DCH, MB와 FK는 기금에 EPSRC 사후에 의해 지원되었다. BL-V와 DS는 각 MRC의 박사 studentships에 의해 지원되었다. KC는 영국 재생 의학 플랫폼에서 자금에 의해 지원되었다.

PHNGAD (폴리 [1,6-hexanodiol / 네오 펜틸 글리콜 / 디 (에틸렌 글리콜) -alt - 아 디프 산] 디올)의 1 합성 <img alt="반응식 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> 반응식 1 : PHNAGD의 합성 PHNAGD의 합성의 도식 표현.. PHNAGD는 1,6-Hexanodiol의 반응, 디 에틸렌 글리콜, n​​eoppentyl 글리콜 및 아 디프 산을 제조 하였다. PHNAGD, 폴리 [1,6-hexanodiol / 네오 펜틸 글리콜 / 디 (에틸렌 글리콜) -alt - 아 디프 산] 디올. <ol><li> 잔류 물을 제거하기 위해 진공 오븐에서 48 시간 동안 40 ° C에서 단량체 1,6 - 헥산 디올, 디 (에틸렌 글리콜) 및 네오 펜틸 글리콜로 열처리를 적용. 진공 상태에서 RT까지 차가운 할 수 있습니다. </li><li> 딘 - 스탁 장치에 교반 막대가 장착되고 연결의 2 구 둥근 바닥 플라스크에 각각의 단량체 및 아 디프 산 (55 밀리몰)의 22 밀리몰 추가. </li><li> 진공 상태에서 전체 어셈블리를 놓고 부드럽게 GLA를 가열플라스크에 화학 물질의 첨가 중에 흡습을 방지하기 위해 6 시간 동안 40 ° C의,시 ssware. </li><li> 드롭에 의해 촉매 티타늄 (IV) 부톡 사이드 0.0055 몰을 드롭 주사기를 통해 추가합니다. </li><li> 24 시간 동안 N 2 분위기에서 180 ° C에서 반응 혼합물을 교반하고, 딘 - 스타크 트랩에서 잔류 물을 수집한다. 제품이 RT로 냉각시킵니다. </li></ol> PU134의 2 합성 <img alt="계획이" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" /> 반응식 2 : PU134의 합성 PU134가 4,4 메틸렌 비스 (페닐 이소시아네이트) 2.0 당량과 PHNGAD 1.0 당량의 반응에 의해 제조 된 폴리 우레탄 (134)의 합성 도식 표현, 1.0을 첨가 하였다. 1,4 - 부탄디올 사슬 연장 제의 당량. <ol><li> (4) 2 당량과 폴리올 PHNGAD 1 당량 (Mn이 1,800 ~ DA, 3.2 밀리몰)을 혼합무수 N에서 &#39;4 메틸렌 비스 (페닐 이소시아네이트) (6.4 밀리몰), N 디메틸 포름 아미드 (12 mL)을 첨가 하였다. </li><li> N 2 분위기 하에서 70 ° C에서 반응 혼합물을 저어. </li><li> , 주사기를 통해 추가 촉매 티타늄 드롭 들러 (IV) 부톡 사이드 (0.8 중량 %). </li><li> 1 시간 후, 사슬 연장 1,4 - 부탄디올 (3.2 밀리몰)의 1 당량을 추가한다. 90 ° C의 온도를 높이고 N 2 분위기 하에서 24 시간 동안 교반한다. </li><li> 반응에 이어, 침전이 일어날 때까지 반응 용액에 적가 하였다 (도 사용될 수 헥산 또는 물), 디 에틸 에테르를 첨가하여 침전에 의해 폴리 우레탄을 모은다. </li><li> 5 분 동안 5,300 XG에 용액을 원심 분리기. </li><li> 뜨는을 가만히 따르다 용매가 증발 할 때까지 진공 오븐에서 40 ° C에서 떨어져 건조. 주 : 위해 반응의 최종 생성물은 분자량 분포에 관한 정확한 파라미터를 갖고 있는지 확인하기 위해, 기능적 GROU중합체 및 융점 및 유리 전이 온도의 PS는 다양한 분석 기술 및 방법은, 겔 투과 크로마토 그래피 또는 피트 르 분광법으로 사용될 수있다. </li></ol> PU134 솔루션의 3 준비 <ol><li> 유리 병에 PU134의 200 mg의 무게를 측정한다. </li><li> 클로로포름, 1 클로로포름과 톨루엔의 조합 : 용매의 수가 2 %의 최종 농도로 희석 한 PU134 비율, 테트라 히드로 푸란과 조합 한 테트라 히드로 푸란과의 dicloromethane : 1 비율. 참고 : 오른쪽 용매의 선거 사용할 수있는 폴리머에 따라 달라집니다. 다른 용매는 중합체의 용해에 영향을 미칠 수있는 다른 비등점을 갖는다. </li><li> 용액을 균질하게하고 침전물이 관찰되지 않을 때까지, 200 MOT / 분의 속도로 진탕을 사용하여 RT에서 20 분 동안 격렬히 흔들어 용액. </li></ol> PU134 유리 슬라이드의 4 코팅 <ol><li> 알자 배치스핀 코터에 ND 15mm이 커버 슬립. </li><li> 피펫을 사용하여 각 PU134 용액 50 μl를 적용합니다. 