넣어. 우물에서 모든 PBS 및 사용까지 보육에 벨크로와 장소를 제거합니다. </li><li> 냉장고에 Matrigel 솔루션을 해동 곧 3D 구조를하기 전에 원하는 농도로 콜라겐 솔루션을 준비합니다. 멸균 0.02 % 아세트산 (최종 농도 3.2 밀리그램 / ML)와 주식 콜라겐을 희석. </li></ol> 참고 : 얼음의 모든 둡니다. <ol start="4"><li> GM과 횟수에 resuspend 세포를 Trypsinize. 보육의 원심 분리기 튜브에 세포를 둡니다. </li><li> 이 콜라겐 솔루션으로 0.5M NaOH를 추가, 당신은 관 (표 1에 따라)에 변경하고자하는 구조의 숫자 콜라겐 주식 솔루션의 원하는 금액을 추가 밝은 분홍색 colo까지R은 7.5의 산도를 나타내는 수 있습니다. 등을 아래로 pipetting하여 부드럽게 섞어서 거품 형성을 피할 수 있습니다. 그런 다음 Matrigel을 추가하고 부드럽게하지만 철저하게 매우 섞는다. 마지막으로, 콜라겐 / Matrigel 혼합물 (해당 금액에 대한 표 1 참조)에 GM을 추가합니다. </li></ol> 참고 사항 : - 얼음의 각 단계를 수행! Matrigel과 콜라겐이 증가하고 온도에서 쉽게 젤 것이다. - 색상이 실제로 분홍색입니다 조심해! 산도의 증가는 빠른 겔화를 유도합니다. - 콜라겐의 최종 농도 : 1.6 MG / ML. <ol start="6"><li> 5 분 1,000 rpm으로 세포의 원하는 금액을 원심 분리기하고 표면에 뜨는을 제거합니다. </li></ol> 참고 : - 구성 당 세포의 숫자가 조정해야하는 세포의 활동에 따라. 일반적으로 세포의 수는 구조 당 750,000 사이 1,250,000 세포를 자리 잡고 있습니다. <ol start="7"><li> 세포 펠렛을 resuspend하는 겔 혼합물의 일부를 사용하여나머지 혼합물에 세포를 전송하고 철저하게 혼합하지만, 기포를 도입하지 않고. </li><li> 보육 및 피펫 벨크로 첫째로 셀 겔 혼합물의 0.35 -0.4 ML의 preheated 잘 접시를보십시오. 그런 다음 첨부 파일 사이트 사이의 중심에서 혼합물을 피펫과 벨크로로 확장하기 시작합니다. 마지막으로, 벨크로 조각 주위에 두 개의 벨크로 앵커와 피펫 사이의 간격을 채우기 위해 젤의 나머지를 사용합니다. </li><li> 젤은 양도 할 정도로 단단한 경우주의 깊게 5-10 분 후 확인 즉, 부드럽게 요리를 누르고 젤이 단단한 경우 시각적으로 검사한다. 그렇다면, 보육에 요리를주의 깊게 배치합니다. 보통, 그들은는 열명 분 후에 픽업 할 수 있습니다. 그런 다음, 1-2의 시간 후, 부드럽게 따뜻한 GM의 4 ML 각 젤을 입혀 라. </li></ol> 참고 :-DO 접시를 다룰 때 어떤 힘차게 운동을하지. <ol start="10"><li> (성장 매체 차별화 매체에 의해 GM 교체여자의 배아 추출물없이) 24 시간 후, 신선한 중간마다 2~3일로 교체하십시오. 일 7 당신이 융합 된 근육 섬유에서 교차 결 개발에 얼룩으로 평가 될 수 있기 때문에, 성숙 지향적 인 근육 섬유를 얻은 것입니다. </li></ol> 3. 전기 자극 <ol><li> 안전 캐비닛의 UV 이하 70 %의 에탄올과 이후 건조에 사용하기 전에 ionoptix 판에서 전극을 (시약 및 장비의 표를 참조) 소독하다. 구조와 문화 판 위에 전극으로 판을 놓고 문화 판과 보육에 송금 뚜껑을 다룹니다. 적절한 케이블로 Ionoptix C-맥박 조정 장치를 연결합니다. </li></ol> 참고 : 전극은 근육 구조 (그림 2)와 함께 병렬 배치됩니다. <ol start="2"><li> 예상대로 자극 프로토콜을 적용합니다. </li></ol> 참고 : 우리는 일반적으로 4 V / CM을 사용하여2Hz의 주파수에서 6ms 펄스. 문화 매체에게 자극 동안은 24 시간을 변경합니다.

