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

我々は、筋生検材料の少量の試料からのヒト筋肉由来前駆体の選択的濃縮のためimmunomagentic選別手順を記載している。この手法は、線維芽細胞にヒト筋肉由来の文化の損失を克服するための、だけでなく、筋肉由来前駆細胞の異なる集団のユニークな振る舞いを理解するための私たちの研究室では非常に貴重なてきた。精製した後筋原細胞は、タンパク質および/または遺伝子発現の変化を調?...

骨格筋の修復および再生は2,3筋原幹細胞、衛星細胞1の作用を必要とする。 インビボでこれらの細胞はあらゆるmyofibreの筋細胞膜と基底膜の間に位置して可逆的に静止状態で存在するが、増殖するように活性化され、ヒューズ及び筋肉組織として分化するが、損傷を受けた修理および3を再生する。衛星細胞は、酵素消化4を用いて、若年および高齢のヒト筋肉生検試料から単離することができ、それらの筋形成特性は、その後、初代培養5に研究することができる。細胞集団の両方の収率および純度に関して、この分離プロセスの効率は、使用される方法に依存し、サンプル間で変化し得る。酵素消化から得られた二つの主要な接着細胞型は、CD56 + /デスミン細胞、およびμとして最初に同定された衛 ​​星細胞（現在称される筋原細胞又は筋前駆細胞）、あるCD56として識別SCLE由来線維芽細胞、 -とTE7 +細胞5。線維芽細胞は、急速な増殖率を持っており、筋原細胞のような細胞間接触の際に不可逆的な増殖停止および最終分化を受けない。したがって、混合集団に、線維芽細胞は、文化を支配する筋原細胞をオーバーランすることができる。 線維芽細胞は、多くの場合、しかし、それ自体で研究の価値がある細胞のような線維芽細胞への関心の高まりは、それらが筋肉修復6時の筋原細胞との協力的な役割を持っていることが示されている、特にとして、今そこにある、筋肉の生物学者のための刺激として見てきた。ヒト筋肉異なる細胞型の単離および精製は、このように、培養中の両方の細胞型の先天性挙動を調査しようとしている方法論の重要な考慮事項である。蛍光活性化細胞選別（FACS）は、細胞は、さらなる研究のために選別することができる方法、および/または、計数し分析した。 FACSは、確実に、ヒト筋原細胞を濃縮することが示されているが、その後の培養のための細胞の収率は、これまで7高くはなかった。そのような老化4に関連した衛星細胞由来の筋原細胞と非常に貧しい増殖や分化などの体細胞の限定された複製の可能性を考えると、より穏やかなアプローチが必要とされる。シングル筋線維培養は別のは、あまり積極的で、彼らのsublaminalニッチと文化8,9におけるそれらの活性化の後にまだ常駐マウス衛星細胞を得る手段を提供。しかし、これは、この技術がヒト筋肉由来細胞を研究することに興味の多くの研究室にアクセス可能でないかもしれないことを意味する（繊維はめったに腱の腱から得ることができないため）、ヒト筋生検材料からしばしば不可能である。また、単繊維の技術は非常に限られた細胞数を提供する。 ここでは、GENに基づいて並べ替えのシステムを説明TLEの酵素コラゲナーゼを用いて細胞の消化と高純度（&gt; 95％筋原細胞）および収率（〜2.8×10 6±8.87×10 5細胞/両方を与えソーティング（MACS）、磁気活性化細胞の2連続ラウンドが続くディスパーゼ培養での実験のためのg組織）。 CD56は、in situ 10 およびインビトロ 11 におけるヒト衛星細胞の同定のためのゴールドスタンダード表面マーカーとみなされ、ビーズ結合のための理想的な表面マーカー候補を提供している。このアプローチのCD56に酸化鉄および多糖を含む超常磁性ビーズに結合した抗体を細胞に結合され、強磁場12,13に配置された高勾配磁気細胞分離カラムに通した。分離カラムは、強い磁場勾配を生成するそれらの表面に向かって磁力線を集中させる役割を果たす強磁性スチールウールや鉄球の行列（〜4tesla）14が充填されている。これらの列では、少しでも磁気細胞は、その表面14に引きつけられ吸着される。結合していない（CD56 - ）は、磁気マイクロビーズで標識されたCD56 +細胞は、磁界12,15から除去されるまで保持されているのに対し、細胞がカラムを通過する。 選別プロセスの任意の段階からの細胞懸濁液は、さらなる実験のために所望の密度で播種することができる。細胞成分は免疫細胞化学を用いて同定することができる特定の介入後、広視野または共焦点蛍光顕微鏡を用いて画像化し、任意の画像内のすべての標識細胞の急速な客観的測定を可能にする画像解析手法を用いて定量的に分析した。筋原細胞一方で、ヒト線維芽細胞は容易に脂肪細胞への分化転換-私たちの研究室では、そのCD56を実証するために、画像解析16に続いて、この二重の免疫磁気ソーティングアプローチを使用していた衛星由来のsがこの脂肪生成変換5に対して非常に耐性がある。