비율에 비례하는 양 표면을 유지해야 커버 슬립 크기에 따라 PU134 용액의 양을 조정한다. </li><li> 23의 X g에서 7 초 동안 각 coverslip에 스핀. </li><li> 멸균하기 전에 최소 24 시간 동안 실온에서 공기 건조 커버 슬립. </li></ol> 커버 슬립의 5 조사 <ol><li> 12 분 동안 실험실 조사 장치를 사용하여 회색의 열 용량을 적용하여 중합체 코팅 된 커버 슬립을 감마 - 조사한다. </li><li> 16 분 각 팀의 30 W, UV 전구를 사용하여 폴리머 코팅 된 커버 슬립을 조사 자외선. </li><li> 슬라이드 크기에 따라 적합한 조직 배양 접시에 고분자 코팅 된 커버 슬립을 놓습니다. </li></ol> 6 주사 전자 현미경 <ol><li> 압력이 5 × 10-1 밀리바의 분위기에서 200 초간 스퍼터링에 의해 금 코트 폴리머 커버 슬립. </li><li> microg 캡처이차 전자 촬상 모드에서의 20 kV의 가속 전압에서 주 사형 전자 현미경을 사용하여 코팅 된 커버 슬립의 중합체 raphs. </li></ol> 7 원자 힘 현미경 관찰 <ol><li> 중합체 표면의 20 × 20 ​​μm 인 영역을 스캔. </li><li> 1.32 Hz에서에서 1.60 Hz로 스캔 속도를 설정합니다. </li><li> 스캔 한 지역에서 512 X 512 픽셀의 해상도를 설정합니다. </li><li> 루트 평균 화상 데이터 플레인에서 찍은 높이 편차의 평균을 이용하여 코팅의 제곱 (RMS 또는 자승)를 계산 의미로 표현 : <img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" /> 여기서 닫아 현재 Z 값이며, N은 주어진 지역 내 점의 수이다. </li><li> 이용한 화상의 7.5 편차를 계산하거나, 표면 평균 거칠기 (라) <img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" /> 어디 Z (X)는 t을 설명하는 기능입니다그 높이 (Z) 및 평가 길이 &quot;L&quot;을 통해 샘플의 위치 (X)의 관점에서 분석 프로파일 표면. RA가 중심면에 대하여 표면의 평균 값을 나타낸다. </li></ol> 8 세포 배양 및 분화 <ol><li> 농경 건초 및 17에 기재된 바와 같이 인간 배아 줄기 세포주 (hESC의) H9 차별화. </li><li> 해리 시약을 이용하여 세포를 분리하고 (18,19)에 대해 설명 Szkolnicka으로 무 혈청 배지의 존재하에 분화 과정의 일 9시 PU134 코팅 슬라이드 위에 그들을 replate. 주 : 효소 적 세포 해리의 사용은 물리적 셀 박리하는 것이 바람직하다. </li></ol> 9 사이토 크롬 P450 기능 분석 <ol><li> 제조업체의 지침에 따라 CYP3A 활동을 측정한다. </li><li> 세포없이 hESC의 파생 일 13 일 19에서 간세포, 미디어를 품어 - 음성 대조군으로, 적절한 SUBSTR로37 ° C에서 5 시간 동안 먹었다. </li><li> , 셀 및 미디어에서 상층 액을 수집 제조업체의 지침에 따라 분석을 수행한다. </li><li> 표면적 (cm 2) ~ CYP 활성 및 정규화의 상대적인 수준을 측정한다. </li></ol> (10) 면역 염색 <ol><li> 워시 hESC의 1 분 동안 PBS로 간세포를 유도, 두 번 반복합니다. </li><li> 10 분 동안 -20 ° C에 차가운 얼음 백퍼센트 세포를 해결하기 위해 메탄올, 장소를 추가합니다. </li><li> 5 분 동안 PBS로 세포를 씻으 두 번 반복합니다. </li><li> PBS / T (0.1 % 트윈) RT에서 1 시간 / 10 % BSA와 함께 세포를 배양한다. </li><li> PBST 솔루션을 기음과 PBS / T (0.1 % 트윈) / 1 % BSA에 희석 적절한 일차 항체를 추가, 부드러운 교반 O / N로 4 ° C에서 배양한다. </li><li> 5 분 동안 PBS / T (0.1 % 트윈) / 1 % BSA와 세포를 세척하고 세 번 반복합니다. </li><li> , PBS / T (0.1 % 트윈) / 1 % BSA에 적합한 항체를 희석하여 세포에 추가하고 교반과 함께 1 시간 동안 RT에서 어두운 곳에서 배양. </li><li> 5 분 동안 PBS로 세포를 세척하고 세 번 반복합니다. </li><li> 각 웰에 기포의 수를 줄이기 위해 부드럽게 커버 슬립을 추가 (1000 DAPI 일을 포함) MOWIOL 488를 추가합니다. 스토어는 어둠 속에서 4 ° C에서 세포를 고정. 적절한 필터 및 형광 램프 현미경을 사용하여 염색 관찰한다. 최적화 된 일차 및 이차 항체는 표 1에 나열되어 있습니다. </li></ol>

