The authors wish to thank Carl Hobbs and Lindsey Majoram for their technical assistance, and Professor Pat Doherty for use of microscopy facilities. Dr. Agley was supported by a studentship from King&#8217;s College London. Funding from the Spurrell Trust is also acknowledged.

<ol>
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The authors declare that they have no competing financial interests.

我们已经描述了immunomagentic分拣过程对人类肌源性前体从肌肉活检材料小样本的选择性富集。这种技术是非常宝贵的在我们的实验室克服人类肌源性文化的流失，成纤维细胞，同时也为了解肌源性祖细胞不同群体的独特的行为。一旦纯化的生肌细胞可以追究变化的蛋白质和/或基因表达，或者用于下行实验。 除了细胞纯化，我们也详细了快速而直接的荧光显微镜分析的显微照?...

骨骼肌的修复和再生，需要卫星细胞1，肌原干细胞2,3。的作用在体内位于每肌纤维的肌膜和基底层之间的可逆静止状态存在这些细胞中，但被活化增殖，保险丝和区分肌肉组织被破坏，修复和再生3。卫星细胞可以使用酶消化4年轻人和老年人人体肌肉活检标本及其生肌性能隔离随后可以在研究原代培养5。这种隔离方法的关于细胞群体的产量和纯度的效率取决于所使用的方法，可从样品变化进行采样。从酶消化得到的两个主要贴壁细胞类型的卫星细胞（现称为肌细胞或肌肉前体细胞），初步确定为CD56 + /结蛋白细胞，万亩SCLE衍生的成纤维细胞，鉴定为CD56 -和TE7 +细胞5。的成纤维细胞具有快速的增殖速率和不发生不可逆的生长停滞和终末分化时像肌原细胞的细胞 - 细胞接触;因此，在混合人群，成纤维细胞可能溢出肌细胞主导文化。 成纤维细胞经常被视为刺激肌肉生物学家，然而，现在有在成纤维细胞作为值得研究的在他们自己的权利的越来越大的兴趣，特别是因为它们已被证明在肌肉修复6有一个与肌细胞协同作用。从人的肌肉的不同类型的细胞的分离和纯化由此试图调查中培养两种细胞类型的先天行为时一个重要的考虑因素的方法。荧光激活细胞分选（FACS）是一种方法，通过它的细胞可以被分类为进一步研究和/或计数并分析。 FACS已表明可靠丰富人类肌细胞，但细胞的产量进行后续培养迄今尚未高7。定的体细胞，例如与衰老4相关的卫星细胞来源的肌细胞和非常差的增殖和分化的有限复制潜力，更温和的方法是必需的。单肌纤维的文化提供了一个不太积极的，是指获得鼠卫星细胞在他们sublaminal利基及其文化8,9激活后仍驻留的。然而，这常常是不可能的，从人的肌肉活检材料（因为纤维很少能由肌腱到肌腱获得），这意味着，这种技术可能无法访问到兴趣研究人类肌源性细胞许多研究实验室。另外，单纤维的技术仅提供了非常有限的细胞数。 在这里，我们将介绍基于发电机排序系统利用胶原酶细胞TLE酶消化和分散酶，随后通过两个连续的轮磁激活细胞分选（MACS），它不仅提供了高纯度（&gt; 95％生肌细胞）和产率（〜2.8×10 6±8.87×10 5个细胞/ g组织），用于在培养实验。 CD56被认为是金标准表面标记为人类卫星细胞的原位 10和体外 11的识别和提供用于珠附着了理想表面标记候选。在此方法中CD56抗体缀合的氧化铁和超顺磁珠含有多糖类的通过一个高梯度磁性细胞分离柱放置在强磁场12,13结合到细胞和通过。分离柱充满从而起到集中磁力线走向其表面产生强磁场梯度（〜4tesla）铁磁性钢丝绒或铁球矩阵14。在这些列中，即使稍微磁性的细胞被吸引和吸附到其表面14。未结合（CD56 - ）细胞通过柱子而CD56 +细胞标记与磁微珠被保留，直到去除来自磁场12,15。 从分类处理的任何阶段的细胞悬浮液可以在进一步的实验所需的密度进行电镀。以下给定的介入​​的细胞成分可使用免疫细胞化学来鉴定，成像用宽视场或共聚焦荧光显微镜和分析定量使用图像分析方法，它允许在任何给定的图像中的所有标记细胞的快速客观测量。在我们的实验室，我们已经用这种双重免疫排序方式依次通过图像分析16表明CD56 -人成纤维细胞转分化容易进入脂肪细胞，而细胞肌卫星产地s为这种脂肪细胞转化5高度耐药。

