Name: generating primary fibroblast cultures from mouse ear and tail tissues
Uploaded: 2016-01-10T13:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

마우스는 euthanization까지 제도적 지침 (기관 동물 관리 및 사용위원회 (IACUC) 가이드 라인 싱가포르 국립 대학과 실험 동물 연구를위한 국가 자문위원회 (NACLAR) 지침)을 준수 무균 상태에서 보관 하였다. 1. 마우스 <ol><li> 해당 유전 적 배경 중 하나 마우스를 주문하십시오. 이 프로토콜은 하나의 C57BL / 6 마​​우스 유래의 조직에 기초한다. </li></ol> 완전 배지 2. 준비 <ol><li> 10 % 소 태아 혈청 (FCS), 50 μM 2- 머 캅토 에탄올, 100 μM 아스파라긴, 2 mM의 글루타민, 1 % 페니실린 - 스트렙토 마이신 용액 로즈웰 파크 메모리얼 연구소 (RPMI) 1640 배지에하기 성분을 첨가함으로써 완전 배지를 준비한다. </li></ol> 효소 솔루션 3. 준비 <ol><li> 15 ML 원뿔 바닥 튜브 콜라게나 D 솔루션을 준비합니다. <ol><li> 콜라게나 디 디의 10 mg의 무게4 용액에 ssolve 콜라게나 D 완전한 매체. </li></ol></li><li> 1.7 ml의 microcentrifuge 관에서 프로 나아 솔루션을 준비합니다. <ol><li> 프로 나제​​ 10 mg의 무게. </li><li> 1 M 트리스 완충액 (pH 8.0)의 5 μl를 추가합니다. 의 0.5 M EDTA (pH 8.0) 1 μl를 추가합니다. </li><li> 멸균 물 494 μL와 최고. </li><li> 30 분 동안 수욕에서 37 ℃에서 프로 나제​​ 용액을 부화. </li></ol></li></ol> 콜라게나 D-프로 나제​​ 믹스 (≤2 꼬리) 4. 준비 참고 : 무균 세포 배양 후드에서 다음 단계를 수행합니다. <ol><li> 콜라게나 D 솔루션의 4 ml의 프로 나제​​ 솔루션의 250 μl를 추가합니다. </li><li> 멸균 15ml의 원뿔형 바닥 튜브에 0.2 μm의 주사기 필터를 통해 콜라게나 D-프로 나제​​ 혼합물을 전달한다. </li></ol> 귀와 꼬리 조직에서 섬유 아세포의 5 추출 <ol><li> 해당 기관의 지침에 따라 쥐를 안락사.</li><li> 세포 배양 후드에서 멸균 수술기구 (가위와 집게를) 놓습니다. </li><li> 두 10cm 세포 배양 접시에 각각 10 ml의에게 완전한 매체를 추가합니다. </li><li> 귀 (~ 1cm 반경)와 가위와 마우스 (꼬리의 끝에서) 꼬리 5cm를 잘라 멸균 50 ML 원뿔 바닥 튜브에 40 ml의 70 % 에탄올에 5 분 동안 품어. </li><li> 5 분 동안 후드에서 개방 10cm 세포 배양 접시에 배치하여 공기 건조 귀와 꼬리. 일단 단계 5.3에 설명 된대로 전송 귀 두 개의 배양 접시에 꼬리 조각은 &quot;귀&quot;를 10 ml의에게 완전한 매체를 포함하는 &quot;꼬리&quot;를 표시, 건조. </li><li> 귀와 꼬리 사용하여 가위로 머리를 제거합니다. </li><li> 가위를 사용하여 크기가 3mm보다 작은 조각으로 귀와 꼬리를 잘라. </li><li> &quot;귀&quot;와 &quot;꼬리&quot;라고 표시된 1.8 ml의 cryotube 튜브로 절단 된 조직을 전송하고 유리 병에 1.8 ml의 마크에 도달하기 위해 볼륨에 대한 충분한 콜라게나 D-프로 나제​​ 솔루션을 추가 할 수 있습니다. </li><li> 피쉐이커에 수평으로 cryotube 튜브를 레이스와 37 ℃에서 90 분 동안 200 rpm으로 샘플을 흔들. </li><li> 90 분 부화 후, 진탕 기에서 cryotube 튜브를 제거하고 후드에 튜브를 배치합니다. </li><li> 두 10cm 세포 배양 접시, 표시 &quot;귀&quot;와 &quot;꼬리&quot;각에 10 ml의에게 완전한 매체를 추가하고 각 접시에 70 μm의 셀 스트레이너를 넣어. </li><li> &gt; 5 분 10 ML의 주사기 플런저를 사용하여 조직을 분쇄 강제 단계 5.11에서 제조 된 따라 표시된 요리에 70 μm의 셀 스트레이너의 소화 귀와 꼬리 조직을 놓고. 