건강 연구의 캐나다 연구소 (CIHR), 자연 과학 및 공학 연구위원회 (NSERC) : 저자는 다음과 같은 자금 조달에게 감사의 말씀을 전합니다.

1. LabVIEW 프로그램의 개발 <ol><li> 유량 제어기 (MKS M100B 매스 플로 컨트롤러) 정확한 가스 유량을 보장하기 위해 헬륨 가스 라인에 부착된다. 이 장치는 0과 5 V. LabVIEW 프로그램의 제어 루프는 임의의 주어진 시간에 필요한 전압을 결정 사이의 범위, 입력 전압에 의해 제어된다. 컴퓨터와의 사이에 인터페이싱 유량 제어기는 데이터 수집 장치 (NI의 USB-6009)에 의해 수행되고, 12 V 전원 공급 유량 제어기에 전원을 제공한다. </li><li> 두 개의 위치 선형 슬라이드는 6 - 웰 플레이트, 각 방향에 대해 하나의 움직임을 제어하기 위해 사용된다. 각 어셈블리는 스테핑 모터와 결합 된 MA15 시리즈 Velmex Unislide 어셈블리로 구성, 두 어셈블리는 모터 컨트롤러 스테핑 하나 Velmex VXM에 의해 제어된다. 제어기는 각각의 모터의 설명서 조깅 허용 또는 외부 시리얼 명령 문자열을 사용할 수의 조합은 특정 움직임을 허용 Patterns. 여러 패턴만을 원하는 토치 속도뿐만 아니라 경로 치수를 제공하기 위해 사용자가 요구하는 LabVIEW 프로그램으로 프로그래밍 될 수있다. </li></ol> 2. 장비의 제조 <ol><li> 헬륨 실린더와 레귤레이터를 모두 엽니 다. </li><li> 모터 컨트롤러의 전원을 켭니다. </li><li> LabVIEW 프로그램을 열고 모든 조건을 확인합니다. </li><li> 플랫폼이 중심이되어 있는지 확인합니다. 확인 각 모터의 단계에서 보면 각 캐리지가 무대 한쪽 끝 근처에 있지 않은지 확인합니다. 프로그램 중심 캐리지 있고 또는 수동으로 모터 제어기의 전면에 조깅 컨트롤을 사용하여 조정하거나. </li><li> 이 날의 첫 번째 실행 인 경우에는 설치 프로그램이 제대로 작동 할 수 있도록, 세포없이 한 번 프로그램을 실행하는 것이 좋습니다. </li></ol> 3. 가스 제트 모세관 노즐 높이의 준비 <ol><li> 홀더에서 모세 혈관을 제거하고 높이 다이얼을 제로. </li><li> 6 잘 플레이트 (을)를 배치N 플랫폼. </li><li> 교체하여 커버 슬립을 돌 때까지 우물의 바닥에 가스 제트 모세관을 낮 춥니 다. 홀더에 모세관을 고정합니다. </li><li> 높이 다이얼을 사용하여 모세관 3mm를 올립니다. 모세관베이스에서이 높이를 표시합니다. </li><li> 안전한 높이로 모세 혈관을 마련하고 6 잘 플레이트를 제거합니다. </li></ol> 4. 