Name: purification and characterization of extracellular vesicles from human adipose-derived mesenchymal stem cells
Uploaded: 2024-05-03T13:50:00.000+00:00
Duration: 4 min 54 s
Description: Read this Scientific Article Protocol about Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs at JoVE.com

这项工作得到了中国国家自然科学基金 （82202473） 的部分支持。

协议步骤的概述如图 1 所示。材料 表中列出了研究中使用的试剂和设备的详细信息。
1. 培养基制备
<ol><li>使用 450 mL 基础培养基、50 mL 不含外泌体的胎牛血清和 5 mL 三联抗生素制备不含外泌体的 ADSC 培养基。</li>
</ol>2. ADSC 的复苏和培养
注意:复苏 ADSC。
<ol><li>事先打开超净台的紫外线辐射并消毒 30 分钟。</li>
	<li>事先在 37 °C 水浴中预热培养基。</li>
	<li>从液氮中检索 2 管冷冻的 ADSC （P3），并在 37 °C 恒温水浴中搅拌直至完全解冻。 注意: 冷冻管的开口不应与水接触，以防止污染。</li>
	<li>使用 75% 酒精对冻存管进行消毒，然后将其移至超净台上。使用移液管吸出细胞悬液，并将其放入 15 mL 离心管中。</li>
	<li>将离心管放入低速离心机中，并在 4 °C 下以 250 x g 离心 5 分钟。</li>
	<li>准备四个 10 cm 培养皿，并将 9 mL 培养基放入每个培养皿中。标记名称、细胞类型、世代和实验日期，然后在 37 °C 恒温细胞培养箱中预热。</li>
	<li>对离心管进行消毒，并将其移至超净台上。用移液管去除上清液。</li>
	<li>加入 4 mL 培养基，轻轻搅拌溶液（1 x 105 个细胞/mL），直至其均匀分散。 注:提取上清液时注意不要去除细胞沉淀。</li>
	<li>向每个培养皿中加入 1 mL 细胞悬液。用十字法摇晃盘子。 注意:将板放在水平面上，并以交叉模式搅拌（向上、向下、向左和向右移动）重复 5-6 次。避免圆周运动，因为它们可能会导致细胞在中心积聚。</li>
	<li>用光学显微镜以 40 倍放大倍率观察细胞，然后将培养皿放入培养箱中。 注意:观察细胞的主要目的是确定它们是否均匀分布，因为分布不均匀会影响后续生长。</li>
	<li>为细胞准备交换培养基。<ol><li>事先打开超净台的紫外线辐射并消毒 30 分钟。</li>
		<li>事先在 37 °C 水浴中预热培养基。</li>
		<li>在光学显微镜下以 40 倍放大倍率观察细胞状态。汇合度约为 50%。 注意:汇合水平计算为细胞粘附在壁上并完全拉伸后细胞占据的面积与培养皿表面积的比率。</li>
		<li>消毒后将培养皿转移到超净台（生物安全 2 级）上，并去除培养基。</li>
		<li>用 2 mL PBS 溶液轻轻冲洗每个培养皿两次。</li>
		<li>向每个培养皿中加入 10 mL 培养基。将培养皿转移到培养箱中。</li>
	</ol></li>
</ol>3. 收集 ADSCs 上清液并提取 EV
<ol><li>事先打开超净台的紫外线辐射并消毒 30 分钟。</li>
	<li>在显微镜下观察细胞状态。汇合度约为 80%。</li>
	<li>消毒后将培养皿转移到超净台上。用移液管小心收集上清液，并将其放入 50 mL 离心管中。 注意:细胞可以冷冻或丢弃。如果不需要无菌，可以在手术台上执行以下步骤。此时，可以暂停实验，然后稍后重新启动。如果实验停止，请将上清液储存在 4 °C 冰箱中不超过一周。不建议长期存放，因为它可能会影响 EV 的活动。最佳方法是立即提取 EV。</li>
	<li>离心（300 x g，10 分钟，在室温下）以去除悬浮细胞。用移液管收集上清液。 注意:不要倒。留下少许上清液，以防止沉淀被吸走。</li>
	<li>离心以去除细胞碎片（2000 x g ，10 分钟，4 °C），然后继续用移液管收集上清液。 注意:关键点与步骤 3.4 相同。</li>
	<li>用 0.22 um 膜过滤上清液，以去除大颗粒和细胞碎片。 注:过滤前用 2 mL 无菌 PBS 过滤上清液以润湿滤膜。</li>
	<li>在超速离心机中离心（10,000 x g ，30分钟，4°C）。</li>
	<li>接下来，在 4 °C 下以 1,00,000 x g 离心 70 分钟，以去除沉淀并收集上清液。 注意:通过多次离心纯化获得的 EV 以提高其纯度。肉眼可能无法看到颗粒。因此，轻柔操作移液器并在底部留下少许上清液以避免沉淀至关重要。</li>
	<li>用移液管去除上清液。用 PBS 重悬沉淀并离心（1,00,000 x g，70 分钟，4 °C）。 注意:肉眼可能无法看到颗粒。因此，当用移液管吸出上清液时，温和彻底地去除上清液以最大限度地提高 EV 的纯度至关重要。</li>
	<li>去除上清液并获得 EVs 沉淀。用 1 mL PBS 重悬，将其转移到 1 mL 离心管中，并储存在 4 °C 冰箱中以供后续检测。 注意:肉眼可能无法看到颗粒。因此，当用移液管吸出上清液时，温和彻底地去除上清液以最大限度地提高 EV 的纯度至关重要。如果长时间不需要样品，请将其存放在 -80 °C 冰箱中。</li>
</ol>

