우리는 T. 헌트 M. Rosenbluth, 그들의 조언과 유용한 토론 램 연구소 감사드립니다. 우리는 G. 스피너의 지원과 기술 조지아 연구소의 전자 및 나노 테크놀로지 연구소를 인정합니다. 이 작품에 대한 재정 지원은 NIH 교부금 K08-HL093360, UCSF REAC 상, NIH의 Nanomedicine 개발 센터 수상 PN2EY018244, 그리고 애틀랜타의 어린이 건강의 내피 세포 생물학을위한 센터에서 자금 지원에 의해 제공되었다.

1. 내피 Microdevice의 제작 <ol><li> 외부 마스크 공급 업체로 microfluidic 장치의 컴퓨터 보조 설계 (CAD) 도면을 제출하여 photomask를 만듭니다. 사용한 마스크는 소다 석회 유리에 크롬 층으로 구성되었다. 이 경우에는 microfluidic 채널 폭 30 μm의했습니다. </li><li> 15 분 동안 고생과 베어 실리콘 웨이퍼 (황산과 과산화수소의 10시 1분 비율)을 청소하고 30 초 동안 불화 수 소산​​ 산성에서 찍었 다. 약 10 초 동안 deionized (DI) 물로 씻어. </li><li> 30 μm의 높이로 웨이퍼쪽으로 스핀 coater를 이용하여 스핀 Microchem SU-8 2,025 포토 레지스트. SU-8 2025의 경우 3000 RPM의 회전 속도 권장합니다. SU-8의 다른 viscosities이 높이를 달성하는 가능합니다. SU-8 사용에 대한 전체 안내를보실 수 있습니다 <a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> 95 ° C에서 열판에 SU-8로 웨이퍼를 놓으십시오5 분 초과 용매를 운전합니다. 참고 : 오븐은 열판 다르게 웨이퍼를 건조하게 될 것입니다 것은 권장되지 않습니다. </li><li> 웨이퍼 위에 원하는 기능 형상과 마스크를 삽입하고, 자외선 (160 MJ / ㎝ 2로 노출 마스크 aligner (칼 조사할, MA-6)에서) 365 nm의에서 측정. 이 십자가는 포토 레지스트를 연결. </li><li> 추가 SU-8의 중합을 촉진하기 위해 추가로 5 분 95 ° C에서 열판 다시 웨이퍼를 놓습니다. </li><li> 비 교차 연결된 SU-8을 제거하려면 4 분간 PGMEA (프로필렌 글리콜 메틸 에테르 아세테이트)로 주로 구성되어 SU-8의 개발자의 웨이퍼를 담궈. </li><li> 10의 100 % 이소 프로필 알콜 (IPA)와 함께 새로 개발된 웨이퍼를 씻어. 웨이퍼 그런 다음 용매 몇 분 동안 깨끗한 퓸 후드의 웨이퍼에서 증발 수있게하거나 가압 질소를 사용하여 건조 수 있습니다. </li><li> 테이프 배양 접시에 패턴 SU-8로 건조 웨이퍼의 가장자리는 사전하기운동을 빼낼. </li><li> 웨이퍼 전체 코팅을 보장하기 위해 페트리 접시 위에, 그리고 소용돌이 모양의 웨이퍼와 커버, 피펫을 사용하여 웨이퍼에 Sigmacote 1 ML을 적용, 덮개를 제거하고 모든 용매가 증발된 때까지 웨이퍼 몇 분 동안 건조 수 있습니다. </li></ol> 2. PDMS (Polydimethylsiloxane) 준비 <ol><li> 믹스 PDMS 폴리머와 경화 에이전트 10시 1분 비율 (W / W)에서하고 진공 건조기를 사용하여 기포를 제거합니다. 가스를 제거하다 PDMS에 필요한 시간의 길이는 가능한 진공 시스템의 강도에 따라 다르지만 일반적으로 몇 분 거리에 1 시간 범위. 내용은없고 이전에 부어 PDMS, 고분자의 60 ML의 총 볼륨으로 인해 저는 수십 인치 배양 접시 권장합니다. </li><li> 약 5mm 두께의 웨이퍼에 혼합물을 부어. 또한 PDMS의 얇은 시트를 만드는 평평한 바닥 접시 위에 두께 약 1mm를 붓는다. 60 하룻밤 오븐에 ° C로 치료하자. </li><li> 칼이나 메스를 사용하여 C 주위를 잘라PDMS 장치를 ured 및 웨이퍼에서 제거할 수 있습니다. 