The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

나트륨 나프탈렌 나이드 활성화 솔루션 및 표면 활성화 1. 준비 참고 : 통풍이 잘되는 흄 후드에서 반응을 수행하십시오. 가연성 용제 및 금속 나트륨과 같은 부식성 금속을 처리하는 일반적인 규칙을 따르십시오. 나프탈​​렌은 매우 적은 양이라도 매우 불쾌한 냄새 (좀약)를 가지고! 달리 명시하지 않으면 반응은 실온에서 수행된다. 아 지드 화 나트륨은 매우 독성이다! THF 분 자체 (부피)를 통해 약 20 %를 저장했다 (99.9 %은, 재료의 목록 참조). 나트륨에 비해 눈에 띄는 수분 함량과 THF를 증류. 물에 미량 존재하는 경우 나트륨 나프탈렌 나이드의 형성이 발생하지 않는다. <ol><li> (3 Å 분 자체상에서 건조 THF) 20 ㎖, 테트라 히드로 푸란, 나프탈렌 1.4 g (10.9 mmol)의 용액을 위해, PTFE- 구비 한 스크류 캡 100 ㎖ 유리 병에 0.25 g (10.9 mmol)의 나트륨 금속을 추가 코팅 된 자기 교반 막대. 참고 : Dissolu을(- 40 ° C에서 35) 기 크게 작은 조각 적당한 가열로 나트륨을 절단함으로써 향상 될 수있다. 최종 용액을 어두운 약간 녹색 컬러를 가지며 엄격 건조 조건하에 저장 될 수있다. </li><li> 0.5 mm 두께의 박막 재료 12mm 직경의 PTFE 디스크를 펀칭. 마크 한면 (예를 들어, 편지 우표 펀치 사용) 이소프로판올와 청소를. </li><li> 2 분 사용 집게 - 1의 활성화 용액 (1.1 단계)에서 개별적으로 PTFE-샘플을 품어. 참고 : 어두운 갈색 흰색에서 색상 변화가 성공적인 치료를 나타냅니다. </li><li> 그 후 이소프로판올로 두 번 THF와 다음 씻어. 참고 : 나프탈렌 나이드 용액이 소진 될 때 물 밝은 갈색으로 색상이 변경됩니다. (아마도 금속 나트륨을 포함하는) 미사용 나프탈렌 나이드 용액을 천천히 폐기 전에 이소프로판올을 첨가하여 분해한다. </li><li> 과산화수소 (30 %) 3 시간 동안 트리클로로 아세트산 (/ V W)의 20 %를 함유하는 샘플을 산화 처리. 빨래물과 건조와. 표면은 지금 약간의 갈색 외관을 가지고있다. 주 : 활성화 및 산화 결과를 표면의 대폭 증가 습윤성. 이 연구 결과는 이전에 14 자세히 조사 하였다. </li><li> 2 시간 동안 건조 THF 50 % (v / v)의 헥사 메틸렌 디 이소시아네이트 (HMDI)와 산화 디스크를 치료 THF로 씻어 건조 둡니다. </li><li> 3 시간 건조 - 2 물에 이소시아네이트 베어링 샘플을 가수 분해. 주 : 최종 아미노 - 작용 PTFE 표면 훨씬 안정 이소시아네이트 - 함유 샘플보다 더욱 많은 결합 가교제 표준과 호환된다. </li></ol> 2. 펩타이드 고정화 <ol><li> 탄산 완충액, pH가 9에서 50 mM의 20 % (v / v)의 디 에틸렌 글리콜 디 글리시 딜 에테르 (디 에폭시)의 용액을 준비한다. </li><li> 개별적으로 24 웰 플레이트에 표시된면이 위로와 민화 디스크를 놓고 에폭시 용액 1.5 ml를 추가합니다. 