Name: isolation and culture of human adipose-derived stem cells with an innovative xenogeneic-free method for human therapy
Uploaded: 2023-02-03T14:50:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy at JoVE.com

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hASCs是从一名健康女性的腹部脂肪组织中分离出来的，该女性在瑞士洛桑CHUV洛桑大学医院使用腹部自体皮瓣（深下上腹部穿支肌皮瓣，[DIEP]）进行了乳房重建。皮瓣的废弃部分和脂肪组织是在患者签署知情同意书后获得的。根据《赫尔辛基宣言》，所有协议均由医院的生物银行部门DAL（编号314 GGC）和伦理委员会审查和批准。
注意：所有步骤必须在层流罩下和无菌条件下进行?...

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The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Cowper, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+as+a+functional+substitute+for+fetal+bovine+serum+in+the+culture+of+human+adipose+derived+stromal/stem+cells.">Human platelet lysate as a functional substitute for fetal bovine serum in the culture of human adipose derived stromal/stem cells.</a> Cells. 8 (7), 724 (2019).</li><li>Kim, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+the+treatment+and+prevention+of+graft-versus-host+disease:+Experiments+and+practice.">Mesenchymal stem cells for the treatment and prevention of graft-versus-host disease: Experiments and practice.</a> Annals of Hematology. 92 (10), 1295-1308 (2013).</li><li>Palombella, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+metal+micro+and+nano-particles+on+hASCs:+An+in+vitro+model.">Effects of metal micro and nano-particles on hASCs: An in vitro model.</a> Nanomaterials. 7 (8), 212 (2017).</li><li>Han, S., Sun, H. 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N., Papamichail, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+medium+composition+and+translational+adult+bone+marrow-derived+stem+cell+research.">Cell culture medium composition and translational adult bone marrow-derived stem cell research.</a> Stem Cells. 24 (5), 1409-1410 (2006).</li><li>Perez-Ilzarbe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ex+vivo+expansion+culture+conditions+of+mesenchymal+stem+cells+for+human+cell+therapy.">Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy.</a> Transfusion. 49 (9), 1901-1910 (2009).</li><li>Tsuji, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+cell-detaching+methods+on+the+viability+and+cell+surface+antigen+expression+of+synovial+mesenchymal+stem+cells.">Effects of different cell-detaching methods on the viability and cell surface antigen expression of synovial mesenchymal stem cells.</a> Cell Transplantation. 26 (6), 1089-1102 (2017).</li><li>Shah, F. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+mesenchymal+stem+cell+expansion+in+animal+serum-free+medium+supplemented+with+autologous+human+platelet+lysate.">Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate.</a> Transfusion. 49 (12), 2680-2685 (2009).</li><li>Castegnaro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+platelet+lysate+on+the+functional+and+molecular+characteristics+of+mesenchymal+stem+cells+isolated+from+adipose+tissue.">Effect of platelet lysate on the functional and molecular characteristics of mesenchymal stem cells isolated from adipose tissue.</a> Current Stem Cell Research &amp; Therapy. 6 (2), 105-114 (2011).</li><li>Palombella, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+and+meta-analysis+on+the+use+of+human+platelet+lysate+for+mesenchymal+stem+cell+cultures:+Comparison+with+fetal+bovine+serum+and+considerations+on+the+production+protocol.">Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: Comparison with fetal bovine serum and considerations on the production protocol.</a> Stem Cell Research &amp; Therapy. 13 (1), 142 (2022).</li><li>Palombella, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+as+a+potential+clinical-translatable+supplement+to+support+the+neurotrophic+properties+of+human+adipose-derived+stem+cells.">Human platelet lysate as a potential clinical-translatable supplement to support the neurotrophic properties of human adipose-derived stem cells.</a> Stem Cell Research &amp; Therapy. 11 (1), 432 (2020).</li><li>Kr&#228;henb&#252;hl, S. 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脂肪来源的干细胞因其丰富、快速且经济实惠的分离方法、高体外/体内增殖率和干性/分化特性而在过去十年中引起了转化研究的兴趣18，19，20。因此，hASCs被认为是再生医学中基于细胞的策略的绝佳候选者21。从脂肪组织中分离后，hASC需要在体外扩增，并且在某些情况下，在用于特定治?...