또한, 장치보다 약간 큰 PDMS의 얇은 시트를 잘라. </li><li> 1.0 밀리미터 구멍 펀치를 사용하여 PDMS 장치의 유입구와 배출구 구멍을 만듭니다. 이 중 핀 바이스, 해리스 유니 코어, 또는 이와 유사한 장치를 사용하여 수행할 수 있습니다. </li><li> 장치의 표면과 스카치 테이프를 사용하여 시트를 청소합니다. </li><li> 플라즈마 클리너 사용, PDMS 장치에서 30 s에 대한 산소 플라즈마에 PDMS 시트의 표면을 쉽게받을 수 있습니다. 산소 플라즈마는 신체 접촉으로 데려 결합 PDMS의 노출된 표면에 반응 수종을 만듭니다. </li><li> PBS의 인간 혈장 50 μg / ML fibronectin 가득 바​​람에 무딘 점 바늘 주사기에 연결된 대형 튜브의 작은 길이 (몇 센티미터),에 작은 튜브를 연결합니다. 관 그리고 바늘이 마찰 맞춤 튜브 사이에 만들어 졌는지와 같은 크기입니다. 작은 PDMS microdevice의 입구에있는 튜브와를 삽입pply 주사기에 압력 fibronectin 솔루션을 완벽하게 채널을 기입하고 배출구 포트에 작은 100 μL 방울을 만들려면 채널이 젖었 머물 수 있도록. 40-60 분 동안 37 ° C에서 장치를 품어. </li><li> 무딘 포인트 바늘로 PBS로 가득 신선한 주사기를 연결합니다. PBS로 장치를 헹굴 수있는 주사기에 압력을 적용합니다. </li></ol> 3. 내피 세포와 Microfluidic 장치를 퍼뜨리고 <ol><li> 8퍼센트 dextran과 내피 성장 미디어에서 인간 제대 정맥 내피 세포 (HUVECs)의 1,000,000 세포 / ML을 준비합니다. 500,000까지 2,000,000 세포 / ML의 범위가 성공적으로 사용되었습니다. 셀 로딩 매체에 dextran의 추가들은 fibronectin - 코팅 microfluidic 시스템 입력으로 내피 세포의 속도가 감소 유체의 점도를 증가시킵니다. 이것은 차례로, 세포가 microdevice 내에 성공적으로 준수 문화 것이라는 확률이 높아집니다. </li><li> C단계 2.7에서와 같이 새로운 주사기와 튜브를 onnect. 하지만 긴 튜브로 (길이 약 1 미터). </li><li> 이 시스템의 성능을 최적화하기 위해서는 전체를 관류 시스템에 누출이나 거품을 방지하기 위해이 시점에서 중요하다, 솔루션 또는 미디어의 거품의 존재의 누설이 흐름을 변경하고 내피 세포의 성공적인 시딩를 방지할 수 있습니다. 따라서 튜브가 꽉 피팅은 어떤 연결 사이의 누설을 제거하는 것이 필수적이다. 세포 microdevice 도입하고 전체를 관류 시스템은 공기 방울의 도입을 피하기 위해 microfluidic에 튜브를 장착하기 전에 셀 솔루션으로 끝났다해야하기 전에 주사기 및 / 또는 튜빙의 거품은 제거되어야합니다. </li><li> 주사기 펌프를 사용하여, 37에서 2 시간 동안 1.23 μl / 분 용적식 속도로 PDMS 장치에 세포 현탁액을 불어 넣다 ° C에서 5 % CO 2. 주사기 펌프에 있었을 때 최적의 결과를 달성했다<html> PDMS 장치로 같은 높이. 입구 들어, 인큐베이터 안에 낫게 수있는 긴 튜브는 세포와 미디어가 적절 장치와 접촉 오기 전에 워밍업하는 보장합니다. 저희 시스템에서는 1.23 μl / 분의 체적 유량은 중 ~ 1 다인 / 전체 혈액을 사용하는 채널의 cm 2. 벽면 전단 응력에 대응하는 가장 작은 microchannels에 ~ 1mm / s의 강물의 한복판 속도를 대략 </li><li> 1.23 μl / 분 비율로 2~8일에 대해 동일한 주사기 펌프 및 긴 튜브, perfuse 신선한 성장 미디어를 사용합니다. 성공적인 장치는 24-48 시간 이내에 장치의 내부에 성장 내피 세포 단일층을해야합니다. 