<ol>
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근육 조직의 공학 재생 의학의 약물 검사에 대한 질병 모델 등과 고기 생산을위한 사용하기위한 잠재력이 있습니다. 그러나 이러한 응용 프로그램에 대한 요구 사항은 다양합니다. 콜라겐은 세포 정렬이 가능하기 때문에 이전의 2D 연구 12 결정 myoblast의 전구 세포는 지하 막 파생 된 단백질의 존재를 필요로하기 때문에 우리는 콜라겐과 matrigel의 조합과 협력하기로 결정했습니다. 또한, 섬...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> Matrigel - 성장 인자가 감소 </td><td> Beckton 및 디킨슨 </td><td></td><td></td></tr><tr><td> DMEM (높은 혈당) * </td><td> Gibco </td><td> 42,430 </td><td></td></tr><tr><td> 고급 DMEM </td><td> Gibco </td><td> 12,491 </td><td></td></tr><tr><td> 말 혈청 </td><td> Gibco </td><td> 65050-122 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> Greiner </td><td> 758075 </td><td></td></tr><tr><td> 0.45와 0.22 μm 주사기 필터 * </td><td> Whatmann (Schleicher와 Scheull) </td><td> 10462100 </td><td></td></tr><tr><td> L-글루타민 </td><td> Gibco </td><td> 25030024 </td><td></td></tr><tR&gt; <td> 페니실린 / 스트렙토 마이신 </td><td> Gibco </td><td> 10378016 </td><td></td></tr><tr><td> Amphotericin </td><td> Gibco </td><td> 15290-018 </td><td></td></tr><tr><td> 문화 플라스틱 </td><td> Greiner </td><td></td><td> 문화 플라스크와 피펫을 포함 </td></tr><tr><td> 칙의 배아 추출물 </td><td> 미국 생물 </td><td> C3999 </td><td></td></tr><tr><td> 파스퇴르 피펫 * </td><td> Hilgenberg </td><td></td><td> 면, 오픈 팁 L과 수축이있는 파스퇴르 (Pasteur) 피펫, : 0,9의 팁 직경 230mm - 1,1 mm </td></tr><tr><td> 파스퇴르 피펫 * </td><td> Hilgenberg </td><td></td><td> 면, 오픈 팁 L과 수축이있는 파스퇴르 (Pasteur) 피펫, : 1,4의 팁 직경 230mm - 1,6 mm </td></tr><tr><td> 파스퇴르 피펫 </td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> Collagenase 유형 I* </td><td> 시그마 </td><td> C0130-16 </td><td></td></tr><tr><td> 40 μm 셀 스트레이너 * </td><td> BD 팔콘 </td><td> 352340 </td><td></td></tr><tr><td> 19G 바늘 </td><td></td><td></td><td></td></tr><tr><td> 엘라스토머 </td><td> 다우 코닝 기업 </td><td> 3097358-1004 </td><td> Silastic MDX 4-4210 # </td></tr><tr><td> 에이전트 치료 </td><td> 다우 코닝 기업 </td><td></td><td> Silastic MDX 4-4210 # </td></tr><tr><td> 벨크로 </td><td> 일반 상점 </td><td></td><td> 당신은 정기적으로 상점에서이을 구입하실 수 있습니다 만 부드러운면을 사용하여 </td></tr><tr><td> 콜라겐 유형 I, 쥐 꼬리 </td><td> BD Biosciences </td><td> 3544236 </td><td></td></tr><tr><td> C-페이스 EP 문화 맥박 조정 장치 </td><td> Ionoptix </td><td></td><td></td></tr><tr><td> 전기 자극을위한 6도 문화 요리 </td><td> 왜냐면kton 디킨슨 - 매 </td><td> BD 팔콘 # 353846 </td><td></td></tr><tr><td> C-접시 배양 접시 전극 </td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> * 세포의 분리 (포인트 1.1) 필요한 # 함께 한 키트에 </td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

