저자는 기술 지원을 크리스 모로을 인정하고 싶습니다. 자금 조달은 NIH SBIR (R43)에 의해 제공되었다 1R43GM101859-01 (MPI) GRANT10940803을 부여합니다.

1. 문화 제조 예 셀로 <ol><li> 사용 준비가 될 때까지 액체 질소에 NIH/3T3 세포 주식을 저장합니다. </li><li> 50 ㎍ / ml의 스트렙토 마이신을 10 % 소 태아 혈청, 10 mM의 HEPES 완충액, 50 단위 / ㎖ 페니실린 및 보충 된 둘 베코 변형 이글 중간 (DMEM)를 사용 NIH/3T3 세포에 대한 완전한 미디어를 준비한다. </li><li> 37 ℃에서 진탕 수조에서 2 ~ 3 분 동안 세포를 해동 </li><li> 3 분 1,500 XG에 완전한 미디어와 원심 분리기 5 ㎖의 세포를 Resuspend. </li><li> 세포 펠렛을 방해하지 않고 세포의 상층 액을 제거합니다. </li><li> 미디어 5ml에 세포를 재현 탁하고 혈구 세포를 사용하여 계산. <ol><li> 0.5 ML의 microcentrifuge 관에 세포 현탁액과 장소의 10 μl를 제거합니다. </li><li> 세포 현탁액에 트리 판 블루의 10 μl를 추가하고 피펫의 끝으로 저어. </li><li> 피펫 혈구 실에 염색 된 세포의 10 μL. </li><li> 배에서 혈구 세포를 계산확대. <ol><li> 9 큰 사각형의 4의 살아있는 세포 (작은 흰색 공)을 계산합니다. 만 트리 판 블루 염색에서 진한 파란색을 표시하지 않는 살아있는 세포를 계산합니다. </li><li> 세포 본래 세포 현탁액 × 104 세포 / ml의 수의 결과, 대형 사각형 당 세포의 평균 수를 계산한다. </li><li> 1 배 각각 T25 플라스크에 5 ㎖의 총 5 개의 세포 / ml의 밀도로 시드 세포. </li></ol></li><li> 시작 실험 전에 70 %와 80 %의 포화 상태 사이에 문화의 세포. 필요에 따라 2-3 일마다 미디어를 새로 고치거나. </li></ol></li></ol> 2. 인쇄 표면 처리 <ol><li> 마크 조직 문화 처리 된 폴리스티렌 (TCTPS) 12 웰 플레이트의 하단에 마커로 사각형 프린트 헤드 (x 19mm 7mm)의 크기. </li><li> 피펫 (50) 사각형 영역에 소 태아 혈청의 μL 및 팁 전체 영역에 그것을 확산. </li><li> 페트리 접시를 남겨살균 바이오 안전성 후드 O / N의 혈청 자리가 완전히 건조 할 수 있도록합니다. 이 접시는 사용하기 더 이상 2 일 이상 전에 준비를해야합니다. </li></ol> 3. 머지 인쇄 <ol><li> 증류수로 프린트 헤드를 씻어. <ol><li> 증류수로 60mm 페트리 접시를 채우고 표면에 프린트 헤드를 고정. </li><li> 공기 펌프를 사용하여 150 μL / 분에서 2 분 동안 각 라인을 통해 증류수 흐름. </li><li> 페트리 접시에서 물을 버리고 깨끗한 물을 채운다. </li><li> 물에서 아래로 3 회를 프린트 헤드를 이동합니다. </li></ol></li><li> 사전 준비를 12 웰 플레이트에서 혈청 코팅 자리에 프린트 헤드 중심을 도킹합니다. </li><li> 프라임 사전 예열 완료 미디어, 채널 당 300 μL와 프린트 헤드. </li><li> 인쇄 표면에 50,000 세포 / ml의 농도와 60 μL / 분, 채널 당 100 μL에서 세포를 중단. </li><li> 에 도킹 왼쪽 프린트 헤드를 올려 놓고표면 및 세포 배양 인큐베이터에서 매니 폴드는 2 시간 동안 5 % CO 2와 37 ° C로 설정합니다. </li><li> 2 시간 배양 한 후, 프린트 헤드를 제거하고 배양 접시에 뚜껑을 놓습니다. 프린트 헤드를 제거하는 시간은 0 시간 시점으로 간주됩니다. </li></ol> 4. 표준 세포 배양 <ol><li> 2 단계에서 제조 된 혈청 발견 TCTPS 12 - 웰 플레이트 중 하나에 미리 예열 미디어 1 ML을 추가합니다. </li><li> 12 잘 TCTPS 판의 하나의 우물에 2 단계 피펫에 남아있는 세포 현탁액 1 ㎖를 가져 가라. 이 접시에 50,000 세포를 포함하는 문화를 생성합니다. </li><li> 2 시간 동안 37 ° C에서 인큐베이터에서 세포 배양 접시를 놓습니다. </li><li> 2 시간 배양 타이밍을 시작하면; 인쇄 및 시드 샘플 사이의 일관성을 위해이는 0 시간 시점으로 간주됩니다. </li></ol> 5. 문화의 시각화 <ol><li> 0, 2, 24, 두 가지 방법에서 각각 48 시간 걸릴 세포 D 후표준 거꾸로 현미경을 사용하여 위 및 이미지 escribed. <ol><li> 요오드화 프로피 듐 만 사균의 핵으로 혼입되어 적색 형광 염색을 이용하여 세포를 염색. <ol><li> 부드럽게 칼슘과 이물질을 제거하기 위해 마그네슘 (PBS + +)와 인산염 완충 생리 식염수 1 ㎖로 세포를 3 회 헹군다. </li><li> 각 배양 용기에 미리 예열 PBS + +의 1 ML을 추가합니다. </li><li> 각 배양 용기에 프로피 디움 요오드 원액 (1 ㎎ / ㎖) 1 μl를 추가합니다. </li><li> 10 분 동안 37 ° C에서의 propidium 요오드화 염색 세포를 품어. </li></ol></li><li> 이미지 반전에서 10X 및 40X 현미경 배율을 이용하여 세포. </li></ol></li><li> 적절한 생물 학적 용기에 세포를 폐기하십시오. </li></ol>