샘플의 전체 범위를 확인합니다. 2 시간 동안 품어 두 씻어시간 물에 한 번 탄산 버퍼. </li><li> 신선한 24 웰 플레이트의 각 우물의 바닥에, NaN가 3 0.01 %를 포함하는 50 밀리미터에서 0.5 ㎎ / ㎖ 펩타이드 (예를 들어, REDV) 탄산 완충액, pH가 9의 50 μl를 추가 조심스럽게 에폭시 작용 디스크의 상승 여력이 배치 -down 펩타이드 솔루션의 드롭에 (즉, 아래 좌우로 표시). 우물의 바닥과 PTFE 디스크 사이의 공간이 완전히 모세관 현상으로 침수되어 있는지 확인합니다. </li><li> 습식 챔버에 품어 (예를 들어, 꽉 닫는 뚜껑있는 밀폐 플라스틱 상자) 가습 분위기 하룻밤 적어도 3 시간 또는 위해 (바닥에 젖은 티슈 페이퍼를 배치하여). 주 : REDV 모티프뿐만 특성화 간주 될 수이기는하지만 스크램블링 또는 역방향 아미노산 서열은 부가적인 대조군으로서 포함 할 수있다. </li><li> 물로 3 회 세척하고 적어도 30 분 동안 50 % 이소프로판올 / 물에 소독. 시드, RINS 셀에 앞서멸균 인산 완충 식염수 (PBS)에서 샘플을 전자. </li></ol> 3. 셀 시드 <ol><li> 표준 세포 배양 절차 (17, 18)를 사용하여 HUVECs를 성장. </li><li> 24 웰 플레이트에서 수정 된면이 위로와 펩티드 결합 된 샘플을 놓습니다. 대조군으로 치료 PTFE 디스크를 사용합니다. </li><li> 종자 5 × 10 4 웰 당 2 ml의 배지에서 HUVECs를하고 인큐베이터에서 37 ° C에서 4 시간 5 % CO 2 품어. </li><li> 디스크를 제거하고 부착되지 않은 세포를 제거하고 신선한 24 웰 플레이트로 전송 조심스럽게 씻어냅니다. 원하는 기간 동안 새로운 배지 2 ㎖를 추가하고 배양 (예를 들어, 24 시간, w 1 등). 이틀 매체 변경합니다. </li><li> 디메틸 설폭 사이드 (DMSO)에 1 ㎎ / ㎖ 칼 세인 AM의 스톡 용액을 제조 하였다. </li><li> 성장 후, 세포 배지를 제거하고 PBS로 세척 한 PBS 함유 μL / mL의 칼 세인 AM 얼룩을 추가 (예컨대, PBS 용액 당 원액 1 μL). 부드럽게 교반과 incubat어둠 속에서 45 분 동안 37 ° C에서 전자. </li><li> 표준 FITC 필터가 장착 된 형광 현미경에 현미경을 (예 488 nm의, 엠 515 ㎚) 즉시 PBS로 세척합니다. 100 배의 배율을 사용합니다. 수정되지 않은에서 삼중뿐만 아니라 처리 된 샘플을 수행합니다. </li><li> ImageJ에 소프트웨어를 사용하면 세포에 의해 식민지 영역을 결정합니다. 또는 수동으로 세포를 계산 또는 ImageJ에 사용합니다. 두 방법 모두 각각의 표면에 부착 및 세포의 성장에 대한 유사한 정보를 제공합니다. </li></ol>