셀 스트레이너에서 세포를 씻어 매체에 가끔 셀 스트레이너를 흔들어. </li><li> &quot;귀&quot;와 &quot;꼬리&quot;라는 레이블이 붙은 두 개의 15 ML 원뿔 바닥 튜브에 각각의 접시에서 세포 현탁액을 피펫. 추가로 10 ml의 완전한 매체와 요리와 스트레이너를 세척하고 해당 15 ML 원뿔 바닥 튜브에 매체를 추가 할 수 있습니다. </li><li> 세포를 스핀 다운~ 580 XG에 7 분 및 냉장 세포 원심 분리기를 사용하여 4 ° C의 서스펜션. </li><li> 상등액을 제거 15ml의 원뿔형 바닥 튜브에 세포 펠렛을 10 ㎖를 완전 배지를 추가하고 세포를 재현 탁. </li><li> 단계를 반복 5.14 및 5.15. </li></ol> 세포 혼합물 6. 배양 <ol><li> 상층 액을 제거합니다. 세포 펠렛은 그대로 남아 있는지 확인합니다. </li><li> 10ml에 세포 전체 매체를 다시 일시 중지하고 &quot;귀&quot;와 &quot;꼬리&quot;라는 레이블이 10cm 세포 배양 접시에 각각의 혼합물을 추가합니다. </li><li> 문화 : 10 μL 암포 테리 신 B 용액 (250 μg의 / ㎖ 원액)를 추가합니다. </li><li> 가습 5 % CO2 인큐베이터에서 37 ℃에서 세포를 배양한다. </li><li> 셋째 날, 10 ml의 신선한 완전한 파편 제거 암포 테리 신 B의 10 μl를 포함하는 매체 (그림 1, 2)와 매체를 교체합니다. </li></ol> 섬유 아세포의 7 하위 문화 <ol><li> 갔지N 문화는 약 70 % 컨 플루 (일 문화의 3 ~ 4) 매체를 제거하고 5 ml의 멸균 1X 인산 완충 식염수 (PBS)로 세포를 씻어 도달한다. </li><li> PBS를 제거하고 세포에 2 ㎖의 멸균 1X 트립신 EDTA 솔루션을 추가 할 수 있습니다. </li><li> 가습 5 % CO2 인큐베이터에서 37 ℃에서 5 분 동안 세포를 배양한다. </li><li> 5 분 후, 부드럽게 배양 접시를 누르고 세포에 6 ml의에게 완전한 매체를 추가합니다. </li><li> 15ml의 원뿔형 바닥 튜브에 세포 현탁액을 옮기고 냉장 세포 원심 분리기를 사용하여 450 ~ XG, 4 ° C에서 5 분 동안 튜브를 스핀. </li><li> 상층 액을 제거하고 부드럽게 5 ml의 완전 배지에서 세포 펠렛을 다시 일시 중지합니다. </li><li> 10cm 세포 배양 접시에 시드 2 × 105 세포 및 7.6 단계 7.1을 반복하기 전의 3-4일위한 가습 5 % CO2 인큐베이터에서 37 ℃에서 세포를 배양한다. </li></ol> 출하를위한 귀 8. 준비 참고 : T를 수행그는 무균 세포 배양 후드에서 다음 단계. <ol><li> 해당 기관의 지침에 따라 쥐를 안락사. </li><li> 세포 배양 후드로 멸균 가위와 집게를 놓습니다. </li><li> 50 ML 원뿔 바닥 튜브에 50 ㎖를 완전 배지를 추가합니다. </li><li> 가위와 마우스의 귀 (~ 1 센티미터 반경)를 잘라. </li><li> 사용 집게 (3 단계에서 제조 된) 50 ML 원뿔 바닥 튜브에 귀를 전송합니다. </li><li> 해당 상자에 실온에서 출하하기 전에 파라 필름으로 50 ML 원뿔 바닥 튜브를 밀봉합니다. </li></ol>

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The authors declare no conflict of financial interests.

