APS는 데브라 국제 및 영국 피부 재단이 지원됩니다. 던디 파트너십 박사 프로그램의 대학 - YZN는 * STAR에 의해 지원됩니다.

이 연구는 헬싱키 원칙의 선언에 따라 수행하고 적절한 윤리위원회의 승인을 받았다. 1. 미디어 및 시약의 준비 <ol><li> L-아스 코르 빈산 2 - 인산 주식의 200 배의 준비 <ol><li> 둘 베코의 수정 이글의 중간 (DMEM) 용액 5 ㎖ 당 L-아스 코르 빈산 2 - 인산의 29 밀리그램을 녹이고 0.22 ㎛의 멤브레인 필터를 통해 필터링합니다. 상점 -20 ° C에서 0.25 ㎖의 멸균 분주로 </li><li> L의 0.1 ㎜의 최종 농도를 위해 필요한 일에 섬유 아세포 미디어 (1 % L-글루타민 및 10 % 소 태아 혈청 DMEM)의 각 50ml로 200X L-아스 코르 빈산 2 - 인산 재고 0.25 ㎖의 분취 량을 쓰 -아스 코르 빈산 2 - 인산. </li></ol></li><li> 각질 세포 성장 배지의 제조 <ol><li> 기본 SCC 각질 세포의 분리 및 배양은 이전에 21 설명 하였다. 각질 세포 성장 배지의 제조가 기술되고내부뿐만 아니라. </li><li> 다음과 같이 간단히, 미디어를 준비합니다 DMEM 300 ㎖ 및 10 % FBS로 보충 된 햄 F-12을 100 ㎖ 0.4 ㎎ / ㎖의 하이드로 코티손 5 ㎎ / ㎖ 인슐린 10 NG / ML EGF 5 ㎎ / ㎖의 트랜스페린 8.4 NG / ML 콜레라 독소 13 NG / ML의 liothyronine 1X 페니실린 - 스트렙토 마이신 솔루션 </li></ol></li></ol> 2. L-아스 코르 빈산 2 - 인산 보충 미디어에서 섬유 아세포 유래의 기본 ​​매트릭스의 체외 건설 <ol><li> 6 - 웰 플레이트 L-아스 코르 빈산 2 - 인산의 보충 섬유 아세포 미디어 (20,000 세포 / cm 2) 잘 당 종자 20 만 섬유 아세포. 미디어의 2-5 ㎖로 2-3 일마다 재 공급. (주 : 재 이송 주파수 및 음량은 다른 세포의 개별적인 필요에 맞게 조절 될 수있다 &quot;토론&quot;참조.). </li><li> 세포 외 기질 (extracellular matrix)에 포함 된 세포의 두꺼운 층은 육안으로 볼 수 육주, (끝에 형성 할 것이다 <stroNG&gt; 그림 1). , 조직 문화 판에서이 계층을 풀어 부드럽게 1 ㎖의 마이크로 피펫 팁과 매트릭스의 주위를 긁어 후도의 중심을 향해 행렬의 가장자리를 밀어. 이 세포의 층과 매트릭스 (기본 매트릭스)은 플레이트 표면에서 쉽게 분리되어야하고, 지금은 미디어에 떠있는. 그 자체로까지 접어 방지하기 위해 미디어 밖으로 기본 행렬을 확산. </li><li> 기본 행렬 플로트하자 2-3 일마다 미디어를 변경, 5 일 동안 리모델링. 인해 인장 강도와 매트릭스 사이에 본질적인 개조, 행렬 크게 수축 작지만 두꺼운 기본 행렬로 축소되고, 침입 분석에 이용 될 준비가된다. </li></ol> 3. 종양 SCC 각질과 침략 분석 <ol><li> 무딘 집게로 조심스럽게 기본 행렬을 들고 나일론 그물로 전송할 수 있습니다. 일단 나일론 그물에, 1 ㎖의 마이크로 피펫을 사용하여 가능한 한 평면 거짓말을 부드럽게 매트릭스를 확산팁과 무딘 집게. </li><li> 한쪽 끝에서 멸균 바셀린 소량의 번짐에 의해 멸균 클론 실린더를 준비합니다. </li><li> 아래 바셀린면, 네이티브 매트릭스 클론 실린더를 놓습니다. 이 기본 행렬과 클론 링이 단단히 밀착을 보장하는 것입니다. </li><li> 클론 실린더 (각질 세포 성장 배지 100 μL에 25 셀)에 CSCC 세포를 추가합니다. </li><li> CSCC 세포가 기본 행렬에 정착했을 때 6 시간 후 클론 실린더를 제거합니다. </li><li> 각부 스테인리스 철망 지지체 위에 공기 - 액체 계면에 네이티브 매트릭스 및 CSCC 세포와 나일론 네트 리프트. </li><li> 미디어 레벨이 기본 행렬의 바닥에 닿을 때까지 아스코르브 산 보충 된 각질 세포 성장 배지를 추가한다. </li><li> 7시에 2-3 일마다 수확 미디어를 변경하고 CSCC 세포의 14 일 후 파종. </li></ol> 4. 3D 문화의 수확 및 조직학 준비 <ol><li> 4 % 파에 수정실온에서 하룻밤 aformaldehyde. </li><li> 절단 된면이 바깥쪽으로 향하도록 포르말린 고정 파라핀 블록 왁스 샘플 및 소스를 양분. </li><li> 또는 OCT에서 양분 샘플을 포함하고 스냅 동결을 즉시 냉동 조직 블록에 대한 액체 질소에. </li><li> 마이크로톰 및 왁스 제거에 4 μm의 섹션 (필요한 경우) 컷. 표준 헤 마톡 실린 및 에오신을 사용하여 얼룩. CSCC 세포를 시각화하기 위해, 케라틴 14 대한 마우스 단일 클론 항체 (LL001는 사내) immunohistology 염색에 사용될 수있다. </li></ol>