셀 Permeabilization 프로토콜 <ol><li> 커버 유리에 합류하는 농경 점착성 세포주는 표준 배양 기술을 사용하여 6 - 웰 플레이트에 미끄러진다. HeLa 세포의 경우, 씨앗 6 - 웰 웰 당 2 × 10 5 세포와 접시와는 치료 전 48 시간 동안 배양한다. </li><li> 원하는 벡터를 함유 용액의 적절한 볼륨을 준비합니다. hrGFPII-1, 1X PBS에서 50 NG / μL의 농도는 HeLa 세포에서 강한 형광 신호 형질 전환 후 24 시간을 제공하십시오. 덱스 트란 연구의 경우, 10 kDa의 녹색 형광 덱스 트란 80 ㎍ / ml를 추천합니다. 금후,이 용액을 칭한다의 &quot;벡터 솔루션&quot;. </li><li> 우물에서 세포 배양 배지를 제거하고 1X PBS로 3 회 씻어. </li><li> 우물에 4.2 단계에서 제조 된 벡터 솔루션의 1,370 μl를 피펫. 이 약 1.3 mm의 액체 깊이에서 발생한다. </li><li> 플랫폼의 중심에있는 벡터를 함유하는 용액을 잘 배치. 슬라이드 위에 3mm의 노즐 높이를 나타내는 이전에 표시된 수준으로 가스 분사 노즐을 낮추. </li><li> LabVIEW 프로그램에서 헬륨 유량을 설정하고 사용하기 원하는 움직임 패턴을 선택한다. 때 permeabilization 프로토콜을 시작할 준비가 &quot;실행&quot;을 선택합니다. 유량 제어기는 필요한 유량을 제공하기 위해 준비하는 동안 초기 지연 기간이있을 것이다. 적절한 유동 속도에 도달하면, LabVIEW 프로그램은 원하는 permeabilization 패턴에 잘 이동할 스테퍼 모터를 활성화 할 것이다. 플랫폼이 정지되면, 약 30 초를 기다려 벡터 솔루션을 제거합니다.참고 : 최적 permeabilization는 모세관의 직경에 따라, 노즐 출구에서 100 ~ 200 ㎩하에 동압 근처에 발생한다. </li><li> 1X PBS로 잘 세 번 씻으십시오. </li><li> 신선한 문화 매체와 잘 기입하십시오. </li><li> 실험의 원하는 번호를 4.6 - 반복 4.3 단계를 반복합니다. 참고 : 그들을 통해 실행되는 가스 제트없이 벡터 솔루션을 충만 우물로 구성 제어 샘플을 포함해야합니다. 다음 단계를 4.5과 4.6로 진행 약 1 분간 제어 우물에 솔루션을 둡니다. </li><li> hrGFP 형질 전환 실험을 실행하는 경우, 형질 전환 물질이 세포에 의해 복사 할 수 있도록 24 시간 동안 인큐베이터 6 - 웰 플레이트를 반환합니다. 덱스 트란 permeabilization 실험을 실행하면, 15 분 동안 인큐베이터 6 잘 플레이트를 반환합니다. </li></ol> 5. 셀 이미징 <ol><li> 원하는 경우, 직전 이미징, 세포 죽음을 평가하는 적절한 죽은 / 라이브 얼룩 counterstain. </li><li> approp와 이미지 세포형질 전환 / permeabilization 효능을 조사하기 riate 현미경. </li></ol>