여기에서 우리는 귀를 쥐의 꼬리에서 차 섬유 아세포 문화를 확립하기, 간단한 저렴하고 빠른 실험 절차를 제공합니다. 추출은 조직 3 일 후 격리 내에 부착하고 신속하게 분할하는 섬유 아 세포에서 발생한다. 일차 전지의 중요한 한계는 노화, 영구 성장 정지 15이다. 섬유 아세포는 노화되기 전에 프로토콜을 사용하여, 섬유 아세포 배양 크기 (2-3 배 증가) 및 확장 오류 증가, 세포의 평탄?...

차 세포는 시험관 조건에서 조직 배양 생활에서 파생됩니다. 그것은 일반적으로 일차 전지가 더 밀접하게 생리 학적 상태와 그들이 불멸 또는 종양 세포 라인 1보다 시작된 조직의 유전 적 배경을 닮은 것으로 가정한다. 그 때문에, 일차 세포는 생물학적 질문 2,3-을 연구하는데 유용한 모델을 나타낸다. 그러나 무한정 성장 설립 세포 라인과는 달리, 일차 전지는 결국 문화의 노화를 받아야 자주 다시 설정해야합니다. 일반적으로 사용되는 세포는 일차 섬유 아세포, 상피 세포, 내피 세포, T 세포, B 세포, 골수 유래 대 식세포 (BMDM) 골수 유래의 수상 세포 (BMDC)를 포함한다. 섬유 아세포는 종종 일차 세포 배양 모델로서 이용된다. 그들은 다른 일차 전지를 통해 주요 장점을 제공합니다. 세포 배양은 쉽게 쉽게 유지, 설립 및 더의 purifica을 필요로하지 않습니다문화 이전에 세포의 기. 그들은 빠른 초기 확산 및 전문 매체 또는 정품 인증 프로토콜에 대한 요구 사항이 있습니다. 섬유 아세포를 효율적으로 생물학적, 화학적, 물리적 프로토콜을 사용하여 형질 4,5- 수있다. 세포 배양을 확립하기 전에 실온에서 최대 10 일 동안 귀를 저장하는 가능성이있다. 섬유 아세포 문화는 세포질 프로세스의 시각화에 도움과 유도 만능 줄기 (IPS) 세포를 6으로 재 프로그래밍에 적합하다. 섬유 아세포는 콜라겐 단백질과 세포 외 기질 (7)을 분비 결합 조직의 중요한 세포이다. 그들은 여러 조직 8 구조적 틀을 제공하고 상처 치유 및 조직 수선에 9,10- 필수적인 역할을한다. 여기, 우리는 섬유 아세포 귀에서 문화와 마우스 (11)의 꼬리를 확립하기 간단하고 빠른 (&lt;4 시간) 프로토콜을 설명합니다. 이 프로토콜은 최소한의 마우스가 필요합니다경험 (12,13 다른 프로토콜과 대조적으로) 조직을 수확하는 최대 10 일 동안 실온에서 저장 매체에 귀에서 배양을 확립하는데 사용될 수있다.

<table><tbody><tr><td>RPMI-1640</td><td>HyClone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Asparagine</td><td>Sigma-Aldrich</td><td>A4159</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>HyClone</td><td>SV30010</td><td></td></tr><tr><td>Ethanol</td><td>Merck Millipore</td><td>107017</td><td>Absolute for analysis</td></tr><tr><td>Collagenase D</td><td>Roche Diagnostics</td><td>11088866001</td><td>From &lt;em&gt;Clostridium histolyticum&lt;/em&gt;, lyophilized, non-sterile</td></tr><tr><td>Pronase protease</td><td>Merck Millipore</td><td>53702</td><td>From &lt;em&gt;Streptomyces griseus&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>Tris buffer (pH 8)</td><td>1st BASE</td><td>1415</td><td>Ultra pure grade</td></tr><tr><td>0.5M EDTA (pH 8)</td><td>1st BASE</td><td>BUF-1053</td><td>Biotechnology grade</td></tr><tr><td>10X Phosphate Buffered Saline (PBS)</td><td>1st BASE</td><td>BUF-2040-10X4L</td><td>Ultra pure grade</td></tr><tr><td>Trypsin-EDTA solution 10X</td><td>Sigma-Aldrich</td><td>59418C-100ML</td><td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2492-20ml</td><td>250 &amp;mu;g/ml in deionized water, sterile-filtered</td></tr><tr><td>Scissors</td><td>Aesculap</td><td></td><td></td></tr><tr><td>Forcep</td><td>Aesculap</td><td>AE-BD312R</td><td></td></tr><tr><td>0.2 &amp;mu;M syringe filter</td><td>Sartorius Stedim</td><td>16534</td><td></td></tr><tr><td>70 &amp;mu;M cell strainer</td><td>SPL</td><td>93070</td><td></td></tr><tr><td>Syringe plunger</td><td>Terumo</td><td>SS+10L</td><td></td></tr><tr><td>Cryovial tube</td><td>NUNC</td><td>368362</td><td></td></tr><tr><td>1.7 ml microcentrifuge tube</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Greiner</td><td>664160</td><td>Cell culture treated dish&amp;nbsp;</td></tr><tr><td>15 ml conical bottom tube</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>50 ml conical bottom tube</td><td>Greiner</td><td>227261</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>1002</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Incubation shaker</td><td>Sartorius Stedim</td><td></td><td>Certomat-BS1</td></tr><tr><td>Zeiss Axiovert 25 light microscope</td><td>Carl Zeiss AG</td><td></td><td></td></tr></tbody></table>

