이 작품은 JLI과 국립 연구소 히스 부여 R01AR056624의에 미국 심장 협회 Predoctoral 원정대에 의해 지원되었다.

A. 단일 폴리머 Electrospinning <ol><li> 전에 electrospinning 솔루션을 준비하기 위해 몇 일 동안 37 ° C를 용해하여 탈이온수에 photoinitiator의 0.5 % WT의 솔루션, Irgacure 2959 (I2959)를 확인하십시오. photoreactive 폴리머가 사용되고 있지 않으면이 단계는 필요하지 않습니다. </li><li> 이 WT % MeHA 3 WT %의 최종 농도와 솔루션을 준비하는 methacrylated hyaluronic 산 (MeHA, 버딕 외를 참조하십시오. 합성), 폴리 (에틸렌 옥사이드) (PEO, 900 KDA), 그리고 탈이온수에 I2959을 결합 PEO, 그리고 0.05 % WT의 I2959. 그것이 명확 때까지 솔루션을 혼합하는 소용돌이를 사용합니다. 폴리머 종류와 농도뿐만 아니라 용제 사용은 원하는 발판 특성에 따라이 단계에서 수정할 수 있습니다. </li><li> 주사기로 솔루션을 전송하고 끝까지 18 게이지, 길이 6 인치, 뭉툭한 끝 바늘을 첨부합니다. </li><li> 1.2 ML / HR의 속도로 추출 프로그램에 주사기 펌프. 장치에 주사기와 바늘을 삽입합니다. </li><li> 수집 장치에 높은 전압 전원의 접지 리드를 연결합니다. 무작위로 배포 섬유가 평면 금속 접시를 사용하여 수집하거나, 정렬 섬유를 수집하는 ~ 10m / s의 회전 심봉를 사용할 수 있습니다. 바늘에 긍정적으로 부과 리드를 연결합니다. electrospinning 장치의 구조도 1 그림을 참조하십시오. </li><li> 둘 사이에 15cm 거리가되는 등 바늘이나 수집 장치를 조정합니다. </li><li> 주사기 펌프의 흐름을 시작합니다. 액체가 바늘 끝 시각 경우, 전원 켜고 22 KV로 전압을 설정합니다. </li><li> 컬렉션 (박막 몇 분 거리에 두꺼운 매트를 위해 최대 24 시간) 완료 후 수집 장치에서 비계를 제거하고 그것은 용매의 완전한 제거를 위해 밤새 진공 아래에 저장합니다. </li><li> 스캐닝 전자 현미경 (SEM)를 사용하여 섬유 형태를 시각화, 대표 발판 양쪽 정렬이 아닌 정렬된 구조에 대한 그림 1에 표시됩니다. </li></ol> 참고 : 샘플 유량, 수집 장치로 거리와 전압이 폴리머와 용매 조합에 의존하고 있으며 일반적으로 SEM과 발판의 형태를 관찰하여 각 시스템에 최적화된해야합니다. B.의 Photocrosslinking 및 Photopatterning <ol><li> 발판 매트에서 5mm X 5mm 샘플을 잘라 버릴거야. </li><li> 유리 슬라이드를 덮고 호일에있는 각 발판을 놓는 발판에서 직접 photomask를 삽입, 깨끗한 유리 슬라이드로 덮개와 바인더 클립 양쪽을 클리핑하여 crosslinking 준비. 참고 : 네 샘플을 한번에 할 수 있으며, 패턴이 원하는되지 않은 경우 패턴이없는 투명성이 crosslinking 사용할 수 있습니다. </li><li> 정리 발판은 질소 챔버에 설정할 수 있습니다. crosslinking을 억제 할 수 산소에서 만들 무료 유지하는 것이 중요합니다. </li><li> ~ 10 MW / cm 5 분 collimating 어댑터 2 365 NM 조명 아래에서 질소 챔버에 넣어 비계 설치. 그림 2에서 설계도를 참조하십시오. </li><li> 각각의 발판과 12 잘 접시에 장소를 제거합니다. </li><li> 각 음, parafilm 37 24 시간 동안 ° C의 물 장소 증발을 방지하기 위해 접시에 탈이온수 2 ML을 추가합니다. 물이 세 번 변경합니다. </li><li> 가벼운 현미경 기공 형성 (그림 2의 예제를 참조) 시각화. 