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저자는 더 경쟁 재정적 이해가없는 선언합니다.

불활성 가스 제트 permeabilization은 자기편 세포의 형질 전환을위한 유용한 기술이다. 그것은 잠재적으로 유해한 화학 물질이나 바이러스 벡터에 대한 필요성을 없애 기계적 힘의 용도로 세포에 생물 분자를 전송하는 기능을 제공한다. 이 기술은 잠재적으로 연구자와 임상의에게 정확하게 세포를 형질 효율적이고 비교적 간단한 방법을 제공 할 수 있습니다. permeabilization의 선택은 또한 연구자는 여...

생물 의약의 발전과 세포 역학의 이해, 세포로 생체 분자의 전달은 많은 연구 분야 및 의료 치료에 중요한되고있다. 다른 기술은 세포막을 통해 세포의 세포질에 외래 분자를 도입하기 위해 개발되었다. 바이러스, 화학 또는 기계적 방법 : 이들은 일반적으로 분류 될 수있다. 바이러스 성 기술은 바이러스 벡터를 사용하여 RNA 및 DNA로 유전 물질을 형질있다. 바이러스 성 기술은 그러나 그들은 면역 및 염증 반응 2에 대한 가능성을 가지고 할, 특정 세포 라인에 높은 효율을 가지고하는 경향이있다. 일반 화학 형질 전환 방법은 인산 칼슘 3, 세균 외독소 4 리포 펙 (5)과 강수량 (가) 있습니다. 이러한 기법은 효과적 일 수있다, 그러나, 특정 문제는 세포 독성 및 비 특이성으로 발생한다. 또한, 칼슘 인산염 같은 기법전자 강수량은 거의 일차 전지 6에서 최대 70 %에 있지만, 특정 세포주에 형질 전환 효율이 발견되었습니다. 증가하는 연구 노력은 특히 임상과 DNA의 기능에 대한 연구를위한 다양한 치료를 설계하는 경우, 일차 전지에 투입되는 등이 주입니다. 기계적 자극을 통해 세포막의 일시적인 중단이 세포로 외래 분자를 도입하기위한 대안이다. 기술은 다음을 포함한다 : 분자 (7)의 미세 주입, 막 8,9을 방해하는 전기장을 사용하여 일렉트로, sonoporation와 결합 입자를 촬영 &quot;유전자 총&quot;에 의해 설명되는 것과 같이 세포막 10-12, 입자 충격을 방해하기 위해 초음파를 사용하여 셀 (13) 내로의 유전자, 및 더 최근에, 시간적으로 포유 동물 세포에게 14,15 투과 표시되었습니다 유체 전단 응력의 ​​적용. 하지만 정비공알 방법은 앞서 언급 한 몇 가지 문제를 방지, 그들은 일반적으로 낮은 효율성과 매우 복잡하고 전문적인 설정 함께 제공됩니다. 대기압 글로우 방전 토치 (APGD-t)는 표면 기능화 및 생체 외 (16)에 부착 세포를 분리의 초기 목표로 맥길로 개발되었다. Serendipitously, 사용되는 라이브 / 죽은 얼룩은 어떻게 든 permeabilizing 에이전트가 첨가하지 않고 세포에 의해 촬영되고 있음을 발견했다. 또한,이 단지 토치 경로와 일치하는 일이 우물의 제한 지역에서 발생한 것 같았다. APGD-T의 permeabilization 기능의 조사는 계속 제어 연구에서, 그것은 여자가없는 플라즈마의 캐리어 불활성 가스 제트는 또한 세포를 투과 할 수 있었다 것으로 나타났습니다. 이것은 플라즈마 제트에 의해 생성 된 반응 종 일시적 막의 기능을 손상 초기 가설 모순 및 제안 단순히 기계적 것을칼의 힘은 세포 permeabilization의 원인이하기에 충분했다. 여기에서 연구를 시도하고 정량화 permeabilization의 불활성 가스 제트의 효율성뿐만 아니라 형질 전환 기능 (16)에 의해 모양의 특징을 우리의 실험실에서 계속했다. 이러한 연구를 통해, 그 미세 기공은 세포막에 팩트 형태로 수행하는 것이 발견되었으며, 이러한 기공은 약 5 초 이내에 15 봉인하는 경향이있다. 우리는 병아리 태아의 융모 막 생체 외 및 생체 내에서 기술의 유틸리티를 증명하고있다.

<table border="1">
	<tbody>
		<tr>
			<td>Vitality hrGFPII-1</td>
			<td>Agilent Technologies Canada</td>
			<td>240143-57</td>
			<td>Green fluorescent protein for transfection experiments on HeLa cells</td>
		</tr>
		<tr>
			<td>Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable</td>
			<td>Life Technologies Inc.</td>
			<td>D22910</td>
			<td>dextran for permeabilization experiments</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Life Technologies Inc.</td>
			<td>L3224</td>
			<td>for counterstaining of mammalian cells</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>for mounting slides when imaging</td>
		</tr>
		<tr>
			<td>Ultra High Purity Helium</td>
			<td>Praxair Canada Inc.</td>
			<td>HE 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone DMEM/ High Glucose Media - 500 ml</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td>for HeLa cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1x</td>
			<td></td>
			<td></td>
			<td>Produced in lab</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. List of required reagents</td>
		</tr>
		<tr>
			<td>6-Well Clear TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>#1 22 x 22 Coverslips</td>
			<td>Fisher</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td>WPI Sizing Information</td>
		</tr>
		<tr>
			<td>ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100</td>
		</tr>
		<tr>
			<td>Mass-Flo Controller</td>
			<td>MKS</td>
			<td>M100B01314CS1BV</td>
			<td>Mass flow controller, Series M100B, requires an external 12 V power supply</td>
		</tr>
		<tr>
			<td>USB-6009 DAQ Device</td>
			<td>National Instruments</td>
			<td>779026-01</td>
			<td></td>
		</tr>
		<tr>
			<td>UniSlide Assembly with stepping motor and controller</td>
			<td>Velmex</td>
			<td></td>
			<td>Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8&#176; stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. List of required equipment</td>
		</tr>
	</tbody>
</table>