설계 근육 조직은 재생 의료 및 육류 대체 1로 압력 궤양을 공부하는 예를 체외에서 질병 모델로 사용하기위한 조직의 생산을 포함하는 여러 가지 목적으로 사용 할 수 있습니다. 첫 번째보고 3D 근육 구조는 몇 년 전에 만든 분야의 개척자 Vandenburgh 및 동료 2,3 아르되었습니다. 근육 조직 공학에서 만든 발전 생화학 요인, 줄기 세포 및 전구 세포에 대한 지식의 광대 한 이득의 결과뿐만 아니라하지만, 물리적 요인이 세포 행동 제어에 필수적인 역할을하는 연구자들에 의해 얻은 통찰력의 특정 기반에 있고 조직 개발. 첨단 엔지니어링 근육은 현재 셀 인구 히드로 겔 구조로 구성되어 구성합니다. 우리가 실험실에서이 일반적으로 C2C12, 미 손쥐 뒷다리 근육에서 분리 손쥐 myoblast의 전구 세포, 또는 손쥐 myoblast 세포 라인으로 구성콜라겐 / Matrigel의 혼합물로 xed 두 개의 고정 지점 사이의 도금, 근육 인대를 모방. 다른 세포는,뿐 아니라 L6 쥐 myoblasts 4, 신생아 근육 유래 전구 세포 5, 그러한 인간의 6도 유도 만능 줄기 세포 (IPS 세포) 7과 같은 다른 종에서 성인 근육 조직에서 파생 된 세포와 같은 예를 들어 다른 세포 라인을 고려 될 수있다 . 셀 수축성은 구조 8,9와 문화 약 1 주일 후에 근육 전구 세포의 분화의 긴 축을 따라 세포의 정렬을 발생합니다. 또한, 전기 자극의 응용 프로그램이 어느 정도 8로 차별화 과정을 향상시킬 수 있습니다. 때문에 그 제한된 크기 (8 X 2 X 0.5 mm)의 전체 조직은 예 생존, 분화 및 세포 정렬을 모니터링 할 공 촛점 현미경을 사용하여 분석 할 수 있습니다. 특정 응용 프로그램에 enginee에 대한 요구 사항을 따라붉은 근육 조직은 경우에 따라 다릅니다, 다른 종에 고기 대체 번역이 필요한 역할을하는 동안 재생 의료에 대한 예를 들어 사용, 조직 크기와 vascularization의 최대 크기 조절이 필요합니다.

최종 제품은 그림 3에 표시된 근육 구조 될 것입니다. 조직의 크기는 길이 약 8mm, 폭 2mm와 두께 0.5 mm 될 것입니다. 차별화 동안 전기 자극은 마이 오신 중쇄 isoforms의 표현을 변경되지만, 차별화 매체 (8)에 의해 유도로 크게 차별화 프로세스를 개선하지 않지만, 전기 자극은 근육의 기능을 확인하기 위해 절차의 끝 부분에 적용 할 수 왜냐하면 완전히 개발 sarcomeres와 근육이 전기 펄스에...

Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

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

설계 근육 조직은 질병 모델로 또한 고기에 대한 대체 소스로, 재생 의료에 큰 가능성이 있습니다. 여기 마우스 myoblast의 전구 세포, 및 전기 펄스에 의해 자극에서이 경우에, 근육 구조의 기술에 대해 설명합니다.

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 ...

engineering-skeletal-muscle-tissues-from-murine-myoblast-progenitor

Research

JoVE Journal

Bioengineering

20.8K 조회수.  Eindhoven University of Technology, The Netherlands. 설계 근육 조직은 질병 모델로 또한 고기에 대한 대체 소스로, 재생 의료에 큰 가능성이 있습니다. 여기 마우스 myoblast의 전구 세포, 및 전기 펄스에 의해 자극에서이 경우에, 근육 구조의 기술에 대해 설명합니다.

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

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

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

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 ...

연구

생체공학

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

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

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 ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

마우스 배아 줄기 세포에서 신경 조직 공학을위한 폴리의 전기 방사 섬유 발판 (글리세롤 dodecanedioate)

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

투과성이 골격 근육 섬유에 의해 생성 된 최대 아이소 포스의 측정

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

심장 근육 세포 기반 액츄에이터 및 자체 안정화 비 오로봇 - 1 부

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

심장 근육 세포 기반 액츄에이터 및 자체 안정화 바이오 오봇 - 제 2 부

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

공학에 대 한 미세 Bioprinting 나가도록 조직 및 Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Decellularization 식물 조직의 조직 공학 응용 프로그램에 대 한 두 가지 방법

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells&#039; Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...