SynVivo 기술은 NHLBI에서 부여 # 2R44HL076034에서 개발되었다.

1. 못쓰게 SynVivo - SMN 미세 유체 장치 <ol><li> 장치의 각 포트 (입구 / 출구)는 두 개의 병렬 포트로 구성되어 있습니다 - 표면 코팅 부분에 흐르는 하나의 (부착 분자, 성장 행렬 등) 및 / 또는 파종 및 분석 (그림 1A를 실행하는 기타 세포 ). </li><li> 완전 멸균 탈 (DI) 물을 포함하는 페트리 접시에 SynVivo - SMN 마이크로 유체 장치 (그림 1B)를 물속에 진공 데시 케이 터에 접시를 놓습니다. 모든 공기는 장치의 채널에서 제거 될 때까지 건조기가 실행될. 이것은 약 15 분 소요됩니다. </li><li> 물에서 장치를 제거하기 전에, 미세 포인트 집게로 장치의 각 포트에 물을 준비하는 타이곤 튜브 (0.06 OD &quot;0.02의 ID&quot;)를 배치합니다. 튜브의 길이는 약 1 인치이어야한다. 이 디바이스는 이제 물에서 제거 할 수 있습니다. 그림 1C는 배포 장치의 이미지를 보여줍니다튜브에 전자. </li></ol> 2. 원하는 단백질과 미세 유체 장치를 코팅 (예를 들어, 아비딘) <ol><li> 피펫을 사용하여, 하나의 흡입구 튜브의 기지 주변의 물 (약 100 μL)의 하락을 배치합니다. 조심스럽게 주요 장치에 사용되는 튜브를 제거합니다. 물방울은 장치에 들어가는 공기를 방지 할 것이다. </li><li> 20 ㎍ / ㎖의 농도에서 아비딘로드 1 ML의 주사기를 준비합니다. 24 G 스테인레스 스틸 바늘과 튜브에 주사기를 연결합니다. 장치의 입구 포트 중 하나에 튜브를 삽입한다. 턱 클램프로 활용되고 있지 유입구 클램프. </li><li> 장치의 완전한 재관류를 허용하도록 10 분 동안 1 μL / 분의 유속으로 주입 아비딘. 유동 시간의 끝에서, 턱 클램프 튜브 클램프하고 밤새 39 ° C에서 장치를 배치. </li></ol> 3. 접착 실험에 대한 바이오틴 입자를 흐르는 <ol><li> 장치에 허용실온에 와서. 동력 스테이지 및 고성능 카메라를 구비 한 반전 된 형광 현미경에 기기를 배치. </li><li> 인산 완충액 생리 식염수 (PBS) 중 5 × 106 입자 / ml의 농도로 2 μM 바이오틴 입자의 용액을 제조 하였다. 1 ML의 주사기로 입자를 넣습니다. 만 PBS의 제 1 ML의 주사기를 준비합니다. 주사기 펌프 각 주사기를 넣고 바늘과 튜브에 연결합니다. </li><li> 피펫을 사용하여 흡입구 튜브의 기지 주변의 물 (약 100 μL)의 하락을 배치합니다. 조심스럽게 코트 장치에 사용되는 튜브를 제거합니다. 물방울은 장치에 들어가는 공기를 방지 할 것이다. </li><li> 조심스럽게 입구 포트에 각각 단계 3.2 바이오틴 입자와 PBS에 대한 튜브의 삽입합니다. 그림 2A는 셋업의 이미지를 보여줍니다. </li><li> 2.5 μL / 분의 유속으로 바이오틴 화 입자를 주입하기 시작한다. 현미경의 유입 포트를 모니터링합니다. 첫 번째 기호입자, 타이머를 시작하고 3 분 동안 흐름을 계속합니다. </li><li> 동시에 2.5 μL / 분의 유속으로 PBS의 흐름을 응시하면서 3 분의 끝에서, 바이오틴 화 입자의 흐름을 멈춘다. PBS는 언 바운드 입자를 씻어 3 분의 장치에 흐름을 허용합니다. </li></ol> 4. 이미지 획득 및 이미징 소프트웨어 (NIKON 요소)를 사용하여이자 (AOI) 측정의 지역 만들기 <ol><li> 전체 장치의 이미지를 얻기 위해 이미징 소프트웨어의 &quot;스캔 큰 이미지&quot;기능을 사용합니다. </li><li> 장치에 순차적으로 번호 분기점과 채널의 두 배 직경 원형 AOI를 만듭니다. 채널 직경 100 μm의 때문에이 경우에는 200 ㎛로 AOI 직경을 설정한다. </li><li> MS 엑셀 시트에 각 AOI에있는 입자의 수​​를 내보낼 이미징 소프트웨어의 자동 카운트 기능을 사용합니다. </li><li> 마찬가지로, 전체 입자의 수​​를 내보낼 자동 카운트 기능을 사용장치. </li></ol> 5. 전산 유체 역학 (CFD) 모델을 사용하여 입자 플럭스 분석 <ol><li> CFD 시뮬레이션은 SynVivo - SMN 장치 토폴로지를위한 상용 소프트웨어 (CFD-ACE +, ESI 주식 회사)를 사용하여 실행됩니다. 결과는 실험적 관측을 분석 데이터베이스에 저장된다. 벽 전단 속도, 속도, 입자 플럭스, 장치의 부착에 대한 시뮬레이션 결과 정보를 저장합니다. </li><li> 시뮬레이션 결과는 관련 입구 입자 농도에 기초하여 각각의 AOI 들어가는 입자의 개수를 결정하는 데 사용된다. </li></ol> 6. 생성 전단 접착지도 <ol><li> 수학 식 6.1에 도시 된 바와 같이 분기점에서 흐르는 입자로의 분기점에 부착 된 입자를 분할함으로써 밀착성의 %를 계산한다. <img alt="식 (1)" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> 부착 입자 흐르는 입자의 수​​는 P로부터 획득되는 곳rotocol은 각각 4.3과 5.2 단계를 반복합니다. </li><li> 단계 (5.1)에서의 데이터베이스로부터 얻어진 네트워크 및 수학 식 6.1로부터 얻어진 % 접착 값의 각 분기점에서 전단 속도를 사용하여 전단 접착지도를 플롯. </li></ol>

