我们想感谢提供帮助滑液存储和离心罗宾奈（塔夫茨医学中心），智也内村和Dana凯恩斯（塔夫茨大学）。这项工作是由NIH（1R01AR059106 - 01A1为LZ）

一个培养人关节软骨细胞的三维系统在滑液在这项工作中，我们人体关节软骨细胞封装在使用修改后的制造商建议的封装协议（ 龙沙 ，和31）的海藻酸钠珠。使用这些三维结构，我们已经发展为培养细胞中含有人体滑液的不同比例的中等文化制度和评估软骨基因表达的这些三维结构。 1。准备人关节软骨细胞三维（3D）封装（HAC） <ol><li>解冻在37℃水浴1分钟的小瓶人类关节软骨细胞（HAC）（龙沙）（通道2）（1毫升）。 </li><li>混合软骨细胞的生长介质（龙沙）在3000 RPM为3-5分钟，收集细胞离心1毫升。弃上清。 </li><li>重悬细胞沉淀（龙沙）在软骨细胞的生长介质。 </li><li>在组织一个10厘米的板细胞培养板（BD公司），和软骨细胞的生长介质（龙沙）培养，直至细胞融合。 HACS应使用一个通道数量不高于3或4（P3或P4）。 </li><li>洗净板155MM氯化钠代替标准的PBS由trypsinzation收集细胞海藻酸钠珠封装，HACS。一旦细胞的分离，降速为5min 3000RPM的细胞。这些细胞是现在准备3D封装。 </li></ol> 2。封装成3D珠HACS 重悬在1.2％的海藻酸钠溶液（Sigma公司）在8x10 的 5细胞/ ml密度HACS 。手机号码之前确定封装，使用标准的细胞计数。这是非常重要拌匀，以确保即使细胞分布在珠。 <ol><li>移液器的HAC /海藻酸钠溶液混合成12毫升注射器22号针头（泰科医疗公司）。 </li><li>同时，准备与102米的50 mL烧杯中号氯化钙2卷5倍的HAC / aginate解决方案。烧杯内放置一个搅拌棒和102毫米氯化钙溶液慢慢搅拌（约150转）。 </li><li>保持约6英寸以上的氯化钙溶液表面的注射器提示和氯化钙溶液逐滴添加到HAC /海藻酸钠混合物。在一般情况下，海藻酸钠珠2毫米直径约10 4细胞/珠的封装密度。注：高度的氯化钙溶液的注射器是很重要的。更短的距离将在泪珠形球面形珠，而不是结果，造成不均匀的机械完整性和细胞封装。 </li><li>让珠搅拌的氯化钙 20-25分钟的解决方案。虽然其他海藻酸钠封装协议表明了10分钟的孵育时间要短得多，我们发现， 与氯化钙溶液培养时间较长的是增强了完整性广告。 </li><li>除去氯化钙溶液，洗2-3次2-3卷氯化钠珠，然后一次在软骨细胞分化培养基（龙沙）。虽然其他褐藻胶珠封装协议涉及上述洗涤程序使用一个过滤器，我们发现，海藻酸钠珠往往成为被困在过滤器和干出来的，从而降低了封装效率。我们发现最有效的珠子解决之前丢弃到烧杯底部的解决方案。 </li><li>使用一个标准的铲转移到培养皿的珠子。我们通常文化为软骨细胞的生长介质的2天3厘米的12％珠，让软骨调整他们在种植前开关滑液，软骨细胞的分化培养基（龙沙）中期封装过程。 </li></ol> 3。滑液培养基培养软骨细胞<ol><li>新鲜滑液可以得到一个Outpatient诊所（我们获得塔夫茨医学中心滑液）。滑液可以在15毫升猎鹰管转移到实验室，并立即为15分钟3000转速离心去除细胞碎片。分装细胞滑液到1.5 ml离心管，以避免重复冷冻解冻。上清可存放于-80 ° C，直到使用。 </li><li>此前培养，在不同体积比混合滑液与软骨细胞的分化培养基（清洁发展机制，龙沙），一个100mm的抗坏血酸的恒定浓度和9 毫米氯化钙（氯化钙这种微量确保海藻酸钠的完整性珠）。 </li><li>保持在37℃培养箱摇晃平台上板（摇摆频率：约75次/分），以帮助在海藻酸钠珠养分分布的影响，并减少珠聚集。这是我们这些软骨细胞培养一个较长时间（4周），特别是作为重要的。请注意虽然软骨细胞的生长和分化的媒体（龙沙）含有两种常用抗生素庆大霉素和两性霉素，我们并没有补充任何抗生素，当我们用滑液的软骨细​​胞培养的不同比例。曾观察到没有污染。 </li><li>更改媒体混合物，每2-3天。滑液的粘度一直封装在褐藻胶珠培养软骨细胞，在长期的文化的一个主要障碍。我们发现，最有效的滑液培养基稀释102毫米氯化钙2（50％V / V），这也加强了海藻酸钠珠的完整性。这样，中期可以慢慢用吸管被删除，不损坏珠。 </li></ol> 4。在褐藻胶珠的收获HACS基因表达分析从基因表达分析海藻酸钠珠HACS收获时必须特别小心。 <ol><li>洗净海藻酸钠珠3 TIMES在102毫米氯化钙2，每次约5分钟。 </li><li>检索在4-5倍的珠子量增加了55毫米NaCitrate的软骨细胞。