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.

小鼠饲养在无病原体条件符合机构的指导方针，直到安乐死（机构动物护理和使用委员会（IACUC）的指导方针，在新加坡国立大学和国家咨询委员会的实验动物研究（NACLAR）的指导方针）。 1.鼠标<ol><li>订购一个鼠标适当的遗传背景。这个协议是基于组织从一个C57BL / 6小鼠衍生的。 </li></ol> 2.准备完全培养基<ol><li> 10％胎牛血清（FCS），50μM2-巯基乙醇，100微米的天冬酰胺，2mM谷氨酰胺，1％青霉素，链霉素溶液:通过添加以下组件来罗斯威尔公园纪念研究所（RPMI）1640培养基准备完全培养基。 </li></ol> 3.准备酶溶液<ol><li>准备在一个15毫升的锥形底部管胶原酶D溶液。 <ol><li>称取10毫克胶原酶D.迪在4毫升ssolve胶原酶d完成网上平台。 </li></ol></li><li>准备在1.7 ml离心管链霉蛋白酶的解决方案。 <ol><li>称取10毫克链霉的。 </li><li>加入5微升的1M Tris缓冲液（pH 8.0）。加入1微升的0.5M EDTA（pH 8.0）中。 </li><li>加满494微升无菌水。 </li><li>孵育在37℃的水浴中的链霉蛋白酶溶液30分钟。 </li></ol></li></ol> 4.准备胶原酶D-链霉混合（≤2反面） 注意:请在无菌细胞培养罩随后的步骤。 <ol><li>加入250微升链霉蛋白酶溶液至4毫升胶原酶D溶液。 </li><li>通过0.2微米的针筒式过滤器的胶原酶D-链霉蛋白酶混合物到无菌的15毫升锥形底部管。 </li></ol> 5.提取成纤维细胞，从耳朵和尾巴组织<ol><li>根据相应的制度指引安乐死小鼠。</li><li>放置在细胞培养罩蒸压手术器械（剪刀和镊子）。 </li><li>加入10 mL完全培养基分为两10厘米细胞培养每个菜肴。 </li><li>切耳（〜1厘米半径）和5厘米的尾巴（从尾部的尖端）的小鼠用剪刀和在无菌50ml锥形底部管孵育5分钟在40毫升70％的乙醇。 </li><li>风干耳朵和尾巴通过将它们放置在一个开放的10个厘米细胞培养皿在引擎盖5分钟。一旦干燥，如在步骤5.3中所述转印耳和尾件两个培养皿贴上"耳朵"和含有10ml完全培养基"尾巴"。 </li><li>从耳朵和尾巴用剪刀取下头发。 </li><li>切耳朵和尾巴成片超过3毫米大小用剪刀小。 </li><li>转移切割组织进入标有"耳朵"和"尾"1.8毫升冻存管小瓶和补充足够的胶原D-链霉蛋白酶溶液的体积达到1.8 ml刻度的小瓶。 </li><li> P水平花边冻存管瓶在振荡器上，在37℃下摇晃样品以200rpm进行90分钟。 </li><li>经过90分钟孵化，请从摇床冻存管小瓶，并将小瓶在通风橱中。 </li><li>加入10 mL完全培养基分为两10厘米细胞培养皿，标有"耳朵"和"尾"每一个，并把一个70微米的细胞过滤的每一道菜。 </li><li>将被消化的耳朵和尾巴组织在70微米的细胞过滤在步骤5.11编制的相应标记菜肴和强制使用10毫升注射器推杆&gt; 5分钟研磨组织。偶尔摇动细胞过滤培养基中洗细胞出细胞滤网。 </li><li>移液器从每道菜的细胞悬液到标有"耳朵"和"尾"两个15毫升锥形底管。洗净的菜，过滤与另外10 ml完全介质和介质添加到相应的15毫升锥形底管。 </li><li>降速细胞悬浮液在580〜xg离心7分钟，并使用冷冻细胞离心机4℃。 </li><li>除去上清液，加入10 ml完全培养基将细胞沉淀在15毫升的锥形底部管和悬浮细胞。 </li><li>重复步骤5.14和5.15。 </li></ol> 6.培养细胞混合物的<ol><li>除去上清液。确保细胞沉淀未受干扰。 </li><li>重悬的细胞在10ml完全培养基和相应混合物添加到标记的"耳朵"和"尾"两个10厘米细胞培养皿。 </li><li>加10微升两性霉素B溶液（原液:250微克/毫升）的培养。 </li><li>孵育在湿润的5％CO 2培养箱细胞在37℃。 </li><li>在第三天，用含有10微升两性霉素B以除去碎片10 ml的新鲜完全培养基（ 图1和2）替代培养基。 </li></ol>成纤维细胞7次文化<ol><li> WHEN培养达到约70％汇合（3-4培养天）取出介质，并用5毫升无菌1×磷酸盐缓冲盐水（PBS）洗细胞。 </li><li>除去PBS中并加入2毫升无菌1×胰蛋白酶-EDTA溶液到细胞中。 </li><li>在湿润的5％CO 2培养箱孵育细胞5分钟，在37℃。 </li><li> 5分钟后，轻轻拍打培养皿，并添加6 ml完全培养基的细胞中。 </li><li>细胞悬液转移到15毫升锥形底部管和使用冷冻细胞离心机旋转离心管5分钟，在450〜XG和4℃。 </li><li>除去上清液，并轻轻重新悬浮在5ml完全培养基将细胞沉淀。 </li><li>种子2×10 5个细胞中有10厘米细胞培养皿并重复步骤7.1至7.6之前孵育细胞，在37℃在湿润的5％CO 2培养箱3-4天。 </li></ol> 8.准备耳朵装运注:执行牛逼他在无菌细胞培养罩后续步骤。 <ol><li>根据相应的制度指引安乐死小鼠。 </li><li>放置蒸压剪刀和镊子进入细胞培养罩。 </li><li>加入50 ml完全培养基到50毫升锥形底部管。 </li><li>切割的小鼠用剪刀耳（〜1厘米半径）。 </li><li>转移耳朵入50ml锥形底部管（如制备步骤3）使用镊子。 </li><li>在适当的箱海运在室温之前密封50毫升锥形筒底部用封口膜。 </li></ol>