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저자가 공개하는 게 없다.

때문에 본 실험의 특성상 완료에 필요한 전체 시간은 최대 2 개월 일 수있다. 이 시간 동안, 최대한의주의와 무균 조직 배양 방법은 미생물 오염을 방지하기 위해 사용되어야합니다. 이외에도 하이드 록시 프롤린 및 히드 록시 리신의 콜라겐의 합성에 보조 인자로서의 역할에서, 아스코르브 산 (22)은 섬유 아세포에서 콜라겐 특정 mRNA 발현을 자극...

3 차원 조직 배양에서 생체 물질의 사용은 2 차원 접착 및 플라스틱 기재로 효과적으로 요약 한보다 생체 내 환경에 더 가깝다 생리적 조건 하에서 실험실에서 세포 행동을 연구하는 연구자 사용할 수있다. 특히, 커다란 진보는 앞으로 공기 - 액체 인터페이스 1-4에서 3 차원 배양 방법의 도입으로 계층화 상피 세포를 모델링되었습니다. 이러한 기술은 충실하게 각질 세포 분화와 이러한 프로세스를 공부하는 연구자에 대한 유연성과 정확도를 가능하게 종양 세포 침입을 모방. 기질 환경을 모방하는 생체 재료 기판의 선택은 주로 I 콜라겐 유형, Engelbreth-홀름 - 떼 마우스 육종 매트릭스와 디 epidermized 진피의 사용을 포함하고있다. 예를 들어, 암 관련 섬유 아 세포는 각막 상피 상호 작용 6,7를 통해 암 침공 5, 개시 및 진행에 기여하는 것으로 나타났다 갔지N 같은 기판에서 성장. 피부의 기질 환경을 흉내 낸위한 황금 표준은 이러한 기술을 사용하여 가장 크고 가장 널리 연구 층상 상피 세포는 인간의 진피 (DED)를 해제 epidermized 할 간주됩니다. DED의 제조는 인간 사체 피부 3,4에서 트립신 또는 물리적 탈퇴를 통해 표피의 제거를 포함한다. 그러나, 피부에 대한 접근은 임상 기관과 관련이없는 실험실을 위해 매우 어려울 수 있으며, 병 진피 얻을 거의 불가능하다. 대안으로, 실험실 자주 I 콜라겐 (쥐의 꼬리에서 분리) 및 / 또는 Engelbreth-홀름 - 떼 마우스 육종 매트릭스 타입의 조합을 사용합니다. 그 콜라겐 8 Nageotte에 의해 1927 년 발견 쉽게 아세트산 염 침전을 이용하여 분리 할 수 있습니다 후, 조직 배양에의 응용은 이후 Huzel에 의해 개척되었다라와 동료 9. 콜라겐 코팅은 에르와 Gey 9 심문으로 29 균주 및 조직 절편의 세포 배양을위한 유리에 우수한 것으로 판명되었다. 현재 조직 배양에 사용되는 콜라겐의 주요 유형은 쥐 꼬리, 힘줄에서 고립되고, 일반적으로 상용 소스에서 구입한다. 그러나 충실한 기판 재현부의 단점은 쥐의 꼬리 콜라겐은 인간의 콜라겐, 또는 타입 I과 III 콜라겐이 주요 성분으로 존재하는 인간의 진피, 동일하지 않은, 고립 된 쥐의 꼬리 콜라겐은 변함없이 조각 것입니다. Engelbreth-홀름 - 떼 마우스 육종 행렬은 배양 Engelbreth-홀름 - 떼 마우스 육종 세포 (10)에 의해 분비되는 젤라틴 단백질의 혼합물이다. 주요 성분은 라미닌, IV 형 콜라겐, 헤파린 황산 프로테오글리칸, entactin 및 nidogen하고이 단백질의 정확한 비율은 배치에서 배치에 따라 다를 것입니다. 이외에도 구조 프로에게에서teins,이 행렬은 또한 성장 인자 β, 표피 성장 인자, 인슐린 - 유사 성장 인자 1, 소 섬유 아세포 성장 인자, 및 셀룰러 동작 11,12을 변화시키는 혈소판 유래 성장 인자가 바뀌는 등의 성장 인자의 유의 수준을 포함한다. Engelbreth-홀름 - 떼 마우스 육종 행렬의 깎아 지른듯한 복잡쪽으로 향하고, 1,851 단백질의 총 최근 프로테오믹스 연구 (13)에서 확인되었다. 