这项工作由Carraresi基金会和MiND FoodS Hub ID:1176436资助。所有作者都是COST Action CA16119体外3-D全细胞指导和健身（CellFit）的成员。

所有研究均由米兰大学伦理委员会审查和批准。所有动物实验均按照美国国立卫生研究院（NIH）发布的《实验动物护理和使用指南》进行。从健康的成年个体中分离人类细胞已获得米兰Ospedale Maggiore Policlinico伦理委员会的批准。我们研究中的所有方法都是根据批准的指南进行的。
1. 皮肤成纤维细胞分离
注意:下面描述的所有程序都可以应用于从不同哺乳动物物种（包括小鼠，猪和人类）中分离的成纤维细胞。从7周龄的雄性小鼠中分离出鼠细胞，并在当地屠宰场收集猪皮组织。在书面知情同意后，从成年患者中分离出人类细胞。
<ol><li>准备0.1%猪明胶溶液。<ol>
<li>称取0.1克猪明胶，将其溶解在100毫升水中。使用前通过高压灭菌对明胶溶液进行灭菌。</li>
		<li>通过加入1.5mL制备的溶液，用0.1%猪明胶涂覆35mm培养皿。在室温下孵育2小时。</li>
	</ol>
</li>
	<li>切下长约2-5厘米的哺乳动物（小鼠，猪和人类）皮肤活检，并将其置于含有2%抗生素抗真菌溶液的Dulbecco磷酸盐缓冲盐水（PBS）中。储存在+ 4°C直至使用。 注意:活检收集必须在同意的情况下进行，并在伦理委员会批准后，根据既定指南进行。</li>
	<li>在含有2%抗生素抗真菌剂溶液的新鲜无菌PBS中广泛洗涤收集的活检液三次。</li>
	<li>收集最后一次洗涤的活检，并将其放入无菌的100毫米培养皿中。使用无菌手术刀将它们切成约2毫米3 大小的碎片。</li>
	<li>在2小时孵育结束时，从35mm培养皿中除去多余的明胶溶液（在步骤1.1.2中描述），并使用无菌手术镊子立即将5-6个皮肤碎片放入每个预涂的培养皿中。</li>
	<li>通过在每个片段上加入100μL成纤维细胞分离培养基液滴（表1）来润湿片段。在37°C下在5%CO2 培养箱中培养。 注意:为防止培养基蒸发，请将35 mm培养皿置于含有无菌水的100 mm或更大的培养皿中。确保盖上两个培养皿。</li>
	<li>培养24小时后，检查35mm培养皿中培养基的量。如果需要，加入500μL成纤维细胞分离培养基以保持碎片湿润。</li>
	<li>小心地取出培养基，并使用移液器至少每2天培养一次。</li>
	<li>当成纤维细胞开始从放置在35mm培养皿（在步骤1.5中描述）的皮肤碎片中生长出来并开始形成细胞单层（通常为6天）时。使用无菌手术镊子去除皮片，并在2 mL成纤维细胞分离培养基中培养。</li>
	<li>继续在37°C下在5%CO2 培养箱中培养细胞单层，直到80%汇合，每隔一天刷新培养基。</li>
</ol>2. 成纤维细胞原代细胞系培养
<ol><li>当成纤维细胞达到80%汇合时，小心地取出成纤维细胞分离培养基，并用含有1%抗生素抗真菌溶液的3mLPBS洗涤细胞三次。</li>
	<li>对于细胞分离，在培养皿中加入600μL0.25%胰蛋白酶- EDTA溶液，并在37°C下孵育3-5分钟。</li>
	<li>当细胞开始从培养皿中分离时，加入5.4mL成纤维细胞培养基以中和胰蛋白酶（表1）。</li>
	<li>通过重复和温和的移液去除细胞。在新培养皿（不含明胶）中培养细胞，将传代比保持在1:2和1:4之间（取决于生长速率）。 注:无需离心。</li>
	<li>将细胞保持在培养物中，每2天更换一次培养基，直到它们达到80%汇合并传代。 注意:每周繁殖两次成纤维细胞，以保持剧烈生长。</li>
</ol>3. 成纤维细胞暴露于5-氮杂-CR
<ol><li>准备新鲜的1 mM 5-氮杂-CR储备溶液。<ol>
<li>称取2.44毫克5-氮杂-CR并将其溶解在10毫升DMEM高血糖中。通过涡旋重悬粉末。用0.22μm过滤器对溶液进行灭菌。 注意:5-氮杂-CR储备溶液必须在使用前立即制备。</li>
		<li>通过在1mL成纤维细胞培养基中稀释1μL5-氮杂-CR储备溶液（3.1.1.）来制备5-氮杂-CR工作溶液。 注:5-氮杂-CR工作溶液的浓度为1μM1，2，3，8，9。</li>
	</ol>
</li>
	<li>胰蛋白酶消化细胞如前所述（2.1.-2.3.）并通过重复和轻柔的移液来移出细胞。</li>
	<li>收集细胞悬浮液并将其转移到锥形管中。</li>
	<li>在室温下，在光学显微镜下使用计数室对细胞进行计数。计算重新悬浮细胞所需的培养基体积，以在补充有1μM 5-氮杂-CR的30μL成纤维细胞培养基中获得4×104 细胞（参见步骤3.1.2）。 注意:要使用的配方取决于腔室的具体类型。 <img alt="Equation 1" src="/files/ftp_upload/61655/61655equ01v3.jpg">
</li>
	<li>在室温下以150 ×g 离心细胞悬浮液5分钟。除去上清液并用补充有1μM 5-氮杂-CR的成纤维细胞培养基重悬沉淀（参见步骤3.1.2）。对于要使用的成纤维细胞培养基的体积，请参见步骤3.4。 注意:作为阴性对照，将细胞重悬于不含5-氮杂-CR的成纤维细胞培养基中相同浓度，并在PTFE粉末中进行细胞包封（步骤4.1.-4.13）。</li>
</ol>4. PTFE微生物反应器中的成纤维细胞封装
<ol><li>用聚四氟乙烯（PTFE）粉末填充35毫米培养皿以产生床（图1A）。 注意:使用35毫米细菌培养皿，以避免液体大理石粘附。为了获得薄的疏水性和多孔外壳，使用平均粒径为1μm的PTFE粉末，并以最大研磨量为2.0 NPIRI生产。这允许创建透气的液体大理石。此外，半透明涂层有助于实时观察细胞聚集过程 较大的粒径导致高多分散性，从而导致蒸发升高，变形和球形损失，以及微生物反应器的过早溶解。</li>
	<li>将含有4 x 104个细胞 的30μL单滴（参见步骤3.4.- 3.5.）分配到粉末床上（图1B）。</li>
	<li>以圆周运动轻轻旋转35毫米培养皿，以确保PFTE粉末完全覆盖液滴表面，形成液体大理石微生物反应器（图1C）。</li>
