이 작품은 재정적으로 스위스 국립 과학 재단 (National Science Foundation) (- 스마트 재료 NRP 62)에 의해 지원되었다. 또한 B. Hanselmann, K. 켈, U. 쉬츠와 B Leuthold의 지원이 있습니다 인정했다.

1. 플라즈마 개시 중합 <ol><li> 단량체 용액의 준비. <ol><li> 200 ml의 물과 분액 깔때기에 헥산 (100 ㎖)로 3 회 세척; HEMA (0.718 몰 100 ml)에 녹이고. 염화나트륨으로 포화 수성 상을 디 에틸 에테르 (50ml)로 HEMA를 추출한다. 황산 마그네슘, 유기 상을 건조하고, 진공에서 용매 (100 밀리바, 40 ° C)를 제거합니다. 감압 증류 HEMA (15 밀리바, 99 ° C). </li><li> 섹션 1.1.1에서 생성 억제제없는 HEMA의 0.62 M의 메탄올 용액을 준비합니다. 한 구 플라스크에 상기 용액 30 ㎖를 넣고 1 시간 동안 용액을 통해 아르곤을 버블 링하여 산소를 제거한다. </li></ol></li><li> 표면에 의한 중합. <ol><li> 포지션 서로 옆 플라즈마 챔버로이 폴리 카보네이트 막 (도 2). 기체 상태를 가리키는 멤브레인의 반짝이는면을 넣습니다. </li><li> 아 플라즈마 챔버를 연결5 분 동안 고등학교의 진공 (20 밀리바). 진공 밸브를 닫고 아르곤과 산소 가스에 연결하고 15 SCCM 아르곤 2.5 sccm의 산소와 2 시간 동안이 혼합물을 실을 제거하는 다른 밸브를 엽니 다. </li><li> 플라즈마를 시작하고 (폴리 카보네이트 멤브레인 : 12 W) 원하는 전원에 전력을 감소하고, 플라즈마와 4 분의 세포막을 취급합니다. 대응하는 밸브를 개방하여 챔버로 단량체 용액을 연결한다. 플라즈마 전원을 끄고 챔버 대피. </li><li> 해당 밸브를 개방하여 챔버와 단량체 용액을 연결하고 챔버에 솔루션을 붓는다. 막은 단량체 용액으로 덮여 있는지 확인합니다. 아르곤에 연결된 밸브를 열고 20 ° C (에어컨 룸)에서 12 시간 동안 반응 혼합물을 저장합니다. </li><li> 단량체 용액을 제거합니다. 5 분간 초음파 욕조에서 메탄올로 세포막을 씻으십시오. 물 세척 절차를 반복합니다. </li><li> 에서 멤브레인 건조진공 2 시간 동안 분 자체에. </li></ol></li></ol> 2. 코팅 막의 Postmodification <ol><li> 급 - butylmetylether 및 디메틸 아미노 피리딘 (DMAP) (33 ㎎, 0.27 밀리몰), N, N-디시 클로 헥실 카르 보디이 미드 (DCC) (0.27 밀리몰 55 MG) 스피로 벤조 피란 하나의 용액 (도 1) (0.27 밀리몰 100 ㎎)을 준비 (TBME) (12 ㎖). </li><li> 둥근 바닥 플라스크에 보호 교반기 바 및 보호 그리드를 배치합니다. 플라스크를 건조 아르곤 플라스크 홍수. </li><li> 도막이어서 플라스크에 상기 용액을 붓는다. </li><li> 실온에서 12 시간 동안 온화하게 교반 하였다. </li><li> 솔루션을 제거하고 5 분간 초음파 욕조에 급 butylmetylether를 가진 막을 씻는다. 에탄올과 물로 세척 절차를 반복합니다. </li><li> 2 시간 동안 분 자체에 진공에서 막을 건조. </li></ol> 3. 표면 장력 측정 <ol><l난&gt; 금속 O-링의 표준 테이프로 막 수정. O-링과 접촉하지 막 표면의 일부에 nanopure 물 (3.3 μL)의 드롭 위치를 지정합니다. 멤브레인의 5 가지 지점에서 접촉각 (CA)를 측정한다. </li><li> 샘플의 장기간 안정성을 테스트하기 위해, 세 가지 다른 공 후의 막의 스폿, 1, 2, 3, 7, 14, 21 일에 접촉각을 측정한다. </li></ol> 4. 광 변색 막 투과성 시험 <ol><li> 물 프란츠 확산 셀 (12 ㎖)의 수용체 챔버를 입력합니다. </li><li> 프란츠 확산 셀 멤브레인을 고정합니다. 막 수용체 실의 물과의 접촉에 있는지 확인합니다. 수성 카페인 용액 (; 3.0 ㎖를 20 ㎜)와 기증자 실 (막 위에 챔버)를 입력합니다. 백색광으로 도너 챔버 (도 3)의 상부로부터 분리막을 조사. 수용체 세포에서 샘플 (200 μL)를 수집, FO200 ㎚의 공경 R 트랙 진 폴리 카보네이트 멤브레인은, 샘플마다 10 분을 모은다. </li><li> 4.2 단계에 설명 된대로 실험을 반복합니다. 하지만 전체 투과성 시험 중 UV 광 (366 nm의, 80 W / m 2)를 가진 막을 조사. </li><li> 수집 된 샘플의 카페인 농도의 결정. <ol><li> UV / 비스 분광계를 사용하여 (ML 0.05 ㎎ / 1.5 밀리미터 / L 사이의) 15 다른 카페인 농도 보정 곡선을 그린다. 293 nm에서 보정합니다. </li><li> 검량선을 사용하여 수집 된 각각의 샘플의 농도를 결정. </li><li> 대 결정 농도를 그린다. 수집 된 샘플의 시간. 점을 선형 적합을 확인하고 기울기 ΔC를 결정합니다. </li></ol></li></ol>

