我们感谢为我们提供与本议定书​​的第一个版本博士史蒂芬Austad。这项工作是由从美国国立卫生研究院和埃利森医学基金会VG的赠款和支持

1。开始之前<ol><li>用70％乙醇消毒的剪刀和镊子。 </li><li>置于30 mL烧杯内的小型磁力搅拌器，盖上两层铝箔和高压灭菌。 </li><li>无菌水，准备一个28 Wunsch算法Liberase Blendzyme 3单位/毫升原液。在-20 ° C，0.5毫升分装和冻结解冻之前，每次使用新的等分。解冻后的解决方案可能会出现阴天。旋涡的解决方案，直到它变得清晰。 </li><li>热身细胞培养基。 </li></ol> 2。动物样品制备 注意 ：野生动物可能含有病原体，如狂犬病毒。始终意识到开放锐器。 <ol><li>安乐死的动物，胴体放置在4 ° C。这是最好立即使用的胴体，但是，如果这是不实际的的，例如，如果动物是在外地收集，尸体可能会在24小时内使用。 24小时后的细胞产量下降。 </li><li>动物解剖手术套件或化学罩（而不是在组织培养罩）。 </li><li>腋下区是一个便捷的网站收集皮肤样本，腋下的皮肤较薄，含脂肪少，和皮毛密度较小。用70％乙醇的清洁切口部位。确保毛皮是用乙醇浸泡。 </li><li>剃须用锋利的手术刀切开现场周围的皮毛。剃须比预期的切口部位的面积较大。尽量减少对皮肤的削减。喷雾用70％乙醇的面积，并让它干。 </li><li>为了收集皮肤样本消费约1厘米2的一个片段。捏皮肤组织钳，用剪刀剪开。尽量不减少与皮肤的脂肪层，脂肪干扰胶原酶消化。要收集的肺样本，用70％乙醇洗胸部，胸部用剪刀打开一个T形切口，除了皮肤的皮瓣拉的皮肤。用70％乙醇洗开的肌肉面积，并让干燥。切肋骨，用无菌镊子和剪刀（用于较大的动物骨铣刀）。使用无菌技术，以避免污染五脏六腑。请勿触摸内部器官与仪器接触动物皮毛。切约1厘米，用无菌镊子和剪刀 2肺片段。 </li><li>广场组织碎片，用无菌PBS 50 mL管，以避免干燥。 </li><li> 50 mL管外组织碎片用乙醇清洗，并把他们组织培养罩。 </li><li>妥善地处理动物尸体。 </li></ol> 3。提取细胞<ol><li>转移到一个10厘米的组织培养盘用无菌手术刀的组织碎片。不要太多PBS样品转移。 </li><li> 〜1毫米，使用两个手术刀片切成组织。使用两个叶片，用剪刀的动作，从中心出发，除了拉。保持组织揉成团，不切一块一块。当切割是足够的皮肤像腻子，它不会单独成片，但会拉伸薄。肺组织是比较容易切割，将分成小块。 </li><li>使用手术刀，转移到无菌的30 mL烧杯中，用无菌搅拌棒切割组织。用于切割组织与10毫升的DMEM/F12媒体与文施0.14单位/毫升Liberase Blendzyme 3，1X抗生素/ antimycotic板洗净，和解决方案添加到30毫升的烧杯组织碎片。 </li><li>用无菌铝箔盖住烧杯中，并在37 ° C孵育，慢慢地搅拌，30至90分钟。的潜伏期的长短取决于品种及组织类型。小心不要过度消化组织。组织碎片时，仍处于消化结束，获得最佳产量。皮肤需要更长的时间消化比肺癌。大型动物的皮肤需要更长的时间来消化。检查消化后30分钟，然后每10分钟。当皮肤消化完成后，媒体变得混浊和皮肤相互分离的片段，并件的边缘变得“模糊”。肺片段改变颜色由红色变为白色，并开始形成粘纤维时，应停止龙消化。 </li><li>吸取的解决方案，含有组织碎片向上和向下突破的团块。传输的解决方案，以50毫升无菌试管。如果碎片移动10毫升吸管切割和消化容易做得很好。冲洗烧杯3次，与10毫升与15％FBS，抗生素/ antimycotic 1X温暖的DMEM/F12媒体和媒体组织碎片添加50毫升管。关闭50毫升管和颠倒几次混合。 FBS在媒体将停止Liberase消化。 </li><li>摆动桶组织文化离心5分钟旋转524克。去除上清。在温暖的DMEM/F12媒体10毫升与15％FBS，抗生素1X / antimycotic的重悬沉淀。吸管悬挂最大的力量来打破组织块。 </li><li>添加15％胎牛血清，1X抗生素/ antimycotic摆动桶组织文化离心5分钟在524克，混合和离心机的DMEM/F12媒体的另一个30毫升。重复一个更多的时间，以消除Liberase痕迹。 </li><li>重悬在10毫升的DMEM/F12媒体颗粒，含15％FBS 1X抗生素/ antimycotic和3％转移到10 cm组织培养皿中并放置在一个组织文化的孵化器在37 ° C，5％CO 2，O 2 。 </li><li>检查板每天都为成纤维细胞和媒体的色彩。如果隔离是成功的，成纤维细胞爬出来的组织碎片和附加板（图1）。成纤维细胞开始退出在2-5天内组织碎片。 </li><li>如果媒体改变颜色为黄色，这表明潜在污染或细胞的挤迫情况。在高倍显微镜下检查板。如果存在的细菌，真菌，或蠕虫丢弃板（这是很少与实验动物的问题，但是可能会发生时从野外收集的样本）。如果没有污染的存在，但媒体变了颜色，这是造成要么太多的细胞或组织放置在同一盘菜太多片段。如果超过60％的板是由附加的成纤维细胞覆盖，改变盘上的媒体，并转移到一个新的板块与新媒体的组织碎片。如果没有很多的成纤维细胞附着板，改变媒体和分裂组织块2-4板块。 </li><li> 7天之后，如果媒体没有改变早些时候，改变媒体和组织碎片转移到与新媒体的新盘。 </li><li>孵育一个额外的7天的细胞和组织碎片。第14天，已经退出所有可行​​的成纤维细胞的组织碎片。 </li><li>从细胞分离开始的14天之后，摒弃旧的媒体和组织碎片，收获15％胎牛血清，1X青霉素/链霉素，非必需氨基酸的细胞和一个新的板块板他们在5 × 10 5细胞/板EMEM ，和丙酮酸钠。 EMEM媒体将支持只有和其他类型的细胞会死亡或停止增殖的成纤维细胞的生长。 </li><li>细胞达到80％-90％汇合后，冻结等分细胞以供将来使用。 </li><li>当细胞达到80％-90％汇合，继续培养细胞分裂他们在5 × 10 5细胞/板。 </li></ol> 4。代表性的成果正常成纤维细胞具有显着突起（lamellipodia）（图2）的大细胞。成纤维细胞生长在一个单层。健康成长的文化包含1-10％M期细胞，四舍五入超过板面升高的细胞，但不能脱离板（图3）确认。通常情况下，与5X10 5细胞接种10厘米的菜在3-4天相连。倍增时间物种之间的差别很大，可能是一些长寿命的啮齿类动物 1 。当细胞填补他们逮捕在G1期的扩散板。典型的成纤维细胞融合板包含一个紧凑层细胞（图4）。当细胞达到90％汇合，他们准备分裂。如果需要的话，细胞可以保持长时间与常规媒体的变化（每周一次或两次）被逮捕的合流板。 <img src="/files/ftp_upload/2033/2033fig1.jpg" alt="图1" /> 图1。被改变成纤维细胞从老鼠的皮肤（A）和肺癌（二）隔离。媒体之前，到第7天的图片删除独立的细胞和碎片。 <img src="/files/ftp_upload/2033/2033fig2.jpg" alt="图2" /> 图2。小鼠肺成纤维细胞。 <img src="/files/ftp_upload/2033/2033fig3.jpg" alt="图3" /> 图3。一个领域的小鼠成纤维细胞含有细胞在M级，10倍的放大倍率拍摄。 <img src="/files/ftp_upload/2033/2033fig4.jpg" alt="图4" /> 图4。一个小鼠成纤维细胞的融合板，10倍的放大倍率拍摄 。