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저자는 시약 및 / 또는이 원고에 사용되는 악기를 생산하고 사치 미세 유체의 직원 및 주주입니다.

여기에 설명 된 3D 미세 인쇄 기술은 잘 채워진 액체로 세포 microfluidically 프린팅 배열 고유 능력 잠긴 표면 즉. 잠긴 표면에 인쇄에 의해, 세포 마이크로 어레이에서는 세포의 생리 학적으로 관련된 세포 표현형뿐만 아니라 단일 웰도 4의 하단에 세포를 다중화 할 수있는 능력을 유지하는 것을 제조 할 수있다. microfluidically 세포 결과를 인쇄하는 본 연구 쇼의 결과 유사한 세포 ?...

제약 산업의 최근 발전은 약물 검사 및 cytotoxicological 분석 1,2,3의 약물 발견 과정에서 세포 마이크로 어레이를 사용하여 증가 된 관심을 이끌고있다. 세포의 마이크로 어레이를 사용하여 시험 관내 높은 처리량 분석 및 스크리닝 방법의 개발이 신속하고 비용 효과적인 약물 후보의 개발뿐만 아니라, 미리 셀 1,4의 기본적인 이해를 용이하게한다. 세포 검사에 대한 전통적인 접근 방식은 기존의 잘 플레이트 플랫폼을 사용한다; 그러나이 접근법으로 인해 높은 비용, 제한된 처리량 및 세포 기능에 대한 정량적 정보 1,5 제한적 기능으로 제한된다. 때문에 이러한 제한, 세포 마이크로 어레이 기술의 연구는 분자 생물학적 특성, 조직 공학, 약물 검사의 1,6에 급성장하고 있습니다. 셀룰러 마이크로 어레이의 장점은 더 작은 샘플 사용, 최소한의 영향을 포함세포의 표현형의 이질성 정보를 마스킹, 그리고 더 높은 처리량 응용 프로그램 -1,7,8,8에 대한 분석을 자동화하는 가장 중요한 기능입니다. 제약 산업은 현재 마이크로 웰 플레이트 9 약물 검사에 대한 2 차원 세포 단층 배양과 높은 처리량 세포 기반 스크리닝 분석을 이용한다. 마이크로 플레이트의 우물에서 다중 세포는 독특한 실험 옵션을 더 높은 처리량에 대한 가능성을 제공합니다. 또한, 세포 마이크로 어레이에 대한 현재의 기술은 세포가 극적으로 생체 내에서 10, 11에서 세포의 표현형을 변경할 수있는 건조 할 수 있습니다. 이러한 문제를 극복하기 위해, MFCA는 설계하고도 1에 도시된다. MFCA 차원 유체의 디자인이 욕에 하강과 시일을 형성하는 표면에 대해 압축하는도 1의 프린트 헤드 칩을 가능하게한다. 프린트 헤드의 부드러운 실리콘 팁처럼 동작가스켓 가역 씰을 형성한다. MFCA 기술은 고유 세포 배양 및 조직 슬라이스 시스템 모두에 필요한 잠긴 표면과의 인터페이스에 적합하고, 대부분의 다른 접근 방식을 가진 어렵거나 불가능하다. 핀 또는 잉크젯 인쇄가 작동하지 않습니다, 그리고 2 차원 마이크로 유체 장치는 증착 또는 개별 관광 명소의 큰 배열과 인터페이스에 적합하지 않습니다. 또한, 실험 소형화 및 지역화로 - 세포 마이크로 어레이 - MFCA는 높은 처리량 세포 기반 스크리닝 분석과 관련된 주요 문제를 극복한다. CFM은 표면 (12, 13)에 미세한 반점 위치에 사이클 작은 양의 유체 샘플에 3D 채널 네트워크를 사용합니다. 흐름과 함께 인쇄함으로써, 생체 분자, 세포 및 다른 시약은 현재 셀의 인쇄 기술을 방해하는 공기에의 노출없이 민감한 생체 분자와 세포의 인쇄를 가능하게 프린팅 프로세스에 걸쳐 액체 환경에서 유지된다. 이 어레이 표면에 캡처 메커니즘이 이러한 하이 브리 도마 또는 제공 상청액 같은 조질 물질로부터 직접 인쇄하는 것도 가능하다. 이 원고의 목적은 표면에 상세히 두 세포 유형의 잠긴 인쇄를 설명하는 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="571">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Continuous Flow Microspotter</td>
			<td style="width:71px;">Wasatch Microfluidics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NIH/3T3 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dubbleco&#39;s Modified Eagle Medium</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">11965-092</td>
			<td>base media for cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">HEPES buffer</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">15630-080</td>
			<td>cell media additive (control pH)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Sodium pyruvate</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">11360-070</td>
			<td>cell media additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>cell media additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Trypan blue</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">15250-061</td>
