Name: mechanical micronization of lipoaspirates for regenerative therapy
Uploaded: 2019-03-15T13:00:00
Description: Watch this Scientific Journal Video about Mechanical Micronization of Lipoaspirates for Regenerative Therapy at JoVE.com

这项工作得到了中国国家自然科学基金 (81471881、81471881、81471881)、中国广东省自然科学基金 (2014A030310155) 和南方医院院长基金会 (2014B009、2015Z002, 2015Z002, 2016B001)。

这项研究得到了中国广州南方医院伦理评审委员会的批准。脂肪组织是从健康的捐献者那里收集的, 他们书面知情同意参加这项研究。所有动物实验均由南方医院机构动物护理和使用委员会批准, 并按照国家卫生和医学研究委员会 (中国) 的指导方针进行。
1. ecm只能用凝胶制备
<ol><li>收获脂肪。<ol>
<li>用3毫米多端口插管对人体进行吸脂, 其中包含几个直径为1毫米的尖侧孔, 吸?...

<ol>
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基于干细胞的再生疗法在不同的疾病中显示出巨大的潜在优势。Asc 是优秀的治疗候选, 因为它们易于获得, 并具有组织修复和新组织再生的能力15。然而, 扩大其临床应用是有局限性的, 因为需要复杂的程序来分离细胞和胶原酶进行加工.因此, 必须开发一种简单的技术, 在不使用胶原酶的情况下获得干细胞。
在这项研究中, 我们提出了一个纯?...

干细胞疗法为组织修复和再生提供了一个范式转变, 以便它们可以为各种疾病提供替代治疗方案1。干细胞 (如诱导多能干细胞和胚胎干细胞) 具有巨大的治疗潜力, 但由于细胞调节和伦理考虑而受到限制。脂肪源性间充质基质干细胞 (Asc) 很容易从脂质中获得, 不受同样的限制;因此, 它已成为实用再生医学的理想细胞类型2。此外, 它们是非免疫原性的, 从自体脂肪3中获得丰富的资源。
目前, Asc 主要是通过胶原酶介导的脂肪组织消化获得的。脂肪组织的基质血管分数 (SVF) 包含 Asc、内皮祖细胞、周细胞和免疫细胞。虽然酶化获得高密度的 Svf/asc 被证明具有有益的效果, 但一些国家的立法对使用胶原酶4的细胞基产品的临床应用进行了严格的规范。用胶原酶消化脂肪组织30分钟至1小时以获得 SVF 细胞会增加制备过程中的外源材料和生物污染的风险。粘附培养和 Asc 的纯化需要数天到数周的时间, 需要特定的实验室设备。此外, 在大多数研究中, SVF 细胞和 Asc 被用于悬浮液。如果没有细胞外基质 (ECM) 或其他载体的保护, 游离细胞易受攻击, 导致注射后细胞保留不良, 并损害治疗结果5。所有这些原因都限制了干细胞治疗的进一步应用。
为了从没有胶原酶介导消化的脂肪组织中获得 asc, 开发了几种机械加工程序, 包括离心、机械切碎、切碎、粉碎、粉碎和切碎.,8,9. 这些方法被认为通过机械破坏成熟的脂肪细胞及其含油囊来使组织和 asc 凝结。此外, 这些含有高浓度 asc 的制剂在动物模型 8、9、10中显示出相当大的治疗潜力。
2013年, Tonnard等人引进了纳米管接枝技术, 该技术涉及通过注射器间处理生产乳化的聚脂.注射器间移位产生的剪切力可以选择性地破坏成熟的脂肪细胞。根据他们的发现, 我们开发了一种纯机械的处理方法, 去除了脂质中的大部分脂质和液体, 只留下 SVF 细胞和分馏 ecm, 即 ECM/SVF-gel12。在此, 我们描述了人源性脂肪组织产生 ECM/SVF-gel 的机械过程的细节。

<table border="0" cellpadding="0" cellspacing="0" style="width:733px;" width="734">
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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488-conjugated isolectin GS-IB4</td>
			<td style="width:232px;">Molecular Probes</td>
			<td style="width:119px;">I21411</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">guinea pig anti-mouse perilipin</td>
			<td>Progen</td>
			<td>GP29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Thermofisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">wide tip pipet</td>
			<td>Celltreat</td>
			<td>229211B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>TCS SP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nude nice&#160;</td>
			<td>Southern Mdical University</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">light microscope&#160;</td>
			<td>Olympus</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL tube</td>
			<td>Cornig</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile bag</td>
			<td>Laishi</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microtome</td>
			<td>Leica&#160;</td>
			<td>CM1900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td>Heraus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanical micronization of lipoaspirates for regenerative therapy

