Name: idg-sw3 cell culture in a three-dimensional extracellular matrix
Uploaded: 2023-11-13T14:10:00
Description: Watch this Scientific Journal Video about IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix at JoVE.com

我们感谢 Lynda F. Bonewald 博士赠送 IDG-SW3 细胞系。这项工作得到了国家自然科学基金（82070902、82100935和81700778）和上海市“科技创新”扬帆工程（21YF1442000）的支持。

该方案适用于在24孔板的四个孔中培养细胞。如果制备多个样品或板，则应相应增加试剂的量。
1.胶原蛋白I混合物的制备
注：胶原蛋白I和基底膜基质凝胶在室温下快速。因此，胶原蛋白应在冰上处理（2°C至8°C）。除非另有说明，否则所有使用的吸头和管子都必须预冷。所有程序都应在安全罩中执行。
<ol><li>将所有试剂和离心管放在冰上。...

<ol>
	<li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amazing+osteocyte.">The amazing osteocyte.</a> Journal of Bone and Mineral Research. 26 (2), 229-238 (2011).</li><li>Dallas, S. L., Prideaux, M., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteocyte:+An+endocrine+cell+...+and+more.">The osteocyte: An endocrine cell ... and more.</a> Endocrine Reviews. 34 (5), 658-690 (2013).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+osteocyte+in+bone+and+nonbone+disease.">The role of the osteocyte in bone and nonbone disease.</a> Endocrinology and Metabolism Clinics of North America. 46 (1), 1-18 (2017).</li><li>Woo, S. M., Rosser, J., Dusevich, V., Kalajzic, I., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo.">Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo.</a> Journal of Bone and Mineral Research. 26 (11), 2634-2646 (2011).</li><li>Wang, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+osteocyte+dendrite+formation+by+Sp7+and+its+target+gene+osteocrin.">Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin.</a> Nature Communications. 12 (1), 6271 (2021).</li><li>Chicatun, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoid-mimicking+dense+collagen/chitosan+hybrid+gels.">Osteoid-mimicking dense collagen/chitosan hybrid gels.</a> Biomacromolecules. 12 (8), 2946-2956 (2011).</li><li>Kawasaki, K., Buchanan, A. V., Weiss, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+in+humans:+making+the+hard+choices+in+life.">Biomineralization in humans: making the hard choices in life.</a> Annual Review of Genetics. 43, 119-142 (2009).</li><li>Bosman, F. T., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+and+composition+of+the+extracellular+matrix.">Functional structure and composition of the extracellular matrix.</a> Journal of Pathology. 200 (4), 423-428 (2003).</li><li>Papadopoulos, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+fluorescence+assay+for+the+determination+of+lymphocyte-mediated+cytotoxicity+using+flow+cytometry.">An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry.</a> Journal of Immunological Methods. 177 (1-2), 101-111 (1994).</li><li>Staines, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypomorphic+conditional+deletion+of+E11/Podoplanin+reveals+a+role+in+osteocyte+dendrite+elongation.">Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.</a> Journal of Cellular Physiology. 232 (11), 3006-3019 (2017).</li><li>Feng, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism.">Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.</a> Nature Genetics. 38 (11), 1310-1315 (2006).</li><li>Winkler, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte+control+of+bone+formation+via+sclerostin,+a+novel+BMP+antagonist.">Osteocyte control of bone formation via sclerostin, a novel BMP antagonist.</a> EMBO Journal. 22 (23), 6267-6276 (2003).</li><li>Riminucci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-23+in+fibrous+dysplasia+of+bone+and+its+relationship+to+renal+phosphate+wasting.">FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting.</a> Journal of Clinical Investigation. 112 (5), 683-692 (2003).</li><li>Dallas, S. L., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+transition+from+osteoblast+to+osteocyte.">Dynamics of the transition from osteoblast to osteocyte.</a> Annals of the New York Academy of Sciences. 1192, 437-443 (2010).</li><li>Robin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+3D+osteoblast+migration+and+bone+apatite+during+in+vitro+early+osteocytogenesis.">Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis.</a> Bone. 88, 146-156 (2016).</li><li>Chen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+mineralization+capacity+of+IDG-SW3+cells+in+3D+collagen+hydrogel+for+bone+healing+in+estrogen-deficient+mice.">High mineralization capacity of IDG-SW3 cells in 3D collagen hydrogel for bone healing in estrogen-deficient mice.</a> Frontiers in Bioengineering and Biotechnology. 8, 864 (2020).</li></ol>

