Name: clinical protocol of producing adipose tissue-derived stromal vascular fraction for potential cartilage regeneration
Uploaded: 2018-09-29T14:00:00
Description: Watch this Scientific Journal Video about Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration at JoVE.com

作者确认了嘉强诊所员工的支持, 以及 Jaepil/李国宝的形象设计。这项工作得到了 MSIT (NRF-2017M3A9E4078014) 资助的 NRF 生物 & 医疗技术发展计划的研究补助金的支持;以及由科学和信息和通信技术部 (数字 NRF-2017R1A2B4002315 和 NRF-2016R1C1B2010308) 资助的韩国国家研究基金会 (NRF)。

明知大学机构审查委员会委员会 (MJUIRB) 放弃了批准和同意报告下列案例报告。此外, 这项临床议定书符合《赫尔辛基宣言》和 KFDA 的规章准则。对于这些程序, 知情同意是从病人那里得到的。
1. 吸脂
注意: 用无菌技术进行。
<ol><li>使用以下包含条件: (1) 3 级 OA 的 MRI 证据;(2) 男性或女性;(3) 18 岁以上;(4) 足够 (100-110 克) 脂肪组织吸脂;(5) 不愿意进行手术干?...

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在 2001年, 祖克。用胶原酶分解胶原基质, 从脂肪组织中分离出干细胞6。随后, 该组发现, 这些从脂肪组织中分离出来的干细胞可以转化为软骨和胚层的其他组织, 证明这些干细胞是以间充质为来源的。
同样, 本文所提出的程序也是将类似方法应用于人类患者的一种改进的协议。该协议的主要修改是将闭式系统注射器纳入 lipoaspirates 的获取和处理, 以便尽...