이전 실험 합류 monolayers 적절하게 기기에 걸쳐 14 세포 - 세포 분기점에서 VE-cadherin을 표현하는 것으로 나타났습니다. </li><li> 실험을위한 시스템으로 혈액이나 세포 현탁액을 주사. </li></ol> 4. 대표 결과 _content가 &quot;&gt;이 프로토콜을 사용하여 표준 리소그래피 microfabrication 기법은 생리학 microvasculature (그림 1A)의 sizescale을 모방 microfluidic 채널을 생성하는 데 필요한 금형을 만드는 데 사용됩니다. 최적화된 관류 기법을 사용하여 내피 세포가 종자 및 confluently 문화 microfluidic 시스템이 투명으로 세포 시딩의 24 ~ 48 시간 내 microfluidic 시스템 (그림 1B)의 전체 내부 표면은., 전체 microdevice는 이미징 및 데이터 수집을위한 brightfield / 형광 현미경 스테이지에 둘 수 있습니다. 우리의 시스템은 다음 sickled 붉은 세포 탈선 백혈구 및 내피 접착의 증가 강성은 microvascular 장애물에 기여하는 등 낫 세포 질환과 같은 biophysical 속성, 변경 포함 hematologic 질환을 연구에 적용할 수 있습니다. 임상적으로 승인된 약물 치료, hydroxyurea는 증상이 있지만 D를 amelioratesmicrovascular 흐름에 irect 효과는 알 수 없습니다. 우리의 분석은 계정의 셀 강성과 접착 모두로 소요되며, hydroxyurea가 크게 낫 세포 질환 (그림 2)에서 흐름을 ameliorates 것을 보여줍니다. 이 시스템은 이상적인 혈액 세포가 microvasculature에서 서로와 내피 세포와 상호 작용하는 모든 hematologic 과정을 공부하고 적합으로 낫 세포 질병은 microvasculature - 온 - 칩위한 응용 프로그램의 한 예입니다. 더 기본적인 애플 리케이션이 많은 사람 중에서 백혈구 생물학과 조혈 줄기 세포 생물학을 포함하면서 기타 임상 관련 애플 리케이션은 염증성 질환, 패혈증 / 폐 상해, thrombotic microangiopathies, 말라리아, 그리고 암 전이를 포함합니다. <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="그림 1"/> 그림 1.) endothelialization 전에 초기 PDMS microfluidic 장치. 여기 microdevice는 재치가 주입된다H 식품 시스템의 sizescale 및 전반적인 디자인을 설명하기 착색. B) Brightfield 현미경은 microfluidic 체제가 완전히 여기에서 설명한 프로토콜을 사용하여 세포 시딩 48 시간 이내에 endothelialized되어 보여줍니다. <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="그림 2"/> 그림 2.) microchannels있는 microvasculature - 온 - 칩은 사후 모세관 venules (30 μm의), 대부분 낫을 세포 microvascular 폐쇄 이벤트 사이트의 크기를 대략. hydroxyurea를받는 낫 셀 환자에서와 hydroxyurea를받지 못한 환자의 전체 혈액은 두 개의 서로 다른 microvasculature - 온 - 칩 디바이스를 통해 흘러됩니다. endothelialized microchannels 내에서 상대적으로 쉽게 hydroxyurea 흐름을 받고 낫 셀 환자에서 B) 전체 혈액. C) 같은 hemodynamic 조건에서 hydroxyurea를받지 못하고 낫 셀 환자에서 전체 혈액 그러나, microch와 훨씬 더 부진한 흐름을 전시하고annel의 방해. 아래 채널이 완전히없이 흐름에 방해되고 다른 endothelialized microchannels의 흐름 판매율은 hydroxyurea 조건에 비해 현저히 낮은됩니다. 세 가지 이미지 스케일 바는 30 μm의입니다.