generating primary fibroblast cultures from mouse ear and tail tissues

차 세포는 조직에서 직접 파생 된 설립 세포주에 비해 생체 내에서 세포의 생리 학적 상태를보다 잘 나타내는 것으로 생각된다. 그러나, 일차 세포 배양 물은 일반적으로 제한된 수명을 가지며 빈번하게 재 확립 될 필요가있다. 섬유 아세포는 일차 전지의 쉽게 접근 할 수있는 소스입니다. 여기, 우리가 귀 마우스의 꼬리에서 차 섬유 아세포 문화를 확립하기 간단하고 빠른 실험 절차에 대해 설명합니다. 프로토콜은 최대 10 일 동안 실온에서 저장 귀에서 일차 섬유 아세포 배양을 확립하는데 사용될 수있다. 프로토콜에 따라 신중하게되면 오염은 일부 경우에 장시간 저장된 비 멸균 조직의 사용에도 불구하고 발생하기 어렵다. 섬유 아세포 문화에 빠르​​게 증식과 복제 성 노화가 진행되기 전에 상당한 숫자로 확장 할 수 있습니다.

조직 파편의 상당한 양의 조직 결과에서 섬유 아세포의 추출 (도 1). 조직 파편과는 대조적으로, 섬유 아세포는 1 일, 문화의 3 사이의 조직 배양 플라스틱 표면에 부착. 섬유 아세포 배양 매체는 안전하게 크게 문화 (그림 2)에서 파편 현재의 수준을 감소해야 문화의 3 일에 변경 될 수 있습니다. 섬유 아세포는 가늘고 긴 형태와 명확하게 볼 세포질 (그림 1과 2)를</stron...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

우리는 귀 및 쥐의 꼬리에서 일차 섬유 아세포를 생성하기위한 간단하고 신속한 실험 절차를 설명한다. 절차는 특별한 동물 훈련을 필요로하지 않고, 최대 10 일 동안 실온에서 저장 귀에서 배양 섬유 아세포의 생성을 위해 사용될 수있다.

마우스 귀와 꼬리 조직에서 생성 차 섬유 아세포 배양

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

generating-primary-fibroblast-cultures-from-mouse-ear-and-tail-tissues

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

33.8K 조회수.  Yong Loo Lin School of Medicine, National University of Singapore.  우리는 귀 및 쥐의 꼬리에서 일차 섬유 아세포를 생성하기위한 간단하고 신속한 실험 절차를 설명한다. 절차는 특별한 동물 훈련을 필요로하지 않고, 최대 10 일 동안 실온에서 저장 귀에서 배양 섬유 아세포의 생성을 위해 사용될 수있다. 

비디오: Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Gata4, Mef2c 및 Tbx5의 최적 비율을 표현하는 폴리 시스 트론 구조를 사용하여 유도 된 심근의 향상 발전

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

연구

발생학

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

1 차적인 뮤린 멜라닌세포 및 섬유아세포 배양물의 급속한 생성

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

암 연구학

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

생체공학

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

리니지 Reprogramming 성인 마우스 구와 Erythroid 창시자에의 직접

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

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

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

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

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

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

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

설치류에서 기본 성인 Fibroblast 문화를 구축

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

생물학

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

섬유아세포, 선별 애플리케이션을 위한 인간 공학형 결합 조직

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

초음파 증강 원발성 섬유아세포 분리

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...