间充质干细胞 (MSCs) 具有再生软骨1、2、3、4、5、6的能力。它们可以很容易地从各种来源获得: 骨髓, 脐带血, 脂肪组织在许多其他。在这些来源中, 脂肪组织是唯一的来源, 可以获得足够数量的 MSCs, 没有任何文化扩张, 以再生软骨在临床设置7,8。自体骨髓基质血管分数 (SVF) 也很容易得到。然而, 非培养的骨髓中的干细胞数量非常低7,8。脐带血可能含有足够数量的 MSCs。然而, 脐带血不是一个现成的自体 SVF 的来源。
许多方法来处理脂肪组织获得 SVF 可用于临床应用。其中, 采用胶原酶法从脂肪组织中获得骨髓间充质干细胞, 并由祖克等进行开发和证实。5,6、很好接受。采用胶原酶法对骨科临床应用进行了改进。为了适用于临床设置, 系统必须是一个封闭的系统, 以保持不育, 同时保持方便。本文提出的一个特殊修改涉及 lipoaspirates 的均匀化。小尺寸 lipoaspirates 的消化速度比较大的大, 导致脂肪组织的不均匀分解。此外, 这些较大尺寸的 lipoaspirates 可能产生纤维组织, 可以堵塞注射器和针头, 同时执行联合注射9,10。为了防止这些问题, lipoaspirates 可以通过在胶原酶的孵化前切割和切碎 lipoaspirates 来进行匀质。所产生的脂肪组织衍生的 SVF 可能含有更均匀的细胞外基质 (ECM) 相比, lipoaspirates 是不均匀11。SVF 中所含的分解 ECM 可以作为脚手架12使用。
在 2009年, 韩国食品和药物管理局 (KFDA) 在医疗设施内进行了13的最小处理, 从而允许自体脂肪组织衍生 SVF。此后, 自体脂肪组织衍生 SVF 作为一种潜在的药物, 以改善骨关节炎 (OA) 患者的膝关节功能可能再生软骨样组织10,14,15,16,17,18. 
在 2011年, 白细胞首次显示脂肪组织衍生的干细胞 (陶瓷) 中含有的脂组织衍生 SVF 可以改善膝关节功能, 可能再生的软骨样组织的人 OA 患者注入血小板丰富血浆 (PRP) 14。此外, 巴基斯坦等人在2013年报告的安全数据涉及91名病人。该安全数据报告的平均功效率为 67%15。随后, 白等其他研究显示, 由于半月板撕裂和髌骨髌骨10、16、17 等患者的软骨样组织再生, 膝关节功能有可能改善. ,18。根据报道的文章, 据了解, 本条所述协议处理的100克脂肪组织中所含的干细胞数量可能从 100万-4000万不等, 视病人的特征8而定,19,20,21,22,23。
在这里, 我们提出了一个人体膝关节 OA 的临床协议, 使用自体脂肪组织衍生 SVF 与 HA 和 PRP 活化氯化钙。该临床议定书的第一个版本, 涉及一个封闭的, 手动系统, 以维持不孕, 报告了 2011年14。相同的协议被优化, 保持不孕, 并报告了2013年和 201610,15。在此, 提出了优化协议。该协议的示意图概述如图 1所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58363/58363fig1.jpg"> 图 1: 协议的示意图概述.<a href="https://www.jove.com/files/ftp_upload/58363/58363fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:941px;" width="941">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Material</td>
			<td style="width:221px;"></td>
			<td style="width:156px;"></td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5% Betadine (povidone-iodine)&#160;</td>
			<td>Firson Co., Ltd.</td>
			<td style="width:156px;">657400260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% Lidocaine&#160;</td>
			<td>Daehan Pharmaceutical Co.</td>
			<td style="width:156px;">670603480</td>
			<td></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;">Tumescent solution&#160;</td>
			<td style="width:221px;">Myungmoon Pharm. Co. Ltd.</td>
			<td style="width:156px;">N01BB01</td>
			<td style="width:303px;">The solution was composed of 500 mL normal saline, 40 mL 2% lidocaine, 20 mL 0.5% marcaine, and 0.5 mL epinephrine 1:1000.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Liberase TL and TM research grade&#160;</td>
			<td style="width:221px;">Roche Applied Science</td>
			<td style="width:156px;">5401020001</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D5LR</td>
			<td>Dahan Pharm. Co., Ltd.</td>
			<td style="width:156px;">645101072</td>
			<td>Dextrose 5% in lactated Ringer&#39;s solution&#160;</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;">Anticoagulant citrate dextrose solution&#160;</td>
			<td>Fenwal, Inc.</td>
			<td style="width:156px;">NDC:0942-0641</td>
			<td style="width:303px;">The solution was composed of 0.8% citric acid, 
			0.22% sodium citrate, and 0.223% dextrose.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3% (w/v) Calcium chloride&#160;</td>
			<td>Choongwae Pharmaceutical Co.</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5% (w/v) HA (Hyaluronic acid )</td>
			<td>Dongkwang pharm. Co., Ltd.</td>
			<td style="width:156px;">645902030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Ropivacaine</td>
			<td>Huons Co., Ltd.</td>
			<td style="width:156px;">670600150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Equipment</td>
			<td style="width:221px;"></td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3.0 mm Cannula&#160;</td>
			<td style="width:221px;">WOOJU Medical Instruments Co.</td>
			<td style="width:156px;">ML30200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mL Luer-Lock syringe</td>
			<td style="width:221px;">BD (Becton Dickinson)&#160;</td>
			<td style="width:156px;">309653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Barrel Kit&#160;</td>
			<td>CPL Co., Ltd.</td>
			<td style="width:156px;">30-0827044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue homogenizer that contains blades</td>
			<td>CPL Co., Ltd.</td>
			<td style="width:156px;">30-0827045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotating incubator mixer</td>
			<td style="width:221px;">Medikan Co., Ltd</td>
			<td style="width:156px;">MS02060092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Centrifuge</td>
			<td style="width:221px;">Hanil Scientific Inc.</td>
			<td style="width:156px;">CE1133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Magnetic Resonance Imaging</td>
			<td style="width:221px;">Philips Medical Systems Inc.</td>
			<td style="width:156px;">18068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Ultrasound Imaging System</td>
			<td style="width:221px;">Samsung Medison co., Ltd</td>
			<td style="width:156px;">CT-LK-V10-ICM-09.05.2007</td>
			<td></td>
		</tr>
	</tbody>
</table>

clinical protocol of producing adipose tissue-derived stromal vascular fraction for potential cartilage regeneration

