This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

원주민 수용성 콜라겐 솔루션 1. 준비 <ol><li> 준비 또는 내가 1-3 밀리그램 / 등 Piez 등에 의해보고 된 표준 격리 프로토콜을 사용하여 ML. (15)에 쥐의 꼬리에서 콜라겐 산성화, 가용성, 유형의 200 mg의 구입 </li><li> 변성 콜라겐 용액의 목적하는 최종 부피를 기준으로 출발 물질의 양을 규모. 약 수용성 천연 콜라겐의 200 mg의에서, 3 ㎎ / ㎖에서 25 ~ 30 메틸화 ㎖의 석신 콜라겐 솔루션 25 ~ 30 ml의 각합니다. </li></ol> 2. 콜라겐 메틸화 <ol><li> 빙냉 멸균 물을 0.5 mL의 밀리그램 /의 농도 네이티브, 산성화 (PH 2-3) 콜라겐 용액을 100 mg의 희석 및 겔화를 방지하기 위해 상기 용액에 얼음을 보관. </li><li> 1 N NaOH를 몇 방울 9-10 콜라겐 용액의 pH를 조정하고, 30 분 동안 실온에서 교반한다. 흐린 될 해결책의 원인이 콜라겐 석출물을 관찰한다. </lI&gt; <li> 25 분 동안 3,000 × g에서 침전 된 콜라겐 솔루션을 스핀 다운. 분명, 젤 같은 침전물은 튜브의 바닥에서 볼 수 있어야합니다. 대기음이 제대로 뜨는 유체 폐기하십시오. </li><li> 0.1 N HCl을 메탄올 200ml에 침전 된 콜라겐을 재현 탁하고, 메틸화 반응은 4 일 동안 실온에서 교반하면서 발생할 수 있도록. 콜라겐은 용해되지 않지만 솔루션은 흐린 것 아주 작은 볼 수 조각으로 깨지한다. </li><li> 메틸화 후, 원심 분리기 25 분간 3,000 × g에서 용액은 콜라겐 메틸화 펠렛. 대기음이와 산성화 메탄올 뜨는 폐기하십시오. </li><li> 반복적 피펫으로 약 3 ㎎ / ㎖의 농도를 제공 멸균 PBS 25 ㎖에 메틸화 콜라겐을 녹이고 60 μm의 셀 스트레이너를 통해 용액을 필터. 1 N의 NaOH를 20 μL 씩 7.3-7.4를 사용하여 용액의 pH를 조정한다. </li><li> the concentratio 평가상업적 래트 콜라겐 ELISA 키트 또는 하이드 록시 프롤린 분석 키트를 사용하여 용액의 N. 3 밀리그램 / 멸균 PBS와 용액에 대한 해결책을 희석. </li><li> 신중 병 바닥에 클로로포름 3 ㎖를 적층, 스크류 캡 유리 병에 전송하여 메틸화 된 콜라겐 용액을 소독 병은 4 ℃에서 O / N을 설정 한 다음, 무균 적으로 분리하고 저장하도록 허용 메틸화 콜라겐 상부층. </li><li> 한 달 이내 사용을위한 4 ° C에서 솔루션을 저장합니다. </li></ol> 3. 콜라겐 Succinylation <ol><li> 빙냉 멸균 물을 0.5 mL의 밀리그램 /의 농도 네이티브, 산성화 (PH 2-3) 콜라겐 용액의 다른 100 mg의 희석 및 겔화를 방지하기 위해 상기 용액에 얼음을 보관. </li><li> 1 N NaOH를 몇 방울 9-10 콜라겐 용액의 pH를 조정하고, 30 분 동안 실온에서 교반한다. 콜라겐은 흐린되기 위해 용액을 일으키는 석출한다. </li><li> SUC 40 mg의 해산(콜라겐 용액 부피의 1/20 번째)을 아세톤 10ml에 cinic 무수물 (콜라겐 밀리그램 당 0.4 mg)을 얻었다. 천천히 (약 0.5 ml의 단위) 지속적으로 pH를 모니터링하면서 교반 콜라겐 솔루션이 혼합물을 추가합니다. pH가 9.0에 근접함에 1 N NaOH를 1 ~ 2 방울을 첨가하여 pH를 9.0 위를 유지한다. </li><li> 아세톤 숙신산 무수물 모두를 추가 한 후 120 분 동안 실온에서 계속 교반 하였다. 