이 작품은 플로리다 국제 대학의 생명 공학 부서의 기능을 사용하여 실시 하였다.

1. 전기 방사 수집기 설치 <ol><li> 사각형 조각으로 알루미늄 호일을 잘라. </li><li> 직사각형의 스트립에 사각형 조각을 접어 테이프로 평평한 금속판 (그림 1)에 수직으로 장착. 주 : 섬유 매트의 사이즈는 스트립의 길이 및 폭에 따라 다르다. 필요에 따라서, 스트립의 크기는 조정될 수있다. </li></ol> 2. 고분자 용액의 제조 <ol><li> PGD​​ 폴리머를 얻기 위해 100 시간 동안 120 ° C에서 비커 1:1 몰비 글리세롤과 도데 산 (DDA)를 섞는다. </li><li> 15 ML 튜브에 1.5:3:95.5의 중량 비율로 65 % 에탄올에 폴리 (에틸렌 옥사이드) (PEO)와 젤라틴을 녹여 뚜껑을 강화하고 교반하면서 1 시간 동안 60 ° C의 오븐에서 혼합물을 가열 그것은 균일 용액 (기초 용액)이 될 때까지. </li><li> 전기 방사를 들어, PGD 중합체 4:6 중량비 기저 용액을 혼합한다. 참고 : PGD 농도는 양이G 섬유 직경에 효과. 30 % -50 %의 퍼센트 PGD는 더 이상 증가 된 섬유 직경 5cm 이상의 섬유를 생산할 좋다. </li><li> 고분자 용액에 0.1 % 리보플라빈을 넣고 잘 섞는다. </li></ol> 3. 전기 방사 <ol><li> 18 G에 5 ㎖의 표준 주사기로 폴리머 솔루션을 공급 스테인리스 바늘을 무디게. </li><li> 주사기 펌프에 주사기를 삽입합니다. </li><li> 금속판과 바늘 양전하 리드에 고전압 전원의 접지 리드를 부착. </li><li> 15cm에 바늘과 알루미늄 호일 지구 사이의 거리를 조정합니다. </li><li> 스트립의 전면에 섬유의 응집을 방지하기 위해 수평으로 약 15 °의 각도에서 주사기 펌프를 배치. </li><li> 주사기 펌프의 전원을 켜고 0.6 ㎖ / 시간에 펌프의 유량을 조정합니다. </li><li> 높은 전압의 전원을 켜고 14.6 kV의에 동작 전압을 설정합니다. </li></ol> 4.섬유 처리 <ol><li> 수집이 완료된 후, 가교, 60 분 동안 UV 광에 섬유 매트를 노출. </li><li> 100mm 페트리 접시에 알루미늄 호일 스트립으로부터 섬유 매트를 이동 및 살균을 또 다른 20 분 동안 UV 광에 노출. </li><li> 생물 안전 캐비닛에서 수술 블레이드와 동일한 크기의 원형 조각으로 섬유 매트를 잘라 24 - 웰 플레이트에 조각을 배치합니다. </li><li> 휴대 미리 파종 처리를 위해, 인산염 완충 생리 식염수 (PBS)의 1 ml의 섬유 샘플을 담가 하룻밤 37 ° C에서 알을 품다. </li><li> 다음 날, 대기음 PBS는주의 깊게, 각 웰 (재료 테이블의 레시피 참조) 분화 배지 1 ㎖ (DMEM/F12, N2, 및 FGF2)를 추가하고 3 시간 동안 37 ° C에서 알을 품다. </li><li> 기음 차별화 매체주의 깊게 각 섬유 샘플 리겔 0.2 mL를 넣고 30 분 동안 37 ° C에서 알을 품다. 참고 : 라미닌 또한 섬유 코팅에 사용될 수있다. 20 ㎍ / ml의 라​​미닌의 0.2 ML을 추가섬유 및 3 시간 동안 실온에서 배양한다. </li><li> 조심스럽게 각 우물에서 초과 리​​겔를 제거합니다. 