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CFD 연구 법인의 후원이 기사에 대한 게재료.

병렬 플레이트 흐름 챔버, 세포 - 세포 및 세포 입자의 상호 작용에 중요한 통찰력을 제공하는 동안, 이러한 높은 시약의 소비와 전단 접착 맵을 생성하기 위해 여러 실험을 실행에 대한 필요성과 같은 몇 가지 제한으로 고통. SynVivo 합성 미세 혈관 네트워크 (SynVivo-SMNs)의 사용은 생체 내 조건에서 모방 조건 번의 실험에서 전단 접착 맵의 생성을 가능하게한다. 또한, 시약의 상당한 절감 (&gt;...

세포 - 세포 및 입자 세포의 상호 작용을 연구하는 현재의 분석은 일반적으로 입자 또는 세포가 단백질 행렬 또는 자기편 세포 배양되는 정적 잘 플레이트 형식을 포함한다. 특정 배양 시간의 끝에서, 접착 입자 또는 세포의 숫자는 한 현미경을 사용하여 정량화된다. 이러한 분석은 이러한 상호 작용의 뒤에 생화학 적 프로세스에 중요한 통찰력을 제공하더라도, 키 제한 (미세 혈관의 일반) 생리 학적 유체 흐름의 부족 및 입자 부착에 미치는 영향이다. 이러한 한계를 극복하기 위하여, 시험 관내 유동 챔버는 최근 몇 년 동안 개발되었다. 이러한 흐름 챔버의 일반적인 요소는 생체 내 2 혈관에서 관찰 벽 전단 속도에 맞게 낮은 레이놀즈 수에서 관류 투명 장치이다. 혈관벽은 유량 (C)의 한 표면 상에 생체 분자의 코팅 또는 세포의 성장 중 하나에 의해 모델링된다hamber 3. 입자 4-7 또는 8-16 세포는 다양한 전단 속도에 따라 입자를 부착의 수를 계량하는 유량의 원하는 범위에 유입된다. 그러나, 생화학 적 현상을 연구하고 검증하는 평행 평판 유동 챔버의 사용은 약간 고가이며 시간 소모적이다. 이것은 여러 실험 부착 입자 / 세포의 수 대 유체 전단의지도를 생성하기 위해 수행 될 필요가 있다는 사실에 주로 기인한다. 또한, 플레이트 흐름 챔버는 그들의 큰 크기 (높이&gt; 250 μm의 폭&gt; 1mm)에 시약의 많은 양을 필요로한다. 마지막으로, 이러한 장치는 정확하게 생체에 존재하는 기하학적 특징 (예를 들면, 분기점) 및 유동 조건 (예를 들어, 흐름을 발산 대 수렴)를 모델링하지 않습니다. 리소그래피 기반 미세 17-19 최근 진보는 랩 온어 칩의 필드를 가속화장치 20-21. 이러한 장치는 마이크로 미터 정권의 차원과 병렬 플레이트 흐름 챔버의 소형 버전을 개발하는 수단이되고있다. 치수의 감소는 또한 시약, 세포 나 실험에 필요한 입자의 양 측면에서 상당한 이점을 얻을 수 있습니다. 그러나, 현재 사용 가능한 장치의 주요 제한은 생체 내에서 관찰 복잡한 미세 혈관을 모방하지 않는 미세 혈관을 모델링하는 선형 채널의 사용이다. 우리는 최근 생체 조건의 합성 표현의 결과로 처분 할 수있는 플라스틱 기판에 미세 혈관 네트워크를 재현하기위한 새로운 방법을 개발했습니다. 이 장치는 SynVivo 합성 미세 혈관 네트워크 (SMN)이 소프트 리소그래피 공정을 기반 PDMS를 사용하여 개발 불린다. SynVivo-SMN 장치가 셀 / 파티클 부착 (22)의 전단 접착력 맵을 얻기 위해 사용될 수 있으며, 연구는 약물 전달 (23)과 H를 타겟팅생체 데이터를 24 ~ 25에 대해 유효성이 확인 된 번가. 이 논문에서, 우리는 1 ~ 5 ㎕를가함으로써 자원과 시간을 크게 절감의 결과로 작은 볼륨에서 하나의 실험에서 전단 접착지도의 생성을 가능하게하는 프로토콜을 제시한다.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr>
			<td>SynVivo-SMN</td>
			<td>CFD Research</td>
			<td>SMN-001</td>
			<td>Exclusive at CFDRC</td>
		</tr>
		<tr>
			<td>CFD-ACE+</td>
			<td>ESI Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Invitrogen</td>
			<td>43-4401</td>
			<td>Any avidin source will work for this assay</td>
		</tr>
		<tr>
			<td>Biotinylated Particles</td>
			<td>Polysciences</td>
			<td>24173-1</td>
			<td>Any source of biotinylated particles will work for the assay</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>63018-044</td>
			<td>Size is typical for use with SynVivo-SMN</td>
		</tr>
		<tr>
			<td>NIKON Elements</td>
			<td>NIKON Instruments</td>
			<td>N/A</td>
			<td>Any other imaging software can be used</td>
		</tr>
	</tbody>
</table>

