이 작품은 NIH 연구 보조금 R01AR062207, R01AR061484, R56AI100901, K01AR053210 및 (CJ 리우에) 류마티스 연구 재단 질환 표적 연구 보조금에 의해 부분적으로 지원되었다.

동물에 관련된 수술의 모든 통증과 수술로 인한 불편을 최소화하기위한 노력으로 지역 기관의 동물 관리 및 윤리위원회의 승인을 받아야한다. 1. 준비하기 <ol><li> 수술 약 25 g의 체중과 8-12주 세 남성 C57BL / 6 마​​우스를 선택합니다. </li><li> 자일 라진 (5 ㎎ / kg)과 케타민 (40 ㎎ / kg)을 모두 함유하는 칵테일을 복강 내 주사를 통해 동물을 마취. </li><li> 면도칼로 무릎을 면도 후 수술 동물을 드레이프와 베타 딘, 알코올 (3x)로 수술 부위를 소독하고 연고와 마우스의 눈을 포함한다. </li></ol> 2. 수술 과정 <ol><li> 무릎 관절을 노출 1cm 길이 중간 파라 슬개골 절개를합니다. </li><li> 슬개골 및 슬개 인대의 측면 전위를 부드럽게 무릎 관절을 엽니 다. <ol><li> 길이 방향으로 무릎 관절 캡슐을 잘라2.1 단계에서 중간 파라 슬개골 절개를 통해. </li><li> 집게와 무릎 관절 캡슐을 해부하다. </li><li> 왼손으로 뒷발의 말단 부분을 쥐고, 집게로 슬개골 및 슬개 인대의 측면 전위를 수행합니다. 부드럽게 뒷다리를 잡고 발에 외상을 방지해야합니다. 공동 캡슐 해부 더 나은, 더 적은 힘은 전위를 만들기 위해 필요합니다. </li></ol></li><li> 연골 표면의 건조를 방지하기 위해 작업 중 관절 연골의 표면에 멸균 식염수 드립. </li><li> 경골 고원에 내측 반월 상 연골을 고정하는 중간 meniscotibial 인대를 잘라. 내측 반월 상 연골 아래에있는 연골을 손상하지 마십시오. <ol><li> 대퇴골 내측 관절 융기 및 경골의 내측 고원 사이에 위치한다 내측 반월 상 연골을 확인합니다. </li><li> 경골 과간 융기와 내측 반월 상 연골의 측면을 연결하는 meniscotibial 인대를 확인합니다. </li><li> 하이를 잡고ND 손으로 부드럽게 포, 및 제 10 외과 블레이드를 사용하여 조심스럽게 중간 meniscotibial 인대를 잘라. 연골 및 다른 인대 관절에 해치다가 발생하지 않도록하십시오. 많은 경우에, 커버 지방 패드쪽으로 당길 수 있지만 인대를 식별하기 위해 제거되어야 할 필요는 없다. </li></ol></li><li> 6-0 흡수성 봉합사와 무릎 관절 캡슐을 닫습니다. </li><li> 6-0 실크 봉합사로 피부를 닫습니다. </li></ol> 3. 수술 후 관리 <ol><li> 수술 후 통증을 최소화하기 위해 각 마우스의 수술 부위에 0.25 %의 부피 바카 인의 한 방울을 적용합니다. </li><li> 물과 음식을 무료로 운영 쥐를 둡니다. </li></ol> 4. 외과 DMM 모델의 조직 학적 점수 <ol><li> 지정된 시점에서 쥐를 희생 수술 후 (예를 들어, 사주, 8 주 및 12 주. 여기에 우리가 대표로 팔주의 결과를 보여 주었다). </li><li> 전체 무릎 관절은, 탈회, embe에게 고정되어파라핀으로 dded 다음 5 μm의 간격으로 순차적으로 단면. <ol><li> 10 번 블레이드와 전체 뒷다리를 잘라. </li><li> 조심스럽게 두 다리의 피부와 근육을 해부하고, RT에서 3 일 동안 4 % PFA에 샘플을 고정합니다. </li><li> PFA를 제거하고 물 샘플을 청소합니다. 그 후 2 주 동안 4 ° C에서 EDTA의 샘플을 석회. </li><li> 에탄올 그라데이션의 샘플을 탈수. 구체적으로, 다음 에탄올을 변경 및 O / N. 70 % 에탄올의 새로운 세트에서 샘플을 유지, 1 시간 동안 70 % 에탄올에서 샘플을 계속 그 후, 80 %의 샘플을 넣어 90 %의 에탄올 결과, 및 O / N. 100 % 에탄올 다음에, 각각 1 시간 동안 그들을 유지 </li><li> 에탄올을 제거하십시오. 1 시간 동안 oxylene에 샘플을 유지하고 oxylene의 새로운 세트로 변경합니다. 이 단계의 3 배를 반복합니다. </li><li> 라이카 삽입 센터를 사용하여 파라핀 금형에 샘플을 포함합니다. 그 후, 샘플을 회전 마이크로톰 (Leica의 RM225을 사용하여 5 ㎛의 절편으로 아르5, 독일) 다음 유리 슬라이드에 수집. </li><li> 직렬 시상 부분은 내측 관절 돌기의 중심으로 측면 외과의 중심에서 지역에 걸쳐있는 각각의 샘플에 대해 잘립니다. </li></ol></li><li> Safranin O 염색이 수행 이전 25 설명한대로 OARSI 채점 시스템을 통해 채점 및 통계 분석에 의해 이어진다. <ol><li> 첫째, oxylene에 슬라이드를 deparaffinize, 증류수에 에탄올 구배를 수화. 그 후, 슬라이드는 Weigert의 철은 Hematoxylin 솔루션, 0.05 % 빠른 그린 (FCF) 솔루션, 1 % 아세트산 용액, 0.1 % Safranin O 솔루션을 통해 염색된다. 수지 매체를 사용하여 슬라이드를 장착합니다. </li><li> 조직 학적 점수에 대한 통계 분석을 수행, 연골과 연골 구조의 파괴의 비율에 프로테오글리칸 (붉은 색)의 손실에 따라 Safranin O 스테인드 슬라이드를 점수. </li></ol></li></ol>