在过去的几十年中，对创新治疗的需求不断增加，这引起了转化医学领域的重大努力和资源投资1。基于细胞的产品与细胞来源、制造过程（分离、扩增或基因修饰）和非细胞补充剂（酶、生长因子、培养补充剂和抗生素）确定的风险相关，这些风险因素取决于具体的治疗适应症。最终产品的质量、安全性和有效性可能受到上述要素的深刻影响2.干细胞疗法需要遵守生物安全原则;细胞培养中动物源性产品致病传播的潜在风险可能存在问题，对制造过程中引入的任何产品进行彻底测试至关重要3.
分离人脂肪来源干细胞（hASCs）的传统方法包括用胶原酶进行酶消化，然后通过离心4进行洗涤步骤。虽然酶分离通常被认为在细胞产量和活力方面比其他机械技术更有效，但所使用的动物源性成分，如胶原酶，被美国食品和药物管理局认为不仅仅是最低限度的操纵。这意味着免疫反应或疾病传播的风险显着增加，从而限制了hASC治疗在临床环境中的转化5，6。
基于胰蛋白酶的消化是另一种分离ASC的酶方案。已经描述了不同的技术，在胰蛋白酶浓度、离心速度和孵育时间方面略有修改。不幸的是，这种方法没有得到很好的描述，并且文献中缺乏比较，特别是与机械隔离方案7的比较。然而，就该方法的可翻译性而言，胰蛋白酶具有与胶原酶相同的缺点。
基于机械力且不添加酶的替代分离方法涉及脂肪组织碎片的高速离心（800 x g 或 1，280 x g，15 分钟）。然后，将沉淀与红细胞裂解缓冲液孵育（5分钟），然后在培养基中重悬之前以600×g进行另一个离心步骤。尽管与外植体方法相比，在第一天分离的细胞数量更多，但之前的一项研究表明，在培养8的第二周之后，增殖较低或不存在。
除此之外，使用异源添加培养基（例如用作细胞培养的生长因子补充剂的胎牛血清（FBS））的进一步操作与宿主患者的免疫反应性和暴露于病毒，细菌或朊病毒感染的风险增加有关9，10。免疫反应和荨麻疹样皮疹的发展已经在接受用FBS11产生的几剂量间充质干细胞的个体中描述过。此外，FBS会受到批次间差异的影响，这可能会影响最终产品质量12。
根据良好生产规范 （GMP） 指南，应避免酶促组织解离，并应用无异基因补充剂替代 FBS。这些步骤以及更可靠和可重复的方案对于支持细胞疗法的应用至关重要3，13。
在这种情况下，人血小板裂解物（hPL）已被建议作为FBS的替代品，因为它是一种无细胞，含蛋白质，富含生长因子的补充剂，并且较早在临床级基于细胞的产品中引入，作为 体外 细胞培养和扩增的生长培养基的添加剂14，15。由于hPL是一种人源性产品，因此在用于临床应用的hASC的 体外 培养过程中，它经常被用作FBS的替代品，从而减少了与FBS可翻译性相关的免疫反应和感染问题15，16。
尽管生产成本较高，但已经证明，与FBS相比，hPL支持许多细胞类型的细胞活力，增加增殖，延缓衰老，确保基因组稳定性，即使在晚期细胞传代中也能保存细胞免疫表型;所有这些要素都支持向这种文化补充11的转变。
这项工作的目的是开发一种标准化方案，以使用完整的无动物方法分离和培养hASC，与经典FBS培养的hASC相比，无需改变细胞生理学和干性特性（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL tubes</td>
			<td>euroclone</td>
			<td>et5015b</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD105</td>
			<td>BD Biosciences</td>
			<td>BD560839</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>BD Biosciences</td>
			<td>BD555821</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>BD Biosciences</td>
			<td>BD555482</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD73</td>
			<td>BD Biosciences</td>
			<td>BD561254</td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution (FACS buffer)</td>
			<td>Miltenyi</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 apparatus (flow cytometry instrument)</td>
			<td>BD accuri</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Burker chamber</td>
			<td>Blaubrand</td>
			<td>717810</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing container</td>
			<td>corning</td>
			<td>CLS432002</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter 96 AQueous One Solution Cell Proliferation Assay</td>
			<td>Promega</td>
			<td>G3582</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell Freezing container</td>
			<td>Corning</td>
			<td>CLS432002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>clearline</td>
			<td>390701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfide</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>disposabile blade scalpel</td>
			<td>paragon</td>
			<td>bs 2982</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>GIBCO</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Platelet Lysate FD (GMP grade)</td>
			<td>Stemulate</td>
			<td>PL-NH-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite F50 spectrophotometer</td>
			<td>Tecan</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope with 4x and 10x magnification objectives</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 10 cm</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpels</td>
			<td>Reda</td>
			<td>07104-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissors</td>
			<td>Bochem</td>
			<td>4071</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td>Bochem</td>
			<td>1152</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging bucket centrifuge</td>
			<td>Sigma</td>
			<td>3-16K</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>Greiner bio-one</td>
			<td>6910170</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLe (animal free trypsin substitute)</td>
			<td>GIBCO</td>
			<td>12604-013</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of human adipose-derived stem cells with an innovative xenogeneic-free method for human therapy