	<li>使用1，000μL移液器吸头拾取液体大理石微生物反应器，在边缘切割，以适应大理石的直径（图1D，E）。将液体大理石微生物反应器板到干净的细菌培养皿上以稳定其（图1F）。 注意:要产生摩擦力以抓住吸头内的大理石，请切割直径约略小于液体大理石直径的移液器吸头。</li>
	<li>将液体大理石微生物反应器从培养皿转移到96孔板（一个大理石/孔）中（图1G）。</li>
	<li>从孔的边缘慢慢加入100μL培养基。微生物反应器开始漂浮在培养基顶部（图1H）。 注:由于PTFE疏水性的破坏，微生物反应器在直接液体接触时破裂。作为替代方法，液体大理石微生物反应器可以单独放置在35mm细菌培养皿中。在这种情况下，为了防止液体大理石蒸发，必须将含有微生物反应器的35毫米培养皿插入100毫米培养皿中，事先用无菌水等分</li>
	<li>将液体大理石微生物反应器在37°C下在5%CO2培养箱中孵育18小时，2，3，8，9。 注意:1μm的PTFE粒径可以确保内部液体与周围环境之间的最佳气体交换。</li>
	<li>在5-aza-CR孵育18小时后，使用在边缘切割的1，000μL移液器尖端收集液体大理石微生物反应器（参见步骤4.5）。</li>
	<li>将微生物反应器置于新的35mm细菌培养皿中（图1D-F）。</li>
	<li>用针刺穿液体大理石并打破它。</li>
	<li>在立体显微镜下，用200μL移液器尖端回收形成的球体，在边缘切割（图1I，J）。 注意:包裹在PTFE中的表观遗传擦除的细胞形成3D球形结构（每个液体大理石中都有一个聚集体）。</li>
	<li>为了评估对5-aza-CR响应的多能态的获得，通过定性PCR检查多能性相关基因表达OCT4，NANOG，REX1和SOX2的发作（表2）。</li>
	<li>继续执行协议的第二步，如下所述。</li>
</ol>5. 在表观遗传学擦除细胞的PTFE微生物反应器中培养
<ol><li>准备新鲜的ESC培养基（表1）。</li>
	<li>在含有ESC培养基的培养皿中转移类器官以洗涤5-氮杂-CR残留物（参见步骤5.1.-5.2）。</li>
	<li>准备含有PTFE粉末床的新的35毫米细菌培养皿（另见步骤4.1.</li>
	<li>使用200μL移液器吸头将单个类器官在30μL ESC培养基的液滴中分配到粉末床上，在边缘切割（参见步骤4.9）。5.3.).</li>
	<li>以圆周运动轻轻旋转35mm培养皿以形成新的液体大理石微生物反应器，使用1，000μL移液器尖端拾取它，在边缘切割，并将新形成的微生物反应器放入96孔板（一个大理石/孔）的孔中（参见步骤4.3.-4.6）。</li>
	<li>要漂浮微生物反应器，从孔的边缘加入100μL培养基以缓慢沐浴大理石（见注4.7）。</li>
	<li>将液体大理石微生物反应器在37°C下在5%CO2 培养箱中培养所需的时间。按照5.3.-5.7中描述的步骤，每隔一天更换一次培养基。 注意:在本手稿中，提供了28天的类器官培养获得的结果。但是，如果需要，可以进行更长的培养期。</li>
</ol>

<ol>
	<li>Pennarossa, 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=Brief+demethylation+step+allows+the+conversion+of+adult+human+skin+fibroblasts+into+insulin-secreting+cells.">Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 8948-8953 (2013).</li><li>Pennarossa, 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=Reprogramming+of+pig+dermal+fibroblast+into+insulin+secreting+cells+by+a+brief+exposure+to+5-aza-cytidine.">Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine.</a> Stem Cell Reviews and Reports. 10 (1), 31-43 (2014).</li><li>Brevini, T. A. L., 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=Morphological+and+molecular+changes+of+human+granulosa+cells+exposed+to+5-azacytidine+and+addressed+toward+muscular+differentiation.">Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation.</a> Stem Cell Reviews and Reports. 10 (5), 633-642 (2014).</li><li>Mirakhori, F., Zeynali, B., Kiani, S., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+azacytidine+step+allows+the+conversion+of+suspension+human+fibroblasts+into+neural+progenitor-like+cells.">Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells.</a> Cell Journal. 17 (1), 153-158 (2015).</li><li>Tan, S. J., 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=Muscle+tissue+engineering+and+regeneration+through+epigenetic+reprogramming+and+scaffold+manipulation.">Muscle tissue engineering and regeneration through epigenetic reprogramming and scaffold manipulation.