석신 콜라겐이 용해으로 명확하게하는 솔루션을 관찰한다. 정기적으로는 9.0 이상으로 유지하기 위해 pH를 확인합니다. </li><li> 1 N HCl을 20 μL 씩 증분하여 상기 용액의 pH를 4.0으로 조정한다. 석신 콜라겐 침전으로, 다시 흐린 솔루션을 관찰한다. </li><li> succinylation 후, 원심 분리기 25 분 3,000 × g에서 솔루션은 석신 콜라겐 펠렛합니다. 기음은 미 반응 숙신산 무수물로 산성화 뜨는 폐기하십시오. </li><li> 숙시 콜라겐을 녹여멸균 PBS 25 ㎖, 60 μm의 셀 스트레이너를 통해 용액을 피펫 팅을 반복하여, ㎖ / 약 3의 Mg 농도를 제공하고, 필터. 1 N의 NaOH를 20 μL 씩 7.3-7.4를 사용하여 용액의 pH를 조정한다. </li><li> 상업적 래트 콜라겐 ELISA 키트 또는 하이드 록시 프롤린 분석 키트를 사용하여 용액의 농도를 평가한다. 3 밀리그램 / 멸균 PBS와 용액에 대한 해결책을 희석. </li><li> 신중 병 바닥에 클로로포름 3 ㎖를 적층, 스크류 캡 유리 병에 전사함으로써 석신 콜라겐 용액을 소독 병을 4 ℃에서 O / N을 설정 한 다음, 무균 적으로 제거하고 저장하도록 허용 the 석신 콜라겐입니다 상단 층. </li><li> 한 달 이내 사용을위한 4 ° C에서 솔루션을 저장합니다. </li></ol> 콜라겐 수정 4. 검증 <ol><li> 1, 기본 메틸화 및 석신 콜라겐 솔루션의 각 1 ㎖의 샘플을 준비하고 진한 희석수소 이온 적정 멸균 순수 ml의 0.1 밀리그램 /의 entration. 또한 적어도 4 시간마다 10 kDa의 컷오프 membraneusing 3 솔루션의 변화를 통해 물에 대한 투석에 의해 버퍼 적어도 1000 배 희석. </li><li> 수산화 나트륨과 염산 소량의 7.3 용액의 pH를 조절합니다. 네이티브 메틸화 및 숙시 콜라겐 솔루션 적정 곡선을 만들 Tanford (16)에 의해 설명 된 바와 같이 임의의 기준으로 pH가 7.3을 사용하여, 각각의 샘플에 수소 이온 적정을 수행한다. </li><li> 산의 볼륨이 결합 된 H + 분자 당 이온의 수 대 추가 당 pH의 변화를 그린다. 높은 산도 &quot;아미노&quot;범위 중성점 향해 석신 콜라겐 시프트 (아민 기의 손실)를 표시해야하고, 낮은 pH &quot;카르복시&quot;범위는 메틸화 콜라겐 좌측 시프트 (카르복실기의 손실을 표시해야 ) 및 석신 콜라겐 우측 시프트 (카르복실기의 이득S), 천연 콜라겐에 비해. </li><li> 표준 프로토콜 17,18 다음, 2,4,6- 트리니트로 산 (TNBA)를 사용하여 비색법에 의해 대체 succinylation 네이티브 콜라겐 아미노기의 %를 결정함으로써 succinylation 반응의 효능을 평가한다. </li></ol> 미세 유체 장치 및 셀 시드 5. 제작 <ol><li> 표준 방법 (9)를 사용하여 미세 유체 장치를 제조. 100 ㎛, 높이 0.4 ~ 1.5 mm 폭, 세포 성장을위한 긴 채널 1~10mm로 미세 세포 배양 챔버를 만들 포토 리소그래피를 사용하여 정의 실리콘에 SU-8 마스터에서 PDMS의 복제 성형을 사용합니다. </li><li> 장치와 슬라이드 유리의 표면을 산화시키기 위해 플라즈마 세정기를 사용하고 본드 함께 누른다. 적어도 30 분 동안 UV 광에 노출하여 장치를 멸균 한 후, 멸균 PBS 중 50 μg의 / ml의 피브로넥틴을 가진 챔버를 채우고 45 분 동안 37 ℃에서 배양한다. </li&gt; <li> 표현형 또는 분화 상태의 안정화를위한 콜라겐 젤을 필요로 같은 차 쥐 또는 인간의 간세포와 같은 세포와 장치를 시드. 