한 번 분화 배지 2 ㎖로 씻어. 참고 : 섬유 샘플은 세포 배양을위한 준비가되어 있습니다. </li></ol> 섬유에 5. 셀 시드 <ol><li> MES 세포 배양 접시에 Accutase 1 mL를 넣고 37 ℃에서 10 분 동안 배양 </li><li> 10 분 후, MES 세포는 배양 접시에서 분리됩니다. 플레이트에 차별화 매체의 4 ML을 추가합니다. 식민지 (약 15 배)을 휴식을 위아래로 15 ML 튜브 및 피펫 세포에 떠있는 MES 세포를 수집합니다. </li><li> 분화 배지 4 ㎖에 5 분 후 다시 중단 세포 400 XG에 원심 분리기. </li><li> 혈구를 사용하여 세포를 카운트. 15 ML 튜브에 세포 현탁액 200 μl를 전송하고 차별화 매체와 그것을 10 배 희석. 장소에 커버 슬립과 혈구에 챔버에 희석 세포 현탁액의 15 μl를 전송합니다. 계산1mm의 중앙 광장과 현미경으로 혈구의 네 모서리의 사각형에있는 세포. 참고 : ㎖를 희석 계수 × 10 4 × 평방 당 평균 수를 = 셀 당 </li><li> 각 웰에 약 5 × 4 MES 세포를 놓습니다. 천천히 섬유 매트를 물기로부터 용액을 방지하기 위해, 대신에 웰의 측면으로 미끄러 져 건, 섬유 샘플들의 중간에 세포 현탁액을 드롭. </li><li> 물론 각각 차별화 매체의 1 ML을 추가하고, 37 ° C에서 인큐베이터에서 접시를 유지하고 5 % CO 2 세포 부착, 성장 및 분화를 허용 할 수 있습니다. </li><li> 또한 각에서 기존 매체를 대기음 매일 신선한 분화 배지 1 ㎖로 대체합니다. </li></ol> 6. 세포 생존 <ol><li> 각 우물에서 기존 매체를 대기음. </li><li> 문화 매체와 레사 주린 형광 시약의 1 / 10 번째 볼륨을 혼합하고 각 웰에 1 ㎖를 추가합니다. </li><li> Incub37 ° C와 직사광선으로부터 보호 4 시간, 5 %의 CO 2를 먹었다. </li><li> 4 시간 후, 96 - 웰 플레이트에 각 웰로부터 시약 100 μL 3X를 전송 한 다음 샘플 560EX nm/590EM 필터 설정을 사용하여 형광 분광 광도계로 측정 할 준비가되었다. </li></ol> 7. 리얼 타임 PCR <ol><li> 배양 14 일 후, 샘플을 용해 버퍼를 추가하고 섬유 샘플의 세포로부터 전체 RNA를 분리. </li><li> 역전사 20 ㎕의 반응 비늘의 cDNA를 합성하는 총 RNA의 4 μl를 사용합니다. </li><li> 10 ㎕의 마스터 믹스 (2 배), 0.2 ㎕의 염료, 8 μL의 nuclease가없는 물, 1 μL의 cDNA를, 1 μL 프라이머 (본 연구에서 사용 된 프라이머는 표 1에 나열되어 있습니다) : 각각의 광 튜브에 PCR 반응 혼합물을 준비합니다. </li><li> : PCR 프로그램을 설정합니다. 95 ° C의 2시 20분 분, 1주기 나. 95 ° C 3 초 → 60 ° C에서 1 분, 40주기 다. 95 ° C 15 초, 1주기 D. 60 ° C에서 1분, 1 사이클 전자. 95 ° C 15 초, 1주기. 상대적인 유전자 발현 수준을 결정하기 위해 비교 C T 방식을 선택한다. </li><li> PCR이 완료되면, StepOne 소프트웨어로 실시간 PCR 결과를 분석하고 Excel로 결과를 보냅니다. </li></ol>