</a> Scientific Reports. 5, 16333 (2015).</li><li>Brevini, T. A. L., Pennarossa, G., Acocella, F., Brizzola, S., Zenobi, A., Gandolfi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+conversion+of+adult+dog+skin+fibroblasts+into+insulin-secreting+cells.">Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells.</a> Veterinary Journal. 211, 52-56 (2016).</li><li>Chandrakanthan, V., 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=PDGF-AB+and+5-Azacytidine+induce+conversion+of+somatic+cells+into+tissue-regenerative+multipotent+stem+cells.">PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (16), 2306-2315 (2016).</li><li>Manzoni, E. F. 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=5-azacytidine+affects+TET2+and+histone+transcription+and+reshapes+morphology+of+human+skin+fibroblasts.">5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.</a> Scientific Reports. 6, 37017 (2016).</li><li>Pennarossa, 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=Epigenetic+erasing+and+pancreatic+differentiation+of+dermal+fibroblasts+into+insulin-producing+cells+are+boosted+by+the+use+of+low-stiffness+substrate.">Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate.</a> Stem Cell reviews and reports. 14 (3), 398-411 (2018).</li><li>Pennarossa, 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=Use+of+a+PTFE+micro-bioreactor+to+promote+3D+cell+rearrangement+and+maintain+high+plasticity+in+epigenetically+erased+fibroblasts.">Use of a PTFE micro-bioreactor to promote 3D cell rearrangement and maintain high plasticity in epigenetically erased fibroblasts.</a> Stem Cell Reviews and Reports. 15 (1), 82-92 (2019).</li><li>Constantinides, P. G., Jones, P. A., Gevers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+striated+muscle+cells+from+non-myoblast+precursors+following+5-azacytidine+treatment.">Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment.</a> Nature. 267 (5609), 364-366 (1977).</li><li>Taylor, S. M., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+new+phenotypes+induced+in+10T1/2+and+3T3+cells+treated+with+5-azacytidine.">Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.</a> Cell. 17 (4), 771-779 (1979).</li><li>Taylor, S. M., Constantinides, P. A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Azacytidine,+DNA+methylation,+and+differentiation.">5-Azacytidine, DNA methylation, and differentiation.</a> Current Topics in Microbiology and Immunology. 108, 115-127 (1984).</li><li>Jones, P. A. <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+5-azacytidine+and+its+2'-deoxyderivative+on+cell+differentiation+and+DNA+methylation.">Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation.</a> Pharmacology &amp; Therapeutics. 