作者要感谢biolitec研究有限公司（德国耶拿）对好心为我们提供了脂质体mTHPC配方，并特别感谢苏珊娜格拉夫和阿诺Wiehe工厂对他们的帮助和技术专长。我们还要感谢凯尔斯丁里迪，谁产生的结果有很大一部分，是coresponsible其出版11。 这项工作是由来自Schweizerischer社团组织Balgrist，苏黎世大学，苏黎世Krebsliga以及从沃尔特湖和约翰娜·沃尔夫基金会，瑞士苏黎世的赠款，并从EuroNanoMed ERA-NET / SNF瑞士拨款补助支持美国国家科学基金会31NM30-一分之十三万一千零四。这项工作也是由HSM的坎顿苏黎世的肌肉骨骼肿瘤（高度专业化医学）计划的支持。

1。 mTHPC在相应的低的吸收和高转移居屋和143B的OS细胞系的比较<ol><li>制备含有DMEM，含F12，并且在一个比4.5:4.5:1热灭活的胎牛血清的细胞培养基。 注意：在PS mTHPC的脂质体制剂溶解在水中以1.5毫克/毫升mTHPC的最终浓度。 </li><li>板0.2×10 6个居屋和143B细胞/孔在6孔板中（一式三份对各条件），并让细胞贴壁过夜。 注意：该数量必须调节到个体的细胞系。 80-90％A汇合是在实验时最合适。 </li><li>在接下来的一天，孵育细胞mTHPC固定浓度不同的时间段（时间依赖性摄取），或者与在一个固定的时间周期（剂量依赖性摄取）增加mTHPC的浓度。 </li><li>孵育细胞mTHPC具体时间Points或mTHPC的浓度。 注：在本研究中，居屋和143B细胞系孵育0.6微克/毫升mTHPC为0，2.5，5，或10小时，或用0，0.6，2.5和10微克/毫升mTHPC 5小时。所施加的时间周期和剂量可能取决于所使用的细胞株不同而不同。 </li><li>记住，总是在黑暗或暗光条件下工作，防止细胞的直接曝光。 </li><li>将细胞在指定的条件下孵育后，吸出培养基，用PBS洗细胞3次。接着，取下电池用0.5ml胰蛋白酶/ EDTA和计数细胞。 </li><li>脱离后，离心细胞，在400×g离心5分钟，吸出培养基，重悬细胞于PBS中，以200,000细胞/ ml的终密度。 </li><li>移液管将100μl所获得的细胞悬浮液（ 即 20,000个细胞）在96孔板中，并测量这些细胞在荧光分光光度计的荧光。设置S表示mTHPC荧光测量：417 nm处的激发和652 nm处的发射。 </li><li>为了计算每个细胞的内化mTHPC的量，归一化的相对荧光单位（RFU）的细胞数目和细胞的体积，如下所示： </li><li>从0-4微克/毫升（使用一式三份测量）使用不同浓度的mTHPC在PBS中制备的标准曲线。吸取100微升每种稀释液在96孔板中，并测量使用根据步骤1.6中描述的设置的孔的荧光。 </li><li>通过mTHPC，这给mTHPC的浓度（以μg/ ml）的中井的线性标准曲线的斜率除以RFU（为20,000个细胞获得）。 </li><li>除以10这mTHPC浓度得到mTHPC在一个很好的总量（以μg，相当于100微升细胞悬浮液），并通过20,000个细胞的体积除以该值。 </li><li>计算的公式计算，一个球体的体积：（4/3×π）×3条规则。通过测量20个胰蛋白酶消化细胞的直径，在显微镜下，并除以2这个值取得的半径 r。 </li><li>最后，规范所有的值对那些10小时培养（剂量依赖性摄取）或10微克/毫升mTHPC（随时间变化的吸收），这是设置为100％，处理后得到的HOS细胞。 </li></ol> 2。 PS 体外的光毒性的测定<ol><li>种子3000个细胞/孔在96孔板（一式三份对mTHPC的每个浓度使用），并让他们附着过夜。同样准备一个额外的96孔板中，如下所述处理细胞，但随后把它们放在一边的相位衬度成像。 注意：数字必须调整每个单独的细胞系。 50-60％在实验时的汇合是理想的。 <ol><li>记住要添加单元格对应的暗毒性控制每个mTHPC浓度。该本身是细胞，将在选定的mTHPC浓度的存在或不存在下温育，但不会被点亮。 </li></ol></li><li>在接下来的一天，孵育细胞取决于细胞系不同mTHPC（0，0.0001，0.001，0.01，0.03，0.075，0.15，0.3，0.6，1.25，2.5，5和10微克/毫升）的浓度和让他们在黑暗中5小时。 <ol><li>参见1.3.2节 注意：孵育时间前照明依赖于细胞系和所使用的PS，因此最好是在单独的实验中，测试不同浓度和不同的时间点。 </li></ol></li><li>在温育指定的时间内，用PBS洗细胞两次，并没有mTHPC添加新的新鲜培养基中。 </li><li>照亮细胞用652纳米二极管激光器，具体为mTHPC吸收光谱（ 见图1）。 <ol><li>激光设置为21.88毫瓦/厘米2的电源在13.5厘米DISTA的高度考从细胞中，以获得5焦耳/厘米2的能量剂量。照亮细胞为230秒。不亮的暗毒性对照细胞（有，无mTHPC）。 注意：一定要戴眼镜以保护眼睛免受用于PS的激发激光。 </li></ol></li><li>光照后，保持细胞在黑暗中，在37℃24小时。随后，添加水溶性四氮唑（WST-1）试剂（10μl/100微升培养基）。 </li><li>在加入WST-1试剂后三小时，测量96孔板415nm处的各个孔的吸光度。 </li><li>为了计算存活细胞的百分比，设定为无mTHPC处理的细胞为每个条件为100％（ 即照明系统，无mTHPC和nonilluminated，没有mTHPC）中得到的值的平均值。 </li></ol> 3。的细胞数量估计由细胞计数<ol><li>种子20,000个细胞/我们LL在24孔板中，并让他们坚持过夜。 </li><li>于室温孵育5小时以mTHPC（0.001，0.003，0.15，0.6和1.25微克/毫升），并如上所述照射它们。 </li><li>收集培养基，一旦用PBS以400×g离心洗细胞，胰蛋白酶消化它们和将它们收集通过离心分离5分钟。 </li><li>离心后，吸出培养基并悬浮细胞于200μl的新鲜培养基。 </li><li>细胞计数在纽鲍尔室。 </li></ol>

<ol>
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作者宣称没有竞争的经济利益或利益冲突。

为了实现在响应PDT最佳的细胞毒性，关键是要选择合适的激光设置和孵育时间。这里所描述的过程是一致的，高效的，确定的PS吸收和量化PDT诱导的细胞毒性体外 。使用PS mTHPC的特定吸收波长，蜂窝PS摄取可以以直接的方式来确定，并且PS可以被激活，以产生细胞毒性的活性氧物种。 利用这种体外设置中，所有的参数，如聚苯乙烯浓度，药物的光的时间间隔和激?...

骨肉瘤（OS）的艺术处理今天的状态，原发性骨肿瘤，包括为新辅助化疗和手术的组合。这个治疗方案显示增加的患者从使用化疗前约20％的局部疾病的存活率，到目前的60-70％3,4。然而，在过去的二十年中，操作系统患者局部病变的总生存期已趋于稳定4，5。此外，这些患者中30％-40％在确诊后3年内复发和转移性疾病继续有20-30％4一个贫穷的生存，6，7。为了改善这些患者的预后，新的治疗策略需要发展。 光动力疗法（PDT），一个相当新颖的抗癌疗法，采用特定波长光的一个photosensi的激励光敏剂（PS），其中其注射到血液中后积聚在肿瘤细胞。在PS的激光激发产生的氧自由基在氧的存在下，其诱导肿瘤细胞和细胞死亡的细胞毒反应。除了这个主要机制，另外两个PDT诱发的生物学过程有助于减少肿瘤的生长：PDT引起血管收缩和血栓形成的肿瘤微血管，因而局部缺氧及缺氧的肿瘤内，导致肿瘤梗死。最后，PDT受伤和死亡的肿瘤细胞引发局部免疫反应，一个很独特的PDT的功能。这涉及补体系统和抗原的活化呈递树突状细胞8。因此，条件是肿瘤抗原与淋巴样细胞，随后激活的演示文稿中创建，导致肿瘤特异性免疫。 到目前为止，PDT已被用于治疗多种类型的软组织肿瘤和hyperpl亚洲，如光化性角化病，Barrett食管，支气管内肿瘤，膀胱癌，基底细胞癌，和姑息治疗头颈部癌2。治疗是已知的诱导局部，大规模坏死，只有很少的副作用，因此具有选择性地根除肿瘤组织的潜力。尽管有这些优势，PDT的应用程序保持技术上比化疗药物的管理更加严格。为了达到最大的效力，在PS的浓度，光照射时间和总的光能量传递需要被优化。这可以在体内实验来完成，但是，由于相对大量的需要被优化的参数的初步确定体外最佳条件下，它是更有效的。 在下面描述的实验中，我们使用PS 5,10,15,20 -四（间-羟基苯基）叶绿素测试PDT的体外功效奥林简称mTHPC（ 图1A）。 mTHPC是在医药品Foscan，这是目前在临床上对姑息治疗头部和颈部癌症的使用的活性物质。它是最有效的PS中的一个，在低浓度下已经大规模诱导的细胞损伤，并且它被证明是优于其它PS在组织穿透9,10的条款。它的光吸收光谱（ 图1B）示出了两个显着的峰，一个在417 nm和第二在652纳米，这是用于积累PS的组织定位和对PDT诱导，分别。 目前，脂质体制剂用于mTHPC正在开发中。在这里，我们描述的程序来量化这种脂质体制剂的摄取，并且在两个人的OS细胞株进行PDT;低转移性居屋和高转移性143B细胞。一些这里介绍的数据已报告前面11。钍这里描述É方法让我们来研究PDT疗效转移表型的影响。 143B细胞原位注射到免疫缺陷的SCID小鼠的后肢引起intratibial转移性原发性肿瘤，模型密切模仿人类转移性疾病。因此，建议在体外实验中是完全合适的，以评估在体内实验中用于以后的最佳PDT设置。

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:251px;">PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-883</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HAM F12</td>
			<td>PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">heat-inactivated fetal calf serum</td>
			<td>GIBCO, Basel, Switzerland</td>
			<td style="width:127px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:291px;">mTHPC</td>
			<td style="width:251px;">biolitec research GmbH, Jena, Germany</td>
			<td style="width:127px;"></td>
			<td style="width:287px;">As this liposomal formulation is originating from R &#38; D 
			no catalogue number is available at the moment. Stock: 1.5mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &#62;99% purity)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectramax Gemini XS plate reader</td>
			<td style="width:251px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:127px;">ID# 861</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Microscope Zeiss Observer.Z1</td>
			<td style="width:251px;">Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ceralas PDT 652 nm Laser</td>
			<td style="width:251px;">Biolitec AG, Jena, Germany</td>
			<td style="width:127px;">LD652nm2W400u</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Water-soluble tetrazolium (WST) reagent</td>
			<td style="width:251px;">Roche Diagnostics AG, Rotkreuz, Switzerland</td>
			<td style="width:127px;">1644807</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

近年来，出现了在寻找更有效的治疗对癌症具有较少的全身性副作用的困难。因此，光动力疗法是一种新的方法对多种肿瘤有选择性的治疗。 光动力疗法（PDT），使一个无毒的光敏剂（PS），其中，经活化的光在氧的存在下的特定波长的光，产生氧自由基，引发细胞毒性反应1的使用。尽管其批准几乎是二十年前由美国FDA，PDT是时下唯一用于治疗癌症类型（皮肤，膀胱）和nononcological疾病（牛皮癣，光化性角化病）2的数量有限。 利用光动力疗法的主要优点是能够执行一个局部治疗，以防止全身性副作用的能力。此外，它可以让肿瘤在细腻的部位治疗（ 如周围神经或血管）。这里，一个intraoperatPDT的IVE应用被认为是在骨肉瘤（OS），肿瘤的骨，针对原发肿瘤的卫星遗留在肿瘤外科手术切除肿瘤后，周围的组织。治疗的目标是减少复发的数量以及减少为（术后）转移的风险。 在本研究中，我们提出在体外 PDT程序以建立有效的治疗方法，用于重现人类疾病中确立intratibial OS的小鼠模型中广泛使用的操作系统的细胞系的最优PDT设置。在PS mTHPC的吸收进行了检查用分光光度计和光毒性被挑起与mTHPC的激光激发在652 nm到诱导细胞死亡评估用WST-1法和存活细胞计数。所建立的技术使我们能够确定最佳PDT设置在动物模型中未来的研究。他们是一个简单而快速的工具，用于PDT的疗效评价<em&gt;在体外体内应用程序之前。

与此报告的技术，我们研究mTHPC为基础的PDT在人类OS细胞。首先，mTHPC的时间和剂量依赖性摄取研究在低转移性居屋和在高转移性143B OS细胞系。 mTHPC摄取可以通过测量mTHPC的荧光用荧光分光光度计（ 图2中 ，再现从雷迪等人 11的许可）进行评估。 图2A示出mTHPC的时间依赖性摄取。将细胞悬浮液的荧光强度表示mTHPC的胞内水平。 图2B示出了剂量依赖?...

Watch this Scientific Journal Video about Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro at JoVE.com

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

这里所报告的协议允许光动力疗法（PDT）中的细胞系和的PDT设置施加治疗在动物模型中之前，优化的效果进行评价。

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 ...

cytotoxic-efficacy-photodynamic-therapy-osteosarcoma-cells

Research

JoVE Journal

Medicine

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

12.7K 次观看.  Balgrist University Hospital, Zurich, Switzerland. 这里所报告的协议允许光动力疗法（PDT）中的细胞系和的PDT设置施加治疗在动物模型中之前，优化的效果进行评价。 

视频: Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

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

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 and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the 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 ...

科研

医学

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

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 ...

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 ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

一种使用加速度计量化上肢表现在日常生活的方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

一种体外光动力治疗方法的研究

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.