重要的是，海藻酸钠珠完全沉浸在NaCitrate解决方案。 Shake或岩石为20-30分钟。 </li><li>离心细胞，弃上清。 </li><li> RT - PCR分析，重悬细胞沉淀在细胞裂解液（QIAGEN RNA提取试剂盒），并着手RNA纯化。 </li></ol> 5。修复组织学分析海藻酸钠珠HACS 特别注意将要采取的收获从组织学分析3D珠HACS。 <ol><li>洗海藻酸钠珠在102毫米氯化钙2 3次，每次约5分钟。为了彻底洗去粘稠滑液，我们发现最有效的软骨细胞的分化培养基（龙沙）冲洗过夜，在37℃培养箱（摇摆频率中摇晃：约75时间S /分钟）。软骨细胞的生长介质中使用，以确保在这场旷日持久的洗的软骨细胞的生存和允许的最大的固色剂的渗透。 </li><li>珠固定在70％的乙醇过夜，并进行组织学分析。要执行DAPI染色，孵化用DAPI溶液（500ng/ml）的海藻酸钠珠温和的摇摆下1小时，3次用102毫米氯化钙 2每次15分钟。大埤图像，然后可以荧光显微镜下观察。珠子也可以阿尔辛蓝和H＆E染色切片。 </li></ol> 6。代表性的成果我们在滑液的高百分比的人软骨细胞的三维培养方法是描绘在图1所示的示意图。人类软骨细胞在海藻酸钠珠封装后，他们被允许在培养基中不同程度的滑液比增长。由于滑液的粘度，它是必不可少的文化的软骨细胞不断摇摆条件下，以防止软骨结构的聚集和确保的营养素的均匀分布。它也是必不可少的海藻酸钠珠洗前广泛检索的软骨细胞，所以，固定或裂解液可以穿透珠（图1）。明场（BF）的软骨细胞在褐藻胶珠图像如图2所示。 DAPI染色进行确认usniform分布在珠的细胞（图2）。在为期21天的文化时期的结束，QRT - PCR基因表达分析。参照基因GAPDH被用于所有PCR的正常化，因为它被确定为最可靠的参考基因的qPCR分析软骨细胞32。如图就是一个例子。 3，在这里我们从人类汇集从六个骨关节炎患者滑液关节软骨细胞培养的结果进行分析。符合事实，从软骨细胞龙沙已在2D文化的扩大，这将不可避免地导致脱分化 33，我们发现，0天软骨细胞软骨基质基因表达的最低水平（图3 ）。三维培养软骨细胞与软骨细胞分化培养基（龙沙）或滑液培养基显著增加软骨的胶原蛋白，蛋白聚糖和MMP13，这表明第21天（图）34的软骨细胞重新分化的基因表达。在媒体的百分比增加滑液导致软骨基质标志物Ⅱ型胶原和蛋白聚糖mRNA表达（图3A和3B）相媲美的水平。此外，在100％滑液培养软骨细胞软骨降解酶MMP13 mRNA表达减少甚至表现出比那些在培养中单（图3c）。有趣的是，细胞死亡指标caspase 3的表达水平逐渐下降与增加滑液的比率，表明即Synovial液培养，导致细胞凋亡水平下降（图3D）。因此，我们的结果表明，在一个3D的环境中滑液的高层次培养人关节软骨细胞是一种可行的技术。 表格和图表<img alt="图1。" src="/files/ftp_upload/3587/3587fig1.jpg" /> 图1。示意图文化在3D海藻酸钠珠滑液的高百分比的人关节软骨细胞的方法。首先，软骨细胞和海藻酸钠溶液混合。当应用于下降明智的氯化钙溶液中，软骨细胞固定内钙交联海藻酸钠水凝胶珠。这些三维结构，然后培养软骨细胞的分化培养基（龙沙）在不同的人体滑液比。摇摆条件下培养21天之后，含有细胞的海藻酸钠珠洗广泛的基因后，细胞检索表达分析。 <img alt="图2。" src="/files/ftp_upload/3587/3587fig2.jpg" /> 图2。明场（BF）和人关节软骨细胞的DAPI 0％，30％，50％，70％和100％滑液封装的文化图像。软骨细胞在培养条件下，在所有形状的球形。明场（BF）和大埤图像叠加确认软骨细胞的位置和分布。 Insets的，放大的图像。箭镞，BF和DAPI染色图像的软骨细胞共定位。 <img alt="图3。" src="/files/ftp_upload/3587/3587fig3.jpg" /> 图3。QRT - PCR封装人类关节软骨细胞的分析0天（D0）经过21天的培养基中培养（D21），辅以不同比率的滑液（SF）（0％，30％，50％ ，70 ％和100％）。 4个独立样本的结果如下所示。 GAPDH被用作内部参考所有PCR的。 <strONG&gt;一个。 Ⅱ型胶原mRNA的表达，B。蛋白聚糖mRNA的表达，C。 MMP13 mRNA的表达，D。 Caspase 3的mRNA表达。被评定为第0天样品和21天0％和100％的样品滑液（SF）的培养，使用InStat的软件有统计学意义。 *表示P &lt;0.05;

