This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (26461185 and 25460137).

注：此研究是通过适当的伦理委员会。 人肺成纤维细胞1.原代培养<ol><li>收集肺组织： <ol><li>得到直接从外科手术室人肺组织样本。收集来自癌性和非癌肺组织约1cm 3块，以收集作为远离肿瘤尽可能的非癌性样品。 </li><li>暂停在Dulbecco改良的Eagle培养基（DMEM）中补充有100单位/ ml青霉素，100微克/毫升链霉素和0.25微克/毫升两性霉素B（无血清培养基）的样品。保持在无菌50ml试管中悬浮的样品并转移到实验室。 注：成纤维细胞是高度洄游和增殖与其它类型的细胞，如上皮细胞，内皮细胞和平滑肌细胞。该生长方法利用了成纤维细胞的这些特征，并且outgro从组织切片翼细胞丰富的成纤维细胞。经过多次传代细胞，其他细胞类型不能存活和繁殖的DMEM，用10％胎牛血清（FBS），导致纯化的成纤维细胞培养物。所述细胞可用于3-7通道主培养以下。 </li></ol></li><li>植文化和调节细胞生长： <ol><li>执行使用以前报道的协议进行了修改5成纤维细胞原代培养。 </li><li>在实验室中，放置在无培养基10cm的组织培养皿的组织样品切成小切片〜2-3毫米大小用无菌镊子和手术刀。在此过程中，应避免使部分干燥，否则细胞生长的能力受到损害。 </li></ol></li><li>分离上皮细胞层和结缔组织的： <ol><li>泡切的组织切片在含2000的PU /毫升分散酶I培养基和培养16小时，在4℃下&lt;/ li&gt; <li>的组织切片转移到培养皿，并且从相邻的结缔组织分离上皮细胞层。处理在清洁台上的部分，以避免微生物的污染。 注意：如果需要上皮细胞原代培养，执行步骤1.3。如果只成纤维细胞将被分离，则省略步骤1.3，因为在随后的外植体培养和细胞传代是不利的上皮细胞，其产生纯化的成纤维细胞种群。 </li></ol></li><li>附件组织切片： <ol><li>剁碎组织成使用两个手术刀1毫米件。如果这两块不足够小，它们分离更容​​易地从皿中，细胞将无法长大于餐具表面。 </li><li>放置小肺组织切片彼此分开，以维持周围的每一块的空白区域。使用手术刀刀片，以促进附着的组织切片的组织培养皿的表面上的划痕。 注意：抓似乎也促进细胞生长，提供轨道沿着细胞可以迁移。 <ol><li>或者，将组织糜片段单独成6孔板的每个孔中，并用盖玻片覆盖它们。附上盖玻片使用硅脂该板的表面上。 </li><li>放置硅脂在两个点相隔1.5厘米中使用无菌针每个孔中。组织碎片覆盖增强细胞生长和细胞繁殖以下几个段落的疗效。 </li></ol></li></ol></li><li>随后的外植体文化： <ol><li>轻轻培养基添加到培养皿之前组织切片干燥，作为有效的细胞生长失去大部分如果样品变干。不要将媒体过快的组织切片会脱落。 </li><li>培养的细胞在DMEM含10％FBS。使用足够的培养基刚好盖住组织，但不会太大，它允许浮动，作为额外的浮力促进支队从菜。 </li></ol></li><li>细胞传代： <ol><li>小心轻放培养基在任何时候都为培养皿的重复动作可导致组织切片的脱离。 </li><li>将培养皿在培养箱中在37℃下5-7天。刷新媒体隔日，并在媒体变化最小处理培养皿。长大成纤维细胞从组织切片，在未来几周内的边缘。 注：〜2-3周后，outgrowing成纤维细胞几乎达到汇合，这取决于表面积的量。 </li><li>用1×PBS洗涤细胞，并加入胰蛋白酶1毫升我到板上。 </li><li>胰蛋白酶消化后，分离使用9毫升DMEM中的细胞在补充有10％FBS中。与组织切片收集细胞悬浮在10毫升管的介质，在一起。离心以形成细胞沉淀，重悬所得的颗粒在新的平台。 </li><li>种子的细胞和组织切片到一个新的小路TURE盘，在该点的组织中没有附着到培养皿的同时将细胞附着和生长。次日，通过抽吸除去浮动组织片，并改变介质。连接成纤维细胞繁殖与后续培养。 </li></ol></li></ol>人肺成纤维细胞和癌细胞2.三维共培养<ol><li>制备细胞和试剂： <ol><li>大约用2×10 5的上皮细胞和每孔2.5×10 5个成纤维细胞。制备型胶原IA（3毫克/毫升，pH为3），FBS（100％），重建缓冲液（50mM的NaOH，260毫的NaHCO 3，200mM的HEPES），5×DMEM，DMEM补充有10％FBS的成纤维细胞培养，和三维共培养培养基（1：1的混合物的成纤维细胞的培养基和培养基的癌细胞）。 </li><li>培养的A549肺腺癌细胞，成纤维细胞和A549三维共培养在DMEM补充有10％FBS中。 </li></ol></li><li>胶原凝胶˚Formation： <ol><li>洗的成纤维细胞上10cm皿用1×PBS中培养，并添加胰蛋白酶1毫升我到板上。胰蛋白酶消化为〜5分钟，在37℃后，分离使用9毫升DMEM补充有10％FBS的细胞。收集的细胞悬浮在10毫升管的培养基中，离心它们在200-300×g离心以形成细胞小球。 </li><li>重悬在100％FBS的所得粒料在5×10 5 / ml的密度。 </li><li>准备上冰的胶原凝胶。冷却该反应物，移液器和管子在冰箱中之前执行以下步骤。甚至在冰上，在固化到某种程度，总体积小于所有组分的总和。 </li><li>在每个孔中，通过混合0.5毫升成纤维细胞悬浮液（2.5×10 5个细胞）中的FBS的，2.3毫升IA型胶原，670微升5×DMEM，和330微升重建缓冲液制备胶原凝胶。允许凝胶容易地设定在室温。 <ol><li>大力吸管，以确保均匀的英里夹具。避免在移液过程中产生气泡。 </li></ol></li><li>该混合物（3毫升）添加至6孔板的每个孔中，并允许在孵化器在37℃凝胶化，而不扰动30-60分钟。 </li></ol></li><li>在胶原凝胶癌细胞培养： <ol><li>使用先前报道的协议进行了修改6进行三维共培养。 </li><li>重悬的癌细胞在三维共培养培养基（或在DMEM与A549细胞的情况下，10％FBS）以1×10 5个/ ml的密度。 </li><li>倾2毫升再悬浮细胞溶液（2×10 5个细胞）在每个凝胶的表面上。修改癌细胞或成纤维细胞的细胞数目在共培养取决于实验条件。 </li></ol></li><li>凝胶收缩： <ol><li>过夜孵育凝胶在37℃下这样的癌细胞可以附着在胶原凝胶。分离的每个凝胶，并生成每个“流动文化”以及Øf显示6孔板。 注意：通过使用一个浮培养，在凝胶尺寸的各种变化都受到机械张力和胶原降解，其通过共同培养的癌细胞和成纤维细胞之间的细胞相互作用介导的诱导。 </li><li>使用成角度的21g的针或小抹刀将每个凝胶从井的边缘分开。每2-3天，刷新井用2ml三维共培养培养基。每天测量每个凝胶的尺寸为5天。 </li></ol></li><li>分析胶原凝胶： <ol><li>测量使用胶原定量方法，如Sircol测试每个凝胶的胶原含量。从凝胶5收集的RNA后进行转录分析或定量RT-PCR。 </li></ol></li></ol> 3.气 - 液界面文化入侵检测<ol><li>在37℃下5天培养的胶原凝胶后，通过将它们放置在一个网格（70微米孔径）在新的6-暴露收缩凝胶到空气孔板中。使从用于流式细胞市售的细胞滤网的网格。 </li><li>轻轻地用11毫升三维共培养培养基填充每个孔中。 </li><li>凝胶放置到使用无菌勺网格，并从凝胶中除去任何剩余的介质。在此条件下，浸入在介质中的凝胶，而露出的凝胶，以空气的上表面。定义该培养条件如空气 - 液体界面。 注：后5-7天保持在37℃的空气 - 液体界面培养在培养箱的，侵入性癌症细胞迁移到凝胶。根据细胞类型和作为细胞相互作用的结果，可变度在癌细胞中细胞形态的变化是由空气 - 液体界面培养诱导。 </li><li>用于组织学评价，固定在福尔马林溶液中的凝胶在室温下过夜，并嵌入石蜡中。染色垂直切片（4微米）与HE染色（H＆E）。进行免疫组化分析Òn中的第4。 </li></ol>