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The authors have nothing to disclose.

PTFE 표면 개질 프로토콜의 상세한 설명은도 6에 도시 된 바와 같이 중합체 골격으로부터의 불소의 제거로 시작하는 연속적인 단계로 구성된다. 결과적으로, 층의 공액 탄소 - 탄소 이중 결합의 풍부한 양을 포함하는 형성되고 나프탈​​렌 나이드 치료에 개발 된 어두운 갈색 색상에 따라. 산성 과산화수소와 표준 산화는 옅은 갈색으로 밝게 동행 수산화면,이 이중 결합의 손실을 반?...

즉, 향상된 생체 적합성, 세포 접착 부족, 섬유 성 캡슐화 및 혈전 형성의 유도를 전시하지 않는 몇 시간 동안 승인 된 의학에서 사용되는 다양한 폴리머는 몇 가지를 언급합니다. 바이오 물질 및 생물학적 시스템 사이의 상호 작용은 주로 임플란트의 표면에서 발생한다. 결과적으로, 많은 연구가 영향을받지 않는 재료의 벌크 특성을 남기면서, 원하는 응용에 적합한 특성을 생성하기 위해 표면 개질에 집중하고있다. 생리 학적으로 불활성 중합체 폴리 테트라 플루오로 에틸렌 (PTFE)의 탈장 등 외과 메쉬 한 가장 중요한 의학적 포트 (2), 혈관 이식편 3으로 많은 의료 분야에서 사용된다. 특히 피 접촉 상황에서 PTFE의 소수성은 종종 thromboti 결과 결과 혈소판 혈장 접착 성분 등의 비특이적 흡착을 야기C 이벤트 및 이식 4의 폐쇄. 또한, PTFE는 대부분의 중합체와 같은 바람직한 특징은 혈관 그래프트 (5)의 내면 (내강) 표면에서 혈관 내피 세포 (EC에)의 유용 층의 형성을 유도하는 것 세포 부착 및 커버리지를 지원하지 않는다. 생체 모방 내피는 특히 그 antithrombogenic 특성 6 자연 등가의 많은 기능을 수행 할 것으로 예상된다. 일반적인 생체 모방 변형 전략은 영향을받지 않는 물질 벌크 특성을 남기고 전적으로 세포 접착 성 재료를 부여하는 개념에 기초한다. 또한, 혈소판의 부착을 방지 접착제 (방오) (7) 특성을 도입함으로써 감소 될 수있다. 각종 펩티드 - 대부분의 세포 외 기질 단백질로부터 유래는 - 강하게 인테그린 (8)의 클래스에 속하는 세포 수용체에 결합하여 세포 부착을 향상시키는 것이 기재되어있다. 수이와 관련하여, 예를 세인트 알려진 대부분의 세포 유형과 상호 작용하는 펩타이드의 Arg-의 Gly-의 Asp (RGD)이다. 다른 아미노산 서열이 전적으로 특정 세포에 발현 인테그린에 의해 인식된다. 예를 들어,의 Arg-의 Glu-ASP를-발 (REDV)와 티르 - 일드-의 Gly-빼앗아, 인수 (YIGSR)는 특정 방식으로 9되는 EC에 결합하는 것으로 밝혀졌다. 이러한 펩티드의 공유 고정화 금속 및 폴리머 10, 11를 포함한 본질적으로 비 - 접착 성 물질의 과다에 대해 수행되었다. 다공성 PTFE,보다 정확하게 PTFE (의 ePTFE) 확장 - 폴리에틸렌 테레 프탈레이트 (PET)와 함께 - 혈관의 제조를위한 가장 중요한 물질 12 접목이다. 플라즈마 개질 (13) 또는 광 화학적 방법 (14)에 의해 적절한 치료를위한 확립 된 물리적 기술은, 다공성 및 / 또는 튜브형 구조는 각각 구멍 또는 내강 안에 용이하게 치료할 수없는 사실에 의해 방해된다. 습식 화학PTFE에 있기 때문에 대부분의 화학 공격 15 견디는 fluorin 함유 중합체의 높은 불활성 자연의 어려운 작업입니다. 본 논문에서는 공유 수정 전략 비교적 손쉬운 방법을 설명합니다. PTFE의 본딩을 렌더링하는 과정에서 적응, 작용기는 생물학적 활성 분자의 추가 활용을위한 기준점 역할을 재료 표면에 만들어졌습니다.

