This work was supported by DFG grant EH 192/5-1 (A.E.), the EU (E-rare-2) project Transposmart (A.E.), the UWH Forschungsf&#246;rderung (E.S. and W.Z), and the PhD programme of the University Witten/Herdecke (P.B.). J.L. was supported by a stipend of the Chinese Scholarship council and T.B. and M.G by the Else Kr&#246;ner-Fresenius foundation (EKFS).

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

여기에 제시된 프로토콜은 전술 한 절차에 기초 4,12 인간 아데노 바이러스 형 5에 기초 HCAdV 벡터들의 정화를 허용한다. pAdFTC 플라스미드 내의 HCAdV 게놈은 모든 아데노 바이러스 유전자의 결여 만 5&#39;- 및 3&#39;- LTR에 및 포장 신호를 전달한다. 이 전략에서 HV AdNG163R-2 (4)는 트랜스 효율적인 바이러스 생산에 필요한 모든 유전자를 제공한다. 또는 아데노 관련 바이러스 (AAV) 기...

유전자 치료 응용 것이 바이러스 단백질의 발현, 유전자 자체 또는 수신 바이러스 단백질에 의한 세포 독성 및 면역 부작용을 방지하기 위해 매우 중요하다. 아데노 바이러스 벡터 (ADV)가 널리 도입 유전자 발현 1,2의 영향을 조사하기 위해 다양한 세포에 외래 DNA를 도입하기 위해 사용된다. ADV의 가장 진보 된 버전은 모든 바이러스 코딩 서열 3,4-함으로써 낮은 면역 원성 및 저독성 5-8 결합 35킬로바이트까지 패키징을 제공하는 능력이 결여 된 고용량 아데노 바이러스 벡터 (HCAdV)으로 표시된다. 때문에 높은 포장 용량 그들은 하나의 벡터 용량을 사용하여 크거나 여러 유전자의 전달을 할 수 있습니다. 따라서, 그들은 연구 지역 사회를위한 유용한 도구를 나타냅니다. 대조적으로, 제일 - 또는 VEC를 2 세대 ADV 쉽게 상업용 키트를 사용하여 제조 할 수있다 초기 유전자 E1 및 / 또는 E3 결여 할토르 HCAdV의 게놈 구조와 바이러스 생산은 더 복잡하다. HCAdV 게놈의 건설을위한 시스템은 모든 바이러스 코딩 시퀀스 및 셔틀 플라스미드 pHM5 9-12없는 HCAdV 게놈을 운반하는 플라스미드 pAdFTC을 기반으로합니다. 최대 14 킬로베이스 (KB)의 관심의 모든 유전자가 여러 복제 사이트가 인식에 의해 형벌되는 셔틀 벡터 pHM5에 복제 할 수 있습니다 / 원점 복귀 엔도 뉴 클레아의 사이트를 절단 SceI 제한 I 및 I-CEU I.를 PI- 따라서, 관심의 유전자를 클로닝 연속 PI- 및 I-SceI 제한 I- CEU I 의해 플라스미드에 함유 pAdFTC HCAdV 게놈에 존재하는 동일한 제한 부위에 관한 후속 삽입 소화 해제 될 수있다. pAdFTC에 PI- SceI 제한 I 및 I- CEU 난 부위를 절단 사이에 유전자 삽입 부위는 스터 DNA 및 5 &#39;및 3&#39;반전으로 말단 반복 게놈 패키징에 필요한 비 암호화하는 아데노 바이러스 서열 (LTR에)와 측면으로 접하고단부와 하류 5&#39;ITR 패키징 신호 모두에서. 추가 투표자 DNA는 바이러스 생산시 효율적인 포장을 보장하기 위해 27-36킬로바이트까지 최종 HCAdV 게놈의 최적 크기를 제공합니다. pAdFTC이 (삽입 된 유전자의 크기에 따라 다름)까지 45킬로바이트와 큰 플라스미드와 비교적 긴 DNA 인식 사이트와 유도 엔도 뉴 클레아의 사용을하기 때문에 결합 강한 DNA, 몇 가지 정리 단계 pHM5에서 유전자의 전송시 필요한 전시 pAdFTC에. 전단력을 피하고 취급에주의하는 것이 좋습니다. HCAdV 게놈은 LTR에 5&#39;ITR의 상류에 직접적으로 위치되고 3&#39;ITR (12)의 하류 없음 I 제한 효소 인식 부위에 의해 측면된다. 따라서, HCAdV 없음 내가 HCAdV 생산 세포주에 바이러스 게놈의 후속 형질 다이제스트에 의해 방출 될 수있다. 참고 제한 효소의 사용하지 I REL위한플라스미드 pAdFTC에서 바이러스 게놈의 용이성 삽입 유전자가 없음 I DNA 인식 부위의 결여 된 것을 의미한다. HEK293 세포 기반 생산 세포 (116 세포)를 안정적으로 Cre 호텔 재조합 효소를 표현한다. 바이러스 증폭 셀 (116)은 트랜스 -3,4-의 복제 및 패키징을 위해 필요한 모든 ADV 유전자를 제공하는 헬퍼 바이러스 (HV)와 공동 감염. HV 116 4 세포에서 발현 Cre 호텔 재조합 바이러스에 의해 증폭되는 동안 제거 floxed 포장 신호와 제 세대 ADV이다. 이것은 본래 패키징 신호를 포함 주로 HCAdV 게놈 encapsidated되도록. HCAdV의 사전 증폭 조직 배양 접시의 표면에 성장 (116) 세포에서 시리얼 계대 단계를 수행하여 수행됩니다. 각각의 통과 후 바이러스 입자는 세 개의 연속 동결 - 해동 단계를 수행하여 감염된 세포에서 방출된다. 세포의 수가 증가는 모든 통로로 감염된선행 통로에서 세포 용 해물의 1/3. 마지막 사전 증폭 단계는 대규모 증폭 스피너 플라스크에 정지에서 재배 생산 세포를 감염하는 데 사용됩니다에서 마지막으로 해물. 비리 온은 4,12 구배 염화 세슘 밀도에서 초 원심 분리를 수행하여 현탁 세포로부터 정제된다. 이 절차를 빈 입자와 완벽하게 조립 입자는 두 가지 대역으로 분리된다. 상기 HCAdV을 집중하는 제 2 비 - 점진적으로 초 원심 분리 단계가 수행되는 입자를 포함한다. 이어서 HCAdV 함유 얻어진 밴드 수거하고 생리 완충액에 대해 투석한다. 최종 제제는 벡터의 절대 바이러스 입자 감염성 입자 및 HV 오염 수준의 숫자에 대한 특징이다. 절대 바이러스 입자는 바이러스 입자를 용균 및 260 nm에서의 흡광도를 측정함으로써 또는 정량적 실시간 PCR (qPCR에) (12)을 수행하여 결정될 수있다. 끝까지 마음의 감염ified하게 바이러스 입자는 감염된 세포에서 3 시간의 감염 후 내에 존재 qPCR의 측정 HCAdV 게놈에 의해 결정될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">I-CeuI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td style="width:123px;">R0699S</td>
			<td style="width:315px;">restriction digest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">PI-SceI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0696S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">T4 Ligase</td>
			<td style="width:147px;">New Engand Biolabs</td>
			<td>M0202S</td>
			<td>ligation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">SwaI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0604S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:312px;">NotI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0189S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Calf Intestinal Alkaline Phosphatase (CIP)</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>M0290S</td>
			<td>dephosphorylation of digested plasmids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Hygromycin B</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P02-015</td>
			<td>selection of CRE expressing 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM</td>
			<td style="width:147px;">PAN Biotech</td>
			<td>P04-03590&#160;&#160;&#160;</td>
			<td>Hek293T cell culture medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Minimal Essential Medium (MEM) Eagle</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P04-08500</td>
			<td>116 cell culture medium&#160;&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dulbecco&#8217;s phosphate buffer saline (DPBS)</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P04-36500</td>
			<td>washing of cells, resuspension of cells</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:312px;">250 ml storage bottle</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:123px;">CLS430281-24EA</td>