该协议的一个关键点是步骤1和步骤2必须在冰上进行，以防止自发凝血。在该方法中，胶原蛋白I的最终浓度为1.2 mg/mL。因此，应计算出最佳的 ddH2O 体积，以匹配来自不同制造商的不同胶原蛋白。
在体内，成骨细胞涉及成骨细胞从小梁骨表面到内部的极性运动14。该协议使细胞免于在没有定向极性的基质中生长。当然，在无细胞凝胶基质表面接...

骨细胞是来源于成骨细胞的终末分化细胞 1,2。一旦成骨细胞被类骨质掩埋，它就会发生骨细胞生成并表达 Pdpn 基因形成前骨细胞，表达 Dmp1 基因使类骨质矿化，并表达 Sost 和 Fgf23 基因在骨组织中作为成熟骨细胞发挥作用3.本文介绍了一种3D培养系统，用于鉴定骨细胞生成过程中的树突延伸和标记基因表达。
IDG-SW3 细胞是一种来源于转基因小鼠的永生化原代细胞系，在不同培养基中培养时，可以扩增或复制成骨细胞至晚期骨细胞分化4。与MLO-A5、MLO-Y4等细胞系相比，IDG-SW3细胞中功能蛋白的表达谱、进行钙盐沉积的能力以及对各种激素的反应更可能与骨组织中原代骨细胞相同4。
与 2D 系统相比，3D 培养系统更能模拟体内细胞生长环境，包括营养梯度、低机械刚度和周围机械范围（表 1）。以前在3D系统中培养成骨细胞的大多数方法都使用胶原I作为配方4,5,6中的独特成分，因为胶原I纤维是钙和磷沉积的位点。然而，类骨质中不可或缺的成分，即细胞外基质，含有一大类促进细胞生长、粘附和迁移的细胞因子7,8，并且是透明的，便于成像观察。因此，该协议使用基质胶（以下简称基底膜基质）作为骨细胞发生研究的次要组分。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-ethylenediaminetetraacetic acid</td>
			<td>Hyclone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Corning, NY, USA</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% (w/v) Sodium bicarbonate</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10483-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>homogenizer</td>
			<td>BiHeng&#160; Biotechnology, Shanghai, China</td>
			<td>SKSI</td>
			<td></td>
		</tr>
		<tr>
			<td>laser confocal fluorescence microscopy</td>
			<td>Carl&#160;Zeiss, Oberkochen, Germany</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Cell Imaging kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning, NY, USA</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (10X), no glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>21430079</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30256.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Carl&#160;Zeiss, Oberkochen, Germany</td>
			<td>Zeiss Axio ZOOM.V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray fluorescence</td>
			<td>EDAX, USA</td>
			<td>EAGLE III</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

idg-sw3 cell culture in a three-dimensional extracellular matrix

骨细胞被认为是从成骨细胞终末分化的非增殖细胞。嵌入骨细胞外基质（类骨质）中的成骨细胞表达 Pdpn 基因形成细胞树突并转化为骨前细胞。后来，前骨细胞表达 Dmp1 基因以促进基质矿化，从而转化为成熟的骨细胞。这个过程称为骨细胞生成。IDG-SW3 是一种用于骨细胞生成 体外 研究的知名细胞系。以前的许多方法都使用胶原I作为培养基质的主要或唯一成分。然而，除了I.型胶原外，类骨质还含有一种基础物质，它是促进细胞生长、粘附和迁移的重要成分。此外，基质物质是透明的，这增加了胶原I形成的凝胶的透明度，从而有助于通过成像技术探索枝晶的形成。因此，本文详细介绍了使用细胞外基质和胶原 I 建立 3D 凝胶以用于 IDG-SW3 存活的方案。在这项工作中，分析了骨细胞发生过程中树突的形成和基因表达。成骨培养 7 天后，在荧光共聚焦显微镜下清晰地观察到广泛的树突网络。实时荧光定量PCR结果显示， Pdbn 和 Dmp1 的mRNA水平持续升高3周。在第 4 周，体视显微镜显示一种充满矿物颗粒的不透明凝胶，与 X 射线荧光 （XRF） 测定一致。这些结果表明，该培养基质成功地促进了从成骨细胞到成熟骨细胞的转变。