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Vitronectin+Is+a+Functionally+Defined+Substrate+That+Supports+Human+Embryonic+Stem+Cell+Self-Renewal+via+&#945;V&#946;5+Integrin.">Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via &#945;V&#946;5 Integrin.</a> Stem Cells. 26 (9), 2257-2265 (2008).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+culturing+of+human+embryonic+stem+cells+on+laminin-521/E-cadherin+matrix+in+defined+and+xeno-free+environment.">Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.</a> Nature Communications. 5 (3195), (2014).</li><li>Thaburet, J. -. F. O., Mizomoto, H., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Evaluation+of+the+Wettability+of+Polymer+Libraries.">High-Throughput Evaluation of the Wettability of Polymer Libraries.</a> Macromolecular Rapid Communication. 25 (1), 336-370 (2003).</li><li>Lim, J. Y., Donahue, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Sensing+and+Response+to+Micro-+and+Nanostructured+Surfaces+Produced+by+Chemical+and+Topographic+Patterning.">Cell Sensing and Response to Micro- and Nanostructured Surfaces Produced by Chemical and Topographic Patterning.</a> Tissue Engineering. 13 (8), 1879-1891 (2007).</li><li>Teixeira, A. I., Abrams, G. A., Bertics, P. J., Murphy, C. J., Nealey, P. 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J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Dang, J. M., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+Induction+of+Aligned+Mesenchymal+Stem+Cell+Sheets+by+Culture+on+Thermally+Responsive+Electrospun+Nanofibers.">Myogenic Induction of Aligned Mesenchymal Stem Cell Sheets by Culture on Thermally Responsive Electrospun Nanofibers.</a> Advanced Materials. 19 (19), 2775-2779 (2007).</li><li>Azevedo, E. C., Nascimento, E. M., Chierice, G. 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DCH는 CSO, FibromEd 제품 유한 회사의 이사, 설립자 FibromEd 제품 유한 회사 MB와 JPI의 주주 아르 설립자 주주입니다