骨关节炎 (OA) 是最常见的衰弱性疾病之一。最近, 许多尝试通过使用不同形式的间充质干细胞 (MSCs) 来改善膝关节的功能。在韩国, 由韩国食品和药物管理局 (KFDA) 批准的骨髓浓缩物和脐带血源干细胞用于软骨再生。此外, KFDA 还允许人体脂肪组织衍生的基质血管分数 (SVF) 进行联合注射。自体脂肪组织衍生的 SVF 含有细胞外基质 (ECM) 除了间充质干细胞。ECM 排泄各种细胞因子, 以及由氯化钙活化的透明质酸 (HA) 和富含血小板的血浆 (PRP), 可帮助 MSCs 再生软骨, 改善膝关节功能。本文提出了一种通过在 OA 中再生软骨样组织来改善膝关节功能的方案。《议定书》的结果于2011年首次报告, 随后又出版了一些出版物。该协议涉及吸脂, 以获得与胶原酶混合的自体 lipoaspirates。这种 lipoaspirates 胶原酶的混合物, 然后切割和匀质, 以消除可能堵塞针在注射过程中的大纤维组织。然后, 将该混合物孵化以获得脂肪组织衍生的 SVF。由此产生的脂肪组织衍生的 SVF, 含有脂肪组织衍生的 MSCs 和 ECM 残留物, 被注射到患者的膝盖, 结合 HA 和氯化钙活化 PRP。包括三例患者谁接受了我们的协议, 导致改善膝关节疼痛, 肿胀和运动范围, 以及 MRI 证据的透明软骨样组织。

三例患者 (87 岁女性, 3 期 oa, 68 岁男性, 3 岁 oa), 一名年龄在60年级 oa 的女性, 而没有任何重要的过去病史向临床提供持续性膝痛, 并希望潜在自体脂肪组织衍生 SVF 治疗。所有三名患者的膝盖都经过整形外科医生的检查, 并提供了全膝关节置换术 (TKR), 不愿意进行手术。在手术前, 所有三例患者都接受了多次注射类固醇和 HA, 但没有任何延长的改善。
<p class="jove_content" fo:kee...

Watch this Scientific Journal Video about Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration at JoVE.com