28 (1), 17-27 (1985).</li><li>Palii, S. S., Van Emburgh, B. O., Sankpal, U. T., Brown, K. D., Robertson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+inhibitor+5-Aza-2'-deoxycytidine+induces+reversible+genome-wide+DNA+damage+that+is+distinctly+influenced+by+DNA+methyltransferases+1+and+3B.">DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B.</a> Molecular and Cellular Biology. 28 (2), 752-771 (2008).</li><li>Vining, K. H., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+forces+direct+stem+cell+behaviour+in+development+and+regeneration.">Mechanical forces direct stem cell behaviour in development and regeneration.</a> Nature Reviews. Molecular Cell Biology. 18 (12), 728 (2017).</li><li>Matamoro-Vidal, A., Levayer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+influences+of+mechanical+forces+on+cell+competition.">Multiple influences of mechanical forces on cell competition.</a> Current Biology. 29 (15), 762-774 (2019).</li><li>Yim, E. K., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-dependent+cell+signaling+in+stem+cell+differentiation.">Force-dependent cell signaling in stem cell differentiation.</a> Stem Cell Research &amp; Therapy. 3 (5), 41 (2012).</li><li>Kumar, A., Placone, J. K., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+extracellular+forces+that+determine+cell+fate+and+maintenance.">Understanding the extracellular forces that determine cell fate and maintenance.</a> Development. 144 (23), 4261-4270 (2017).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Current Opinion in Cell Biology. 15 (6), 753-762 (2003).</li><li>Streuli, C. H., 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=Laminin+mediates+tissue-specific+gene+expression+in+mammary+epithelia.">Laminin mediates tissue-specific gene expression in mammary epithelia.</a> The Journal of Cell Biology. 129 (3), 591-603 (1995).</li><li>Christman, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Azacytidine+and+5-aza-2[prime]-deoxycytidine+as+inhibitors+of+DNA+methylation:+mechanistic+studies+and+their+implications+for+cancer+therapy.">5-Azacytidine and 5-aza-2[prime]-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.</a> Oncogene. 21, 5483-5495 (2002).</li><li>Stresemann, C., Lyko, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+action+of+the+DNA+methyltransferase+inhibitors+azacytidine+and+decitabine.">Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine.</a> International Journal of Cancer. 123 (1), 8-13 (2008).</li><li>Ledda, S., Idda, A., Kelly, J., Ariu, F., Bogliolo, L., Bebbere, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+in+vitro+maturation+of+sheep+oocytes+in+a+liquid+marble+microbioreactor.">A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.