			<td>infection of 116 cells grown in suspension</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">500mL PP CentrifugeTubes</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:123px;">CLS431123-36EA</td>
			<td>sedimentation of cells from suspension culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Spinner flask</td>
			<td style="width:147px;">Bellco</td>
			<td style="width:123px;">1965-61030</td>
			<td>growth of 116 cells in suspension</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Ultra Clear Ultracentrifuge tubes</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">344059</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Ultracentrifuge</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">&#160;--</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">SW-41 rotor</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">&#160;--</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:312px;">Spectrum Laboratories Spectrapor Membrane</td>
			<td style="width:147px;">VWR</td>
			<td style="width:123px;">132129</td>
			<td>dialysis tubing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ready-to-use dialysis cassettes&#160;</td>
			<td style="width:147px;">Thermo</td>
			<td style="width:123px;">66383</td>
			<td>dialysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">one shot DH10B electrocompetent E. coli</td>
			<td style="width:147px;">invitrogen</td>
			<td>C4040-52</td>
			<td>transformation of ligation reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">PureYield&#8482; Plasmid Midiprep System</td>
			<td>Promega</td>
			<td>A2495</td>
			<td>midiprep</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">peqGOLD Tissue DNA Mini Kit&#160;</td>
			<td style="width:147px;">Peqlab</td>
			<td>12-3396-02</td>
			<td>isolation of genomic DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SuperFect Transfection Reagent</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:123px;">301305</td>
			<td>tranfection of 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">opti MEM (10 % FBS)&#160;</td>
			<td style="width:147px;">Gibco</td>
			<td>31985-062</td>
			<td>transfection of 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iQ SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>&#160;170-8882</td>
			<td>q-PCR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">CFX 96 C1000 touch&#160;</td>
			<td style="width:147px;">Biorad</td>
			<td style="width:123px;"></td>
			<td>qPCR machine</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Phenol/Chloroform/Isoamyl alcohol&#160;</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">A156.1</td>
			<td>purification of DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Cesium chloride</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">8627.1</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium acetate 99%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">6773.2</td>
			<td>DNA precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">LB medium&#160;</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">X968.3</td>
			<td>bakterial growth medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ethanol&#160; 99,8% pure</td>
			<td style="width:147px;">Carl Roth</td>
			<td>9065.5&#160;</td>
			<td>DNA precipitation and washing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SDS 99,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td>2326.2</td>
			<td>lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EDTA</td>
			<td style="width:147px;">Carl Roth</td>
			<td>8043.2</td>
			<td>lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tris-HCl 99%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">9090.3</td>
			<td>dialysis buffer/ lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">glycerol 99,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3783.1</td>
			<td>dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2 98,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">KK36.2</td>
			<td>dialysis buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3957.1</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">KH2PO4</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3904.2</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">sucrose</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">9286.1</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">Na2HPO4x2H2O</td>
			<td style="width:147px;">Carl Roth</td>
			<td>4984.2</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1,5ml tubes</td>
			<td style="width:147px;">sarstedt</td>
			<td style="width:123px;">72,730,005</td>
			<td>storage of&#160; virus preparations at -80&#176;C</td>
		</tr>
	</tbody>
</table>

