우리는 원고 지원, 기술 지원에 그림과 페어 뷰 영상 서비스와 지원 Jerrald 스펜서 주니어 게리 윌리엄스 모델 생성 및 측정 모니카 Mahre에 대한 지원은 Dionna 도박, 앨리슨 라슨, 그리고 카시아 토레스를 인정하고 싶습니다 미네소타 대학. 기금은 메드 트로닉 사와 연구 계약에서 의학 공학 연구소 (미네소타 대학)로부터 부분에서 수신 된

순서 표 1은 공정 중에 사용 된 자료를 요약 한 것입니다. 그림 1은 프로세스의 개요를 제공합니다. 1. 시편 검사 준비 <ol><li> 자신의 이완 기말 상태에서 포르말린 10 %를 해결 신선하고 연속적으로 관류 고립 된 인간의 마음을 얻을 수 있습니다. </li><li> 포르말린의 대부분을 제거하기 위해 검사 전날 물에 스캔 할 마음을 씻어. </li><li> 스캐너에 향하기 전에, venogram 풍선 카테터 각 심장 내 관상 정맥동 (CS) 정맥 cannulate. 직접적인 시각화 아래의 우수 또는 하대의 정맥 또는 videoscopes의 사용을 통해 CS에 대한 액세스 권한을 얻을 수 있습니다. </li><li> 한번의 CS에 카테터를 고정하기 위해이 venogram 카테터의 풍선을 팽창. </li><li> 있도록 설계되었습니다 스펀지의 상단에 밀봉 고분자 컨테이너 내의 각 마음을 놓으십시오마음은 attitudinally 정확한 해부학 적 위치에 앉을 수 있습니다. </li></ol> 2. 전산화 단층 촬영 검사 <ol><li> 환자가 앙와위와 스캐너의 첫 번째 머리를 누워있는 것처럼 CT 스캐너 침대에 주어진 마음을 놓습니다. </li><li> 대비 하나 식염수 하나 : 두 개의 주입 주사기를 포함 인젝터에 venogram 카테터의 근위 끝을 연결합니다. </li><li> 자동으로 5 ML / s의 속도로 심장 정맥 시스템에 대비 40 mL를 주입. </li><li> 반면 주입이 시작된 후 CT는 심장 8 초를 검색합니다. 0.6 mm 슬라이스 두께가 512 X 512 픽셀의 해상도로 CT 검사를 설정합니다. </li><li> 자동 대비를 플러시 5 ML / s의 속도로 심장 정맥 시스템에 식염수 40 mL를 주입. </li><li> 외장 하드 드라이브에 CT DICOM 이미지를 보냅니다. </li></ol> 3. 재건 및 측정 <ol><li> 모방 자 소프트웨어에 CT DICOM 이미지를 업로드 할 수 있습니다. </li><li> CT imag에 대한 마스크를 생성만 마음 만 대비 선물을 강조하기 위해 높은 하운 단위로 픽셀을 포함 말이지. </li><li> 마스크는 주요 심장 혈관 내 대조를 포함하도록 조직으로 챔버 또는 확산에 누출 된 대비를 제거합니다. </li><li> 수동 프레임에 의해 주어진 정맥 프레임 안에 공기 주머니를 입력합니다. </li><li> 결과 마스크에서 3D 개체를 생성합니다. </li><li> 거친 형상을 제거하기 위해이 객체를 반반하게하고 쌉니다. 비디오 1 공간에서 회전하는 이러한 3D 모델 중 하나를 제공합니다. </li><li> 각 생성 된 3D 모델의 중심선을 생성합니다. </li><li> 이 중심선 사용하여 각 중심부에 각 주요 혈관에 대한 호의 길이, 분기 각도 tortuousity (호의 길이 / 직선 거리) 및 직경을 측정합니다. 우리의 고용 해부학 적 명칭은 그림 2에 표시됩니다. </li></ol>