<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

骨格筋の修復および再生は、常駐筋肉幹細胞である、衛星細胞の作用を必要とする。これらは、酵素消化を用いてヒト筋肉生検試料から単離することができ、それらの筋原性性質は、培養で研究。定量的には、酵素消化から得られた二つの主要な接着細胞型がある：CD56 +として最初に同定された（i）の衛星細胞は、（筋原細胞又は筋前駆細胞とも呼ばれる）、およびそれ以降のCD56 + /デスミン+細胞など、および（ii）muscle-とTE-7 + - CD56として識別由来の線維芽細胞、。線維芽細胞は培養液中で非常に効率的に増殖し、混合細胞集団においてこれらの細胞は、培養を支配する筋原細胞をオーバーランすることができる。培養中の細胞型のいずれかの生来の挙動を調査しようとしたときに、人間の筋肉とは異なる細胞型の単離および精製は、このように重要な方法論的な考慮事項である。ここでは、非難される両方の高純度（&gt; 95％筋原細胞）および良好な収率（〜を与えるコラゲナーゼを用いて細胞の穏やかな酵素消化に基づいて並べ替えや磁気活性化細胞選別（MACS）が続くディスパーゼのシステムをIBE 2.8×10 6±8.87培養での実験のためのin vitroでの 7日後に×10 5細胞/ g組織）。このアプローチは、CD56に対する抗体にコンジュゲートした磁気ビーズのビーズと混合筋肉由来細胞集団をインキュベートした後、磁場も細胞を通過させるに基づいている。細胞カラムを通過妨げられずに通過する- CD56が、一方、マイクロビーズに結合されたCD56 +細胞は、フィールドによって保持されている。選別プロセスの任意の段階からの細胞懸濁液を播種し、培養することができる。所与の介入、細胞の形態以下、核転写因子を含むタンパク質の発現および局在を免疫特異的な抗体で標識し、画像のpを用いて定量することができるrocessingと解析パッケージ。

精製された筋原細胞及び線維芽細胞は、脂肪生成のためのそれらの可能性を評価するための任意の場所の間で7〜30日間の脂肪生成栄養培地、続いて三日間脂肪細胞分化培地で培養することができる。脂肪生成および筋原性系統マーカーのための免疫染色と組み合わせて、精製された細胞集団を用いて、オイルレッドO染色のみ線維芽細胞画分は、脂肪細胞への分化が可能であったことを示した...

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.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

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 ビュー.  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. 

ビデオ: 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.

成人および高齢人間の骨格筋から筋原性前駆細胞/一次筋芽細胞の調製と文化

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

発生生物学

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.

ヒト筋芽細胞の単離、筋分化の評価、および貯蔵操作カルシウムエントリー測定

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.

マウス骨格筋移植からの原発性筋芽生の単離と分化

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.

病気のモデリングのための筋生検からの患者由来の細胞株の単離と不死

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.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

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.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

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).

出生後初期の発達におけるエメリー・ドレイフス型筋ジストロフィーのマウスモデルからの単一の筋繊維の分離と培養

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.

マウスおよびヒト食道から筋線維芽細胞の単離

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.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

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.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

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