<ol>
	<li>Migneco, F., Huang, Y. -. C., Birla, R. K., Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+(glycerol-dodecanoate),+a+biodegradable+polyester+for+medical+devices+and+tissue+engineering+scaffolds.">Poly (glycerol-dodecanoate), a biodegradable polyester for medical devices and tissue engineering scaffolds.</a> Biomaterials. 30, 6479-6484 (2009).</li><li>Reneker, D. H., Yarin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+jets+and+polymer+nanofibers.">Electrospinning jets and polymer nanofibers.</a> Polymer. 49, 2387-2425 (2008).</li><li>Wang, Y., Ameer, G. A., Sheppard, B. J., 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=A+tough+biodegradable+elastomer.">A tough biodegradable elastomer.</a> Nature biotechnology. 20, 602-606 (2002).</li><li>Panunzi, S., De Gaetano, A., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approximate+linear+confidence+and+curvature+of+a+kinetic+model+of+dodecanedioic+acid+in+humans.">Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans.</a> American Journal of Physiology-Endocrinology And Metabolism. 289, (2005).</li><li>Park, 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=Apparatus+for+preparing+electrospun+nanofibers:+designing+an+electrospinning+process+for+nanofiber+fabrication.">Apparatus for preparing electrospun nanofibers: designing an electrospinning process for nanofiber fabrication.</a> Polymer Internationa l. 56, 1361-1366 (2007).</li><li>Barnes, C. P., Sell, S. A., Boland, E. D., Simpson, D. G., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+technology:+designing+the+next+generation+of+tissue+engineering+scaffolds.">Nanofiber technology: designing the next generation of tissue engineering scaffolds.</a> Advanced drug delivery reviews. 59, 1413-1433 (2007).</li><li>Li, W. -. J., Mauck, R. L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds:+production,+characterization,+and+applications+for+tissue+engineering+and+drug+delivery.">Electrospun nanofibrous scaffolds: production, characterization, and applications for tissue engineering and drug delivery.</a> Journal of Biomedical Nanotechnology. 1, 259-275 (2005).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+polymeric+nanofibers+for+tissue+engineering+applications:+a+review.">Electrospinning of polymeric nanofibers for tissue engineering applications: a review.</a> Tissue engineering. 12, 1197-1211 (2006).</li><li>Lim, S. H., Mao, H. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+for+stem+cell+engineering.">Electrospun scaffolds for stem cell engineering.</a> Advanced drug delivery reviews. 61, 1084-1096 (2009).</li><li>Lowery, J. L., Datta, N., Rutledge, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter,+pore+size+and+seeding+method+on+growth+of+human+dermal+fibroblasts+in+electrospun+poly+(epsilon-caprolactone)+fibrous+mats.">Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly (epsilon-caprolactone) fibrous mats.</a> Biomaterials. 31, 491-504 (2010).</li><li>Tillman, B. W., 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=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30, 583-588 (2009).</li><li>Ju, Y. M., Choi, J. S., Atala, A., Yoo, J. J., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayered+scaffold+for+engineering+cellularized+blood+vessels.">Bilayered scaffold for engineering cellularized blood vessels.</a> Biomaterials. 31, 4313-4321 (2010).</li><li>McCullen, S. D., 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=In+situ+collagen+polymerization+of+layered+cell-seeded+electrospun+scaffolds+for+bone+tissue+engineering+applications.">In situ collagen polymerization of layered cell-seeded electrospun scaffolds for bone tissue engineering applications.</a> Tissue Engineering Part C: Methods. 16, 1095-1105 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