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+synovial+fluid+from+injured+knee+joints+on+in+vitro+chondrogenesis.">The effect of synovial fluid from injured knee joints on in vitro chondrogenesis.</a> Tissue Eng. 12, 2957-2964 (2006).</li><li>Skoog, V., Widenfalk, B., Ohlsen, L., Wasteson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+growth+factors+and+synovial+fluid+on+chondrogenesis+in+perichondrium.">The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.</a> Scand. J. Plast. Reconstr. Surg. Hand. Surg.. 24, 89-95 (1990).</li><li>Nuver-Zwart, I., Schalkwijk, J., Joosten, L. A., van den Berg, W. B., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+and+synovial+fluid+cells+on+chondrocyte+metabolism+in+short+term+tissue+culture.">Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.</a> J. Rheumatol. 15, 210-216 (1988).</li><li>Beekhuizen, M., Bastiaansen-Jenniskens, Y. M., Koevoet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+tissue+inhibits+proteoglycan+production+in+human+osteoarthritic+cartilage;+Establishment+and+characterisation+of+a+long-term+coculture.">Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage; Establishment and characterisation of a long-term coculture.</a> Arthritis Rheum. , (2011).</li><li>Rodrigo, J. J., Steadman, J. R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

我们什么都没有透露。

在这份报告中，我们开发了一个人关节软骨细胞培养的方法，允许在一个中型的3D环境，它包含人体滑液的高浓度。滑液的主要成分构成的关节腔，关节软骨细胞所在的自然环境之一。然而，滑液的粘度一直是立体培养软骨细胞的长期的重大挑战。要克服的挑战，保持甚至养分分布在三维构造在粘稠的环境，并防止聚集，我们将不断的运动，温和的摇摆下的软骨结构。为了提高结构的软骨结构完?...