考虑到干细胞疗法的影响越来越大，由于在 体外 操作期间引入动物源性产品，人们对潜在的污染或感染传播提出了生物安全问题。在细胞分离和扩增步骤中常用的异种成分，如胶原酶或胎牛血清，可能与接受患者的免疫反应性或病毒、细菌和朊病毒感染的潜在风险有关。遵循良好生产规范指南，应避免化学组织解离，而胎牛血清（FBS）可以用无异种补充剂代替。此外，为了确保细胞产品的安全性，定义更可靠和可重复的方法非常重要。与胶原酶FBS培养的标准方案相比，我们开发了一种创新的，完全无异种的方法，用于分离和体 外 扩增人脂肪来源的干细胞，而不会改变其特性。在这里，从腹部脂肪组织中分离出人脂肪来源的干细胞（hASCs）。用剪刀/手术刀机械切碎样品，显微解剖并机械分散在10cm培养皿中，并用手术刀切口制备，以促进组织碎片的附着和hASC的迁移。在洗涤步骤之后，选择hASCs，因为它们具有塑料粘附性，无需酶消化。分离的hASC用补充有5%无肝素的人血小板裂解物的培养基培养，并与无动物胰蛋白酶替代品分离。按照良好生产规范（GMP）关于生产用于人类治疗的细胞产品的指示，在任何培养基中均未使用抗生素。

应用上述分离方法，在不使用胶原酶的情况下，成功地从腹部脂肪组织样品中获得hASC。此外，hASCs在完全无异种条件下在hPL存在下扩增，并且没有任何其他动物来源的成分。以下结果支持该协议，并且是从与hPL并行培养的hASC和以FBS作为对照条件中获得的。
在最初的簇出现后，hASC显示出经典的纺锤状形状，并且与存在FBS的对照细胞相比更小，更细长（图3</strong...

Watch this Scientific Journal Video about Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy at JoVE.com

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

在细胞疗法制备/操作步骤中引入的异源性（化学或动物源性）产品与宿主患者免疫反应性和致病性传播的风险增加有关。这里描述了一种完整的无异种方法，用于分离和 体外 扩增人脂肪来源的干细胞。

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 ...

我们用于分离和体外扩增人类脂肪来源干细胞的无外源性方法是在考虑用于转化应用时解决细胞疗法生物安全问题的答案。我们的分离和扩增方法易于复制，快速且技术要求低，因此对每个人都有用。我们研究了基于人类脂肪干细胞的周围神经修复治疗策略。然而，当在不同的临床研究设置中需要新的脂肪干细胞时，我们的技术可以得到广泛应用。我们保持简单，以节省更容易的可生产性。从手术室到细胞培养，应仔细观察无菌情况。对于样品操作和碎片，显微解剖有污染的风险。从腹部整形手术中获得脂肪组织样本后，使用无菌剪刀将其切成约一平方厘米的碎片。用手术刀将每块碎片微观解剖成更小的碎片，并将五个碎片分散在10厘米的培养皿中。对于碎片附着，使用手术刀在塑料表面上创建切口并拖动每个碎片。轻轻地加入 10 毫升完全培养基以覆盖所有片段而不分离它们，并在 37 摄氏度和 5% 二氧化碳下孵育培养皿。每天在光学显微镜下监测人类脂肪来源的干细胞（HASC）的扩增，直到细胞汇合。每72小时用新鲜的完全培养基替换培养基以去除细胞碎片，并让它们生长直到第一次传代。一旦HASC培养物达到70%至80%的汇合度，使用无菌镊子去除并丢弃所有组织碎片。此外，丢弃耗尽的培养基，避免HASC与培养皿分离。用10毫升PBS仔细清洗细胞。去除PBS后，加入一毫升无动物胰蛋白酶，并在37摄氏度下孵育五分钟。在光学显微镜下检查细胞脱落，如果需要，再孵育两分钟。要支撑细胞分离，请轻轻敲击塑料表面。通过加入两毫升完全培养基停止胰蛋白酶作用，并将所有细胞收集在15毫升管中。通过离心除去剩余的胰蛋白酶。弃去上清液后，将细胞悬浮在5毫升完全培养基中，并将细胞悬液转移到新的T25烧瓶中。为了冷冻保存，使用光学显微镜下的细胞计数器计数分离细胞的等分试样。将细胞以300 G离心五分钟。弃去上清液后，将细胞以每毫升10至第六个细胞的密度悬浮在冷冻混合物中，并将一毫升细胞悬液转移到一个冷冻瓶中。将零下80摄氏度的冷冻瓶放入冷冻容器中，每分钟将温度降低一摄氏度，然后将冷冻瓶移至液氮中长期储存。在最初的簇外观中，HASC表现出经典的纺锤状形状，并且与存在FBS的对照细胞相比更小，并且更细长。用两种补充剂培养的HASC之间的细胞免疫表型似乎相似。超过80%至90%的HASC对CD73和CD105呈阳性，对CD34和CD45表示不到5%。最后，增殖测定显示，与FBS对照相比，将人血小板裂解物添加到培养基中时，细胞生长显着增加。最重要的步骤是脂肪碎片的显微解剖及其在培养皿上的手术刀切口附着。根据GMP生物安全标准获得即用型人脂肪来源的干细胞群，保持治疗细胞特性并增强细胞增殖。

Research

JoVE Journal

Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

科研

医学

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

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

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, ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...