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> Cell. 7, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5, 741-747 (2003).</li></ol>

普通小学的成纤维细胞生物学研究癌症细胞株的建立提供了一个绝佳的替代方案。成纤维细胞的一个重要的优势是，他们不进行癌基因和肿瘤抑制基因的突变，并保持完好的细胞周期检查点。这使得一个首选系统研究了细胞周期调控，DNA修复，和凋亡的正常成纤维细胞。这里所描述的协议提供了一个简单的初级成纤维细胞的分离和维护配方。该协议被用来成功地分离出超过20种啮齿类动物细胞。 <...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TM Research Grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05401127001</td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

establishing primary adult fibroblast cultures from rodents

在生物学研究中使用的主要细胞，而不是肿瘤细胞株，的重要性是越来越广泛的认可。原代细胞在细胞周期调控，细胞凋亡的研究中的首选，DNA修复，癌细胞进行，在这些过程中所涉及的基因突变。原电池不能无限期由于复制衰老或aneuploidization开始培养。因此，新的文化需要建立定期。啮齿类动物胚胎成纤维细胞的分离过程是公认的，但隔离成年成纤维细胞培养，往往提出了严峻挑战。成人啮齿类动物从人类疾病小鼠模型中分离出的成纤维细胞，可能是来自人类患者的成纤维细胞进行比较时，他们首选的控制。此外，成人的成纤维细胞与怀孕的女性不能轻易获得的野生啮齿类动物工作时，唯一可用的材料。在这里，我们提供了一个从啮齿类动物的皮肤和肺部成年成纤维细胞的分离和文化协议。我们用这个程序成功地隔离开来超过20个啮齿类动物从实验室小鼠和大鼠的成纤维细胞，如河狸，豪猪，松鼠等野生啮齿类动物。

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents

本文介绍了一种从野生啮齿类动物的皮肤和肺组织的初级成纤维细胞培养的隔离和维护协议。

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 ...

establishing-primary-adult-fibroblast-cultures-from-rodents

Research

JoVE Journal

Biology

49.1K 次观看.  University of Rochester. 本文介绍了一种从野生啮齿类动物的皮肤和肺组织的初级成纤维细胞培养的隔离和维护协议。

视频: Establishing Primary Adult Fibroblast Cultures From Rodents

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

科研

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

快速高密度的人类胚胎干细胞培养成纤维细胞去除

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...