공사장 공중 발판을 이제 사용할 준비가되어 있습니다. </li></ol> 참고 : Photocrosslinking는 고분자의 여러 유형, 그러나 photopatterning에만 photoreactive 폴리머와 함께 사용할 수있는 필요하지 않습니다. 형광 광섬유 시각화와 C. 듀얼 폴리머 Electrospinning <ol><li> 90 % 에탄올에 PEO (200 KDA)의 5 % WT의 솔루션을 준비합니다. 50 ° C 이전 electrospinning 최소한 2 시간 동안 700 rpm으로 저어. </li><li> 주사기로 PEO 솔루션을 전송하고 electrospinning 솔루션 10 MG / ML 농도의 최종 농도에 대한 DAPI를 추가합니다. 빛으로부터 보호하기 위해 알루미늄 호일에 주사기를 넣어. </li><li> electrospinning 솔루션에 25 μm의의 최종 농도에 대한 methacryloxyethyl thiocarbamoyl rhodamine B을 (MeRho, 683.24 g / 몰) 추가는 제외하고, 단계 A2에서 설명하는 동일한 솔루션을 준비합니다. 빛으로부터 보호하기 위해 알루미늄 호일에 주사기를 넣어. </li><li> 조심스럽게 심봉의 표면에 methacrylated 유리 coverslips을 (Khademhosseini 외를 참조하십시오.) 보안에 스카치 테이프를 사용합니다. 참고 : 섬유 methacrylated 유리 coverslips에 닿는 될 것입니다. </li><li> luer 잠금 첨부 파일이있는 주사기 중 하나에 실리콘 튜브의 5mm 긴 조각의 한쪽 끝을 연결하고 바늘로 다른 쪽 끝을 연결합니다. 주사기 펌프에 주사기를 삽입하고 부채질하는 사람에있는 구멍을 통해 바늘을 넣어. 심봉 반대 사이트에서 다른 주사기와 부채질하는 사람과 함께 반복합니다. fanners는 리터 번역심봉의 ength하며 결과 발판 두 고분자의 평등한 분배를 보장하기 위해 사용됩니다. 그림 3에 회로도를 참조하십시오. </li><li> 적절한 표 1에 따라 섹션에 설명된 매개 변수를 조정합니다. 바늘 팁이 심봉 중심 있는지 확인합니다. 표 1. 듀얼 폴리머 Electrospinning 매개 변수 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 주사기 </td><td> 부채질하는 사람으로 니들 팁 (cm) </td><td> 심봉 (CM)에 니들 팁 </td><td> 유량 (ML / HR) </td><td> 적용 전압 (KV) </td></tr><tr><td> MeHA </td><td> 6 </td><td> 15 </td><td> 1.2 </td><td> 22 </td></tr><tr><td> PEO </td><td> 6 </td><td> 10 </td><td> 1.2 </td><td> 15 </td></tr></tbody></table> </li><li> 모든이 제대로 정렬되면 불을 끄고 심봉과 주사기 펌프를 켭니다. 주사기에서 알루미늄 호일을 제거합니다. 유체는 두 바늘의 끝부분에 볼 때, 동시에 fanners의 전원 공급 및 플러그 전원을 켜십시오. </li><li> 수집이 완료되면 전원 공급 장치 및 심봉을 해제하고 fanners를 뽑습니다. 조심스럽게 면도날을 사용하여 coverslips와 테이프를 제거합니다. </li><li> rhodamine 및 DAPI에 대한 필터가 장착된 형광 현미경을 사용하여 섬유를 시각화. 그림 3의 예제를 참조하십시오. 참고 : 시간 ​​(&lt;3 분)의 짧은 기간 동안 electrospun는하지만,이 과정은 특히 유리 coverslips를 사용하지 않고, 두꺼운 구조를 만들려면 시간의 무제한 연장 수있을 때 볼 수있는 형광 섬유는 대부분 명확하다 . </li></ol> 공사장 공중 발판 D.의 심는 셀 <ol><li> 개별로 잘 적절하게 크기 잘 접시의 첨부 electrospun 폴리머 각 coverslip를 놓습니다. 