Name: establishment of a primary culture of patient-derived soft tissue sarcoma
Uploaded: 2018-04-11T13:00:00
Description: Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

作者想感谢 Gráinne 的编辑协助。

sts 细胞与由 sts 患者手术切除的肿瘤肿块分离。该议定书已得到当地道德委员会的批准, 并根据良好的临床实践指导方针和赫尔辛基宣言进行。所有患者都给予书面知情同意, 参加该项研究。手术标本由经验丰富的肉瘤病理学家分析, 并在3小时内进行手术处理。
1. 肿瘤标本的采集与加工:
<ol><li>用100毫升无菌尿液容器中的肉瘤病理学家收集肿瘤标本, 用石蜡膜密封的50毫升 DMEM ...

<ol>
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为了阐明肿瘤的分子背景, 预测预后不佳, 为癌症患者制定新的治疗策略, 需要明确的临床前模型。这对于像 sts 这样的罕见肿瘤尤其重要, 因为它的形态学、侵袭性潜能和临床行为等方面的高异质性挑战了我们对 sts 生物学和患者管理的理解21。此外, 少数商业肉瘤细胞系可限制对这组间质肿瘤的临床前调查18。因此,前体模型代表了 STS 的宝贵研究资源。我?...

软组织肉瘤 (STS) 包括一组骨髓间质来源的异质性病变, 占所有实体肿瘤的 1%,1,2, 新的世界卫生组织分类确认存在超过50不同的子类型3。其中, 最常见的 histotypes 是脂肪肉瘤或肉瘤和平滑肌肉瘤, 占所有成人 STS 的15% 和 11%, 分别为4,5。虽然 STS 可以发展在身体的不同部分, 四肢和腹腔是最常见的网站, 发生在60% 和20% 的情况下, 分别为6。
治疗局部性疾病的基石是广泛的外科手术切除, 而佐剂和新辅助化疗的好处仍然不清楚, 仅在选定的病例中考虑7。在转移设置中, 化疗是治疗的标准, 但结果显示有限的效果8。更好地了解 STS 的分子生物学将有助于改善目前的鉴别诊断, 优化现有的治疗方法, 并确定新的治疗目标。
在这种背景下, 使用永生化细胞系9进行了多年的癌症研究。这些实验模型通常在标准单层支持或植入 immunodeficient 啮齿动物中培养, 以建立体内异种移植模型10。永生化细胞系代表一种易于管理和容易储存的材料, 可以进行大量的研究实验。事实上, 它们是临床前研究的最便宜和最广泛使用的工具11。
然而, 基于细胞线的癌症模型也有一些局限性,例如在单层系统中长时间的培养以及越来越多的通道诱导表型和遗传漂移, 使细胞不太可能重述关键肿瘤特征。此外, 细胞系的培养不能重现细胞间的相互作用和信号分子串扰, 模仿肿瘤的生物学行为和特征的肿瘤微环境。这些问题构成临床前和临床结果之间存在的差距的基础12。
鉴于上述情况, 对患者派生的初级文化的兴趣增加了13。事实上, 切除恶性和正常组织降低了失去癌细胞表型和异质性的风险, 从而提供了更具代表性的肿瘤微环境的起始材料。此外, 使用从手术切除病人身上采集的新鲜肿瘤标本, 我们可以调查单个肿瘤, 并比较患者身体同一部位的不同病变14。基于上述原因, 组织衍生细胞培养物为研究肿瘤病理生理学和药理学15,16,17, 特别是在 STS 中提供了一种有价值的材料, 可用于商业上可用的肉瘤细胞系。受限18。因此, 我们通过手术切除肿瘤组织13,19,20, 优化了建立病人派生的 STS 原细胞培养的方法。我们的方法包括将肿瘤标本分列到小组织片段, 然后用隔夜酶组织离解来获得单个细胞悬浮。第二天, 通过添加新鲜培养基停止酶消化, 并过滤得到的悬浮物以去除组织碎片、细胞团聚体和过量基质物质或碎片。最后, 将一小部分分离的肿瘤细胞 cytospinned 到玻璃滑梯上, 固定存放, 用于下游分析, 目的是通过项细胞形态、免疫组化和分子细胞遗传学评价确定 STS 原代培养的建立。(例如MDM2 放大的鱼分析代表区分良好的肉瘤和去分化肉瘤肉瘤的标准鉴别诊断)4. 其余的细胞被播种成一个标准的单层培养基因表达谱和药理学研究。所有的实验都是使用低通道初级培养, 以避免选择特定的癌症 subclones, 并分析了经验丰富的肉瘤病理学家。因此, 我们创建了一个完全人类 STS前体模型13,19,20。这种高通用性、易于处理的模型可以代表不同类型的临床前研究的有用工具,例如确定新的诊断和预后分子标记, 或加深对其活动和机制的了解。标准和新药在 STS 管理中的作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:599px;" width="597">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:163px;">DMEM High Glucose</td>
			<td style="width:180px;">EuroClone</td>
			<td style="width:111px;">ECB7501L</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:163px;">Fetal bovine serum</td>
			<td style="width:180px;">EuroClone</td>
			<td style="width:111px;">ECS0180D</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:163px;">Glutamine</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:111px;">25030-024</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="112">
			<td height="112" style="height:112px;width:163px;">Paraformaldehyde 4% aqueous solution, EM grade</td>
			<td style="width:180px;">Electron Microscopy Sciences</td>
			<td style="width:111px;">157-4-100</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Penicillin streptomycin</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:111px;">15140122</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:163px;">Triazol reagent</td>
			<td style="width:180px;">Ambion Life-Technologies</td>
			<td style="width:111px;">15596018</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">Trypsin</td>
			<td>EuroClone</td>
			<td style="width:111px;">ECB3052D</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a primary culture of patient-derived soft tissue sarcoma

