소냐 Schrepfer, 레나드 콘라디 및 아르네 한센 독일 Forschungsgemeinschaft (; SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1 DFG)로부터 자금을 받았습니다. 작품은 또한 유럽 연합 (EU)에서 토마스 Eschenhagen (EUGeneHeart 및 Angioscaff)에 보조금 지원했다.

1. EHT 생성 및 문화 약관 섬유소 기반 EHT의 제조는 다른 네 설명된대로 수행되었다. 간단히, 주입 목적으로 섬유소 기반 EHT를 생성하는, 마스터 믹스는 신생아 로사 / 루시페라제 - 루 형질 전환 쥐의 마음 소 피브리노겐 및 Matrigel에서 분리된 세포를 포함하는 준비되었다. 아가로 오스 캐스팅 금형은 6 잘 문화 요리에 위치 테플론 스페이서를 사용하여 준비되었습니다. 아가로 오스의 응고 후, 스페이서가 제거되었으며 실리콘 포스트 랙에은 금형에 도달 게시물 문화 요리에 배치했다. EHT를 생성하려면, mastermix는 간략하게 트롬빈의 계산된 금액과 혼합하여 몰드에 pipetted되었습니다. 섬유소의 polymerisation 후 게시물을 준수 EHTs와 실리콘 랙에은 부드럽게 주문 제작을 사용하여 새 6 - 웰 세포 배양 접시에 전송하고 37 ° C, 7% CO 2 세포 문화 인큐베이터 유지 아가로 오스 주조 금형에서 제거되었습니다 세포 배양 매체. EHT는 일 열까​​지 세포 배양 조건 하에서 유지되었습니다. 이 기간 동안 신생아 쥐의 심장 세포는 길쭉한, 인터커넥트 시작 실리콘 게시물 사이에 강제로 라인을 따라 정렬합니다. 이식 당시, 자발적인 일관된 수축은 실리콘 게시물을 왜곡하고,​​ 관찰되었다. 2. EHT 주입 EHT 주입은 allogeneic 면역 반응을 조사하기 위해 면역 적격 브라운 노르웨이 (BN) 쥐, 또는 immunodeficient 누드 쥐 중 하나에서 수행되었다. 100 무게 BN와 누드 쥐 - 150g 찰스 강 독일 (Sandhofer 웨그 7 D - 97633 Sulzfeld)에서 구입하여 기존의 조건, 공급 표준 쥐 차우와 물 광고 libidum 아래에서 지내게되었습니다. Immunodeficient 쥐는 무균 침구, 버팀대 및 소독 물 사용에 보관되어 있습니다. 독일 윤리 심사위원회는 검토와 동물 절차를 승인했다. 모든 수술기구는 사용하기 전에 소독을하고 있습니다. 주입 절차는 다음과 같이 수행됩니다 : 랫은 유도 챔버를 사용하여 (2.5-3 %) isoflurane과 anesthesized 있습니다. isoflurane으로 인간의 노출은 downdraft 테이블이나 이외의 순환 모자에서 작업으로 줄일 수있다. 체온은 수술하는 동안 유지됩니다. <ol><li> 복부 영역을 면도하고 마취 동안 건조에서 눈을 방지하기 위해 눈 연고를 적용합니다. </li><li> 의 뒷면에있는 쥐를 삽입하고 마취를 유지하기 위해 코와 입을 통해 안면을 넣으십시오. </li><li> Betadine 그리고 80 % 에탄올을 사용하여 복부 지역을 소독. 면도 지역의 중심에서 깨끗한에서 더러운로 면봉은 머리 지역 향해 바깥쪽으로 이동. </li><li> 쥐가 충분히 anesthetized되었는지 확인하는 뒷다리 곤란하게 반사를 확인합니다. </li><li> 동물을 드레이프와 복부를 열려면 두 단계의 피부와 근육을 분리 정중선 복부 절개를 수행합니다. 핫 비드 살균은 피부와 근육 사이에 개방하는 데 사용됩니다. </li><li> 예열 생리 moisturized 분말 무료로 글러브에 장내를 놓습니다. 수분의 손실을 방지하기 위해 내장 주변의 장갑을 접으십시오. </li><li> 지방 조직을 제거하고 비장을 폭로. </li><li> 포셉로 큰 omentum을 시각화 및 확산. </li><li> 조심스럽게 큰 omentum에 실리콘 게시물 및 전송을 중단에서 EHT 잘라. </li><li> EHT 주변의 큰 omentus를 싸서 (Ethicon, Norderstedt, 독일) 2 싱글 7-0 prolene의 봉합을 사용하여 수정 </li><li> 다음 복부에 다시​​ 장내를 이동합니다. </li><li> prewarmed 무균 식염수에 복부를 