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The authors declare that they have no competing financial interests.

的CAF形成周围癌细胞ECM的一个重要组成部分，而不是仅提供一个支架的肿瘤，但也积极参与肿瘤发展7。越来越多的证据揭开CAF或与其相关的分子上的最终预后的影响，突出CAF介导的肿瘤进展8的关键作用。 在以前的研究中，我们采用的方法生长隔离肺的CAF 4。在这个实验中，组织切片的粘附维持到餐具表面是关键的。细胞不能从组织切片漂浮在培养基?...

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells1. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance2.
As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as &#945;-smooth muscle actin (&#945;-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues3. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.
Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs4.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:267px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:471px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DMEM</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:471px;">D5796</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FBS</td>
			<td style="width:297px;">GIBCO</td>
			<td style="width:471px;">10437</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Collagen type IA</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;">CELL-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Reconstitution buffer</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cover slip</td>
			<td style="width:297px;">NUNC</td>
			<td style="width:471px;">174934</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Silicone grease</td>
			<td style="width:297px;">Dow Corning Toray</td>
			<td style="width:471px;">High vacuum grease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dispase I</td>
			<td style="width:297px;">WAKO</td>
			<td style="width:471px;">386-02271</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">6-well plate</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">353046</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell strainer (70 &#956;m)</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">352350</td>
		</tr>
	</tbody>
</table>

three-dimensional co-culture model for tumor-stromal interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

该共培养方法，模仿肿瘤微环境，是一种有用的工具，调查癌细胞和嵌入在胶原凝胶的成纤维细胞之间的相互作用。胶原凝胶收缩，癌细胞的侵袭和形态变化：在以前的研究中，三个参数在这个实验模型进行了评价。还预计使用Ki67的免疫染色4癌细胞增殖。肺组织样品从一个切除肺叶（ 图1A）的癌性和非癌的部分收集。样品切成小片，并在DMEM中培养在补充有10％FBS的（ ?...

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

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

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 ...

three-dimensional-co-culture-model-for-tumor-stromal-interaction

Research

JoVE Journal

Medicine

17.2K 次观看.  The University of Tokyo. Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

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

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

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

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

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

复杂主动脉异常的三维打印

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

三维打印指南模板辅助皮椎间盘（PVP）

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...