우리는 장치 당 14 × 10 6 세포 / ml에 접종 갓 격리 기본 쥐의 간세포 7,19 또는 시판 냉동 보관 차 인간 간세포의 20 μl를 사용합니다. </li><li> 세포가 4-6 시간 동안 부착하고 부착되지 않은 세포를 씻어 내고 성장 매체와 도금 매체를 대체 할 수 있습니다. 확산 전체 셀을 보장하고 세포의 합류 단층을 만들 O / N을 품어. </li></ol> 6. 레이어로 레이어 콜라겐 증착 <ol><li> 층류 조직 배양 후드에서, 각각의 디바이스 당 용액 10 애플리케이션 메틸화 및 석신 콜라겐 용액의 충분한 양뿐만 아니라 미디어의 몇 MLS 준비. 얼음에 솔루션을 유지합니다. 우리는 장치 레이어 당 콜라겐의 20 μL (10 ~ 15 배의 전체 장치 볼륨)를 사용합니다. </li><li> 있다메틸화 (폴리 양이온) 솔루션 ginning 후 20 메틸화의 μL와 함께 장치를 세척 대체 각 응용 프로그램 사이에 1 분을 기다리고, 콜라겐 솔루션을 숙시. 장치를 약 20 분 소요됩니다 솔루션 당 10 회, 총 플래시합니다. 세포가없는 미디어 아르 시간의 양을 최소화하기 위해 신속하게 작동한다. </li><li> 천천히 그 크기에 따라 입구 / 출구에 축적 콜라겐을 준수하십시오. 유체 흐름에 대한 저항성 증가가 미디어 한두번 장치를 세척 한 경우 축성을 계속한다. </li><li> 모든 층을 적용한 후, 새로운 미디어로 두 번 장치를 씻어 인큐베이터로 돌아갑니다. 세포 유형에 따라, 그러한 간세포의 향상된 편광 같은 세포 외 기질 - 유도 된 형태 적 변화는, 몇 시간 내에 볼 수 있어야한다. </li><li> 콜라겐 매트릭스의 존재를 확인하기 위해 표준 방법을 사용하여 (9) 투과형 전자 현미경 장치를 준비 대표배양 된 세포의 상단에 조립. </li></ol> 세포 표현형 및 기능 7. 안정화 <ol><li> 화상 세포 유형에 대한 표준 방법을 사용하여 세포 형태, 생존 및 극성. 간세포를 들어, 콜라겐 침착의 효과를 입증하기위한 정점 편광 위상 현미경에 대한 생존 LIVE / DEAD 염색 및 CMFDA 염료를 사용 14일 위에 이미지를 수집한다. </li><li> 세포 형태에 대해, 린스 PBS 20 μL의, 화상, 위상차 현미경을 사용하여 장치를 주입하기 위해 PBS 20 μL 피펫으로하여 입구를 통해 장치를 세척. </li><li> 라이브 / 죽은 얼룩 들어, 린스 제조업체의 지침에 따라 제조 DAPI와 라이브 / 죽은 염색 용액 20 μl를 주입하는 PBS의 20 μL에 피펫으로 입구를 통해 장치를 세척, 37 ℃에서 30 분 동안 품어, 린스 다시 디바이스 (20)의 PBS μL, 화상 형광 현미경을 사용하여 장치. </li><li> 담즙 날리 들어culi 염색은 제조자의 지시에 따라, 37 ° C에서 30 분 동안 배양하여 제조 DAPI와 2 μM C​​MFDA 염색 용액 20 μL를, 린스 주입하는 PBS 20 μL로 피펫으로 유입구를 통해 장치를 세척하여 장치를 다시 린스 20 μL의 PBS 및 이미지 형광 현미경을 사용하여 장치. </li><li> 소비 된 매체를 수집하고 세포의 기능을 결정하기 위해 배양 기간 동안 적절한 시점에서 세포 대사의 제품을 측정한다. 간세포를 들어, 소비 된 배지에서 알부민 및 우레아의 양을 측정한다. </li><li> 이러한 평가 효소 활동 수준, 항체 염색, 또는 RNA 분석과 같은 세포 자체에 엔드 포인트 기능 분석을 수행합니다. 간세포를 들어, 유도 및 시토크롬 P450 효소 활동이나 위상 공액 II 효소 글루타티온 S 트랜스퍼를 측정한다. </li></ol>