cloning and large-scale production of high-capacity adenoviral vectors based on the human adenovirus type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

복제, 증폭 및 HCAdV 준비 정화용 여기에 대표적인 예는 표시됩니다. 효소 소화 제한에 의해 복제 전략의 개요 (그림 1)과 복제에 대한 대표적인 예와 HCAdV 게놈의 릴리스는 (그림 2)이 제공된다. 소화 나는 PI- SceI 제한 I 및 I-CEU에 의해 pHM5에서 GOI-발현 카세트 및 후속 페놀 - 클로로포름 추출 및 EtOH로 침전의 출시 후 일반적인 제한 패턴 <...

Watch this Scientific Journal Video about Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5 at JoVE.com

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

인간 아데노 바이러스 유형 5를 기반으로 대용량 아데노 바이러스 벡터의 클로닝 및 대규모 생산

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

cloning-large-scale-production-high-capacity-adenoviral-vectors-based

Research

JoVE Journal

Bioengineering

14.3K 조회수.  University Witten/Herdecke. A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans. 

비디오: Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

실험실 규모의 합성 거미의 실크 생산

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

연구

생체공학

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

생리 연구를위한 모든 고체 이온 선택성 전극 (ASSISE)를 기반으로 다중 분석 바이오칩 (MAB)

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

폴리 (ε 카프로 락톤) 전기 방사 원사에 인간 중간 엽 줄기 세포의 첨부 파일을 최적화

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

회전 타원체의 포유 동물 세포의 대규모 확장을위한 지속적인 공급함으로써 영양 규정

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinocyte 확산에 2-및 3 차원의 평가 유형 I 교원 질 기판

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

광학 확산 상관 분광법을 사용하여 인간의 뇌의 대뇌 혈류 기반 휴식 상태 기능 적 연결

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

아데노바이러스 생산을 위한 효율적인 방법

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

대규모로 3차원 재건 된 인간 표피 를 육성

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

미리 형성된 마이크로버블에서 멤브레인 여과 위상 시프트 데카플루오로부탄 나노방울의 생산

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

인간 1차 피부 섬유아세포를 기반으로 한 헌팅턴 병 모델의 마이크로튜블러 플러스 엔드 다이내믹스 시각화

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...