活细胞/死细胞染色后，使用共聚焦激光显微镜观察细胞。所有细胞均为钙黄绿素AM阳性（绿色），现场几乎没有EthD-1阳性细胞（红色），表明该方法制备的凝胶系统非常适合骨细胞生成（图1A，左）。为了更好地确定细胞的空间分布，选择伪彩色图像来显示凝胶不同深度的细胞树突;红色表示凝胶底部的树突，蓝色表示顶部的树突。结果表明，IDG-SW3细胞在该细胞凝胶基质中生...

Watch this Scientific Journal Video about IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix at JoVE.com

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

在这里，我们提出了一种在三维（3D）细胞外基质中培养IDG-SW3细胞的方案。

三维细胞外基质中的 IDG-SW3 细胞培养

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...

我们详细介绍了使用细胞外指标和胶原蛋白指标建立 3D 系统的方案，用于分析通过成骨形成和基因表达。细胞外指标可以增加胶原蛋白单形式凝胶的透明度，适用于探索枝晶形成的成像技术。首先，缓慢地将 0.9 毫升基底膜基质移入冰上新的 15 毫升离心管中，并将其放在一边。接下来，从培养箱中取出 4 毫升补充有干扰素 γ 的完全培养基中的 IDG SW3 培养细胞的 T25 牙线。取出培养基后，用移液管用PBS pH 7.4洗涤细胞。然后用 0.5 毫升 0.25% 胰蛋白酶 EDTA 在 37 摄氏度下对细胞进行胰蛋白酶处理 30 秒。通过添加 3.5 毫升完全培养基灭活胰蛋白酶。将4毫升的摄氏养老金转移到15毫升离心管中。在4摄氏度下以300G旋转细胞悬液5分钟。弃去上清液后，将细胞调色板重悬于冰上一毫升完全培养基中。使用血细胞仪对细胞进行计数，并用完全培养基将最终细胞密度调整为每毫升 10 至第五个细胞的四倍。将0.1毫升制备的细胞悬液添加到保持在冰上的0.9毫升细胞外基质中。轻轻上下移液，充分混合。通过在冰上上下移液，轻轻混合一毫升细胞基质混合物和一毫升先前制备的胶原蛋白。将 0.5 毫升最终混合物移液到 24 孔板的每个孔中，并在 37 摄氏度下孵育 1 小时以形成细胞凝胶混合物。孵育后，在每个孔的细胞凝胶混合物中加入 0.5 毫升成骨培养基。要在 37 摄氏度时开始成骨分化，请将其视为第 0 天。每两天，用新鲜的成骨培养基替换一半的培养基，并继续培养35天。从冰箱中取出钙AM和乙锭一储备溶液，让它们加热至室温。将 4 微升 2 毫摩尔乙锭 1 储备溶液和 5 微升 4 毫摩尔钙 AM 储备溶液加入 2 毫升 DPBS 中，充分混合，得到 2 微摩尔钙 AM 和 4 微摩尔乙锭 1 作为工作溶液。为了在培养的第 1 天和第 7 天进行细胞染色，将 0.5 毫升工作溶液移入 24 孔板的孔中，并在室温下孵育 30 至 45 分钟以对细胞凝胶基质进行染色。将大约 0.5 毫升新鲜的 DPBS 添加到新的 35 毫米玻璃底培养皿中，并盖住培养皿以防止样品污染或干燥。使用肘尖镊子，小心地将染色的细胞凝胶基质转移到含有DPBS的培养皿中，而不会损坏或剪切细胞凝胶基质。在激光共聚焦荧光显微镜下观察标记的细胞。