플러쉬. </li><li> 6-0 prolene 실행 봉합 (Ethicon, Norderstedt, 독일)를 사용하여 복벽의 근육 레이어를 닫습니다. </li><li> 피부를 닫습니다 봉합을 실행 5-0 prolene을 (Ethicon, Norderstedt, 독일)를 사용합니다. </li><li> 쥐가 마취 여전히 있지만, subcutaneously 5 MG / kg Carprofen를 삽입. </li><li> 절차의이 유형, 진통제 (예 : Metamizol 등) 3 일간 게시 이식을위한 진통제에 대한 식수 (100 ML 당 50 MG Metamizol)에 추가됩니다 충분한 진통제를 제공합니다. </li></ol> 3. EHT 다음 이식의 Bioluminescence 이미징 IVIS의 bioimaging 플랫폼 (Xenogen 알라 메다, CA, 미국) 생체내에서 비침습 bioluminescence 이미징에 사용되며 결과는 IVIS 생활 이미지 (Xenogen) 소프트웨어 패키지를 사용하여 분석하고 있습니다. 이미징은 매일 이시간 postoperatively 시작 수행됩니다. 유도 챔버를 사용하여 - (3 % 2.5) isoflurane 함께 쥐를 마취. <ol><li> 널리 프로보 - 요오드, 다음 사용하는 80 % 에탄올을이 단계를 세 번 반복을 사용하여 복부 및 흉부 영역을 소독. </li><li> IP, D - Luciferin (375 MG / kg 체중 Xenogen)을 주사 </li><li> IVIS 이미징 챔버 (최대 4 쥐가 동시에 분석할 수)에 anaesthetized 쥐를 놓습니다. </li><li> 리터의 신호 강도를 평가UC + 광자 / 초 / cm 2 / steridianin 기준에의 관심 영역 (ROI) (이식 후 120분)의 단위와 일 1, 2, 3, 5, 7, 10, 그리고 주 2 EHT 내부 세포, 3 4. 정상 신호 (D - Luciferin 분사 이후 20 분 정도가 나타납니다) 참고. </li></ol> 시간이 지남에 따라 신호 강도를 보여주는 그래프를 (엑셀)를 만듭니다. 4. BLI 결과의 상관 관계와 ELISPOT 면역 적격 BN 및 immunodeficient 누드 쥐의 세포로 생체내 면역 - 응답 EHT 이식 후 공부했다. 받는 동물의 비장이 EHT 이식받는 splenocytes를 분리 5 일 후에 수확했다. 응답 세포가 IFN - γ와 IL - 4 코팅을 사용하여 제조 업체의 프로토콜 (BD Biosciences)에 따라 수행 것처럼 Elispot의 assays 사용 자극기 5 X10 6받는 splenocytes로 EHTs을 (시그마 알드리치, 세인트 루이스, MO, 미국) mitomycin을 저해 조사 접시 TH1을하고, TH2 반응, 각각. IFN - γ 및 IL - 4 명소 immunodeficient 모델에 비해 면역 적격, 모델 상당히 높은되었습니다 (IFN - γ : P = 0.001, IL - 4 : P = 0.023; 학생의 T - 테스트). 5. 대표 결과 : <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="그림 1" /> 그림 1. BLI 결과와 ELISPOT의 상관 관계) 면역 적격 브라운 노르웨이와 immunodeficient 누드 쥐의 생체내 면역 반응의 세포는 이식 후 EHT 공부했다. 받는 사람 splenocytes은 오일 EHT 주입 후에 수확했다. IFN? 및 IL - 4 표현식은 응답 세포로 stimulators 및 5x106받는 splenocytes으로 mitomycin - 저해 EHT를 사용하여 ELISpot의 assays에 의해 평가되었다. Th1과 Th2 반응은 각각 IFNγ와 IL - 4 생산에 의해 입증으로 immunodeficient 모델에 비해 면역 적격 모델에서 상당히 높은되었습니다. B) 룩 + EHTs는 면역 적격 BN의 쥐 또는 immunodeficient 누드 쥐 하나의 큰 omentus로 이식되었다. 세포 생존은 길이 방향 BLI 다음되었다. 이식 EHTs을 거부 동물의 요금이 제공됩니다. 세포가 T 세포 결함 쥐에서 살아남은 반면 모든 BN의 쥐가 빠르게, EHT 이식을 거부했다.