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The authors declare no conflict of financial interests.

在这里，我们提供了一种简单，廉价且快速的实验步骤建立从耳朵和小鼠的尾巴初级成纤维细胞培养物。提取应导致粘附和迅速分裂成纤维细胞内后3天的隔离组织。原代细胞的一个重要的限制是衰老，一个永久生长停滞15。使用的协议，成纤维细胞培养可传代5-6次之前的成纤维细胞变得衰老，通过细胞的扁平化表示，尺寸增加（2-3倍增加）和故障扩大。 当执行的成纤...

主细胞来源于体外条件下活组织进行培养。它通常假设原代细胞更接近生理状态和其原始比永生化或肿瘤细胞系1的组织的遗传背景。出于这个原因，原代细胞代表用于研究生物学问题2,3的有用模型。然而，不同于无限增长建立的细胞系，原代细胞最终经历衰老中培养并需要经常重新建立。 常用的原代细胞包括成纤维细胞，上皮细胞，内皮细胞，T细胞，B细胞，骨髓衍生的巨噬细胞（BMDM）和骨髓来源的树突细胞（BMDC）。成纤维细胞通常用作原代细胞培养模型。它们提供超过其他初级细胞关键优势。细胞培养物容易建立，容易地维持和不需要purifica用前，培养细胞。他们有快速的初始扩散和专门的媒体或活化协议没有要求。成纤维细胞可以使用的生物，化学和物理协议4,5-有效地转染。有一种可能性，以存储耳朵之前建立的细胞培养物达10天室温。成纤维细胞培养有利于胞质突起的可视化和适于重新编程成诱导多能干细胞（iPS）6。 成纤维细胞是结缔组织的重要细胞分泌胶原蛋白和细胞外基质7。它们所提供的结构框架在许多组织8和播放在伤口愈合和组织修复9,10-必不可少的作用。 在这里，我们描述了一个简单而快速（&lt;4小时）协议来建立从耳朵成纤维细胞培养和小鼠11尾。该协议需要最少的鼠标经验收获组织（相对于其它协议12,13），并且可以用于建立从存储在介质在RT至10天耳朵培养物。

<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

原代细胞是直接从组织来源和被认为是更具有代表性的细胞在体内比建立的细胞系的生理状态。然而，原代细胞培养，通常具有有限的寿命，需要频繁重新建立。成纤维细胞是原代细胞的一个方便的来源。在这里，我们讨论一个简单而快速的实验程序，以建立从耳朵和老鼠尾巴初级成纤维细胞培养。该协议可以用于建立初级成纤维细胞培养从存储在RT至10天耳朵。当协议是经过精心接着，污染物是不太可能发生尽管使用储存延长的时间在某些情况下，非无菌的组织。成纤维细胞增殖迅速在培养和接受复制衰老之前可扩展到相当数量。

来自组织的结果的成纤维细胞的一个显著量组织碎片的提取（ 图1）。在对比组织碎片，纤维母细胞附着到1天，3培养的间组织培养塑料表面。成纤维细胞培养物的培养基可以安全地变更上培养，其中应显著减少在培养（图2）的碎片本水平的3天。的成纤维细胞显示的细长形态和一个清晰可见的细胞质（图1和2）。有丝分裂的细胞应该从培养的第3?...

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

我们描述用于产生从耳朵和小鼠的尾部的成纤维细胞初级一个简单而快速的实验程序。该过程不需要特殊的动物训练和可用于成纤维细胞培养的产生从存储在RT至10天耳朵。

从鼠耳和尾组织生成主成纤维细胞培养

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

Research

JoVE Journal

Developmental Biology

33.7K 次观看.  Yong Loo Lin School of Medicine, National University of Singapore. 我们描述用于产生从耳朵和小鼠的尾部的成纤维细胞初级一个简单而快速的实验程序。该过程不需要特殊的动物训练和可用于成纤维细胞培养的产生从存储在RT至10天耳朵。 

视频: 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.

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

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

科研

发育生物学

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.

初级毛质素黑色素细胞和纤维细胞培养的快速生成

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

癌症研究

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.

组织工程血管在小鼠模型的生成和嫁接

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

生物工程

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).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

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.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

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.

建立从小学成人成纤维细胞培养鼠害

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

生物学

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.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

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.

诱导多能干细胞来源于小鼠的生成

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.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

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).

超声波增强原成人成纤维细胞隔离

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