将含有染色细胞凝胶基质的培养皿放在显微镜载物台上。使用 10 倍物镜选择要通过目镜扫描的细胞凝胶基质区域。要设置显微镜扫描，请选择滤光片。使用标准荧光带通滤光片将钙视为绿色，使用碘化丙啶或德克萨斯红色染料滤光片将乙锭视为红色。选择用于扫描的线模式，然后单击采集模式将通风单位设置为 1 au。选择 8 位数据深度和 1024 x 1024 像素的图像分辨率。然后选择行步长并将扫描速度设置为五。要收集Z轴图像堆栈，请通过聚焦样品来定义要扫描的细胞凝胶基质的顶部和底部位置，同时根据绿色通道信号进行连续扫描。指定样品的顶部和底部后，选择所需的扫描框并开始扫描。使用选定的设置从上到下扫描样品，生成图像库。要识别活细胞，请选择一个图像切片。绿色和红色通道分别表示活细胞和死细胞。为了识别细胞树突，选择单个绿色通道，用图像采集软件生成3D重建，一系列彩虹伪彩色图像指示深度信息。她底部的树突显示为红色，而顶部的树突显示为蓝色。在培养的不同日子里，取出培养基并用DPBS洗涤板中的凝胶两次。向孔中加入0.5毫升或4%多聚甲醛的DPBS溶液，使细胞凝胶基质在室温下固定在板中10分钟。如前所述，去除孔中的多聚甲醛，并用板中的DPBS再洗涤细胞凝胶基质两次，并在每个孔中留下0.5毫升DPBS用于成像。将板放在立体显微镜载物台上，并使用0.5倍物镜通过目镜选择最佳位置。图像，在明场下使用自动曝光的板中细胞凝胶基质的全场视图。在培养的第35天，检查液氮水平并启动XRF系统。打开样品室，用肘尖镊子将凝胶从板转移到仪器样品台的中间。关闭样品室并等待 30 分钟，让仪器冷却后再使用。将曝光时间设置为 35 毫秒。光谱范围为零至40千电子伏特，电流为770微安，用于采集设置。移动样品台，选择三到五个扫描位点进行分析。选择元素钙和磷。开始检测并导出结果。在不同的培养日子里。如前所述，用冰冷的DPBS洗涤去除细胞凝胶基质的培养基两次。使用苯酚氯仿异戊醇法从细胞凝胶基质中提取总RNA。然后用随机六聚体引物将两微克总 RNA 逆转录为 CDNA。将试管放在热循环仪上并进行PCR扩增。PCR后，用两个delta CT方法分析倍数变化。在共聚焦激光显微镜下可视化的活死细胞染色显示，所有细胞均为钙AM阳性，几乎没有乙锭一阳性细胞，表明凝胶系统非常适合骨细胞生成。到第七天，细胞树突逐渐延伸到成骨培养基中的网络。第七天培养物的伪彩色图像显示了凝胶底部和顶部不同深度的细胞机器人。在指定时间对24孔板中含有细胞的凝胶和不含细胞的凝胶的全场图像进行成像。与无细胞凝胶基质不同，凝胶基质的透明度继续下降，直到第 35 天变得不透明。第35天不透明凝胶的XRF光谱表明，凝胶中完全充满了钙和磷沉积物。实时荧光定量PCR分析了多个标记基因的表达，从第1天到第21天，鬼臼蛋白和牙本质基质蛋白1的mRNA水平持续升高，第21天后mRNA水平下降。成纤维细胞生长因子 23 和硬化蛋白的 mRNA 水平在所有阶段持续升高。胶原蛋白一和基底膜指标在室温下快速凝胶。因此，除非另有说明，否则所有使用的吸头和管子都必须预冷。人们可以通过成像技术观察成骨过程中任何有趣的事件。在树突形成和伸长过程中更容易定位分子作用。

idg-sw3-cell-culture-in-a-three-dimensional-extracellular-matrix

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...