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우리는 여기에 우리가 관심의 충돌이 없다는 것을 선언합니다.

그것은 마우스의 변형이 DMM 모델 유도를 들어, 마우스의 서로 다른 균주 / 다음 C57BL6, 129/SvInJ 및 FVB 다음, 129/SvEv 균주에서 가장 높은 수준으로, DMM 후 OA의 정도를 변화 한 매우 중요하다고보고 N 26. DMM에 상대적으로 민감하다 우리가 본 연구에 사용 된 마우스 - 형질 전환 유전자의 많은 부분은 이러한 PGRN - /로 C57BL6 마우스에 설정됩니다. 유전자가 같은 FVB / N 마우스로 구분 변형을 기반으?...

또한 퇴행성 관절염으로 알려진 골관절염 (OA)는, 세계 인구의 15 %에 영향을 미치는 미국 내에서 46 만 명 이상, 그리고 윤 활막염, 연골의 변성 및 골극 형성 한 것을 특징으로한다. 그것은 유전 대사, 생체 역학, 생화학 적 요인의 복잡한 상호 작용의 결과 일 수있다. OA의 기본 메커니즘은 사회 과학을 회피하는 것을 계속한다. OA 2,3의 병인을 모방 수있는 수많은 동물 모델은 현재이 있습니다. 그것 때문에 다양한 유전자 변형 마우스의 유용성과 실험의 비용 효율성 양쪽의 생쥐 동물 모델을 확립하는 것이 중요하다. 실험 OA 모델의 여러 종류의 사이에, 내측 반월 상 연골 (DMM) 모델의 수술 유도 불안정 때문에 그것의 좋은 재현성 및 OA를 개발하는 동안 상대적으로 느린 진행의 잘 받아 OA 모델입니다. 이러한 특성은 모두가다른 처리 또는 형질 전환 유전자 3-8 OA 진행의 평가를위한 핵심이었다. 그러나, 수술 OA 모델의 일관성은 수술하는 동안 그 결과 다양한 요인에 의해 영향을받는, 외과 마우스 모델의 적용은 제한된다. Progranulin (PGRN)는 각종 세포에서 발현 다기능 성장 인자이다. 그것은 PGRN 같은 상처 치유 09 종양 10, 11-15 및 염증 등의 각종 생리적 질병 과정에 중요한 역할을하는 것으로 알려져있다. 연구는 또한 PGRN의 부족이 인간과 생쥐 16-18 모두 신경계의 퇴행성 질환을 일으킬 수있는 것으로 나타났습니다. 그것은 PGRN 인간 관절 연골에서 발현되는 것으로 알려져 있으며, 그 수준은 상당히 OA 및 류마티스 관절염 (19)을 가진 환자의 연골 조직에서 증가된다. 또, PGRN 또한 연골 세포 증식 20, 분화 및 연골 내에서 중요한 역할을 담당개발 (21, 22) 중 성장판의 골화. 최근에, 우리는 PGRN가 TNF 수용체에 결합을 통해 TNF-α를 반감과 염증성 관절염 모델 13,14,23,24의 항 염증 기능을 전시하고 있다고 보도했다. 그러나, OA에 PGRN의 역할은, 특히 생체 내에서, 수수께끼로 남아있다. 생쥐 - 여기서, 우리는 수술 DMM 모델을 유도하고, 및 WT-PGRN / DMM 소재 모델을 확립 통해 OA 개발 PGRN의 역할을 조사하는 과정을 제시한다.