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J., 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=A+novel+ultrathin+collagen+nanolayer+assembly+for+3-D+microtissue+engineering:+Layer-by-layer+collagen+deposition+for+long-term+stable+microfluidic+hepatocyte+culture.">A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture.</a> TECHNOLOGY. 2 (01), 67-74 (2014).</li><li>Swierczewska, M., 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=Cellular+response+to+nanoscale+elastin-like+polypeptide+polyelectrolyte+multilayers.">Cellular response to nanoscale elastin-like polypeptide polyelectrolyte multilayers.</a> Acta Biomater. 4 (4), 827-837 (2008).</li><li>Kim, Y., Larkin, A. L., Davis, R. M., Rajagopalan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+in+vitro+liver+sinusoid+mimics+using+chitosan-hyaluronic+acid+polyelectrolyte+multilayers.">The design of in vitro liver sinusoid mimics using chitosan-hyaluronic acid polyelectrolyte multilayers.</a> Tissue Eng Pt A. 16 (9), 2731-2741 (2010).</li><li>Larkin, A. L., Rodrigues, R. R., Murali, T., Rajagopalan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+Multicellular+Organotypic+3D+Liver+Model+with+a+Detachable,+Nanoscale+Polymeric+Space+of+Disse.">Designing a Multicellular Organotypic 3D Liver Model with a Detachable, Nanoscale Polymeric Space of Disse.</a> Tissue Eng Pt C. 19 (11), 875-884 (2013).</li><li>Kidambi, 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=Patterned+Co&#8208;Culture+of+Primary+Hepatocytes+and+Fibroblasts+Using+Polyelectrolyte+Multilayer+Templates.">Patterned Co&#8208;Culture of Primary Hepatocytes and Fibroblasts Using Polyelectrolyte Multilayer Templates.</a> Macromol Biosci. 7 (3), 344-353 (2007).</li><li>Janorkar, A. V., Rajagopalan, P., Yarmush, M. L., Megeed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+elastin-like+polypeptide-polyelectrolyte+complexes+to+control+hepatocyte+morphology+and+function+in+vitro.">The use of elastin-like polypeptide-polyelectrolyte complexes to control hepatocyte morphology and function in vitro.</a> Biomaterials. 29 (6), 625-632 (2008).</li><li>Piez, K. A., Eigner, E. A., Lewis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chromatographic+Separation+and+Amino+Acid+Composition+of+the+Subunits+of+Several+Collagens*.">The Chromatographic Separation and Amino Acid Composition of the Subunits of Several Collagens*.</a> Biochemistry. 2 (1), 58-66 (1963).</li><li>Tanford, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interpretation+of+hydrogen+ion+titration+curves+of+proteins.">The interpretation of hydrogen ion titration curves of proteins.</a> Adv Protein Chem. 17, 69-165 (1962).</li><li>Cayot, P., Tainturier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantification+of+protein+amino+groups+by+the+trinitrobenzenesulfonic+acid+method:+a+reexamination.">The quantification of protein amino groups by the trinitrobenzenesulfonic acid method: a reexamination.</a> Anal Biochem. 249 (2), 184-200 (1997).</li><li>Kakade, M. L., Liener, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+available+lysine+in+proteins.">Determination of available lysine in proteins.</a> Anal Biochem. 27 (2), 273-280 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li></ol>