基于超速离心机从人 ADSC 中分离细胞外囊泡

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作者没有经济利益需要申报。

在正式的实验过程中，有几点对于获得最佳实验结果至关重要。根据我们以前的经验，建议选择第 3-6 代 ADSC，因为它们可以确保最佳的细胞状态。在 P3 之前，红细胞、内皮细胞和其他杂细胞可能没有被筛选出来。P6 之后，细胞可能会逐渐老化，这会影响分泌的 EV 的状态。其次，当细胞汇合度在 80% 和 90% 之间时，必须收集上清液，以确保尽可能多地分泌 EV。为了保持 EV 的最大纯度，研究人员在?...

大多数 ADSC 来源于脂肪组织，并已被证明可促进各种组织和器官的再生和重建，包括心肌、骨骼和皮肤1。最近的研究表明，ADSC 的再生功能可能是由于细胞间接触和细胞旁分泌作用2。旁分泌效应是细胞在短距离内相互作用和传递信息的重要手段，这一功能是通过细胞外囊泡 （EV） 实现的。
EV 是由细胞产生的双层膜结构，直径范围为 40 nm 至 160 nm（平均约为 100 nm）。它们影响不同的细胞功能，例如细胞因子产生、细胞增殖、细胞凋亡和代谢 3,4。已经对 ADSC EV 的功能进行了大量研究，包括促进骨再生、口腔粘膜组织再生、组织移植后脂肪组织存活和皮肤伤口修复 5,6,7,8。ADSC EV 在再生医学中的巨大潜力是显而易见的。从上清液中提取和分离 EV 的方法多种多样，例如基于离心、沉淀、分子大小、亲和力和微流体的技术。超速离心法被广泛认为是分离 EV 的金标准9。用于 EV 分离的超速离心的基本原理是基于这样一个事实，即样品中的颗粒具有不同的沉降系数，导致它们在不同的分离层中沉降和聚集。然而，尚未建立强调超速离心过程中预防措施的详细方案。
因此，本研究客观地概述了 ADSC 培养、上清液收集和 EVs 超速离心程序和关键点，具有逻辑信息流和清晰、正式的语言。这为未来的实验提供了有价值的参考。

<table><tbody><tr><td>Acrodisc Needle Filter of Supor Membrane</td><td>Acros Organics</td><td>4652</td><td>0.22 &amp;mu;m</td></tr><tr><td>Basal Medium For Cell Culture</td><td>OriCell</td><td>BHDM-03011</td><td></td></tr><tr><td>Fetal Bovine Serum Without EXO + Culture Supplement (For Human Adipose-derived Mesenchymal Stem Cells)</td><td>OriCell</td><td>HUXMD-05002</td><td></td></tr><tr><td>Inverted Microscope</td><td>OLYMPUS</td><td>Lx70-S8F2</td><td></td></tr><tr><td>Low Speed Centrifuge</td><td>Anhui USTC Zonkia Scientific Instruments Co.,Ltd.</td><td>SC-3612</td><td></td></tr><tr><td>Normocin</td><td>InvivoGen</td><td>ant-nr-05</td><td></td></tr><tr><td>Optima Max-XP Tabletop Ultracentrifuge</td><td>Beckman Coulter</td><td>393315</td><td></td></tr><tr><td>Penicillin-Streptomycin-Gentamicin Solution (100x)</td><td>Solarbio</td><td>P1410</td><td></td></tr></tbody></table>

purification and characterization of extracellular vesicles from human adipose-derived mesenchymal stem cells