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvascular Research. 82, 210-220 (2011).</li><li>Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+the+microvascular+occlusion+and+thrombosis+that+occur+in+hematologic+diseases+using+microfluidic+technology.">In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.</a> The Journal of Clinical Investigation. , (2011).</li><li>Green, D. A., Murphy, W. G., Uttley, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemolytic+uraemic+syndrome:+prognostic+factors.">Haemolytic uraemic syndrome: prognostic factors.</a> Clinical &#38; Laboratory Haematology. 22, 11-14 (2000).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

우리 endothelialized microdevice 시스템은 생체내 실험에서와 함께 사용할 때 가장 좋은 적합하며, reductionist 접근 방식은 인간과 동물 모델에서 관찰되는 hematologic 프로세스 biophysical 메커니즘을 명료하게하다 도움이 될 수 있습니다. 또한, 우리의 시스템은 제한없이 않습니다. 예를 들어, 우리 microfluidic 채널 단면에서 사각형입니다. 기술적으로 원형 microchannels는 10,11를 조작 수 있지만, ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 무딘 포인트 바늘 </td><td> 확인을 국제 </td><td> 920050-TE </td><td> 정밀 장비 바늘이 20 게이지 X 1 / 2 &quot;, 핑크 </td></tr><tr><td> dextran </td><td> 시그마 - 올드 리치 </td><td> 31,392 </td><td></td></tr><tr><td> Fibronectin </td><td> 시그마 - 올드 리치 </td><td> F0895 </td><td></td></tr><tr><td> 홀 구멍을 뚫는 (핀 바이스) </td><td> 기술 혁신 </td><td></td><td></td></tr><tr><td> 인간의 탯줄 내피 세포 (HUVECs) </td><td> Lonza </td><td> CC-2519 </td><td></td></tr><tr><td> 플라즈마 청소기 </td><td> 혈장 </td><td> PDC-326 </td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) </td><td> 피셔 과학 </td><td> NC9285739 </td><td> Sylgard 184 실리콘 엘라스토머 키트 </td></tr><tr><td> Sigmacote </td><td> 시그마 - 올드 리치 </td><td> SL2 </td><td></td></tr><tr><td> SU-8 2025 </td><td> Microchem </td><td> Y111069 </td><td></td></tr><tr><td> SU-8 개발자 </td><td> Microchem </td><td> Y020100 </td><td></td></tr><tr><td> 주사기 펌프 </td><td> 하버드 장치 </td><td> 70-3008 </td><td> PHD-ULTRA </td></tr><tr><td> 튜빙 (큰) </td><td> 콜 - Parmer의 악기 회사 </td><td> 06418-02 </td><td> Tygonreg microbore 튜빙, 0.020 &quot;ID X 0.060&quot;OD </td></tr><tr><td> 튜빙 (작은) </td><td> 콜 - Parmer의 악기 회사 </td><td> 06417-11 </td><td> PTFE microbore 튜빙, 0.012 &quot;ID X 0.030&quot;OD </td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