generation of shear adhesion map using synvivo synthetic microvascular networks

셀 / 입자 접착 분석은 질병의 병태 생리에 관련된 생화학 적 상호 작용을 이해하는 데 중요하며 새로운 치료제의 개발에 대한 탐구에서 중요한 응용 프로그램이 있습니다. 정적 조건을 이용하여 분석이 생체 내 환경과의 상관 관계를 제한하는 전단에 접착 의존성을 캡처하지 못한다. 생리적 유체 흐름하에 접착력을 정량화 평행 평판 유동 챔버 전단 접착 맵의 생성을위한 다수의 실험이 필요하다. 또, 이들은 생체 내 치수 및 형태를 나타내지 않는 실험 시약 대용량 (~ ml)에 요구한다. 본 연구에서는 미세 유동 장치, SynVivo-SMN 기반 미세 혈관 망을 이용하여 단일 실험에서 전단 접착 맵의 생성을 보여준다. 이 장치는 기하학적 규모, 형태 요소, 흐름의 특징과에있는 세포의 상호 작용을 포함한 생체 내 혈관의 복잡한을 다시체외 형식함으로써 기본에 대한 생물학적 사실적인 환경을 제공하고 세포 행동, 약물 전달 및 약물 발견 연구를 적용했다. 분석은 마이크로 칩의 아비딘 - 코팅 된 표면을 가진 2 μM 바이오틴 - 코팅 입자의 상호 작용을 연구에 의해 입증되었다. 미세 혈관에서 관찰 전단의 전체 범위는 생리적 조건 하에서 입자의 전단지도 대 단일 분석 가능 밀착성이 얻어진다.

도 1a는 SynVivo-SMN 장치의 회로도 및 시야 화상을 나타낸다.도 1b는 유리 슬라이드 상에 장착 SynVivo-SMN 장치.도 1C는 진공 데시 케이 터에 물 프라이밍 다음 튜빙 장치를 도시 나타낸다. 그림 2A는 2 μm의 바이오틴 입자의 결합 다음과 같은 일반적인 아비딘 코팅 SynVivo - SMN 장치를 보여주는 실험 세트입니다. 그림 2B의...

Watch this Scientific Journal Video about Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks at JoVE.com

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

SynVivo 합성 미세 혈관의 네트워크를 사용하여 전단 접착지도의 생성

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

generation-shear-adhesion-map-using-synvivo-synthetic-microvascular

Research

JoVE Journal

Bioengineering

8.8K 조회수.  CFD Research Corporation. Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables. 

비디오: Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

세포 네트워크의 창조를위한 플라즈마 리소그래피 표면 Patterning

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

연구

생체공학

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Hematologic 질환의 Microvascular 상호 작용 유학을위한 Endothelialized Microfluidics

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

낮은 강도의 초음파를 사용하여 접착 종속 신호의 유도

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

매트릭스 수정에 의해 유도 된 세포 접착 및 탈 부착의 실시간 변화를 측정 셀 기판 임피던스와 라이브 세포 이미징을 사용하여

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

살아있는 세포를 엔지니어하는 합성 생물학을 사용하여 해당 프로그램 자료와 인터페이스

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

가 여러 명의 병렬 접시 흐름 챔버를 사용 하 여 스트레스에 노출 된 내 피 세포의 유전자 표정 분석

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

라이브 셀 분석의 전단 응력에 녹 농 균 등 처리량 미세 자동 시스템을 사용 하 여

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

렌티바이러스 벡터 접근법을 이용한 형질전환 쥐 생성

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

세포가 없는 세균 추출물, 리포좀 및 에멀젼 전달을 사용하여 합성 세포를 생산하는 단백질 을 준비합니다.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

글라이딩 키네신 운동성 분석을 이용한 다성분 지질 나노튜브 네트워크 제작

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...