<table><tbody><tr><td>Collagenase&amp;nbsp; D</td><td>Roche&amp;nbsp;</td><td>11088866001</td><td></td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693-1G</td><td>Must be filter sterilized before use</td></tr><tr><td>Trypsin/EDTA</td><td>(Gibco) Invitrogen</td><td>15400-054</td><td></td></tr><tr><td>100 &amp;#956;m cell strainer</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>Collagen solution ( 3 mg/ml in 0.1% acetic acid)</td><td>Sigma, Dorset, UK</td><td>C8919&amp;nbsp;</td><td></td></tr><tr><td>Minisart SRP15 syringe filter (0.2 &amp;#956;m)</td><td>Sartorius</td><td>17573ACK</td><td>Polytetrafluorethylene (PTFE) membrane</td></tr><tr><td>CD56 human Microbeads</td><td>Miltenyi Biotech</td><td>130-050-401</td><td>Be aware of the limited shelf life of microbeads</td></tr><tr><td>Anti- fibroblast Microbeads, human</td><td>Miltenyi Biotech</td><td>130-050-601</td><td></td></tr><tr><td>40 &amp;mu;m Pre-separation filters</td><td>Miltenyi Biotech</td><td>130-041-407</td><td></td></tr><tr><td>Large Cell Collumns</td><td>Miltenyi Biotech</td><td>130-042-202</td><td>These columns come with a flow resistor. Use of the flow resistor is not necessary to obtain the high myogenic purities described here.</td></tr><tr><td>LS columns&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>MiniMacs Seperator</td><td>Miltenyi Biotech</td><td>130-042-102</td><td>This separator fits the large cell column but not the LS column.&amp;nbsp;</td></tr><tr><td>MidiMACS</td><td>Miltenyi Biotech&amp;nbsp;</td><td>130-042-302</td><td></td></tr><tr><td>MACS multistand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td></td><td>Must be filter sterilized before use</td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625</td><td></td></tr><tr><td>Triethyl phosphate</td><td>Sigma</td><td>538728</td><td></td></tr><tr><td>Whatman Paper</td><td>Sigma</td><td>Z241121-1PAK</td><td>No. 42, Ashless. To prepare the filter fold the circular filter paper to make a semi circle, then fold the semi-circle in half again to form a cone shape. Fit the cone into a funnel for filtering.&amp;nbsp;</td></tr><tr><td>ProLong Gold Antifade Reagent</td><td>Molecular Probes, Invitrogen</td><td>P36930</td><td>This can be purchased with or without DAPI and does not quench initial fluorescence.</td></tr><tr><td>AxioVision</td><td>Carl Zeiss</td><td>Contact Zeiss</td><td></td></tr><tr><td>Adobe Photoshop CS5 Extended</td><td>Adobe (purchased from Pugh Computers)</td><td>ADPH16982*&amp;nbsp;</td><td></td></tr></tbody></table>

isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle

修理和骨骼肌的再生需要卫星细胞，这是驻地肌肉干细胞的作用。这些都可以从研究中培养用酶消化人肌肉活检样品和它们的生肌性质进行分离。定量，从酶消化获得的两个主要粘附细胞类型是:（i）卫星细胞（称为生肌细胞或肌细胞前体细胞），初步确定为CD56 +，后来作为CD56 + /结蛋白+细胞和（ii）muscle-成纤维细胞，鉴定为CD56 -和TE-7 +。成纤维细胞增殖非常有效地在文化和混合细胞群，这些细胞可能溢出肌细胞主导文化。从人的肌肉的不同类型的细胞的分离和纯化由此试图调查中培养两种细胞类型的先天行为时一个重要的考虑因素的方法。在这里，我们DESCRIBE基于使用胶原酶细胞轻柔酶消化排序和分散酶，随后通过磁性活化细胞分选（MACS）的一种系统，其不仅提供了高纯度（&gt; 95％生肌细胞）和良好的产率（〜2.8×10 6±8.87 7天后在体外 ），用于在培养实验×10 5个细胞/ g组织。这种方法是基于孵育混合肌源性细胞群与偶联于抗CD56的抗体的磁性微球珠，然后使细胞虽然磁场。 CD56 +细胞势必微珠保留的，而CD56领域-细胞通过列畅通。从分类处理的任何阶段的细胞悬浮液可以被镀和培养。以下给定的介入​​，细胞形态，并且表达和蛋白质，包括核转录因子的定位可以通过使用免疫荧光标记用特异性抗体和图像P进行定量rocessing和分析软件包。

纯化的肌细胞和成纤维细胞可在脂肪形成分化培养基中培养三天之后，从7-30之间的任何地方天成脂营养介质，以评估其潜力脂肪生成。使用纯化的细胞群，在与免疫染色对脂肪形成和生肌谱系标记物组合的油红O染色显示，只有在成纤维细胞级分是能够分化成脂肪细胞（ 图2）的。脂肪由成纤维细胞的大量积累可见肉眼（A组），其完整的转分化显示核PPARγ（ 图2板B＆C和<st...

Watch this Scientific Journal Video about Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle at JoVE.com

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

isolation-quantitative-immunocytochemical-characterization-primary

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

15.9K 次观看.  King's College London. The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells. 

视频: Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

科研

发育生物学

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

人成肌细胞的分离，造血分化的评估和存储操作的钙进入测量

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

医学

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

生物学

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...