우리는 실험 설계와 데이터 분석에 도움이 논의 T. Shatova 감사합니다. G. 파라의 지원과 전문 지식, 기술의 매사 추세 츠 공과 대학에서 코흐 연구소의 핵심 유동 세포 계측법 및 마이크로 기술 연구소의 직원의 인사에 대해 감사의 말을 전한다. 이 작품은 건강 보조금 RC1 EB011187-02, DE013023, DE016516, EB000351의 국립 연구소에 의해 지원되고, 부분적으로 국립 암 연구소 암 센터 지원 (코어)에 의해 P30-CA14051 및 MPP-09Call-랭거-60을 부여했다.

1. 저장 <ol><li> 70 % 에탄올에 저수지, 홀더, O-링과 미세 유체 장치를 저장합니다. 먼지 나 외부 입자에 의해 증발 및 오염을 방지하기 위해 뚜껑이있는 용기 (예를 들어, JAR 또는 비커)를 사용합니다. 셋째로 한 용기 (1), 저수지와 O-링 제 2 용기 (2)의 한 홀더에 장치를 위 (3). </li></ol> 주 : 스토리지 70 % 에탄올의 사용은 불임을 유지하는 것이다. 용액의 성분 만이 에탄올 및 물 (즉 명 변성 에이전트)되는 경우에, 모든 시스템 구성 요소가 완전하게 호환되어야하고 시간이 지남에 따라 열화하지 않을 것이다. <ol start="2"><li> 용기 (2)의 에탄올 용액을 변경하고 (3) 실험을 통해 교차 오염을 방지하고 막힘의 원인이 원하지 않는 입자의 존재를 최소화하기 위해 각 사용하기 전에. </li></ol> 2. 실험 준비 <ol><li> 이쪽 용기 (2) 및 (3) 초음파 배스 FO각 사용하기 전에 R 5​​ ~ 10 분. 이것은 이전의 실험에서 어떤 오염 입자를 제거하는 데 도움이됩니다. </li><li> 70 % 에탄올 용액으로 바이오 안전성 캐비닛 청소 작업 공간. </li><li> 생물 안전 캐비닛 내부를하기 전에 70 %의 에탄올 용액으로 모든 자료 (3 컨테이너 및 핀셋)를 스프레이. </li></ol> 3. 조립 <ol><li> 작업 영역에서 2 ~ 3 저 보풀 와이프를 설정합니다. </li><li> 핀셋과의 컨테이너에서 플라스틱 저수지를 제거하고 내부 표면에서 에탄올 용액의 증발을 촉진하기 위해 잎사귀에 그들을 설정합니다. 부드럽게 표면 저수지 누르거나 에탄올 용액의 제거를 용이하게하기 위해 그들을 통해 공기를 불어. </li><li> 저수지에서 자신의 해당 슬롯에 O-링을 삽입합니다. </li><li> 각각의 용기에서 홀더와 칩을 제거하고 에탄올 (~ 1 ~ 2 분) 증발 할 수 있습니다. </li><li> 홀더에 원하는 칩 앞면 (최대 즉, 액세스 구멍)을 배치 핀셋을 사용합니다. 홀더를 올립니다이 칩은 확인 칩이 홀더에 납작하게되어 있는지 확인하고 필요한 경우 핀셋으로 조정 eyelevel하기로. 중요 : 디바이스 홀더에 적절하게 맞지 않는 경우,이 이후의 단계에서 중단 것이라는 위험이있다. </li><li> 다음, 부드럽게 홀더에 저수지를 배치하고 클립을 맞 춥니 다. O-링은이 과정에서 자신의 슬롯에서 떨어지지 않도록주의하십시오. </li><li> 딸깍 소리가 나면서 고정 될 때까지 조심스럽게 저수지 아래로 누릅니다. 저수지의 양쪽이 안전하고 칩이 올바른 위치에 나타납니다 있는지 확인합니다. </li></ol> 4. 휴대 준비 <ol><li> 부착 세포 (기본 또는 설립 라인)의 경우 : 1-2 일 전에 실험에 플레이트 세포가 실험의 날에는 80 % 이상 합류 없는지 등. </li><li> (PBS 또는 관련 미디어) 서스펜션에 세포를 넣고 1.0 × 10 6 세포 / ml 7 CEL 10 × 1.0의 작동 농도를 목표로LS / ㎖. 