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

在这里, 我们提出了一个协议, 以产生脂肪组织衍生的基质血管分数和它的应用, 以改善膝关节功能的再生软骨样组织的人骨关节炎患者。

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

骨关节炎是老年人口最常见的衰弱性疾病之一。目前，骨关节炎的根本原因还没有明确的治疗方法。此临床协议描述了一种创新程序，通过再生软骨（如组织）来治疗人类膝盖骨关节炎，进行有希望的微创和替代治疗。骨关节炎是老年人口最常见的衰弱性疾病之一。目前，还没有明确的治愈骨关节炎的突出原因。此临床方案是一个创新的程序，通过再生软骨样组织，为人类膝盖骨关节炎提供有希望的、微创的和替代性治疗。将患者带进带生物危害 A 类机罩的手术室，并将患者放在一个超前位置。用5%的β丁-波维丁碘清洁患者的腹部区域，并使用无菌技术将患者覆盖，露出腹部的清洁区域进行抽脂。大约五厘米的横向从脐带，麻醉两个部位，一个在左边，另一个在右侧的切口的凹面。使用两毫升2%利多卡因，无肾上腺素与25量1.5英寸的针头在5毫升注射器中，通过注射每个位点在表皮水平。麻醉切口部位，使用五毫升的肿瘤溶液和10毫升注射器和针头25规格1.5英寸。通过捏皮提高皮下水平的深度，横向将两个 0.5 厘米的切口（大约低于乌姆比利）约 5 厘米。使用数字11刀片，帮助凸起的皮肤穿透到皮下水平，但不穿透腹部壁。通过使用20厘米60米的大麻，麻醉整个下腹部区域的皮下水平，这是抽脂与700至800毫升的肿瘤溶液。在用肿瘤溶液完成整个下腹部的渗透后，通过将连接到60毫升或30毫升注射器的3.0毫米管杆连接到用于手动抽脂或专门设计的3.0毫米管连接到离心机套件，准备抽脂装置。一种由我们自己开发并经FDA CDRH批准的封闭系统注射器，用于保持无菌与真空吸脂机相连。进行抽脂，获得100至110克脂肪组织，不包括肿瘤溶液。进行抽脂时，脂肪组织将连同肿瘤溶液一起获得，但应分离和去除。为了首先通过重力分离肿瘤溶液，离心机套件中的脂肪组织可以转移到60毫升的注射器中，然后放下注射器。例如，注射器的开口部分位于底部。通过等待五到六分钟，脂肪组织和肿瘤溶液将被分离。通过按下注射器柱塞顶部，取出注射器底部的液体。执行上述步骤9至11，直到共100克至110克脂肪组织脂气每膝已累积。在按重力分离肿瘤溶液并每60毫升离心机套件积累50克至55克脂质吸气后，将两个离心机套件放入离心容器桶中，在1600 G下旋转5分钟，凝结脂质吸气，将液体从脂肪组织分离。这个过程进一步凝结脂质吸气，在某些情况下可能会产生脂肪油。注意不要摇晃，拆下离心机套件底部的安全盖和插头。缓慢向下推离离心套件柱塞顶部，拆下底部液体。在单独的60毫升注射器上，溶解10毫克胶原酶，5毫克胶原蛋白酶特异性结缔组织，5毫克胶原酶特异性脂肪组织与50毫升的正常盐水。使用专用连接器将 60 毫升注射器连接到离心机，将大约 25 至 30 毫升的浓缩脂质吸附与溶解的胶原酶、5 毫克专用的结缔组织胶原酶和 5 毫克胶原酶，以一比一、体积与体积的比例混合。使用杆或推杆将 60 毫升注射器和离心机套件之间的含量推，彻底混合以凝结预吸气和胶原酶。将吸脂剂和胶原酶的混合物转移到60毫升注射器。将每个含有混合物的 60 毫升注射器与含有刀片的组织均质器连接。将空的 60 毫升注射器连接到均质器的另一端。通过均质器将混合物推入其他 60 毫升注射器 4 至 6 次，从而切割和切碎脂质吸气。通过专用连接器将均质脂吸气和胶原蛋白混合物转移回 60 毫升离心机套件。将离心机套件放在容器中，放入在 37 摄氏度预热的培养箱中。在 37 摄氏度下用均质混合物孵育两个离心机套件 40 分钟，同时以 45 rpm 转速旋转。孵育40分钟后，以无菌方式将容器从孵化器中取出。然后取出离心机套件并将其放在离心机中。将混合物在800 G下离心5分钟，分离脂肪组织衍生的SVF。离心机后，通过取出柱塞顶部的注射器盖，并通过注射器锁连接将30毫升注射器放在柱塞开口上，从每个离心机套件中去除上一代，包括胶原酶和消化的脂肪组织。缓慢地按下 30 毫升注射器的桶部分，使上看液填充到 30 毫升注射器中。一直按下30毫升注射器筒，一直压到离心机套件底部的最后三到四毫升，只留下最后三到四毫升的脂肪组织衍生SVF。上则被丢弃。从柱塞顶部取出 30 毫升注射器，并给注射器加注 5%葡萄糖乳酸环环器溶液，也称为 D5LR。