해석하고 11의 사용으로 다른 실험을 비교할 때이 행렬의 풍부하고 복잡한 자연의 빛에주의 권고하고있다. 우리의 실험실은 유전 적 피부 질환, 피부 편평 상피 세포 암 (CSCC) 14을 개발하는 경향과 특히에 관심이 있습니다. 열성 이영의 표피 박리증 (RDEB), COL7A1 유전자의 생식 세포 돌연변이 심한 물집이 생기는 질병의 경우 <s&gt; 15 일에서 17 일까지, 우리는 이러한 환자에서 피부의 미세 환경 (18)을 승진 종양이라고 판단했다. 이 연구의 과정에서 우리는 콜라겐 내에서 I / Engelbreth-홀름 - 떼 마우스 육종 행렬을 포함하고 세포의 자신의 네이티브 매트릭스를 평가하는 방법을 조사 피부 섬유 아 세포의 속성을 촉진 종양을 평가 할 수 없습니다. 이를 위해, 우리는 인간의 피부에서 작업하는 19, 20 당량 루시 제르맹의 실험실에서 이전의 기술을 수정했습니다. 제르맹의 기술은 합성 또는 사체 발판의 부재에서 인간의 기본 각질 세포와 섬유 아세포 문화를 사용하여 잘 조직 된 기저막으로 인간의 피부를 재구성 할 수 있었다. 본 논문에서는 피부 종양 기질 미세 환경 (기본 매트릭스)를 요점을 되풀이하는 데 사용되는 단계는 체외에서 기본 간질 섬유 아세포에서 직접 파생는 18에 설명되어 있습니다. 기본 행렬이 P섬유 아 세포의 장기 배양 roduced는 CSCC 세포 침입에 대한 분석에 해당하는 피부로 사용되었다. 우리는 (유형 VII 콜라겐 (C7) 결핍) RDEB 섬유 아세포 또는 retrovirally 종양의 단일 콜라겐의 깊은 효과를 생성하고 보여 표현하는 유형 VII 콜라겐 형질 RDEB 섬유 아세포에서 분비 세포 외 기질 중 하나에서 파생 된 기본 행렬을 사용하여 데이터를 제시 세포 침공.

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			<td class="xl63" height="106" style="height:79.5pt;width:66pt" width="88">4,500 mg/L d-glucose, 110 mg/L sodium pyruvate</td>
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			<td class="xl63" style="width:66pt" width="88">PROL28908.290</td>
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			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Clonal cylinders</td>
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			<td class="xl66" style="width:66pt" width="88">Dimensions were made so that the mesh would fit into 6-well plates</td>
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tissue engineering of tumor stromal microenvironment with application to cancer cell invasion