<ol>
	<li>Eschenhagen, T., Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+myocardial+tissue.">Engineering myocardial tissue.</a> Circ Res. 97, 1220-1231 (2005).</li><li>van der Bogt, K. E., Schrepfer, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transplantation+of+adipose+tissue-+and+bone+marrow-derived+mesenchymal+stem+cells+in+the+infarcted+heart.">Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart.</a> Transplantation. 87, 642-652 (2009).</li><li>Hakamata, Y., Murakami, T., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Firefly+rats"+as+an+organ/cellular+source+for+long-term+in+vivo+bioluminescent+imaging.">"Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging.</a> Transplantation. 81, 1179-1184 (2006).</li><li>Hansen, A., Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Drug+Screening+Platform+Based+on+Engineered+Heart+Tissue.">Development of a Drug Screening Platform Based on Engineered Heart Tissue.</a> Circ Res. , (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

심장 수리를 위해 EHT 기술의 임상 구현하기 전에, 이러한 이식 후 세포와 매트릭스 구성 요소 또는 이식 생존 모두 immunogenicity 같은 근본적인 질문은 실험 모델로 평가해야합니다. 인 - 생체내 bioluminescence 이미징은 이식 후 모니터링 세포 생존에 유용한 새로운 기술을 나타냅니다 . 우리는 성공적으로 들어있는 섬유소 기반 EHT를 생성 긍정 루시페라제 (루크 +) 설립 프로토콜 4 적응?...

<ul><li> 루 - TG (GT (로사) 26Sor - 륙) 11Jmsk 쥐 (NBPR 랫 번호 0299​​, http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li> 보빈 피브리노겐 (재고 솔루션 : NaCl 200 MG / ML 플러스 aprotinin 5 μg / ML의 섬유소 0.9 %, 시그마 F4753) </li><li> Matrigel (BD Bioscience 356235) </li><li> DMEM (Biochrome F0415) </li><li> 호스 혈청 (Gibco 26050) </li><li> 페니실린 / 스트렙토 마이신 (Gibco 15140) </li><li> 인슐린 (시그마 - 알드리치 I9278) </li><li> Aprotinin (시그마 알드리치 A1153) </li><li> D - Luciferin (Biosynth L - 8220) </li><li> 효소 링크된 면역 명소 (ELISpot) 분석 플레이트 (Hassa Laborbedarf, S2EM004M99) </li></ul>

bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (eht)

심장 조직 공학의 다양한 기법을 기본 또는 bioartificial 중 발판 재료, 같은 섬유소 또는 콜라겐 등 hydrogels에 심장 myocytes의 함정 수사와 myocyte monolayers 1 스택을 사용하여 비계 전략을 포함하여 지난 수십 년간 추진하고 있습니다. 이러한 개념은 손상된 심장 기능 (예 : 심근 경색 후)의 복원 또는 실험 모델 (예 : 예측 독성학 및 약물 검사 또는 질병 모델링)로 목표로하고 있습니다. 설계 심장 조직 (EHT)의 주입 후 세포 생존의 정확한 모니터링 이제 bioluminescence 이미징 (BLI) 기법 2 - 생체내에서 사용 가능한되었다. 여기 반딧불 루시페라제 (; 3 로사 / 루시페라제 - 루 TG)의 유비 쿼터스 표현과 유전자 변형 쥐 긴장에서 섬유소 기반 EHT의 생성에 대해 설명합니다. 주입은 주입 EHT 다음받는 유기체의 면역 반응을 평가하기 다른 쥐 종자의 큰 omentum으로 수행됩니다. BLI와 효소 링크된 단백질 스팟 기법 (ELISPOT) 면역 반응의 평가 BLI의 가용성을 확인에 의해 생성된 결과의 비교.

Watch this Scientific Journal Video about Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT at JoVE.com

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

이 비디오의 사용을 보여줍니다<em&gt; 생체내에</em쥐에서 엔지니어링된 심장 조직 이식 (EHT) 이후 면역 반응을 연구하기&gt; bioluminescence 이미징.

엔지니어링된 심장 조직 이식 다음과 같은 면역 반응의 평가 (EHT)에 대한 Bioluminescence 이미징

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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Research

JoVE Journal

Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

9.6K 조회수.  University Heart Center Hamburg. 이 비디오의 사용을 보여줍니다 생체내에 bioluminescence 이미징.

비디오: Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

조직 평가에 대한 자기 공명 Elastography 방법론은 구조 성장하는 기술

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

연구

생체공학

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

트라이 전단지 폴리머 밸브의 상대 유체 역학 평가를위한 프로토콜

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

티슈 엔지니어링 혈관 이식 및 이후의 이식을위한 수술 기법<em&gt; 생체</em&gt; 모니터링

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

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 ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

사용자 지정 레이저 에칭 마스터스에서 조직 문화 금형 설계

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

시드 및 마우스 모델에서 생 합성 조직 설계 Tracheal 이식의 이식

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

쥐의 원주 식도 재건을 위한 조직 공학 이식편

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

생체 내 심근 수리를 위한 인간 iPS 세포에서 파생된 메쉬 모양의 조작된 심장 조직의 준비

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.