참고 : 섬유와 세포 상호 작용이 methacrylated 유리 coverslips에 electrospinning로 쉽게 볼 수 있습니다. 또한, MeRho 다시 섬유 시각화에 사용할 수 있습니다. </li><li> 용매의 완전한 제거 및 부산물 가능한 모든 leachable을 보장하기 위해 하룻밤 PBS에서 공사장 공중 발판을 품어. </li><li> 우물에서 PBS를 제거합니다. </li><li> 공사장 공중 발판을 소독하기 위해 30 분 동안 층류 후드에 살균 램프 하에서 그들을 놓으십시오. 전체 발판이 씨앗을하는 경우, 또 다른 30 ​​분 동안 살균 램프 아래 위에 발판과 장소를 플립. </li><li> 표준 세포 배양을 수행하고 (예 : 6000 전지 cm -2) 원하는 세포 밀도에 집중 세포 현탁액을 준비합니다. 예를 들어, 22mm X 22mm의 coverslip에 대한 세포 현탁액의 100 ML을 사용합니다. 1 시간 동안 인큐베이터에 놓습니다. </li><li> 각 잘하기 위해 세포 배양 매체의 적절한 금액을 추가합니다. 인큐베이터에 다시 공사장 공중 발판을 놓으십시오. </li><li> 상업적으로 사용 가능한 라이브 / 데드 키트를 사용하여 세포를 얼룩. 참고 사항 : DAPI (세포 핵)과 찬란 라벨 phalloidin (굴지 스트레스 섬유)와 같은 세포 얼룩의 다른 유형의 세포를 시각화하기 위해 적용할 수 있습니다. </li><li> 세포 TRITC 및 FITC에 대한 필터가 장착된 형광 현미경을 사용하여 섬유를 시각화. 그림 4의 예제를 참조하십시오. </li></ol> 대표 결과 : <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="그림 1" /> 그림 1. 비 - 정렬 발판 형성 (상단)과 정렬 발판 형성 (아래)의 장치 설정을 보여주는 도식. 발판의 각 유형의 예 스캐닝 전자 현미경 이미지는 표시됩니다. 스케일 바 = 5μm. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="그림 2" /> 그림 2. patterning 방식의 도식. 패턴은 photocrosslinking 동안 광원과 발판 사이 photomask을 배치하고 멀리 unreacted 폴리머를 세척하여 형성됩니다. 기공 형성과 lyophilization 후 공사장 공중 발판의 Photomask 및 SEM 이미지. 스케일 바 = 100 μm의. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">여기를 클릭</a> 그림 2의 더 큰 버전을 볼 수 있습니다. <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="그림 3" border="0" /> 그림 3 여러 고분자 electrospinning 설정하고 필요한 처리 매개 변수의 도식뿐만 아니라,이 섬유 집단 (예 : 빨간색 MeHA 및 파랑 PEO)의 혼합의 대표적인 형광 이미지. 멀티 폴리머 비계를 형성하는 동시에 electrospun되었습니다 . 스케일 바 = 100 μm의. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">여기를 클릭하십시오</a> 큰 versio를보고그림 3 N. <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="그림 4" /> 그림 4. 인간 mesenchymal 줄기 세포와 electrospun 섬유와의 상호 작용의 예를 들어 데드 / 라이브 얼룩 이미지. 스케일 바 = 100 μm의. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">여기를 클릭</a> 그림 4의 더 큰 버전을 볼 수 있습니다.