<table border="0" cellpadding="0" cellspacing="0" width="700">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">PTFE foil 0.5 mm</td>
			<td style="width:119px;">Cadillac Plastic&#160;</td>
			<td style="width:145px;">n/a</td>
			<td style="width:115px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">REDV peptide</td>
			<td>Genecust</td>
			<td>n/a</td>
			<td>custom synthesis &#62;95 % purity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iso-propanol</td>
			<td>Sigma Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tetrahydrofurane (THF)</td>
			<td>Sigma Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dimethylsulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">molecular sieve 3&#197;</td>
			<td>Sigma Aldrich</td>
			<td>208574</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium metal</td>
			<td>Sigma Aldrich</td>
			<td>483745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">naphthalene</td>
			<td>Sigma Aldrich</td>
			<td>147141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrogen peroxide 30 %</td>
			<td>Sigma Aldrich</td>
			<td>95321</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">diethylene glycol diglycidyl ether</td>
			<td>Sigma Aldrich</td>
			<td>17741</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hexamethylene diisocyanate (HMDI)</td>
			<td>Sigma Aldrich</td>
			<td>52650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcein-AM</td>
			<td>Sigma Aldrich</td>
			<td>56496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>71290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well plates</td>
			<td>Greiner-Bio-One</td>
			<td>662 160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td>
			<td>&#160;Nicolet</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorescence microscope Olympus X-70</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">humbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ePTFE vascular graft</td>
			<td>Gore</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

물질이 세포 접착 특성과 표면 부여하는 것은 생체 재료 연구와 조직 공학의 일반적인 전략이다. 이는이 물질이 잘 피할 수 새롭게 합성 된 고분자의 도입과 관련된 특성화 및 법적 문제 때문에 의학의 오랜 사용이 이미 승인 폴리머에 특히 흥미 롭다. 폴리 테트라 플루오로 에틸렌 (PTFE)은 혈관 이식편의 제조에 가장 자주 사용되는 물질 중 하나이지만, 중합체의 기능을 촉진하는 세포 접착 성이 부족하다. 에 내피 세포, 즉, 내피 세포의 융합 층과의 접목 내면 전체에 따르면 주로 인공 인터페이스 혈전을 감소시켜 최적의 성능 키를 간주한다. 본 연구는 펩티드 변성 PTFE에 내피 세포의 성장을 조사하고 변경되지 않은 기판 상에 얻은 결과를 비교한다. 와 커플 링내피 세포 접착 펩타이드의 Arg-Glu가-: ASP 발 (REDV)는 이후 접합 공정 다음에, 시약 나트륨 나프탈렌 나이드를 사용 fluorin 함유 중합체의 활성화를 통해 수행된다. 세포 배양액은 2 주 기간에 걸쳐 증명되는 펩티드를 고정화 재료에 인간 제대 정맥 내피 세포 (HUVEC를) 우수한 세포 성장을 이용하여 달성된다.

중요한 화학 반응 공정의 결과는 IR 스펙트럼 (도 1)에 의해 모니터링 하였다. 나트륨 나프탈렌 나이드와 초기 활성화는 이중 결합을 생성 - 그리고 작은 범위 - OH-기능. 신호를 나타내는 C = C 결합은 거의 독점적으로 수산기 그룹 베어링 표면을 산출 산화에 사라집니다. 또한 표준 접합 단계의 분석은 여기에 표시되지 않습니다. 활성화 및 산화에 의한 색상 변?...

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

향상된 세포 접착 폴리 테트라 플루오로 에틸렌에 젖은 화학 및 펩티드 고정화

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

wet-chemistry-peptide-immobilization-on-polytetrafluoroethylene-for

Research

JoVE Journal

Bioengineering

7.6K 조회수.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

비디오: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

원자 힘 현미경에 의해 영상에 대한 세균 고정

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

연구

생체공학

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

효소 고정 및 안정화를위한 소수성 소금으로 바뀌었 Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

상상 속의 세포 부착 분자의 특성에 대한 비드 집계 분석 실험

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

마이크로 RNA 정량는 DNA 결합 염료 화학, 물방울 디지털 PCR을 사용하여 순환

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

Zebrafish 애벌레의 향상 된 실험적인 조작에 대 한 장기 라이브 이미징 장치

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

생체 외에서 Neuroinflammation의 모델링에 대 한 기본 Glial 세포의 3 차원 하이드로 겔 문화 개선

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

뼈 Morphogenetic 단백질의 2 고체 표면에 화학 클릭 하 여 사이트 감독 동원 정지

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

조직 재생에 대 한 젤라틴의 젖은 회전-기반 성형 과정

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.