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저자는 공개 아무것도 없어.

플라즈마 공정은 이온화 된 아르곤에 의해 야기되는 퍼플 가스를 생성한다. 오렌지 컬러는 누출 원하지 않는 질소의 존재를 나타냅니다. 플라즈마 처리는 표면에 라디칼을 형성 할뿐만 아니라, 막 7,12 에칭하지 않는다. 너무 많으면 에칭 막의 투과성에 영향을 미칠 것이다 이는 상당히 세공 직경을 변경할 수있다. 표시 설정의 제어 된 반응 조건은 상기 플라즈마 - 개시 래프팅 공정의 재?...

재료의 플라즈마 변경은 많은 산업 분야에서 중요한 과정이되고있다. 물질의 대량 속성을 변경하지 않고 표면과 표면의 작용의 청소 플라즈마 처리 표면 과학 1-8에 필수적인 프로세스했다. 고분자의 플라즈마 처리 homolytic 채권 분열을 초래. 이 고분자 재료의 테두리에 라디칼이 풍부한 표면의 형성에 이르게한다. 산소 분자를 포함하는 플라즈마를 이용하여 표면이 산소 풍부되어, 9-11, 따라서 더 친수성. 그러나, 표면의 친수성 시간 ​​12 이상 안정되지 않습니다. 장기간 안정성을 향상시키기 위해, 플라즈마 처리 된 표면은 화학적으로 13-15 후 또는 플라즈마 공정 중에 수정 될 수있다. 이러한 처리는 일반적으로 플라즈마 처리시 기체 상으로 반응성 모노머 종을 첨가함으로써 수행된다, 이러한 단량체는 중합고분자 표면의 생성 라디칼로부터. 화학적 처리는 휘발성 단량체로 수행되는 경우, 중합체는 그라프 플라즈마 수정 후에 일어날 갖는다. 라디칼은 표면 상에 형성 한 후 그 래프팅 제어를 수행하기 위해, 플라즈마 셋업은 제어 된 조건 하에서 12,16 용액 표면에서 플라즈마 - 개시 표면 - 유도 중합을 허용하는 기술된다. 프리젠 테이션은 트랙 양날의 고분자막 12,17의 수정에 초점을 맞추고 있습니다. 이러한 멤브레인의 표면 장력을 수정하여 투과 속도는 12을 변화시킬 수있다. 이 깨끗하고 빠른 프로세스는 고분자막의 벌크 특성을 변경하지 않고 전체 막 표면을 덮 매우 얇은 층 (&lt;5 nm 정도)의 생성을 허용한다. 인하여 플라즈마 공정 동안 테두리에, 트랙 진 멤브레인의 기공 직경은 약간 12 보강. 테두리 비율은 dependi입니다폴리머에 겨와는 선형 동작을. 반응성 관능기를 가진 단량체를 사용하는 경우, 그래프트 폴리머는 추가로 작용 화 될 수있다. 이는 카복실산 작용 스피로 피란와 PHEMA 그래프트 막의 postmodification에 의해 증명된다. UV-광을 조사 할 때 스피로 피란은 메로시 아닌 종으로 변환하는 것으로 알려져 있기 때문에이, 광 변색 표면 초래한다. 스피로 피란 형태는 가시 광선 메로시 아닌 형태 (그림 1) (18, 19)를 조사하여 다시 설정 될 수있다. 메로시 아닌 포름 스피로 피란 상태보다 더 극성이기 때문에, 피막의 표면 장력은 광 (20)로 트리거 될 수있다. 표면 장력의 변화는 수용액쪽으로 막의 투과성 저항에 영향을 미친다. 방법이 빛에 반응 세포막의 투과성 시험을 수행하는 셋업이 표시됩니다 및 투자율 저항의 중요한 변화 (I가 감소97 %로 N 투과성 저항)를 보여줍니다. 이러한 멤브레인은 약물 전달 설정에서 또는 스마트 센싱 시스템에 통합 될 수있다. <img alt="그림 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> 스피로 벤조 피란 화합물 1의 그림 1. 광 이성화.

