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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrodisc Needle Filter of Supor Membrane</td>
			<td>Acros Organics</td>
			<td>4652</td>
			<td>0.22 &#956;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。

purification-characterization-extracellular-vesicles-from-human

Research

JoVE Journal

Medicine

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

539 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

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

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. ...

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 ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

使用创新的无异种方法进行人类治疗，分离和培养人脂肪来源的干细胞

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

评估缺氧人胎盘衍生的 sEV 诱导的小鼠 BBB 破坏

Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated with preeclampsia. The underlying mechanisms of these cerebrovascular complications remain unclear. However, they are linked to placental dysfunction and blood-brain barrier (BBB) disruption. Nevertheless, the connection between these two distant organs is still being determined. Increasing evidence suggests that the placenta releases signaling molecules, including extracellular vesicles, into maternal circulation. Extracellular vesicles are categorized according to their size, with small extracellular vesicles (sEVs smaller than 200 nm in diameter) considered critical signaling particles in both physiological and pathological conditions. In preeclampsia, there is an increased number of circulating sEVs in maternal circulation, the signaling function of which is not well understood. Placental sEVs released in preeclampsia or from normal pregnancy placentas exposed to hypoxia induce brain endothelial dysfunction and disruption of the BBB. In this protocol, we assess whether sEVs isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the BBB in vivo.

作者聚焦:使用缺氧人胎盘来源的小细胞外囊泡对雌性小鼠子痫前期的一个方面进行建模

A protocol is presented to evaluate whether small EVs (sEVs) isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the blood-brain barrier in nonpregnant adult female mice.

缺氧人胎盘的小细胞外囊泡破坏小鼠血脑屏障

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated ...