这项工作是由佛罗里达州，普雷斯顿A.富国小中心的脑肿瘤治疗和美国国立卫生研究院（1R21CA141020-01）脑肿瘤研究中心的支持。

1。准备胶质母细胞瘤单细胞悬液<ol><li>使用的神经球的吸附试验（NSA）如先前所述2,11,12 gliomasphere文化建立和维护。 </li><li>在适当的时间用于传代gliomaspheres的，介质中含有球体除去，放置在一个适当大小的无菌组织培养管中，在室温下5分钟（110克）在800 rpm的转速下离心分离。 </li><li>弃去上清液，和球形的颗粒再悬浮于1毫升的0.05％胰蛋白酶-EDTA和孵育在37℃下在水浴中3-5分钟。 </li><li>然后用等体积的大豆胰蛋白酶抑制剂，停止的胰蛋白酶活性。 </li><li>轻轻地用移液管将细胞悬浮液，以确保均匀和完全中和的胰蛋白酶活性。 </li><li>再次将细胞悬浮液离心5分钟，在800 rpm的转速下。弃去上清液，并将细胞重新悬浮于1ml的NeuroCultNSC基础培养基。 </li><li>单细胞的悬浮液中加入10μl的是，混合与90μl的台盼蓝进行细胞计数。 </li></ol> 2。与细胞微量羧基荧光素琥珀酰亚胺酯（CFSE）绿色荧光染料染色<ol><li>对于每1百万个细胞被染色，1毫升的染色试剂制备通过加入1微升的5mM细胞微量的CFSE染料（分子探针，Invitrogen）中，以1毫升NeuroCult NSC基础培养基，得到的最终染色浓度为5μM 。 </li><li>含有细胞的染色试剂充分混合，直至形成均匀物质，并在室温下孵育10分钟。 </li><li>在培养期间，将冰冷的NeuroCult NSC基础培养基作为淬火溶液制备。 </li><li>要停止的染色过程，约5到10倍体积的冰冷淬火溶液被添加到的CFSE加载细胞。 </li><li>然后将溶液以800rpm离心5分钟，弃去上清液。 </li><li>在日是点，应该出现颗粒的细胞在试管底部具有明亮的绿色色调的颜色，表示染色成功。 </li><li>通过在1-2毫升新鲜NeuroCult NSC基础培养基中重悬沉淀，以800rpm离心5分钟，弃去上清，将细胞洗涤。重复该过程共洗涤三次。 </li><li>第三次洗涤后，将细胞重新悬浮于1毫升NeuroCult NSC基础培养基，并进行细胞计数。 </li><li> CFSE的染色细胞，然后镀在无菌组织培养烧瓶中，在一个适当的密度[50,000个细胞/ ml]，并放置在37℃/ 5％CO 2培养箱中。 </li></ol> 3。 CFSE载入验证<ol><li>可以监视NSA培养接种于细胞，在荧光显微镜下，验证的CFSE的染色。在这个阶段，所有的细胞表现出明亮的绿色的颜色（ 图1）。可替换地，下降的细胞悬浮液CFSE的染色的细胞可以被放置在显微镜载玻片上，用盖玻片覆盖，在荧光显微镜下的可视化细胞。 </li><li>成功CFSE标记通过流式细胞仪检测进一步证实。尽可能多的为1-5×10 5个细胞从每个组是足够的CFSE的染色验证。 CFSE加载细胞和卸载阴性对照细胞分别重悬在1×PBS或NeuroCult NSC基础培养基的总体积为500微升，并放置在单独的FACS管。 </li><li>流式细胞仪是根据它的用户手册说明相关参数调整。的CFSE有一个最大的492 nm和最大发射波长为517 nm波长激发。 </li><li>首先，的卸载控制细胞的运行和收购事件都记录在正向和侧向散射光（FSC与SSC）和直方图，主要的细胞人口的门控信号（ 图2A）。 </li><li>同样，CFSE加载的细胞使用相同的参数进行了分析S用于对照细胞（ 图2B）。 </li></ol> 4。分离慢分裂即CFSE保留细胞人口，用荧光激活细胞分选（FACS） <ol><li>分裂后，CFSE的强度降低，并hGBM细胞形成gliomaspheres，呈现绿色荧光强度的不同水平的（ 图3）的细胞所组成的。当的gliomaspheres已达到约150-200微米的直径（约5〜10天的细胞系生长速率取决于后的CFSE加载）的平均尺寸，球体的培养基中被移除时，放置在一个适当大小的无菌组织培养管中，并以800rpm离心5分钟，在室温下。 </li><li>甲单细胞的悬浮液，在先前的步骤中所描述的方法制备和密度高达每毫升10 7个细胞以制备细胞悬浮液，将细胞重新悬浮在PBS中作细胞计数。 </li><li>船尾呃在适当体积的PBS重悬细胞，加入碘化丙啶（PI，Sigma公司，500微克/毫升）的浓度的1微升/毫升的细胞悬浮液中，排序时/通过流式细胞术分析，以排除死细胞。 </li><li>两个15毫升无菌组织培养管中各含有1ml的完整NeuroCult NSC中等是用来收集的排序条件慢分裂的细胞（CFSE滞留细胞）和快速分裂的细胞（CFSE稀释细胞）。 </li><li>首先，从卸载单元组的细胞悬浮液通过流式细胞仪运行。 </li><li>基于前向散射（FSC）的相对于侧向散射光（SSC）和电压参数，这两个参数的绘制的细胞（事件）被调整以使大多数的细胞被包含在流图。 </li><li>要排除死或受损的细胞，被绘制的细胞基于SSC与PI的反应性和单活细胞群体（PI阴性）被选通。 </li><li>然后，均匀的细胞群体中选择基于forward散射（FSC）与侧向散射光（SSC）。 </li><li>的均相单活细胞种群，然后绘制在直方图中，根据设置在对数刻度上的小区频率随CFSE强度。 CFSE的激光强度被调整，以便放置在0和100之间的负信号。 </li><li>使用相同的参数和类似的门控策略，运行的CFSE标记的细胞通过流式细胞仪和分析（ 图4C）。 </li><li>随后，确定一个缓慢的分裂细胞的人口和总人口（更快速分裂的细胞），根据自己的能力，保留CFSE（5％CFSE 高顶和CFSE 低底的85％，分别）。 </li><li>这些不同的细胞群，然后整理成单独的15毫升无菌组织培养试管包含完整的NSC介质1ml的。 </li><li>条件分类的细胞，然后在800 rpm离心5分钟离心。 </li><li>弃去上清液，将沉淀重悬于在一个合适priate体积的培养基（取决于上孤立的事件的数目，或颗粒尺寸）和细胞计数进行。 </li><li>从不同的排序条件种群的细胞，然后在适当的无菌组织培养烧瓶中50,000个细胞接种于每毫升，并放置在37℃/ 5％CO 2培养箱串联传代或用于下游实验之前为5-10天。 </li></ol> 5。代表性的成果 CFSE的装载后，所有的细胞提出了一种强烈的绿色荧光，如在显微镜下（ 图1）的可视化。绿色调的颜色可以看出，甚至用肉眼（见视频）。这些细胞，用流式细胞仪分析也对照细胞（ 图2）相比，显示高度强烈的绿色荧光。后装电镀CFSE细胞在神经干文化，这些细胞增殖并形成150〜200微米gliomaspheres在5-10天。随着细胞增殖，T<html>荧光染料均匀地分布在部门之间的子细胞，导致的荧光强度与每一个细胞分裂减少了一半。胶质母细胞瘤是功能异质性和细胞增殖在不同的时间尺度上组成的。在本协议中所描述的方法可以凭借自己的能力淡化或保留CFSE，分别为（ 图3中 ，还可以看到视频）识别和分离快，慢分裂的细胞。 流式细胞分析的离解生成与卸载的细胞相比，在从的CFSE加载细胞gliomaspheres演示多样的CFSE保持性质（ 图4）。快速分裂的细胞（最低85％）或缓慢分裂的细胞（5％ - CFSE 高 ）展览gliomapshere形成能力培养的神经球的试验条件下（ 图5）。 gure 1“/&gt; 图1。后立即加载与CFSE染料的游离肿瘤细胞。细胞表现出明亮的绿色，展示成功的标记。放大倍数10倍。 <img src="/files/ftp_upload/3918/3918fig2.jpg" alt="图2" /> 图2。CFSE装载确认使用流式细胞仪A）空载细胞，B细胞）的 CFSE加载的细胞和C）在加载与卸载细胞的CFSE荧光强度比较。请注意，有几个数量级的差异之间的标记和未标记的细胞中的荧光强度。 <img src="/files/ftp_upload/3918/3918fig3.jpg" alt="图3" /> 图3。一位代表gliomasphere从CFSE加载细胞后第6天电镀而得。有些细胞表现出非常明亮的CFSE染色。放大倍数：63×。 <im克SRC =“所有/ files/ftp_upload/3918/3918fig4.jpg”ALT =“图4”/&gt; 图4。代表流式细胞仪图/柱状图隔离的快与慢分裂的细胞，CFSE染色后第6天。）FSC与SSC显示整个细胞群，B）的 SSC与PI反应排除死亡或受损的细胞， C）FSC与SSC栅均匀的活细胞的人口，D）示范证明阴性对照组相比，由一个更大的荧光强度的CFSE标记后6天的的CFSE保持细胞的存在。直方图也显示，CFSE强度随时间衰减。E）直方图显示的选通的策略，以确定的CFSE固定细胞（前5％）和CFSE稀释细胞（底部85％）。 <img src="/files/ftp_upload/3918/3918fig5.jpg" alt="图5" /> 产生图5。代表gliomaspheres <stronG&gt; A）快速分裂的细胞（CFSE 低 ）和B）缓慢分裂的细胞中（CFSE 高 ）。放大倍数：10倍和20倍。C）代表细胞计数获得通过时间或快或慢分裂的细胞衍生的文化。 P &lt;0.05，t检验。 <a href="http://www.jove.com/files/ftp_upload/3918/3918fig5large.jpg" target="_blank">点击这里查看大图</a> 。

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没有利益冲突的声明。

越来越多的研究已经证明一个缓慢的分割的子隔室的细胞在癌症有助于肿瘤的形成和治疗抵抗2-8的重要性。因此，这是至关重要的描述和标准化的实验方法，使此特定的细胞或更普遍的亚群的研究比较亚群具有不同的生长速率。使用的CFSE识别和分离部分的细胞根据其细胞分裂的速度是一个起点，进一步描述这些特定的细胞组分，有助于推进我们的理解，在肿瘤的异质性。在这个协议中，?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> NeuroCult NSC基础培养基（人） </td><td>中</td><td>干细胞技术</td><td> 05750 </td></tr><tr><td> NeuroCult补充NSC增殖（人） </td><td>中补充</td><td>干细胞技术</td><td> 05753 </td></tr><tr><td> ％0.05胰蛋白酶-EDTA </td><td>试剂</td><td> Gibco公司</td><td> 25300-062 </td></tr><tr><td>大豆胰蛋白酶抑制剂</td><td>试剂</td><td>西格玛</td><td> T6522 </td></tr><tr><td>青霉素/链霉素</td><td>试剂</td><td> Gibco公司</td><td> 15140-122 </td></tr><tr><td> T25烧瓶</td><td>培养器皿</td><td> Nalge Nunc公司国际</td><td> 136196 </td></tr><tr><td> T80烧瓶</td><td>培养器皿</td><td> Nalge Nunc公司国际</td><td> 178905 </td></tr><tr><td> 15 mL管</td><td>培养器皿</td><td> BD猎鹰</td><td> 352096 </td></tr><tr><td> 50ml试管</td><td>培养器皿</td><td> BD猎鹰</td><td> 352070 </td></tr><tr><td> EGF </td><td>生长因子</td><td> R＆D </td><td> 2028-EG </td></tr><tr><td> CellTrace CFSE细胞增殖试剂盒</td><td>跟踪染料荧光细胞分裂</td><td> Molecular Probes公司，Invitrogen公司</td><td> C34554 </td></tr></tbody></table>

identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)

肿瘤的异质性表示支持肿瘤的鲁棒性的一个基本特征，并提出了一种中央障碍的发展的治疗策略1。为了克服肿瘤的异质性的问题，它是必不可少的表型，（EPI）的基因和功能的识别和鉴定的肿瘤亚群，推动特定疾病的病理学和代表临床相关指标的检测和工具，使开发。这是目前公认的肿瘤表现出不同的子细胞与细胞分裂的不同频率的分数，慢骑自行车的功能标准与多种癌症，包括脑癌，乳腺癌，皮肤肿瘤形成能力呈正相关胰腺以及白血病2-8。的荧光染料羧基荧光素琥珀酰亚胺基酯（CFSE）已被用于在体外和体内细胞的分裂频率跟踪在血源性吨umors和实体肿瘤如神经母细胞瘤2,7,8。细胞穿透物的非荧光的CFSE的前体药物通过细胞内酯酶转换成荧光的化合物，它被保留在细胞内，由共价结合的蛋白质，通过反应的琥珀酰亚胺基团与细胞内的胺基团，以形成稳定的酰胺键9。荧光染料同样分布之间的子细胞时分裂，导致细胞分裂的荧光强度与每个减半。这使得追踪细胞周期的频率高达八至十个师10轮。用CFSE保留容量与脑肿瘤细胞的识别和分离证明富集在肿瘤干细胞的活性2缓慢循环亚群（顶部5％的染料保持细胞）。 该协议描述了技术CFSE染色细胞内的个别人群的隔离一个的人glio文化母细胞瘤（GBM）衍生的细胞具有不同的部门使用流式细胞仪2。该技术一直凭借自己的能力，保留CFSE标记的识别和分离脑肿瘤慢骑自行车的细胞群。

Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

此视频协议演示的荧光染料羧基荧光素琥珀酰亚胺基酯（CFSE）的应用程序的不同的子种群的细胞分裂的频率的基础上在人成胶质细胞瘤细胞的识别和分离。

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

identification-isolation-slow-dividing-cells-human-glioblastoma-using

Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

14.0K 次观看.  The University of Florida. 此视频协议演示的荧光染料羧基荧光素琥珀酰亚胺基酯（CFSE）的应用程序的不同的子种群的细胞分裂的频率的基础上在人成胶质细胞瘤细胞的识别和分离。

视频: Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

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 ...

科研

医学

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 ...

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

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

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 ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

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 ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

足月胎盘胎膜中原发性人蜕细胞的分离

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...