<table><tbody><tr><td>2-Hydroxyethyl methacrylate, 97%</td><td>Sigma-Aldrich</td><td>128635</td><td></td></tr><tr><td>Hexane 99%</td><td>Biosolve</td><td></td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;, anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methanol, 99%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>14262</td><td>dried over molecular sieves</td></tr><tr><td>N,N-Dicylcohexylcarbodiimide, 99%</td><td>Sigma-Aldrich</td><td>D8002</td><td></td></tr><tr><td>Dimethyl aminopyridine, 99%</td><td>Sigma-Aldrich</td><td>107700</td><td></td></tr><tr><td>Tert-butylmethylether, 98%</td><td>Fluka</td><td>306975</td><td></td></tr><tr><td>Polycarbonate membrane</td><td>Whatman</td><td></td><td>Nanopore Track Etched (TE) (1.0 &amp;mu;m, 0.2 &amp;mu;m, 0.1 &amp;mu;m, 50 nm, 30 nm, and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td></tr><tr><td>Caffeine (reagent plus)</td><td>Sigma-Aldrich</td><td>C0750</td><td></td></tr><tr><td>Franz diffusion cell (12 ml)</td><td>SES-Analysesysteme</td><td>6C010015</td><td>15 mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbar and clamp</td></tr><tr><td>UV-Lamp</td><td></td><td></td><td>UV irradiation (366 nm, 15 W/m&lt;sup&gt;2&lt;/sup&gt;)</td></tr><tr><td>White light lamp</td><td></td><td></td><td>White light irradiation (500 W bulb)</td></tr><tr><td>UV/Vis spectrophotometer</td><td>Varian 50Bio/50MPR</td><td></td><td></td></tr><tr><td>Polyester membranes</td><td>Sterlitech</td><td>PET0225100</td><td>Polyester Membrane Filters, 0.2 &amp;mu;m pore diameter, 25 mm diameter</td></tr><tr><td>Polyvinylidene fluoride membranes</td><td>Millipore</td><td></td><td>PVDF Membranes Durapore (0.22 &amp;mu;m pore diameter; 47 mm membrane diameter)</td></tr><tr><td>Argon (99.9995%)</td><td>Alphagaz</td><td></td><td></td></tr><tr><td>Dressler Cesar RF Power Generator</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>MKS Multi Gas Controller 647C</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>MKS Mass-Flow controllers</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>Vacuubrand RE 2.5 rotary vane vacuum pump</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>Contact angle measurement device</td><td>Kr&amp;uuml;ss</td><td>G10</td><td></td></tr><tr><td>Balances</td><td>Mettler Toledo</td><td>AB204-S and Mettler ME30</td><td></td></tr></tbody></table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