줄기 세포로부터 간세포를 생성하는 데 사용되는 현재의 방법의 대부분은 동물 유래의 미정 행렬에 의존한다. 이러한 기판은 어플리케이션에 상당한 장벽을 나타내는 세포의 기능과 안정성에 영향을 미치는, 비싸고 매우 가변적 일 수있다. 따라서, 우리는 줄기 세포 유래 간세포의 문화를 지원하는 합성 물질에 대한 화면을 수행 하였다. 우리가 식별 한 견고한 간세포 분화 방식과 조합 PHNGAD, MDI ...

생물학적 물질은 널리 능성 줄기 세포의 한 유지 및 분화에 사용되어왔다. 가능하지만, 이러한 생물학적 기질은 종종 정의되지 않은 구성 요소의 무수가 포함되어 있습니다. 마트 리겔은 줄기 세포 배양 및 분화를위한 일반적으로 사용되는 기판이다. 불행하게도, 그 변수 조성물은 세포 표현형 및 기능에 영향을 미친다. 대안 이상의 정의 된 생물학적 매트릭스들의 다양한 2-7을 사용되었지만, 그들의 동물성 또는 나쁨 확장 그들에게 산업 제조에 부적합한 후보를 만든다. 따라서, 줄기 세포 연구의 주요 목표는 정의 된 구성과 신뢰할 수있는 성능으로 합성 대안의 식별이다. 정의되지 않은 세포 배양 기판의 한계를 극복하기위한 시도에서, 화학, 생물학 학제 간의 협력은 세포 표현형을 지원하는 능력과 합성 물질을 확인했다. 신디etic 기판은, 비용 효율적인 확장 성, 그리고 생체 내 환경을 흉내 낸, 복잡한 3 차원 구조로 제조 될 수있다. 이러한 특성으로 인해, 합성 기판은 널리 다양한 유형의 세포의 분화 8-10을 지원하고 구동하기 위해 사용되어왔다. 고급 및 높은 처리량 분석 대형 도서관에서 합성 물질의 신속한 심사를 촉진하고, 생물 의학 연구 및 개발 11-13에서 다양한 응용 프로그램과의 유연한 특성을 가진 새로운 물질을 전달했다. 높은 처리량, 고분자 마이크로 어레이 검사 기술을 활용, 우리는 빠른 속도로 인간의 줄기 세포 유래 간세포의 유지 보수에 적합한 간단한 폴리 우레탄 (PU134)를 확인했다. 이 중합체는 간세포 분화와 기능 14-16와 관련하여 동물 유래의 기재에 우수한 것으로 밝혀졌다. 우리는 이후 효과를 액세스하는 도포 조건, 지형 및 살균 처리를 최적화 한간세포 기능 및 수명을 안정화 중합체 성능. 이 세포 기반의 모델링 및 재생 의학 응용 프로그램에 대한 간세포 생물학의 기초를 이해 관련하여 중요한 의미를 갖는다. 여기에 설명 된 기술은 합성 중합체의 표면이 세포 표현형을 유지하도록 최적화 될 수있는 방법의 예를 나타낸다. 우리는 효율적인 무 혈청 간세포 분화 프로토콜이 기술의 조합이 체외 모델링 및 재생 의료에 사용하기위한 간세포의 생산 확장을 제공하는 잠재력을 가지고 있다고 믿는다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmun B&#252;hler</td>
			<td>KS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>CIS Biointernational</td>
			<td>IBL 637&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating System</td>
			<td>&#160;P-6708</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope&#160;</td>
			<td>Philips</td>
			<td>XL30CPSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope</td>
			<td>DimensionV Nanoscope, VEECO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p4-GLO CYP3A4</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>UV bulb</td>
			<td>ESCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoScope analysis software</td>
			<td>VEECO</td>
			<td></td>
			<td>version 1.20</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>TH45200</td>
			<td>Use Volocity 4 Software</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Corning, UK&#160;</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Scientific Laboratory Supplies</td>
			<td>MIC3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylene glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>93171</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;1,6-hexanediol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>240117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neopentyl glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>408255</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrous N,N-Dimethylformamide</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>676845</td>
			<td></td>
		</tr>
		<tr>
			<td>titanium (IV) butoxide&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>244112</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-butanediol&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>493732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#8217;-Methylenebis(phenyl isocyanate)</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>101688</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofurane</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Bal-Tec SCD 050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Inmunostaining</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (-MgCl2, -CaCl2)</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>&#160;Store at room temperature</td>
		</tr>
		<tr>
			<td>PBST, PBS made up with 0.1% TWEEN 20&#160;</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>EC607&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>CHE5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich, UK</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>MOWIOL 488 DAPI</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>Made up in Tris HCL and glycerol as per manufacturers instructions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell culture and Functional assay</td>
		</tr>
		<tr>
			<td>CYP3A activity pGLO kit</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatozyme</td>
			<td>Gibco</td>
			<td>17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>TryLE express</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/12604013">12604013</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