基质血管分数 (SVF) 已成为各种疾病的再生工具;然而, 立法严格规范了使用胶原酶的细胞产品的临床应用。在这里, 我们提出了一个协议, 以产生注射的混合 SVF 细胞和本机细胞外基质从脂肪组织通过一个纯粹的机械过程。将脂质放入离心机中, 以 1200 x 克的速度旋转 3分钟。中间层被收集并分成两层 (底部是高密度脂肪, 顶部是低密度脂肪)。上层通过注射器间移位直接乳化, 速度为 20 mL/s, 为6倍至8倍。乳化脂肪以 2, 000 x 克离心3分钟, 油层下的粘性物质被收集并定义为细胞外基质 (ecm)//SVF-gel.。收集顶层上的油。在高密度脂肪中加入约5毫升的油, 并通过注射器间移位进行乳化, 速度为 20 mlsx, 速度为6倍至8倍。乳化脂肪以 2, 000 x 克离心 3分钟, 粘性物质也是 ECM/SVF-gel。将 ECM/SVF-gel 移植到裸鼠体内后, 通过组织学检查进行移植和评估。结果表明, 该产品具有再生成正常脂肪组织的潜力。这个过程是一个简单, 有效的机械离解程序, 浓缩 SVF 细胞嵌入在其自然支持 ECM 的再生目的。

在将 Coleman 脂肪加工成 ECM/SVF-gel 后, 废弃油的体积占最终体积的 80%, 在油层下保存的脂肪组织中只有20% 被视为 ECM/SVF-gel (图 1a)。ecmmsvf 凝胶具有光滑的液体状质地, 使其能够穿过27g 细针;然而, 科尔曼脂肪是由一个整体脂肪结构与大纤维, 只能通过 18 G 插管 (图 1b)。
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Watch this Scientific Journal Video about Mechanical Micronization of Lipoaspirates for Regenerative Therapy at JoVE.com

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

再生疗法用脂质吸入剂的机械微化

在这里, 我们提出了一个协议, 以获得基质血管分数从脂肪组织通过一系列的机械过程, 其中包括乳化和多离心。

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...

从脂藻酸盐中获取频闪血管部分或SVFs需要用胶原酶进行消化，这增加了生物污染的风险。一种更好的方法，以获得SVF没有胶原酶应该开发。这些技术使我们能够通过简单的机械过程在其原生细胞外矩阵中获得SVF。演示这个程序的将是来自我们实验室的研究生陈西航。获得脂肪样本后，将脂止汗液转移到一个50毫升的管中，让收获的脂肪平衡10分钟。接下来，使用宽尖移液器将脂肪从管的顶层拉入新的 50 毫升管中，然后通过离心来旋转脂肪。管的上层是科尔曼脂肪分数。将科尔曼脂肪的上部 2/3 转移到新的 15 毫升管中。这部分被认为是低密度脂肪。将科尔曼脂肪的下三分之一转移到第二个15毫升管中。这部分被认为是高密度脂肪。要从低密度脂肪中产生细胞外基质频量血管分数凝胶，请使用两个 20 毫升注射器，由雌性 luer 锁连接器连接，以移位 10 毫升的低密度脂肪。这是摧毁脂肪细胞的关键步骤。执行间环转换的速度和时间有助于提高最终产品的质量。最后一次间移后，通过离心收集脂肪，将顶部油层转移到15毫升管中。然后将粘性中细胞外基质频量血管分数凝胶层转移到一个新的15毫升锥形管中。要从高密度脂肪中产生细胞外基质生成血管分数凝胶，请将低密度油馏分的两毫升添加到高密度脂肪样品的五毫升中。移位混合脂肪，如证明，直到在乳液内观察到絮凝。通过离心收集絮凝物并丢弃顶部油层。然后收集粘性中间层，并混合此分数与低密度细胞外基质频量血管分数凝胶样品。将科尔曼脂肪处理成细胞外基质频血管分数凝胶后，废弃油的体积占最终体积的80%，而油层下保存的脂肪组织只有20%被视为细胞外基质频量血管分数凝胶。细胞外基质频血管分数凝胶具有光滑的液体状纹理，使其通过27量表细针。然而，科尔曼脂肪由一个整体脂肪结构组成，大纤维只能通过18个量表管。移植后的第三天，大量小型脂肪细胞与多个细胞内脂滴，广泛血管化结缔组织，并渗透炎症细胞出现。从第15天开始，炎症细胞的数量开始逐渐减少，脂肪细胞开始成熟。到90日，移植物中大部分血管化结缔组织已被成熟的脂肪细胞所取代。细胞外基质频血管部分凝胶移植也含有一些利平阳性的腺细胞移植三天后，与小尺寸的脂肪细胞，与多个细胞内脂滴开始出现第15天。当天的每个领域 90 个细胞外基质频血管部分凝胶移植部分表现出大量积极腺细胞和新形成的血管。可以进行流细胞测量以确定产品中的细胞群体。扫描电子显微镜可以可视化产品的微观结构。该技术为外科医生和研究人员提供了一种简单的方法，从脂肪组织获得具有高浓度SVF细胞的SVF细胞与原生细胞基质纤维的产品。

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Medicine

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光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

脉络细胞自体移植治疗老年性黄斑变性的再生疗法: 初步体内报告

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Mechanical Ventilation Boot Camp Curriculum

机械通气训练营课程

Medical management of mechanically ventilated patients is challenging to novice providers. Incorrect management of this population may lead to increased ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

分离脂肪衍生的再生细胞用于治疗根治性前列腺切除术后的勃起功能障碍

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...