<ol>
	<li>Burkhardt, J. D., Wilkoff, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventional+electrophysiology+and+cardiac+resynchronization+therapy:+delivering+electrical+therapies+for+heart+failure.">Interventional electrophysiology and cardiac resynchronization therapy: delivering electrical therapies for heart failure.</a> Circ. 115, 2208-2220 (2007).</li><li>Lu, F., Iaizzo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+resynchronization+therapy.">Cardiac resynchronization therapy.</a> Handbook of cardiac physiology and anatomy. , 475-497 (2009).</li><li>Eggen, M. D., Swingen, C. M., Iaizzo, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+diffusion+tensor+MRI+of+human+hearts:+relative+effects+of+specimen+decomposition.">Ex vivo diffusion tensor MRI of human hearts: relative effects of specimen decomposition.</a> Magn. Reson. Med. 67, 1703-1709 (2012).</li><li>Manzke, R., Binner, L., Bornstedt, A., Merkle, N., Lutz, A., Gradinger, R., Rasche, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+coronary+venous+system+in+heart+failure+patients+by+blood+pool+agent+enhanced+whole-heart+MRI.">Assessment of the coronary venous system in heart failure patients by blood pool agent enhanced whole-heart MRI.</a> Eur. Radiol. 21, 799-806 (2010).</li><li>Abbara, S., Cury, R. C., Nieman, K., Reddy, V., Moselewski, F., Schmidt, S., Ferencik, M., Hoffman, U., Brady, T. J., Achenbach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+evaluation+of+cardiac+veins+with+16-MDCT+angiography.">Noninvasive evaluation of cardiac veins with 16-MDCT angiography.</a> AJR. Am. J. Roentgenol. 185, 1001-1006 (2005).</li><li>Gerber, T. C., Sheedy, P. F., Bell, M. R., Hayes, D. L., Rumberger, J. A., Behrenbeck, T., Holmes, D. R., Schwartz, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+coronary+venous+system+using+electron+beam+computed+tomography.">Evaluation of the coronary venous system using electron beam computed tomography.</a> Int. J. Cardiovasc. Imaging. 17, 65-75 (2001).</li><li>Jongbloed, M. R. M., Lamb, H. J., Bax, J. J., Schuijf, J. D., de Roos, A., vander Wall, E. E., Schalij, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+visualization+of+the+cardiac+venous+system+using+multislice+computed+tomography.">Noninvasive visualization of the cardiac venous system using multislice computed tomography.</a> J. Am. Coll. Cardiol. 45, 749-753 (2005).</li><li>Mao, S., Shinbane, J. S., Girky, M. J., Child, J., Carson, S., Oudiz, R. J., Budoff, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+venous+imaging+with+electron+beam+computed+tomographic+angiography:+three-dimensional+mapping+and+relationship+with+coronary+arteries.">Coronary venous imaging with electron beam computed tomographic angiography: three-dimensional mapping and relationship with coronary arteries.</a> Am. Heart J. 150, 315-322 (2005).</li><li>Muhlenbruch, G., Koos, R., Wildberger, J. E., Gunther, R. W., Mahnken, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+the+cardiac+venous+system:+comparison+of+MDCT+and+conventional+angiography.">Imaging of the cardiac venous system: comparison of MDCT and conventional angiography.</a> AJR. Am. J. Roentgenol. 185, 1252-1257 (2005).</li><li>Schaffler, G. J., Groell, R., Peichel, K. H., Rienmuller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+coronary+venous+drainage+system+using+electron-beam+CT.">Imaging the coronary venous drainage system using electron-beam CT.</a> Surg. Radiol. Anat. 22, 35-39 (2000).</li><li>Tada, H., Kurosaki, K., Naito, S., Koyama, K., Itoi, K., Ito, S., Ueda, M., Shinbo, G., Hoshizaki, H., Nogami, A., Oshima, S., Taniguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+visualization+of+the+coronary+venous+system+using+multidetector+row+computed+tomography.">Three-dimensional visualization of the coronary venous system using multidetector row computed tomography.</a> Circ. J. 69, 165-170 (2005).</li><li>Van de Veire, N. R., Schuijf, J. D., Sutter, J. D., Devos, D., Bleeker, G. B., de Roos, A., vander Wall, E. E., Schalij, M. J., Bax, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+visualization+of+the+cardiac+venous+system+in+coronary+artery+disease+patients+using+64-slice+computed+tomography.">Non-invasive visualization of the cardiac venous system in coronary artery disease patients using 64-slice computed tomography.</a> J. Am. Coll. Cardiol. 48, 1832-1838 (2006).</li><li>de Jong, P. A., Mayo, J. R., Golmohammadi, K., Nakano, Y., Lequin, M. H., Tiddens, H. A., Aldrich, J., Coxson, H. O., Sin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cancer+mortality+associated+with+repetitive+computed+tomography+scanning.">Estimation of cancer mortality associated with repetitive computed tomography scanning.</a> Am. J. Respir. Crit. Care Med. 173, 199-203 (2006).</li><li>Martin, D. R., Semelka, R. C., Chapman, A., Peters, H., Finn, P. J., Kalb, B., Thomsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrogenic+systemic+fibrosis+versus+contrast-induced+nephropathy:+risks+and+benefits+of+contrast-enhanced+MR+and+CT+in+renally+impaired+patients.">Nephrogenic systemic fibrosis versus contrast-induced nephropathy: risks and benefits of contrast-enhanced MR and CT in renally impaired patients.</a> J. Magn. Reson. Imaging. 30, 1350-1356 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

우리 연구실은 다양한 해부학 적 조사 연구에 대한 관류 고정 마음 표본의 라이브러리를 개발하고있다. 지금까지, 우리는 240 보존 표본에 있습니다. 우리는이 시편을 준비하는 데 사용한 특정 메서드는 이전에 3 설명하고있다. 본 연구는 인간의 심장 정맥 시스템을 매핑하고 혈관 내 고용 심장 장치의 설계에 사용할 수있는 해부학 적 데이터베이스 개발을위한 새로운 방법을 설명합니다. ...

anatomical reconstructions of the human cardiac venous system using contrast-computed tomography of perfusion-fixed specimens