			<td>stain cell sfor counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Haemocytometer</td>
			<td style="width:71px;">Fisher</td>
			<td align="right" style="width:119px;">267110</td>
			<td>cell chamber to count cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikon Eclipse TS100</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Used to check on cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikon Eclipse TE2000-U</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Used for collecting images</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Phosphate Buffered Saline (with calcium and magnesium)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">14040-133</td>
			<td>rinsing cells before passaging and before staining with PI</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">TrypLE Express&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A12177-01</td>
			<td>used to remove cells from surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the submerged printing of cells onto a modified surface using a continuous flow microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

섬유 아세포 세포주 NIH/3T3 세포 잠긴 표면에 인쇄하거나 시딩 하였다. 세포는 밀리리터 당 5 X 104 세포의 밀도로 성장시켰다. 세포는 기존의 세포 배양 기술을 이용하여 접종 하였다. 세포를 대형, 채널 단백질 및 기타 생체 분자에 사용 CFM보다 (~ 500 μm의) 큰 열두 플로우 셀 프린트 헤드를 사용하여 인쇄 하였다. 세포를 혈청 코팅 표면 상에 인쇄되거나 시딩 하였다. 인쇄 공정은도 1?...

Watch this Scientific Journal Video about The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter at JoVE.com

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

연속 흐름 Microspotter을 사용하여 수정 된 표면에 세포의 침수 인쇄

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

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Research

JoVE Journal

Bioengineering

8.7K 조회수.  Wasatch Microfluidics. This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

비디오: The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

조직 표면 Tensiometry에 의해 집계 응집력의 측정

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

연구

생체공학

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

원자 힘 현미경을 사용하여 살아있는 세포의 기계적 특성을 측정

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

세포의 표면지도 및 Bioprinting에 대한 자동화 된 로봇 분배 기법

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

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 ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

다중 스펙트럼 화상 진찰 교류 세포측정을 사용하여 시험관 내 미세핵 분석기를 능력을 발휘하는 자동화된 방법

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

시간 경과 현미경 검사를 사용하여 대장균에서 생물학적 소음의 지속적인 측정

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

연속 흐름의 제조 및 작동, 투과화 감지 기능이 있는 미세 전기 천공 시스템

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

3D 프린팅 기술을 이용한 혈관 플랩 제작

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...