참고 : 우리는 인해 세포 농도에 전달 성능에 큰 변화가 관찰되지 않았다. </li><li> 실험 목적 물질의 농도를 구하는 분리 튜브에 세포 및 원하는 전달 물질을 혼합한다. 중요 : 설명 배송 방법 전달을 용이하게하기 위해 확산에 의존하기 때문에, 더 높은 물질 농도가 높은 배달을 얻을 것입니다. 가능한 경우, 초기 임상 시험에 대해 원하는 재료의 1 μm의 솔루션을 사용하는 것이 좋습니다. 필요에 따라이 농도는 향후 실험에서 아래로 적정 할 수있다. 이 장치와 함께 사용보고 최저 농도는 10 nM의 21이다. </li></ol> 5. 조작 <ol><li> 피펫 저수지 (현재의 디자인은 최대 150 ㎕의 용량을 가지고)에 세포 전달 물질의 혼합물. 주 : 대부분의 장치 디자인 때문에 채널을 통해 흐름의 방향이 중요하지 않습니다 완전히 되돌릴 수 있습니다. 샘플은 두 개의 저수지 중 하나에로드 할 수있다. </리&gt; <li> 채워진 탱크에 압력 튜브를 연결하고 적절한 밀봉 (손가락 꽉 충분한 경우가 많습니다)를 보장하기 위해 너트를 조입니다. </li><li> 레귤레이터에 원하는 수준으로 압력을 조정합니다. 이것은 세포가 장치를 통해 이동하는 속도를 제어한다. 버튼을 누를 때까지 세포의 흐름이 시작되지 않습니다. </li></ol> 참고 : 이전의 연구에서, 질소 또는 압축 공기가 캐리어 가스로 동일하게 일했다. <ol start="4"><li> 눈 높이에 장치를 올리고 저수지에서 액체가 쉽게 볼 수 있도록 방향을. 참고 : 저장 비우는 전에 시스템을 차단하기 위해 액체 열을 추적합니다. </li><li> 저수지를 가압 세포의 흐름을 시작하는 버튼을 누릅니다. </li><li> 액면은 저장조의 바닥으로부터 약 2mm 일 때, 신속하게 흐름을 중지 0 PSI에 레귤레이터를 돌려. 중요 : 탱크가 비워지기 전에 하나의 흐름을 중단하지 않을 경우, 하나는 collectio에서 샘플을 배출 위험N 저수지. 또한 유체의 열이 감지 할 수있을 정도의 속도로 이동되지 않았거나 초기 유량 (예를 들어 3 배 느린)에 실질적으로 상대적으로 둔화 한 경우, 장착 된 장치가 아마 막혀 교환 할 필요가 있음을 유의하십시오. 막힌 장치를 작동하면 높은 세포의 죽음으로 이어질 수 있습니다. </li><li> 해당 저장소에서 처리 된 세포를 수집하고 원하는 컬렉션 튜브 / 플레이트에 배치합니다. 중요 : porated 세포가 최대 10 분 (20)에 대한 자료를 통풍 관하는 것 등이 단계에서 어떤 버퍼에 수집 된 세포를 희석하지 마십시오. 이 윈도우가 경과 한 후, 원하는 미디어 / 버퍼에 세포를 희석. </li><li> 필요에 따라 5.7 - 더 많은 처리 된 세포를 수집하거나 다른 실험 조건을 실행하려면 반복 5.1 단계를 반복합니다. 칩을 뒤집을 수 있다는 것을 기억하라, 그러므로 샘플 저수지 중 하나에 장착 할 수 있습니다. 그들이 방해 할 경우 필요에 따라 칩을 교환해야합니다. 막힌 장치를 폐기하십시오. </li></ol> 6. 분해 <ol><li&gt; 부드럽게 옆으로 클립 팔을 밀어 주 홀더에서 저수지를 분리합니다. </li><li> (1 절에 설명) 적절한 저장 용기의 각 부분을 놓습니다. </li><li> 폐기를 위해 별도 용기에 사용되는 칩을 놓습니다. </li></ol>

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저자 아르 몬 Sharei, 로버트 랭거 및 Klavs F. 젠슨은이 문서에서 사용되는 마이크로 유체 장치를 생산 SQZ 생명 공학 회사의 주주입니다.