간단한 수집 또는 현재 일부 응용 프로그램에 대해 원하는 길이와 섬유 매트의 크기를 획득하는 제한을 증가 전기 방사에 사용되는 수집 회전의 복잡성의 제한. 또한, 배양 접시 또는 다른 기판에 접지 집에서 섬유를 전송하는 것은 도전이다 5. 이 보고서에서, 접지 된 집에 알루미늄 호일 스트립을 부착하여 간단하게 만든 새로운 디자인의 집은, 한 번에 20cm에서 10cm로 대형 섬유 매트를 ?...

전기 방사는 마이크로 - 투 - 나노 사이즈 섬유 지지체를 생산하는 효과적인 가공 방법의 하나이다. 전기 방사의 기본 원리는, 바늘의 팁과 컬렉터 접지 사이의 높은 전압을인가함으로써, 바늘의 구멍에 유지되어 용액의 테일러 콘을 포함한다. 용액의 정전 반발이 표면 장력을 극복 할 때, 충전 된 유체 제트는 니들 팁 밖으로 배출되고, 용매 증발과 공기를 통해 이동하고, 마지막으로 접지 콜렉터 상에 증착된다. 주사기 펌프은 방적 돌기로부터 나오는 용액의 연속적인 흐름을 제공하고, 전기 방사 섬유 이렇게 여러 사본은 단시간 내에 제조 될 수있다. 집 전체에 도착하는 방사 구금을 떠나는 과정에서 청구 제트는 고분자 용액의 점도 및 표면 장력을 포함 파라미터 electrostati의 개수에 따라 스트레칭 및 채찍 겪을 것이다C의 용액에서 시행하고, 외부 전계의 상호 작용 등이. 전기 방사 공정에서, 콜렉터는 마이크로에 나노 섬유가 증착 될 수있는 전도성 기판으로서 역할을한다. 본 연구에서는 섬유 컬렉터의 새로운 유형을 원하는 크기 (길이 x 폭)와 섬유 매트를 얻을 수 있도록 설계되었습니다. 전통적으로, 알루미늄 박을 집 전체로서 사용하지만, 다른 기판에 평면에서 섬유를 전송하는 것은 곤란하다. 전통적인 집에서 그대로 섬유 매트 수확의 어려움으로 인해 전기 방사 섬유가 콜렉터의 표면에 강하게 부착 사실을 주로했다. 따라서 직사각형 스트립으로 알루미늄 호일 조각을 접는 편평한 금속판에 수직하게 연결하여 집 전체를 변경됨. 전기 방사 섬유 스트립의 끝과 쉽게 다른 substrat로 전송할 수 금속판 사이의 영역에 걸쳐 확장된다전자. 열 가교 탄성 중합체에 대한 관심이 급격히 폴리 (글리세롤 세바 케이트) (PGS) 2002 년 3 가황 고무와 유사하다. PGS 유사 폴리 에스테르를 도입 로버트 랭거의 그룹의 선구적인 작업의 성장, 우리가 성공적으로 개발된다 폴리 (글리세롤 도데) (PGD) 글리세롤과 도데 산의 열 축합하고 독특한 형상 기억 특성 1을 보여 주었다. 딱딱한 합성 물질 폴리 (히드 부티레이트) 또는 폴리 (L-락 타이드) (각각 250 MPA는 660 MPA는,의 영률)와 달리, PGD는 온도가 37 ° 이상 1.08 MPa의 영률과, 고무와 같은 탄성 특성을 나타내고 원위치 말초 신경 (0.45 MPA는)에 가까운 일치 C,. 또, PGD는 생분해 성이며, 분해 시간은 글리세롤 및 도데 칸 이산의 비율을 변화시킴으로써 미세하게 조정될 수있다. 도데 칸 이산이 열두 카본 서브입니다두 터미널 카르 복실 그룹, HOOC (CH 2) 10 COOH와 자세. 세바 신산 및 도데 칸 이산 같은 짝수 디카 르 복실 산 아실-CoA는 아세틸 및 트리 카르 복실 산 (TCA) / (구연산)주기를 입력 대사 될 수있다. 디카 르 복실 산의 대사 생성물, 숙시 닐-CoA를는 gluconeogenetic 전구체와 TCA 사이클 (4)의 중간이다. 따라서, 일부 연구들은 특히 병적 상태에서 장내 및 비경 구 영양에 대한 대체 연료 기질로서 이용 될 수 있다고 제안했다. 유리 전이 온도가 31 ° C이므로, PGD, 따라서 실온에서 체온에서 별개의 기계적 특성을 나타낸다, 독특한 형상 기억 성을 나타낸다. 요컨대, PGD는 신경 조직과 유사한 기계적 성질을 가진 독특한 탄성 특성을 나타내는, 생분해 성 생체 적합성; 따라서 신경 조직 공학 응용 프로그램에 적합한 소재입니다. 이 프로토콜에서, 전기 방사큰 예금 영역에 걸친 긴 섬유는 PGD에서 새로 디자인 된 컬렉터를 통해 제작되었다. 섬유 지지체는 마우스 pluripotent 줄기 세포의 성장과 분화를 지원할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="729">
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		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Glycerol</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7757</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dodecanedioic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Gelatin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly (ehtylene oxide) (PEO)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">182028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Riboflavin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">132350250</td>
			<td style="width:167px;">0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Mouse embryonic stem cells</td>
			<td style="width:175px;">GlobalStem</td>
			<td style="width:119px;">GSC-5002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Matrigel</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:119px;">356234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">DMEM/F12</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:119px;">SH30272.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">N2 supplement&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">17502048</td>
			<td style="width:167px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">FGF2</td>
			<td style="width:175px;">Stemgent</td>
			<td style="width:119px;">03-0002</td>
			<td style="width:167px;">10ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Accutase</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11105-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">10010-031&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Resazurin fluorescence dye&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">62758-13-8&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">SV Total RNA Isolation System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">Z3100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoScript Reverse Transcription System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A5000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoTaq&#129; qPCR Master Mix</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Syringe pump&#160;</td>
			<td style="width:175px;">Fisher scientific</td>
			<td style="width:119px;">14-831-200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:268px;">High voltage power source&#160;</td>
			<td style="width:175px;">Spellman High Voltage Electronics Corporation</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">SL30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">UV light</td>
			<td style="width:175px;">Philips</td>
			<td style="width:119px;">308643</td>
			<td style="width:167px;">15W/G15T8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Synergy HT Multi-Mode Microplate Reader</td>
			<td style="width:175px;">BioTek</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Perkin Elmer GeneAmp PCR System 9600</td>
			<td style="width:175px;">Perkin Elmer</td>
			<td style="width:119px;">8488</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">StepOne Real-time PCR System</td>
			<td style="width:175px;">Applied Biosystems</td>
			<td style="width:119px;">4376357</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