고분자 용매 중합체 코팅 표면의 지형에 영향 폴리 우레탄 (134)은 단독으로 또는 톨루엔 또는 테트라 하이드로 퓨란 또는 디클로로 메탄과 유리 슬라이드가 다양한 제형으로 스핀 코팅과 조합하여, 클로로포름에 용해시켰다. 주사 전자 현미경 (SEM) 및 원자력 현미경 (AFM)을 (도 1) 중합체 코팅의 물리적 성질을 특성화하기 위해 사용되었다. 톨루엔, 클...

Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

이 문서는 장기적 폴리머 코팅 된 표면을 개발에 초점을 맞출 것이다, 줄기 세포의 안정적인 문화는 인간의 간세포를 산출했다.

최적화 된 합성 표면을 간세포 표현형 사용 안정화

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

10.2K 조회수.  University of Edinburgh.  이 문서는 장기적 폴리머 코팅 된 표면을 개발에 초점을 맞출 것이다, 줄기 세포의 안정적인 문화는 인간의 간세포를 산출했다. 

비디오: Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

공유 결합은 자기 조립 단분자막의 접근 방식을 사용하여 표면에 BMP-2의 결합

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

연구

화학

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Nanocellulose, 리그닌의 날조 된 자립형 필름, 및 합성 폴리 양이온 : Biomimicking 나무를 향하여

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

대량 선택 이온 소프트 랜딩에 의해 준비 잘 정의 된 표면의 제자리 SIMS 및 IR 분광법

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

고상 수지 기반의 방법론을 통해 비대칭 페로센 유래 바이오 복합체 시스템 용 합성 방법론

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

합성 단백질의 최적화 : Interpositional 종속 관계 확인이 구조적으로 표시 및 / 또는 기능적으로 연결된 잔류

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

를 사용하여 금 나노 입자의 효율적인 방사성 표지를위한 최적화 된 프로토콜<sup&gt; (125)</sup&gt; I-표시 아 지드 보철 그룹

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

색상 합성 Cathinones의 급속 한 탐지에 대 한 추정 도구로 자리 테스트

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

매트릭스를 통해 합성 고분자의 특성화 지원 레이저 탈 착 이온화 시간의 비행 (TOF MALDI) 질량 분석

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

빛 주도 분자 모터 표면에 단일 분자 이미징

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

대장균-셀 무료 단백질 합성 기반: 강력 하 고 유연 하며 액세스할 수 있는 플랫폼 기술에 대 한 프로토콜

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...