permeabilization of adhered cells using an inert gas jet

다양한 세포 형질 전환 기술이 존재하고 이러한 세 가지 범주로 나눌 수 있습니다 바이러스, 화학, 기계. 이 프로토콜은 시간적으로 세포 내로 일반적으로 비 투과성 거대 분자의 전달을 용이하게 할 수있는 불활성 가스 제트를 사용하여 접착 세포를 투과하는 기계적 방법을 설명한다. 우리는이 기술의 미세 임시 형성의 결과로, 부착 세포의 원형질막에 전단력을 부여하여 작동 믿는다. 이러한 기공이 생성되면, 세포는 유전 물질 및 기타 생체 분자 투과성이다. 관련된 기계적 힘은 기판에서 영구적으로 손상 또는 분리 세포의 위험을 실행 않습니다. 기술이 효과가 불활성 기체 역학의 좁은 범위, 따라서이있다. 불활성 가스 제트는 헬라, HEK293 인간의 복부 대동맥 내피 세포를 포함한 다양한 자기편 세포 라인을 permeabilizing에 효율적으로 입증되었습니다. 이 프로토콜은 P에 적합한우리가 미래의 임상 응용 프로그램에서 잠재적으로 연구에 사용될 수있다 표시, 생체 내에서 증명하고있다으로 관내에는 양쪽 모두에서 부착 세포의 ermeabilization. 또한 가치있는 연구 도구가 될 증명할 수있는, 공간적으로 제한적인 방법으로 세포를 permeabilizing의 장점이 있습니다.

0.86 mm의 내경을 가진 모세관 헬륨 가스 제트를 사용하여 10 kDa의 녹색 형광 덱스 트란과 함께 HeLa 세포의 Permeabilization도 1에 도시된다. 세포는 즉시 세포 죽음을 시각화하는 2 μL / ㎖의 EthD-1 솔루션 (LIVE / DEAD 생존 / 세포 독성 키트)와 permeabilization 후 대조되었다. 즉시 counterstaining 다음, 커버 전표를 장착하고 몇 군데 있었다. 이 그림은 대조 시료에 비해 세 가지 다른 출구 압력에서 헬?...

Watch this Scientific Journal Video about Permeabilization of Adhered Cells Using an Inert Gas Jet at JoVE.com

Permeabilization of Adhered Cells Using an Inert Gas Jet

이 프로토콜은 불활성 가스 제트를 이용한 부착 세포의 임시 permeabilization위한 방법을 설명한다. 이 방법은 세포막을 방해하는 기계적 힘의 이용에 의한 부착 성 포유 동물 세포에 유전 물질과 생체 분자의 전달을 용이하게한다.

불활성 가스 제트를 사용하여 붙음 세포의 Permeabilization

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes ...

permeabilization-of-adhered-cells-using-an-inert-gas-jet

Research

JoVE Journal

Bioengineering

9.6K 조회수.  McGill University.  이 프로토콜은 불활성 가스 제트를 이용한 부착 세포의 임시 permeabilization위한 방법을 설명한다. 이 방법은 세포막을 방해하는 기계적 힘의 이용에 의한 부착 성 포유 동물 세포에 유전 물질과 생체 분자의 전달을 용이하게한다. 

비디오: Permeabilization of Adhered Cells Using an Inert Gas Jet

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

태아의 폐 세포에 Mechanotransduction을 공부하는 실험적 시스템

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

연구

생체공학

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

비행 시간 형 질량 분석과 2D 가스 크로마토 그래피를 사용하여 조류의 열수 액화에서 수성 분수의 질적 특성

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

디지털 홀로그램 현미경 (DHM)를 사용 하 여 낮은 농도에서 미생물 측정

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

충돌기 제트의 두 가지 모드를 사용 하 여 패턴 고해상도: 수요와 근처-필드 전기에

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Vivo에서 부분 간 엔지니어링을에서 위한 쥐를 기초로 새로운 수술 기법

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

파지 디스플레이 펩타이드 라이브러리를 사용하여 섬유아세포 성장 인자 수용체2를 표적으로 하는 소형 펩타이드의 스크리닝 및 식별

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

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 ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

엑소좀 기반 도파민 운반체 시스템의 공식화 및 특성화

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...