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

Electrospinning는 고분자의 섬유 공사장 공중 발판을 준비하는 데 사용되었다. Photocrosslinkable 빛의 노출이 crosslinking에 대해 필요한 설명의 예제로 사용되었던 hyaluronic 산성에 기반 공사장 공중 발판. 같은 MeHA 같은 반응 macromers의 사용으로 이전에 향상된 세포 분포를 보여준 채널은 매크로와 마이크로 다공성 공사장 공중 발판을 형성 photocrosslinking 동안 마스크를 사용하여 공사장 공중 발판에 통합되?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

조직 공학 분야는 진화로 더 복잡한 장기와 조직의 요구 사항 (예, 기계 부품과 vascularity) 주소 위해 더 적합한 소재 및 가공 기술을 생산하기 위해 엄청난 수요가있다. Electrospinning는 원래 세포외 기질의 구조 및 크기의 규모를 모방 섬유 공사장 공중 발판을 만드는 인기있는 기술입니다. 섬유 줄기 세포 분화 (Mauck하여 광범위한 리뷰보기 포함한 세포 동작을 직접하는 데 사용될 수 있기 때문에 이러한 섬유 공사장 공중 발판은 또한 세포 배양 기판으로 유용합니다<em&gt; 외.</em&gt; 및 실<em&gt; 외.</em&gt; 자세한 내용). 이 문서에서는, 우리는 폴리머를 electrospinning의 일반적인 과정을 설명하고 예를 들어, electrospin 가벼운 노출과 crosslinking 수있는 반응 hyaluronic의 산성 (참조 Ifkovits<em&gt; 외.</em&gt; photocrosslinkable 자료에 대한 검토를 위해). 우리는 또한 photopatterning 및 멀티 폴리머 비계 형성과 같은 추가 처리 능력을 소개합니다. Photopatterning는 세포 침투와 조직 분포를 높이기 위해 채널과 다중 스케일 다공성과 공사장 공중 발판을 만드는 데 사용할 수 있습니다. 다중 폴리머 공사장 공중 발판을 더 나은 세포 침투를위한 맞춤형 다공성 포함한 비계의 속성을 (역학 및 파괴), 조정에 유용합니다. 또한, 이러한 기술은 성공적인 조직 설계 구조의 개발에 필요한 단서를 제공 복잡한 공사장 공중 발판을 만드는 폴리머와 반응 macromers의 다양한 배열을 포함하도록 확장될 수 있습니다.

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

조직 공학 및 세포 배양을위한 폴리머를 electrospinning의 과정은이 문서에서 해결되었습니다. 특히 photopatterning 멀티 고분자 electrospinning의 추가 처리 능력과 photoreactive macromers의 electrospinning이 설명되어 있습니다.

조직 공학 및 세포 배양을위한 섬유 고분자의 공사장 공중 발판을 Electrospinning

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

Research

JoVE Journal

Biology

21.4K 조회수.  University of Pennsylvania. 조직 공학 및 세포 배양을위한 폴리머를 electrospinning의 과정은이 문서에서 해결되었습니다. 특히 photopatterning 멀티 고분자 electrospinning의 추가 처리 능력과 photoreactive macromers의 electrospinning이 설명되어 있습니다.

비디오: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

조직 공학 실험 접근법

연구

생물학

Micro-scale Engineering for Cell Biology

세포 생물학을위한 미세 규모 공학

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

입체 세포 배양을위한 칩 크기의 공사장 공중 발판의 Microfabrication

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

인터뷰 : 줄기 세포 연구에 대한 Bioreactors와 분수 - 수정된 3D - 공사장 공중 발판

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

공학 세포 투과 단백질

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

뉴트의 렌즈 재생을 공부 Culturing 이리스 안료 상피 세포에 대한 시스템

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

향상된 Biosafety에 대한 고분자 - complexed Ecotropic Lentivirus과 인간의 세포의 형질 도입

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

세포 배양의 유도와 Hypoxia의 시험

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...