Name: wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion
Uploaded: 2016-08-15T14:00:00.000+00:00
Duration: 6 min 15 s
Description: Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

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><tbody><tr><td>PTFE foil 0.5 mm</td><td>Cadillac Plastic&amp;nbsp;</td><td>n/a</td><td></td></tr><tr><td>REDV peptide</td><td>Genecust</td><td>n/a</td><td>custom synthesis &gt;95% purity</td></tr><tr><td>iso-propanol</td><td>Sigma Aldrich</td><td>34965</td><td></td></tr><tr><td>tetrahydrofurane (THF)</td><td>Sigma Aldrich</td><td>401757</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Sigma Aldrich</td><td>D8418</td><td></td></tr><tr><td>molecular sieve 3 &amp;Aring;</td><td>Sigma Aldrich</td><td>208574</td><td></td></tr><tr><td>sodium metal</td><td>Sigma Aldrich</td><td>483745</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>naphthalene</td><td>Sigma Aldrich</td><td>147141</td><td></td></tr><tr><td>hydrogen peroxide 30%</td><td>Sigma Aldrich</td><td>95321</td><td></td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399&amp;nbsp;</td><td></td></tr><tr><td>diethylene glycol diglycidyl ether</td><td>Sigma Aldrich</td><td>17741</td><td></td></tr><tr><td>hexamethylene diisocyanate (HMDI)</td><td>Sigma Aldrich</td><td>52650</td><td></td></tr><tr><td>Calcein-AM</td><td>Sigma Aldrich</td><td>56496</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014&amp;nbsp;</td><td></td></tr><tr><td>sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td></td></tr><tr><td>24 well plates</td><td>Greiner-Bio-One</td><td>662 160</td><td></td></tr><tr><td>ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td><td>&amp;nbsp;Nicolet</td><td>n/a</td><td></td></tr><tr><td>fluorescence microscope Olympus X-70</td><td>Olympus</td><td>n/a</td><td></td></tr><tr><td>humbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td>n/a</td><td></td></tr><tr><td>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

7.7K 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

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

전자 레인지를 이용한 폴리의 기능화 (에틸렌 글리콜) 및 체인 중합 및 하이드로 겔 형성에있는 사용을위한 온 수지 펩티드

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

연구

화학

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

셀 문화 응용 프로그램에 대 한 단백질 기반 Hydrogels 아키텍처의 쉬운 조작

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

생화학

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

모세관 전기 영동은 실시간으로 키토산 필름에 펩타이드 접목을 모니터링하는

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

단일 분자 힘 분광학에 대 한 단백질의 화학식 동원 정지

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

조직 공학 Thermoresponsive 나노 구조 표면의 제조

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

생체공학

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

AFM 기반 힘 분광학을 위한 단일 분자의 공유 부착

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

생리 활성 단백질 또는 펩 티 드 히드로 광화학을 사용 하 여 생물 학적 응용에 대 한에 패턴화

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

CD47 유래 펩타이드 고정화를 통해 금속 임플란트에 대한 혈액 매개 세포 부착 완화

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

견인력 현미경 검사법의 응용을 위한 하이드로겔 패터닝 기술을 사용하여 세포 접착 제어

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

세포 접착 및 형태학을 위한 Biofunctional Self-Assembling Peptides 평가

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

저자 스포트라이트: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids(대장암 오가노이드를 위한 초단파 펩타이드 매트릭스의 최적화)

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

초단파 자기조립 펩타이드 매트릭스에서 SW1222 세포주의 대장암 오가노이드 제조 및 특성화

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...