人脂肪来源的间充质干细胞 （ADSC） 可以促进各种组织和器官的再生和重建。最近的研究表明，它们的再生功能可能归因于细胞间接触和细胞旁分泌效应。旁分泌效应是细胞在短距离内相互作用和传递信息的重要方式，其中细胞外囊泡 （EV） 作为载体发挥着功能作用。ADSC EV 在再生医学中具有巨大潜力。多项研究报告了这些方法的有效性。目前根据离心、沉淀、分子大小、亲和力和微流体等原理描述了提取和分离 EV 的各种方法。超速离心法被认为是分离 EV 的金标准。尽管如此，仍然缺乏强调超速离心过程中预防措施的细致方案。本研究介绍了 ADSC 培养、上清液收集和 EV 超速离心所涉及的方法和关键步骤。然而，尽管超速离心具有成本效益并且不需要进一步处理，但仍存在一些不可避免的缺点，例如回收率低和 EV 聚集。

首先，对 ADSCs 进行表征和鉴定，包括其形态学10 和表面抗体。根据 图 2A，很明显 ADSCs 呈纺锤形排列，并在密集生长后形成涡旋。将培养的细胞分化为脂肪生成细胞、成骨细胞和成软骨细胞，并用油红 O 、 茜素红 和 阿尔新蓝11 染色。诱导分化实验和流式细胞术验证了它们确实是具有多向分化潜力的 ADSC。然而，22% 的细胞在结果中为 CD45 ?...

Read this Scientific Article Protocol about Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs at JoVE.com

作者聚焦:标准化和改进人 ADSC 细胞外囊泡的提取和纯化

该方案描述了所有程序，从培养人脂肪来源的间充质干细胞 （ADSC） 和收集上清液到使用超速离心提取细胞外囊泡 （EV）。

从人脂肪来源的间充质干细胞中纯化和表征细胞外囊泡

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...

我们的研究重点是从人脂肪来源的间充质干细胞中制备和纯化细胞外囊泡。我们要解决的问题是如何规范这个过程，提高净化效率。目前，用于提取细胞外囊泡的技术很多，如超速离心、超滤、体积排阻色谱、聚合物沉淀、免疫亲和捕获、微流控技术等。然而，不可否认，超速离心法被认为是黄金标准。未来，我们将专注于细胞外囊泡作为载体的应用，包括携带 MRNA、质粒和其他小分子并将它们转染到细胞中。

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Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

590 次观看.  Chinese PLA General Hospital. 我们的研究重点是从人脂肪来源的间充质干细胞中制备和纯化细胞外囊泡。我们要解决的问题是如何规范这个过程，提高净化效率。目前，用于提取细胞外囊泡的技术很多，如超速离心、超滤、体积排阻色谱、聚合物沉淀、免疫亲和捕获、微流控技术等。然而，不可否认，超速离心法被认为是黄金标准。未来，我们将专注于细胞外囊泡作为载体的应用，包括携带 MRNA、?...

视频: 作者聚焦:标准化和改进人 ADSC 细胞外囊泡的提取和纯化

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

科研

生物学

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

医学

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

从脂肪组织和基质血管分数表征中获取间充质干细胞的技术在长期冷冻保存中的应用

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

用于细胞表征和分泌特征的人类脂肪组织微分裂

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

一种从人间充质干细胞中分离小细胞外囊泡的简单台式过滤方法

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

从人脂肪来源的间充质干细胞中纯化和表征细胞外囊泡

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此视频中的章节

从人类Lipoaspirates脂肪来源干细胞的手动隔离

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

从脂肪组织和基质血管分数表征中获取间充质干细胞的技术在长期冷冻保存中的应用

用于细胞表征和分泌特征的人类脂肪组织微分裂

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

一种从人间充质干细胞中分离小细胞外囊泡的简单台式过滤方法

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定