설명 된 실험 절차 (칩 설계 및 동작 속도 이외 즉 인자)의 특정 양상은 시스템에 적용되는 세포 유형 및 전달 재료에 따라 최적화 될 필요가있다. 주소에게 실험을 설계 할 때 고려해야 할 가장 일반적인 요소 중 일부를 다음과 토론. 형광 표지 화합물의 배달 신호를 개선하기 위해, 하나는 배경 형광의 원인을 해결하기 위해 필요합니다. 표면 바인딩 및 엔도 시토 시스...

세포의 세포질에 고분자의 배달 치료 및 연구 응용 프로그램에서 중요한 단계입니다. 단백질 전달 임상 3 및 실험실 모두 4 설정에서 세포 기능에 영향을 미치는 유망한 수단 동안 나노 입자 매개 배달, 예를 들어, 유전자 치료 1,2 잠재적 보이고있다. 이러한 작은 분자 약물, 양자 도트 또는 금 나노 입자와 같은 다른 재료는, 5,6 암 치료제 9 추적 세포 내 라벨 7,8, 단일 분자에 이르기까지 다양한 응용 프로그램에 관심이 있습니다. 세포막은 거대 분자에 크게 불 투과성이다. 많은 기존의 기술은 막 중단 또는 세포 내로 전달을 용이하게하는 고분자 나노 입자 10, 11, 리포좀 (12), 또는 화학 물질 수정 (13)를 사용합니다. 이러한 방법에서, 전달 효율 및 세포 생존율 번째의 구조에 종종 의존전자 표적 분자 및 세포 유형. 이러한 방법은 핵산과 같은 구조적으로 균일 한 물질의 전달에 효율적이 될 수 있지만, 종종 단백질 (14, 15) 및 나노 물질 7과 같은 더 구조적으로 다양한 물질의 전달을 위해 아픈 적합합니다. 또한, 이러한 방법의 대부분에 의존하는 엔도 좀의 파괴 메커니즘은 따라서 소포 구조 16에 갇혀 많은 소재를 떠나, 종종 비효율적이다. 마지막으로, 자주 설립 세포 라인과 함께 사용하기 위해 개발 된 방법은 일차 전지에 잘 번역하지 않습니다. 이러한 일렉트로 17,18 및 sonoporation 19 같은 멤브레인 poration 방법은 일부 애플리케이션에서 매력적인 대안이다 있지만, 이들은 낮은 세포 생존율을 일으키는 것으로 알려져 있으며 목표 전달 물질의 전하에 의해 제한 될 수있다. 세포의 빠른 기계적 변형, 배달 미세 유체 접근 방식은, 최근에이 demonstr세포 리 프로그래밍 (20)과 나노 물질 전달 (21)의 맥락에서 현재의 기술에 장점을 ated. 이 방법은 주위의 버퍼에 존재하는 물질의 세포 내 전달을 용이하게 세포막 O를 기계적 파괴에 의존한다. 이 시스템은 이전에 세포 유형에 도전 (예 일차 면역 세포와 줄기 세포) 및 물질 (예 : 항체와 탄소 나노 튜브)에 전위를 가능하게 보여 주었다. 여기서, 대상 고분자의 세포 내 전달을 위해 이러한 장치를 사용하는 일반적인 절차가 설명된다. 그러나, 그것은 이전에보고되지 않은 응용 프로그램에 대해, 우리의 이전에보고 된 설계 가이드 라인 (20)에 설명 된대로 하나의 조건에 대한 간단한 최적화를 수행 할 것을 권장하며이 절차는 대부분의 세포 유형과 전달 물질에 일반화이다. 지금까지, 시스템은 RNA, DNA, 금 나노 입자, 양자점, 탄소 나노 튜브, 단백질의 전달을 위해 성공적으로 사용되어왔다S 및 덱스 트란 중합체 20,21.