microfabrication 기술의 발전은 마이크로 및 nanoscales 1,2에서 생물 학적 및 생화 학적 실험을 수행을위한 저렴하고 재현성 microfluidic 시스템의 생산을 활성화했습니다. 또한, microfluidics도 구체적으로 인해 쉽게 역동적인 유체 환경과 생물 학적 조건 3-6 제어하는 능력 때문에, 양적 hematologic과 microvascular 프로세스를 분석하는 데 사용되었습니다. 따라서 연구자들은 최근 혈액 세포 deformability, 혈액 세포 집합, microvascular 혈액 흐름과 혈액 세포 - 내피 세포 상호 작용 6-13 공부를 microfluidic 시스템을 사용 해왔다. 그러나 이러한 microfluidic 시스템이 어느 교양 내피 세포를 포함하거나 더 큰 줄 몰랐 microvascular pathologic 프로세스 sizescale 관련된보다. 교양 내피 세포와 microfluidic 플랫폼 정확 recapitulates 세포, 물리적, 그리고 hemodyn 그microcirculation의 amic 환경 microvasculature 참여 hematologic 질환의 근본적인 biophysical pathophysiology 우리의 이해를 촉진하기 위해 필요합니다. 여기서는 hematologic 질환에서 발생 pathologic biophysical microvascular 상호 작용을 연구하기위한 표준 내피 세포 배양 기술과 함께 간단한 단일 마스크 microfabrication 프로세스를 사용하여 microvasculature의 체외 모델에서 &quot;endothelialized&quot;를 만드는 방법을보고합니다. 이것은 &quot;microvasculature - 온 - 칩&quot;단단히 생물학뿐만 아니라 biophysical 조건 제어와 표준 주사기 펌프 및 brightfield / 형광 현미경을 사용하여 운영하고 강력한 분석과 연구자를 제공합니다. 이러한 microcirculatory의 hemodynamic 조건, 내피 세포 유형, 혈액 세포 유형 (들)과 집중 (들), 마약 / 억제 농도 등 같은 매개 변수는 쉽게 제어할 수 있습니다. 따라서 우리의 microsystem 제공양적 microvascular 흐름이 때문에 세포 접착, 집합, 그리고 deformability, 기존 assays 함께 사용할 수없는 기능의 변경 사항에 장애되는 질병 프로세스를 조사하는 방식입니다.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

microvascular 크기의 채널 (&lt;30 μm의)와 microfluidic 장치의 전체 내부 3D 표면에 걸쳐 문화 내피 세포 단일층를하는 방법이 설명되어 있습니다. 이<em&gt; 체외에서</em&gt; microvasculature 모델은 혈액 세포, 내피 세포, 그리고 hematologic 질병 용해 요인 간의 biophysical 상호 작용 연구를 가능하게합니다.

Hematologic 질환의 Microvascular 상호 작용 유학을위한 Endothelialized Microfluidics

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

Research

JoVE Journal

Bioengineering

15.9K 조회수.  Emory University School of Medicine. microvascular 크기의 채널 (

비디오: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

단백질 접기 유학을위한 Microfluidic 믹서

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

연구

생체공학

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

멀티 깊이 원형 단면 Endothelialized 마이크로 채널 - 온 - 칩의 개발을위한 절차

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

라이브 이미징 및 상해 응답의 연구에 대한 마이크로 유체 칩을 사용<em&gt; 초파리</em&gt; 애벌레

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

연속 흐름 Microspotter을 사용하여 수정 된 표면에 세포의 침수 인쇄

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

물방울 미세 유체에 대한 여러 가지 빛깔의 형광 검출 광학 섬유를 사용하여

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

조직 공학 혈관 신생 단위로 쥐 지방 조직 유래 미세 혈관 조각의 분리

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode 배열 마이크로 신경 통신 및 Axonal 신호 전파 공부에 대 한 인터페이스

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

뇌암과 신경 퇴행성 질환을 연구하기위한 인간의 신경 오르가노이드

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...