Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.

IF 5.3 2区 医学 Q2 CELL BIOLOGY Skeletal Muscle Pub Date : 2024-10-11 DOI:10.1186/s13395-024-00355-1
Uxia Gurriaran-Rodriguez, Yves De Repentigny, Rashmi Kothary, Michael A Rudnicki
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Abstract

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

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利用切向流过滤和尺寸排阻色谱法从再生肌肉组织中分离细胞外小泡。
我们最近惊人地发现,肌肉损伤后,Wnt7a 会上调,并从外泌体表面的新生再生肌纤维中分泌,从而在远端引起肌肉再生反应。尽管近年来从不同组织中分离细胞外囊泡(EVs)取得了进展,但仍缺乏从肌肉组织中纯化 EVs 的特定方法。为了消除使用超速离心等传统方法时通常会发现的非EV分泌蛋白和细胞质片段的污染,我们采用了一种结合切向流过滤(TFF)和尺寸排阻色谱(SEC)的方案。我们发现,这种方法可以同时纯化与 EV 结合的 Wnt7a(回流物部分)和游离的非 EV Wnt7a(渗透物部分)。在此,我们介绍了这一优化方案,该方案专为从后肢肌肉外植体中分离 EVs 而设计,不会与其他来源的非 EV 结合蛋白产生交叉污染。该方案的第一步是通过连续离心去除大的EV。然后浓缩细胞外囊泡,并用 TFF 在交换缓冲液中洗涤。最后,用 SEC 去除 TFF 后残留的可溶性蛋白质。总之,该程序可用于从含有来自任何细胞类型或组织的 EVs 的条件培养基或生物流体中分离 EVs,从而提高 EVs 制备的可重复性、效率和纯度。我们的纯化方案可获得保持结构完整性的高纯度 EVs,因此完全符合体外和体内生物活性和分析测试的要求。
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来源期刊
Skeletal Muscle
Skeletal Muscle CELL BIOLOGY-
CiteScore
9.10
自引率
0.00%
发文量
25
审稿时长
12 weeks
期刊介绍: The only open access journal in its field, Skeletal Muscle publishes novel, cutting-edge research and technological advancements that investigate the molecular mechanisms underlying the biology of skeletal muscle. Reflecting the breadth of research in this area, the journal welcomes manuscripts about the development, metabolism, the regulation of mass and function, aging, degeneration, dystrophy and regeneration of skeletal muscle, with an emphasis on understanding adult skeletal muscle, its maintenance, and its interactions with non-muscle cell types and regulatory modulators. Main areas of interest include: -differentiation of skeletal muscle- atrophy and hypertrophy of skeletal muscle- aging of skeletal muscle- regeneration and degeneration of skeletal muscle- biology of satellite and satellite-like cells- dystrophic degeneration of skeletal muscle- energy and glucose homeostasis in skeletal muscle- non-dystrophic genetic diseases of skeletal muscle, such as Spinal Muscular Atrophy and myopathies- maintenance of neuromuscular junctions- roles of ryanodine receptors and calcium signaling in skeletal muscle- roles of nuclear receptors in skeletal muscle- roles of GPCRs and GPCR signaling in skeletal muscle- other relevant aspects of skeletal muscle biology. In addition, articles on translational clinical studies that address molecular and cellular mechanisms of skeletal muscle will be published. Case reports are also encouraged for submission. Skeletal Muscle reflects the breadth of research on skeletal muscle and bridges gaps between diverse areas of science for example cardiac cell biology and neurobiology, which share common features with respect to cell differentiation, excitatory membranes, cell-cell communication, and maintenance. Suitable articles are model and mechanism-driven, and apply statistical principles where appropriate; purely descriptive studies are of lesser interest.
期刊最新文献
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