조직 공학 응용 프로그램의 경우, 생분해 성 및 생체 적합성 지지체의 제조가 가장 바람직하지만, 어려운 작업입니다. 다양한 제조 방법 중에서, 전기 방사는 단순함과 기능성으로 인해 가장 큰 매력입니다. 또한, 전기 방사 나노 섬유는 세포의 생존 및 성장에 대한 추가적인 지원을 보장 천연 세포 외 매트릭스의 크기를 모방. 이 연구는 전기 방사를위한 새로 디자인 컬렉터를 사용하여 폴리 (글리세롤 도데)라는 이름의 새로운 생분해 성 및 생체 적합성 고분자에 대한보다 큰 예금 영역 (PGD) 1 걸친 긴 섬유의 제조의 가능성을 보여 주었다. PGD​​는 따라서 신경 조직 공학 응용 프로그램에 적합합니다, 신경 조직과 유사한 기계적 성질을 가진 독특한 탄성 특성을 나타낸다. 섬유상 비계 재료를 만들기위한 합성 및 제조 셋업이 간단하고 재현성이 높은, 저렴이었다. 생체 적합성의테스트, 마우스 배아 줄기 세포에서 파생 된 세포에 부착하고 전기 방사 PGD 섬유에 성장 수 있습니다. 요약하면,이 프로토콜은 마우스 배아 줄기 세포 유래의 신경 계통 세포의 성장을 지원하기 위해 PGD의 전기 방사 섬유를 제조하기위한 다양한 제조 방법을 제공 하였다.

전기 방사의 주요 구성 요소는도 1에 나타낸다. 대형 섬유 매트는 일반적으로 수직으로 부착 된 알루미늄 호일 스트립 및 편평한 금속판을 얻었다.도 2는 콜렉터 설계 및 전기 방사 섬유 매트를 나타낸다. 폭과 길이는 서로 다른 애플리케이션에 조정될 수있다. PGD​​ 중합체 및 기저 혼합 용액으로 만든 섬유의 길이는 10cm까지이다. 전기 방사 된 섬유의 형태는도 3?...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

폴리 (글리세롤 도데)라는 이름의 새로운 생분해 성 고분자에서 새로운 디자인의 컬렉터를 통해 더 큰 예금 영역에 걸쳐 전기 방사 장 섬유 (PGD)의 합성 및 제조가보고되었다. 마우스 섬유는 다 능성 줄기 세포로부터 유도 된 세포의 성장을지지 할 수 있었다.

마우스 배아 줄기 세포에서 신경 조직 공학을위한 폴리의 전기 방사 섬유 발판 (글리세롤 dodecanedioate)

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

electrospun-fibrous-scaffolds-polyglycerol-dodecanedioate-for

Research

JoVE Journal

Bioengineering

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

10.8K 조회수.  Florida International University.  폴리 (글리세롤 도데)라는 이름의 새로운 생분해 성 고분자에서 새로운 디자인의 컬렉터를 통해 더 큰 예금 영역에 걸쳐 전기 방사 장 섬유 (PGD)의 합성 및 제조가보고되었다. 마우스 섬유는 다 능성 줄기 세포로부터 유도 된 세포의 성장을지지 할 수 있었다. 

비디오: Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

탄성 PGS는 아티 조직 공학 공사장 공중 발판

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

연구

생체공학

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D 섬유소의 준비는 줄기 세포 배양 용도 공사장 공중 발판

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

조직 공학을위한 Electrospun의 Fibres의 Postproduction 처리

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

전기 자극의 손쥐 Myoblast의 전구 세포 및 응용에서 공학 골격근 조직도

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

개발, 확대,<em&gt; 생체 내</em&gt; 인간 배아 줄기 세포 (hESCs에)에서 인간 NK 세포의 모니터링 및 및 유도 된 pluripotent 줄기 세포 (유도 만능)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

섬유 공사장 공중 발판으로 마이크로 스피어를 해제 전기 방사의 성장 인자

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

폴리 (ε 카프로 락톤) 전기 방사 원사에 인간 중간 엽 줄기 세포의 첨부 파일을 최적화

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

섬유 조직의 그라데이션으로 전기 방사 나노 섬유 공사장 공중 발판

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Decellularized 폐 공사장 공중 발판을 사용하여 ESC 파생 마우스기도 상피 세포의 생성

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Decellularization 식물 조직의 조직 공학 응용 프로그램에 대 한 두 가지 방법

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...