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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cell squeezing as a robust, microfluidic intracellular delivery platform

세포의 빠른 기계적인 변형은 고분자 나노 물질의 세포 내 전달을위한 약속, 벡터없는 방법으로 떠오르고있다. 이 기술은 이전에 도전 응용 프로그램을 해결 잠재적 보여 주었다,, 등의 기본 면역 세포, 세포 리 프로그래밍, 탄소 나노 튜브, 양자 도트 배달에 납품 할 수있다. 이 벡터없는 미세 유체 플랫폼은 타겟 재료의 세포질 전달을 용이하게 세포막의 기계적 파괴에 의존한다. 여기서, 우리는 장치의 조립, 세포 준비 및 시스템 운영을 포함하여 이러한 마이크로 유체 장치의 사용의 상세한 방법을 설명합니다. 이 전달 방식은 디바이스 타입 및 이전에보고되지 않은 애플리케이션을위한 작동 조건의 간단한 최적화를 필요로한다. 이 시스템은 전문 버퍼 또는 화학적 변형 / 활용의 단계를 필요로하지 않기 때문에 제공된 지침은 대부분의 세포 유형과 전달 물질에 일반화되어 있습니다. 이 작품은 또한 제공의 장치 성능을 개선하고 막힘, 낮은 전달 효율 및 세포 생존과 관련된 잠재적 인 문제를 문제 촬영하는 방법에 대한 평가.

그림 1은 마이크로 유체 전달 시스템의 개략적 인 설명이 포함되어 있습니다. 2A-B 피규어 찬란 복합 3 kDa의 덱스 트란 (20)의 존재에서 다른 장치의 디자인으로 HeLa 세포 치료의 일반적인 결과를 보여줍니다. 절차가 올바르게 수행 될 경우, 시스템 성능은 디바이스의 종류와 작동 속도에 민감 할 것입니다. 따라서, 하나는 좀 더 복잡한 실험을 진행하기 전에 주어진 ...

Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

세포의 빠른 기계적인 변형은 고분자 나노 물질의 세포 내 전달을위한 약속, 벡터없는 방법으로 떠오르고있다. 이 프로토콜은 다양한 애플리케이션을 위해 시스템을 사용하는 방법에 대한 자세한 정보를 제공.

강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

cell-squeezing-as-robust-microfluidic-intracellular-delivery

Research

JoVE Journal

Bioengineering

12.8K 조회수.  Massachusetts Institute of Technology.  세포의 빠른 기계적인 변형은 고분자 나노 물질의 세포 내 전달을위한 약속, 벡터없는 방법으로 떠오르고있다. 이 프로토콜은 다양한 애플리케이션을 위해 시스템을 사용하는 방법에 대한 자세한 정보를 제공. 

비디오: Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

다른 Lineages로 인간 배아 줄기 세포 (hESC)의 전파 및 차별 화를위한 3D 플랫폼으로서 알긴산의 Microcapsule

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

연구

생체공학

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

마이크로 유체 플랫폼을 사용하여 높은 처리량 단백질 표현 생성기

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

바인더로 박테리아 셀룰로오스를 활용하여 강력한 천연 섬유 프리폼 제조

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

미세 유체 기술은 셀 변형능을 검사 할

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

멀티 장기 칩 - 장기 멀티 조직 공 배양을위한 미세 유체 플랫폼

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

2 층 미세 플랫폼에서 애기 모종의 루트 머리 형태를 이미징

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

꼬마 선 충 에 경도 이미징을 위한 미세 플랫폼

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

단일 셀 게놈 시퀀싱 초고 처리량 미세 플랫폼

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...