상용 가능한 트랙 진 고분자막의 표면 장력을 수정하기 위해, 표면 개시 중합 절차가 제시된다. 막 표면에서 중합은 2 - 히드 록시 에틸 메타 크릴 레이트 (HEMA)의 메탄올 성 용액으로 막 표면을 반응시켜 다음 멤브레인의 플라즈마 처리에 의해 유도된다. 특별한주의 전에 표면에 중합에 플라즈마 처리를위한 처리 파라미터에 관련된다. 예를 들어, 멤브레인의 다른 유형 (예를 들면 폴리 에스텔, 폴리 카보네이트 (polycarbonate), 폴리 불화 비닐 리덴)의 플라즈마 처리의 영향이 연구된다. 또한, 표면 그래프트 막 시간 의존 안정성 접촉각 측정에 의해 도시된다. 이런 방식으로 폴리 (2 - 히드 록시 에틸 메타 크릴 레이트) (PHEMA)를 이식 할 때, 표면은 또한 원하는 물질 카복실산 기능을 가진 중합체의 알코올 잔기의 에스테르 화에 의해 변형 될 수있다.이들 반응 따라서 막 표면의 기능화를 위해 사용될 수있다. 예를 들어, 멤브레인의 표면 장력은 변화 될 수 있거나 제시 광 응답 성 등의 원하는 기능이 삽입 될 수있다. 이것은 광 반응성 막에 이르게 카복실산 작용 스피로 벤조 피란 유닛 PHEMA 반응에 의해 증명된다. 용매의 선택은 postmodification 단계에서 중요한 역할을하고이 글에서 자세히 설명합니다. 그러한 기능화 막의 투과도 측정은 외부 광원 프란츠 셀을 사용하여 수행된다. UV 범위에 가시에서 광의 파장을 변화시킴으로써 수성 카페인 해법의 투자율의 변화가 관찰된다.

에칭 속도는 시간의 다른 기간 후에 막을 계량 선행 될 수있다. 도 4로부터 알 수있는 바와 같이, 에칭 율은 폴리 에스테르, 폴리 불화 비닐 리덴에 대한 다음과 폴리 카보네이트 질량 손실 대 에칭 시간 선형 상관의 기울기로부터 결정될 수 선형 에칭 율을, 세포막. 도 4에 도시 된 바와 같이, 폴리 카보네이트 멤브레인은 모든 세 개의 폴리머 막의 낮은 에칭 속도를 ...

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

플라즈마 유도 중합 조작은 고분자막의 표면에 중합 개시에 기술된다. 광 변색 물질과 이식 고분자의 추가 postmodification는 빛에 반응 세포막의 투과성 측정을 수행하는 프로토콜을 제시한다.

결합 표면의 접목 및 Postmodification 과정에 의한 빛의 응답 멤브레인의 제조

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.7K 조회수.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  플라즈마 유도 중합 조작은 고분자막의 표면에 중합 개시에 기술된다. 광 변색 물질과 이식 고분자의 추가 postmodification는 빛에 반응 세포막의 투과성 측정을 수행하는 프로토콜을 제시한다. 

비디오: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

마이크로 광 및 전기 방사의 조합은 각막 재생을위한 틈새를 장착 멤브레인을 생산하는

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

연구

생체공학

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

브러쉬 모양의 반응 표면 날조 및 Azlactone 기능성된 블록 공동 고분자의 가교 된 영화

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

공학

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

산화 티타늄을 사용하여 수성 환경에서 단백질과 세포를 포토 패터닝<sub&gt; 2</sub&gt; 광촉매

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

용매를 이용한 지질 이중층 (SALB) 방법에 의해 생체막 제작

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

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 ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

폴리 디메틸 실록산 박막을 사용하여 자동화 지질 이중층 제막

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

생체 물리학 적 특성과 기능을 연구하기 위해 멤브레인에서 Septin 어셈블리의 재구성

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

생화학

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

단일 분자 확산 및 폴리머가 밀집된 지질 막에서의 조립

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

G 단백질 결합 수용체 (GPCRs)를 통합 한 모델 지질 막의 건설

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...