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			<td height="21" style="height:21px;width:216px;">No. 10 Surgical blades</td>
			<td style="width:135px;">FEATHER</td>
			<td style="width:119px;">25-2976#10</td>
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			<td height="21" style="height:21px;">6-0 suture</td>
			<td>APPLIED DENTAL</td>
			<td>WG-N53133</td>
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establishment of a surgically-induced model in mice to investigate the protective role of progranulin in osteoarthritis

내측 반월 상 연골의 불안정은 (DMM) 모델은 생체 내에서 골관절염 (OA)의 병인에 많은 관절염 관련 분자의 병태 생리 학적 역할을 연구하기위한 중요한 도구입니다. 그러나, 세부, 특히 쥐에이 복잡한 모델을 구축하기위한 시각화 한 프로토콜은 사용할 수 없습니다. 여기에서 우리는 야생형 및 progranulin (PGRN) 이용했다 - / - 예 생쥐의 DMM의 모델을 유도하기위한 프로토콜,이 수술 유도 모델의 설립 다음 OA의 비교 발병을 소개하는대로 마우스. 생쥐에서 수행 된 작업은 menisco 경골 인대를 잘라 내측 반월 상 연골의 불안정을 야기 관절낭, 또는 DMM 작업을 개설 가짜 운전, 하나 있었다. 관절염의 심각도는 조직 학적 분석 (예 : Safranin O 염색), OA 관련 유전자의 표현, 연골 세포 외 기질 분자의 분해 및 골극 양식을 사용하여 평가 하였다ATION. 마우스,이 수술 유발 모델에서 더 심한 OA 표현형을 주도 PGNR 성장 인자의 손실 - DMM 작업이 성공적으로 야생형 및 PGRN - / 모두 OA의 개시 및 진행을 유도.

DMM의 모델은 성공적으로 생쥐에서 설립 PGRN의 결핍은 수술에 의한 OA 개발을 과장했다. 쥐 - 가짜와 DMM 작업 (그림 1) WT 및 PGRN - / 수행되었다. 8주 동작 후에, 마우스를 희생시키고, Safranin O 염색은 무릎 관절의 부분에서 수행 된, 조직학에 근거 관절염 점수의 통계적 분석 하였다. 도 2a에 도시 된 바와 같이, 유사한 초기 조건 (상부 패널) ?...

Watch this Scientific Journal Video about Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis at JoVE.com

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

우리는 생쥐의 내측 반월 상 연골 (DMM) 모델, 골관절염 (OA) 연구를위한 효과적인 도구의 불안정에 대한 프로토콜을 설명합니다. 또한, 우리는 progranulin가 OA의 병인에 보호 역할을한다는 것을 나타내는,이 모델을 사용하여 progranulincan 과장 OA 개발 및 진행의​​ 부족을 증명하고있다.

관절염에 Progranulin의 보호 역할을 조사하기 위해 마우스의 수술로 유발 된 모델 구축

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

establishment-surgically-induced-model-mice-to-investigate-protective

Research

JoVE Journal

Bioengineering

27.1K 조회수.  NYU Hospital for Joint Diseases.  우리는 생쥐의 내측 반월 상 연골 (DMM) 모델, 골관절염 (OA) 연구를위한 효과적인 도구의 불안정에 대한 프로토콜을 설명합니다. 또한, 우리는 progranulin가 OA의 병인에 보호 역할을한다는 것을 나타내는,이 모델을 사용하여 progranulincan 과장 OA 개발 및 진행의​​ 부족을 증명하고있다. 

비디오: Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Langendorff - perfused 토끼 하트의 Multiparametric 광 매핑

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

연구

생체공학

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

매개 변수화 이완기 충전 형식주의를 통해 Transmitral 흐름의 운동 학적 모델링 기반의 분석에 의하여 세계적인 이완기 기능의 정량화

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

평면 그라데이션 확산 시스템은 3D 콜라겐 매트릭스에 화성을 조사하기 위해

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

<em&gt; 체외</em쥐의 추간판의&gt; 장기 문화 모델

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

Cardiomyocyte 시스템 생물학에서 세포 아키텍처의 역할을 연구 하기 Cardiomyocyte의 구조적으로 현실적인 유한 요소 형상 모델 생성

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry:에 대 한 강력 하 고, 비 침 투 적인 접근 세포 에너지 대사를 조사

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

올레산의 Tripeptide 안정 Nanoemulsion

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...