The authors have nothing to disclose.

얇은 순수 콜라겐 어셈블리 변성 콜라겐의 층별 증착을 이용하여 충전 재료 또는 세포 표면 상에 증착 될 수있다. 이 연구의 결과는 메틸화 네이티브 콜라겐 succinylation 세포 (도 2) 상에 얇은 콜라겐 매트릭스 어셈블리를 증착하기 위해 레이어 - 바이 - 레이어 기술로 사용될 수있다 (도 1) 또는 다른 충전 폴리 양이온과 폴리 음이온 콜라겐 용액을 생성 함을 입증 재료 표면...

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Life Technologies</td>
			<td>A1048301</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Sigma-Aldrich</td>
			<td>C3867-1VL</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>EMD Millipore</td>
			<td>08-115</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>R%D Systems</td>
			<td>3440-100-01</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>succinic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>239690-50G</td>
			<td>succinylation reagent</td>
		</tr>
		<tr>
			<td>anhydrous methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-100ML</td>
			<td>methylation reagent</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331-500ML</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>rat collagen type I ELISA</td>
			<td>Chondrex</td>
			<td>6013</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK008-1KT</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Quickzyme Biosciences</td>
			<td>QZBtotcol1</td>
			<td>option for detecting collagen content</td>
		</tr>
	</tbody>
</table>

layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

네이티브 콜라겐 층별 증착에 사용하기위한 폴리 양이온과 폴리 음이온 콜라겐 용액을 만들 메틸화 및 succinylation를 사용하여 수정 될 수있다. Succinylation가 숙시 닐기로 네이티브 콜라겐의 ε 아미노 그룹을 수정하고 메틸화는 메틸기 (도 1a)와 네이티브 콜라겐 카르복실기를 수정한다. 콜라겐 단백질 아미노산 측쇄에 이러한 변형은 용액의 pH에​​ 대한 적정 곡선을 변경. 메틸화는 ?...

Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Research

JoVE Journal

Bioengineering

9.3K 조회수.  Massachusetts General Hospital, Harvard Medical School. The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

비디오: Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

멀티 채널 Microfluidic 장치에서 분리 비즈와 세포 Dielectrophoresis와 층류를 사용하여

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

연구

생체공학

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

에 대한 간단한 마이크로 유체 장치<em&gt; 생체 내</em의&gt; 이미징<em&gt; C. elegans</em&gt;<em&gt; Drosophila</em&gt;와 Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

미세 패턴 혈관 근육 박막의 확장 문화 미세 유체 Genipin 증착 기술

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

3D 종이 미세 유체 장치를 구축하기 위해 접착제 패터닝을 사용하여

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

단일 또는 공존 화학 / 전기 / 전단 응력 자극에서 세포 응답 유학을위한 미세 유체 장치 설계

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

판막 다층 미세 유체 장치에 대한 여러 단계의 변수 높이 포토 리소그래피

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

면역 측정법에 대한 3 차원 종이 기반 미세 유체 장치의 제작

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

셀 마이그레이션 연구에서 수직 화학 산소 그라디언트에 대한 폴리 디메틸 실록산 - 폴리 카보네이트 미세 유체 장치

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

혈소판 수혈 및 지혈을위한 미세 유체 유량 용기 모델은 실시간으로 혈소판 증착 및 섬유소 형성을 측정합니다

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...