중간 엽 세포를 포함하는 매트릭스에 상피 세포의 3 차원의 Organotypic 문화가 널리 상피 세포의 분화와 침략을 연구하는 데 사용됩니다. I 콜라겐 및 / 또는 Engelbreth-홀름 - 떼 마우스 육종 세포에서 파생 된 매트릭스는 전통적으로 중간 엽 세포 (일반적으로 섬유 아세포) 채워지로 매트릭스 또는 기질 미세 환경을 모델링하는 기판으로 사용 된 쥐의 꼬리 타입. 이러한 행렬을 사용하여 실험이 매우 유익하지만, 그것은 주장 될 수 인해 (예 타입 I 콜라겐에서와 같이) 하나의 단백질 또는 기저막 성분과 같은 마우스로부터 유도 된 매트릭스와 같은 성장 인자 (높은 함량의 최우선 존재 육종 세포),이 기판은 가장 기질 세포 자체에 의해 매트릭스 조성에 기여를 반영하지 않습니다. 종양이 발생하기 쉬운 유전 적 물집 질환 (열성 이영 환자에서 분리 된 주요 피부 섬유 아 세포에 의해 생성 된 기본 행렬을 연구하는표피 박리증), 우리는 종양 세포의 침공을 연구하는 기존의 기본 매트릭스 프로토콜을 적용했다. 섬유 아 세포 배양에 오랜 기간 동안 자신의 행렬을 생성하도록 유도합니다. 이 기본 행렬은 다음 배양 접시로부터 분리되고 전체 공 배양은 공기 - 액체 계면에 발생하기 전에 상피 세포를 그것에 시딩. 세포 분화 및 / 또는 침입는 시간이 지남에 따라 평가 될 수있다. 이 기술은 유일한 단점은 기본 행렬을 생성하는 데 필요한 시간을 장시간 채로 합성 또는 외국 행렬에 대한 필요없이 3D 설정에서 상피 간엽 세포 상호 작용을 평가하는 능력을 제공한다. 여기에 우리가 종양 세포의 침윤을 억제하는 섬유 아 세포에 의해 표현 된 단일 분자의 기능, 유형 VII 콜라겐을 평가하는이 기술의 응용 프로그램을 설명합니다.

<html> 이 기술은 검사 및 다른 3D 간질 환경 하에서 (이 경우 CSCC 소재) 종양 세포의 침습성을 비교 동작의 가능성을 연다. 이 기술을 사용하여, RDEB의 피부 환경을 효과적으로 요약 C7-결핍 기본 행렬뿐만 아니라 생성뿐만 아니라 유전자에 표현 C7 (18)에 설계 한 추가 행렬을 할 수 있습니다. 그림 2에서 보는 바와 같이, RDEB CSCC의 각질 세포의 침공은 C7-결핍 RDEB 제어에 비해 C7-과발?...

Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

조직 공학 섬유 아세포 유래 기본 행렬은 상피 세포의 증식과 분화를 지원하는 기질 기판을 생성하는 새로운 도구입니다. 이곳에서 종양 세포 생물학에 다른 간질 세포 유형의 영향을 평가하기 위하여이 방법을 적용하는 프로토콜이 제시된다.

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

tissue-engineering-tumor-stromal-microenvironment-with-application-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.7K 조회수.  University of Dundee.  조직 공학 섬유 아세포 유래 기본 행렬은 상피 세포의 증식과 분화를 지원하는 기질 기판을 생성하는 새로운 도구입니다. 이곳에서 종양 세포 생물학에 다른 간질 세포 유형의 영향을 평가하기 위하여이 방법을 적용하는 프로토콜이 제시된다. 

비디오: Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

종양 Microenvironment를 재현을위한 마이크로 유체 장치<em&gt; 체외에서</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

연구

생체공학

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

자기 조립 셀룰러이 Biomechanical 분석 및 조직 공학을위한 셀 유래 조직 반지를 조작하는 감독

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

종양 세포 침략에 중간 유체 흐름의 효과를 측정하는 세 가지 차원 세포 배양 모델

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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

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

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

전기 자극의 손쥐 Myoblast의 전구 세포 및 응용에서 공학 골격근 조직도

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

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

엔지니어링 섬유소 기반 조직의 면역 조직 및 제약 조건의 적용에서 생성하고 세포와 콜라겐 (재) 조직을 유도하는 스트레인

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

표면 형광을 가진 조직 매트릭스 구성 요소 및 두 개의 광자 현미경의 장기의 intravital 면역 형광 이미징

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

동심 젤 시스템은 3 차원 세포의 이동에 매트릭스 미세 환경의 생물 물리학 역할을 연구하는

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

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. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

혈관 세포 리모델링의 조사에 생리의 체외 모델 및 응용 프로그램과 함께 병리학 혈액 흐름

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

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종양 Microenvironment를 재현을위한 마이크로 유체 장치