软组织肉瘤 (STS) 是一种非均质恶性肿瘤, 具有诊断、分类和管理的难度。迄今为止, 已经发现了这些罕见的实体肿瘤的50多种组织学亚型。因此, 由于其独特的多样性和低发病率, 我们对这些肿瘤生物学的理解仍然有限。患者源性培养是研究 STS 病理生理学和药理学的理想平台。因此, 我们开发了一个人类临床前模型的 STS 开始从肿瘤标本采集的病人进行手术切除。采用胶原酶消化和过滤分离的方法, 从手术标本中获得患者源性 STS 细胞培养。细胞计数, 种子, 并在标准单层培养14天左右, 然后由下游分析处理。在进行分子或药物分析之前, 通过对 cytomorphologic 特征的评估, 并在可用时免疫组化标记, 确定 STS 初级文化的建立。这个方法代表了一个有用的工具 1) 来研究这些探索性的恶性恶性肿瘤和2的自然历史, 以测试不同药物的影响, 以努力了解更多有关其行动机制。

我们设计了一种简单的方法, 以获得建立一个病人衍生软组织肉瘤的原代培养, 并在此报告一个例子, 取得的结果在一个特定的 STS histotype13。该议定书用于建立不同 STS histotypes 的初级文化, 包括分化良好的肉瘤、去分化肉瘤肉瘤、粘液肉瘤、多形肉瘤、myxofibrosarcoma、未分化多形肉瘤, 要点和硬纤维纤维瘤病。
?...

Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

以下协议的重点是建立一个初级文化的病人衍生软组织肉瘤 (STS)。该模型可以帮助我们更好地了解这些罕见的恶性肿瘤的分子背景和药理学特征, 并可作为进一步研究的出发点, 以改善 STS 管理。

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The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

A Melanoma Patient-Derived Xenograft Model

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Establishment and Culture of Patient-Derived Breast Organoids

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...