通过将装有D5LR的30毫升注射器固定在柱塞开口顶部，将含有3至4毫升脂肪组织衍生SVF的离心机套件填充，并加注D5LR至55毫升。然后取出 30 毫升注射器，盖住柱塞开口，并在 300 G 下再次将离心机套件离心约 4 分钟。重复步骤 17 到 21，总共进行四次洗涤。第四台离心机后，为了获得用于喷射的最终 SVF，请拆下离心机套件底部开口的安全盖和插头，而不摇晃或转动离心机套件。使用专门设计的连接器将 20 毫升注射器连接到离心机套件底部开口。来回多次拉动 20 毫升注射器的柱塞，以摇动已稳定在离心机套件底部的细胞。去除含有 AST 和 ECM 以及其他细胞和组织所需的 SVF 总体积。通常从每个离心机套件中约三到四毫升用于膝关节注射。我们做了30毫升的自体血和2.5毫升的抗凝血酸化脱酸葡萄糖溶液。然后，抽血被转移到60毫升离心机桶包。然后，在730重力下离心5分钟。和上一代被移除，并放入一个新的60毫升离心机桶套件。上那液在1350重力下离心4分钟，获得4.4毫升PRP。用5%的β丁清洁患者的膝盖，并无菌地披上。为三元骨和股骨之间的关节空间，将膝盖前部的关节空间置换。用稀释的利多卡因（lidocaine）表面麻醉注射部位，用四毫升正常盐水稀释1%利多卡因的一毫升，从皮肤到关节胶囊外。然后用稀释的罗皮瓦卡因内麻醉关节胶囊内部，用三毫升的正常盐水稀释一毫升0.75%罗皮瓦卡因。将两毫升HA混合到20毫升注射器中20毫升的SVF中，通过注射器将注射器连接到注射器接头。通过使用注射器注射器连接器，在已经准备并准备并在五毫升注射器中准备的三到四毫升自体 PRP 中加入 0.4 毫升氯化钙。将 3.5 至 4.5 毫升 PRP 氯化钙加入 5 毫升注射器中，将 8 至 10 毫升 HA-SVF 混合物加入 20 毫升注射器中，通过注射器将注射器与注射器接头混合。立即将混合物（约12至15毫升）缓慢注射到膝关节前，使用38毫米18表针，带或不带超声波指导。注射后，通过折叠4x4棉纱布四次，并将胶带放在折叠的4x4纱布上，用压力包扎注射部位。指示患者保持静止60分钟，以允许细胞附着。指示患者在出院后将活动限制在最少一周内。返回诊所，在三周内再注射三次由氯化钙激活的PRP。第三阶段OA的患者接受了协议部分所述的治疗。在 ASV-ECM 混合物注射的第二周后，患者的疼痛和ROM得到改善。到第16周，患者的疼痛和ROM明显改善了超过70%的治疗后MRI，在第16周后采取，显示软骨样组织在膝盖的内侧增加厚度。另一名具有第三阶段OA的患者接受了协议部分所述的治疗。在 ASV-ECM 混合物注射的第二周后，患者的疼痛和ROM得到改善。到第18周，疼痛和ROM明显改善到80%重复的MRI后，第18周显示，软骨样组织的高度在膝盖的前部侧增加。第三阶段OA的第三名患者接受了协议部分所述的治疗。在第二周的 ASV-ECM 混合物注射后，患者的疼痛和ROM在第22周时改善了约50%，疼痛和ROM明显改善超过80%的重复MRI后，第22周显示，在膝盖的中侧软骨样组织的高度增加。目前没有用于治疗OA的治疗方法。然而，使用 ASCs 和均质 ECM 进行干细胞治疗的皮下注射，以及用氯化钙激活的 HA 和类似 PRP，可为当前治疗膝关节 OA 的治疗提供替代方案。

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

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

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. ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

临床检查协议检测非典型和经典刮在绵羊

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

人类肾脏血管周围间质细胞新型临床分级分离方法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

基质血管部分富集脂肪移植治疗症状性端 neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

一种优化的埃文斯蓝协议对小鼠血管泄漏的评估

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...