特定的试剂和设备表： <table border="1"><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>海藻酸钠（褐藻酸的钠盐） </td><td>西格玛</td><td> A2158 - 250G </td><td> 2.4％的溶液储存在40 ° C </td></tr><tr><td>二水氯化钙，颗粒</td><td> JT贝克</td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" >软骨生长介质</td><td width="254" rowspan="2" >龙沙</td><td width="182" > CC - 3156（底座媒体） </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC - 4409（增刊） </td></tr><tr><td width="239" rowspan="2" >软骨分化媒体</td><td width="254" rowspan="2" >大号onza </td><td width="182" > CC - 3226（底座媒体） </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC - 4408（增刊） </td></tr><tr><td>人关节软骨细胞</td><td>龙沙</td><td > CC - 2550 </td><td></td></tr><tr><td> DAPI（4&#39;,6 - Diamidino - 2 -苯基吲哚盐酸盐） </td><td> Sigma - Aldrich公司</td><td > D9542 </td><td></td></tr><tr><td>的RNeasy迷你试剂盒（RNA提取） </td><td> Qiagen公司</td><td > 74104 </td><td></td></tr><tr><td> PCR试剂：SYBR绿色</td><td>广达</td><td > 95053-500 </td><td></td></tr><tr><td> 12毫升注射器</td><td>泰科-肯德尔 - Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22 Gague注射针</td><td>泰科 - 肯德尔L - Monoject </td><td > 8881 </td><td></td></tr><tr><td>显微镜</td><td>奥林巴斯</td><td > IX71 </td><td></td></tr><tr><td>平台摇杆</td><td> Thermoscientific thermolyne </td><td >可变混合</td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> 引物序列 </td><td></td><td ></td><td></td></tr><tr><td>胶原蛋白第IIa进</td><td> 5&#39; - TTC ATC CCA CCC认证的TCT CAC AGT - 3&#39; </td><td ></td><td></td></tr><tr><td>胶原蛋白第IIa -反向</td><td> 5&#39; - CCTCTGCCTTGACCCGAA - 3&#39; </td><td ></td><td></td></tr><tr><td> MMP13，正转</td><td> 5&#39; - TGT GCC铁通铁通CAC ACA GAC的ACT - 3&#39; </td><td ></td><td></td></tr><tr><td> MMP13反向</td><td> 5&#39; - GAG AGC AGA铁通TGA GTC ATT GCC - 3&#39; </td><td ></td><td></td></tr><tr><td> Caspase 3的进</td><td> 5&#39; - TCA TTC AGG TTA CCT的海湾合作​​委员会的GTG GTA - 3&#39; </td><td ></td><td></td></tr><tr><td> Caspase 3的反向</td><td> 5&#39; - TGG ATG AAC CAG GAG共同国家评估台泥TTT -3&#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

软骨破坏的骨性关节炎的病理特征是中央在美国的事业，一个残疾的领先。在成人软骨没有非常有效的再生，在体内，因此，骨关节炎导致不可逆转的软骨丢失及伴有慢性疼痛和行动不便 1,2 。软骨组织工程提供了有希望的潜在的再生和恢复组织功能。这种技术通常涉及到播种天然或人工合成的支架和培养软骨细胞产生的三维构造过了一段时间，在一个平衡的介质中的工程目标的生物化学和生物力学成熟的组织 ，可以在体内3-6成缺损部位移植。软骨组织工程的成功实现，为软骨细胞的生长和外基质沉积的最佳条件是必不可少的。 在本机的关节腔，在北极的软骨ular表面的骨沐浴在滑液。这清晰和粘性流体股骨头关节软骨提供营养，并包含 7,8软骨细胞代谢的重要生长因子，细胞因子和酶。此外，滑液有利于低摩擦的软骨表面之间的运动，主要是通过分泌两个重要组成部分， 透明质酸和 lubricin 9 10。与此相反，组织工程化软骨是最常见的人工培养基培养。虽然这些媒体很可能能够为研究软骨细胞代谢提供更明确的条件，滑液最准确地反映关节软骨细胞，其中居住的自然环境事实上，滑液容易获取和存储的优势，往往可以定期身体补充。一些团体补充滑液在培养基中的人类，牛，兔和狗的彗星hondrocytes，但主要用于滑液（低于20％）11-25只低的水平。虽然鸡，马和人类的软骨细胞已在滑液的百分比较高的中等培养，这些文化系统二维26-28。在这里，我们目前在3D系统培养人关节软骨细胞的方法，我们与滑液的高（可达100％）的比例超过21天的期限。这样做，我们克服了滑液的高粘度提出的一个主要障碍。该系统提供学习人体软骨在滑液在3D设置，可进一步与其他两个重要因素（氧张力和机械负荷）29,30，软骨构成的自然环境，模仿自然的环境相结合的可能性软骨生长。此外，该系统也可用于化验滑液对软骨细胞的活动，并为发展中国家提供了一个平台软骨再生技术和关节炎的治疗方案。

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

一个3D系统培养人关节软骨在滑液的高层次描述。滑液反映关节软骨的最自然的微环境，并可以很容易地获得和存储。因此，该系统可用于软骨再生研究和筛选疗法治疗关节炎。

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

a-3d-system-for-culturing-human-articular-chondrocytes-synovial

Research

JoVE Journal

Biology

20.7K 次观看.  Tufts University School of Medicine. 一个3D系统培养人关节软骨在滑液的高层次描述。滑液反映关节软骨的最自然的微环境，并可以很容易地获得和存储。因此，该系统可用于软骨再生研究和筛选疗法治疗关节炎。

视频: A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

科研

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...