인간의 심장 정맥 시스템 내에서의 복잡성과 상대적인 변화의 상세한 이해는 이러한 혈관에 대한 액세스 권한을 필요로 심장 장치의 개발에 매우​​ 중요합니다. 예를 들어, 심장 정맥 해부학 따라서 인간의 심장 정맥 시스템에 대한 해부학 적 매개 변수의 데이터베이스의 개발 CRT 전달의 설계에 도움이 될 수 있습니다 심장 재 동기화 치료의 적절한 배달 (CRT) 1 키 제한 중 하나가 될 것으로 알려져 있습니다 이러한 한계를 극복하기위한 장치. 이 연구 프로젝트에서는 해부학 매개 변수는 대조 전산화 단층 촬영 (CT) 영상 및 모델링 소프트웨어 (Materialise, 루벵, 벨기에)를 사용하여 정맥 시스템의 3D 재구성에서 하였다. 호의 길이, tortuousity, 분기 각도, 관상 동맥 부비동 개구부까지의 거리, 그리고 혈관 직경 : 다음 매개 변수는 각 정맥에 대한 평가 하였다. CRT는 PATI에 대한 잠재적 인 치료전기 dyssynchrony에 함께 행군. 심장 마비 환자의 약 20 %는 CRT이 혜택을 누릴 수 있습니다. 전기 dyssynchrony에 이전 또는 이후 심장의 정상적인 전도 통로보다 심근 활성화 및 계약의 부분을 의미한다. CRT에서 심근의 dyssynchronous 지역은 전기 자극으로 처리됩니다. CRT 페이싱은 일반적으로 더 많은 동기화 리듬을 생산하는 우심방 (RA), 우심실 (RV), 그리고 좌심실 (LV)을 자극 페이싱 리드를 포함한다. LV 리드는 일반적으로 최근의 심근 활성화의 사이트를 오버레이 할 목적으로, 심장 혈관 내에 이식합니다. 우리는 모델이 얻은 분석은 그 환자, 학생, 임상 및 의료 기기 디자이너를위한 해부학 교육을 촉진 것이라고 믿는다. 여기에 사용 된 방법론은 또한 다음과 같은 우리 인간의 마음 표본의 다른 해부학 적 기능을 연구하기 위하여 이용 될 수있다관상 동맥. 또한 본 연구의 교육적 가치를 장려하기 위해, 우리는 우리의 무료 액세스 웹 사이트에 정맥 모델을 공유했습니다 <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu / 아틀라스</a> .

표 2는 42 인간의 마음 표본의 주요 심장 혈관에 대한 평균 해부학 매개 변수를 제공합니다. 모든 심장 표본 한 뒤 심실 정맥 (PIV)와 전방 심실 정맥 (AIV)를 포함되어 있습니다. 다른 마음이 하나 있었하지 않을 수 있습니다 반면 일부 표본, LV (PVLV), postero - 측면 정맥 (PLV), 왼쪽 측면 정맥 (LLV) 및 / 또는 antero - 측면 정맥 (ALV) 하나 이상의 후방 정맥을 포함 이러한 특정 정맥의 두 가지가 ?...

Watch this Scientific Journal Video about Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens at JoVE.com

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

본 연구의 목적은 다시하고 대조 전산화 단층 촬영 검사에서 생성 3D 재구성을 사용하여 인간의 심장 정맥 시스템의 해부학에 액세스 할 수 있습니다.

콘트라스트 계산하기 관류 고정 시편의 단층 촬영을 이용하여 인간의 심장 정맥 시스템의 해부학 적 재건

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

anatomical-reconstructions-human-cardiac-venous-system-using-contrast

Research

JoVE Journal

Bioengineering

11.7K 조회수.  University of Minnesota. 본 연구의 목적은 다시하고 대조 전산화 단층 촬영 검사에서 생성 3D 재구성을 사용하여 인간의 심장 정맥 시스템의 해부학에 액세스 할 수 있습니다.

비디오: Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

전기 화학 센서를 사용하여 세균의 검출 및 식별

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

연구

생체공학

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

소형 조직 배양 시스템에서 마우스 심장 밸브를 배양

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

정의 인간 공학 심장 조직의 건설 심장 세포 치료의 메커니즘을 연구하는

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

심근 구조, 기능의 생체 양적 평가에서 관류 및 생존 능력은 심장 마이크로 컴퓨터 단층 촬영을 사용하여

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

특별 한 광학 전송 인간의 혈 청에 심장 Biomarkers 척도를 사용 하 여

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

효율적인 생성 및 급지대 무료 Ipsc CRISPR Cas9 시스템을 사용 하 여 인간의 췌 장 세포에서의 편집

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

비 계-무료 3 차원 심장 조직 창조의 그물 형 기반 방법

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

엄밀한 포함의 고정 및 스테인드, 전체, 마이크로 단층에 의해 섹션-무료 3D 조직학에 대 한 밀리미터 단위 표본

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

교란 성 포유류 심장의 경부 흡광도 분광학을 사용하여 세포 대사를 모니터링하기위한 심장 내 측 발사 라이트 카테터

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

인간 섬유근육 조직을 시각화하기 위해 인산화산 제제를 사용한 마이크로 X선 컴퓨터 단층 촬영 사용

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...