</a> Journal of Assisted Reproduction and Genetics. 33 (4), 513-518 (2016).</li><li>Vadivelu, R. 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=Generation+of+three-dimensional+multiple+spheroid+model+of+olfactory+ensheathing+cells+using+floating+liquid+marbles.">Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles.</a> Scientific Reports. 5, 15083 (2015).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marble+as+bioreactor+for+engineering+three-dimensional+toroid+tissues.">Liquid Marble as bioreactor for engineering three-dimensional toroid tissues.</a> Scientific Reports. 7 (1), 12388 (2017).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marbles+as+bioreactors+for+the+study+of+three-dimensional+cell+interactions.">Liquid Marbles as bioreactors for the study of three-dimensional cell interactions.</a> Biomedical Microdevices. 19 (2), 31 (2017).</li><li>Varelas, X., 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=TAZ+controls+Smad+nucleocytoplasmic+shuttling+and+regulates+human+embryonic+stem-cell+self-renewal.">TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal.</a> Nature Cell Biology. 10 (7), 837-848 (2008).</li><li>Panciera, T., 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=Induction+of+Expandable+Tissue-Specific+Stem/Progenitor+Cells+through+Transient+Expression+of+YAP/TAZ.">Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.</a> Cell Stem Cell. 19 (6), 725-737 (2016).</li><li>Pennarossa, G., Paffoni, A., Ragni, G., Gandolfi, F., Brevini, T. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+signaling-directed+YAP/TAZ+regulation+encourages+3D+spheroid+colony+formation+and+boosts+plasticity+of+parthenogenetic+stem+cells.">Rho signaling-directed YAP/TAZ regulation encourages 3D spheroid colony formation and boosts plasticity of parthenogenetic stem cells.</a> Advances in Experimental Medicine and Biology. 1237, 49-60 (2020).</li></ol>

作者没有什么可透露的。

在过去的几十年里，一些研究的重点是制定策略，使终末分化的细胞恢复到不那么投入和更高的宽容状态。这里描述的方案允许从成体成熟的终末分化细胞开始产生和长期维持多能细胞。该方法结合了两个独立的步骤，涉及诱导高允许状态，这是通过化学表观遗传擦除实现的，并使用3D培养系统确保其后续维护。
在PTFE包封的细胞中观察到的3D球体结构的形成（图2）与其他研究一致，表明PTFE能够有效地促进细胞聚集，促进嗅鞘细胞（OEC）球体结构25 的建立或3D环形聚集体26的形成。这些形态学变化与多能性相关基因表达的开始平行（图3， 图4， 图5），与2D后5-aza-CR细胞相比，3D后5-aza-CR细胞中的水平显着更高（图3， 图4， 图5）。一致地，3D Post 5-aza-CR细胞比2D Post 5-aza-CR细胞显示出更高的DNA低甲基化（图6）。总体而言，这些结果表明5-氮杂-CR诱导高可塑性状态的能力，无论使用的细胞培养系统如何。然而，使用PTFE微生物反应器显着促进细胞实现的化学诱导多能性状态，该反应器可增强多能性基因转录并增加5-aza-CR去甲基化效果。更有趣的是，只有3D Post 5-aza-CR细胞在随后的所有培养期间稳定地保留了获得的3D球形结构（图2）并保持多能性相关基因的高表达水平（图3， 图4， 图5）以及低DNA甲基化水平（图6）。总而言之，这里报道的代表性结果表明，这种两步策略是高效和稳健的，使用PTFE微生物反应器不仅可以提高高可塑性，还可以使其在哺乳动物物种中稳定，长期地维持。我们最近证明，这些有益作用与河马信号通路的激活及其机械转导相关线索10有关，这些线索先前已被证明在细胞多能性的主动调节中具有关键作用28，29，30。
成功手术的两个最关键步骤是始终在37°C下严格维持细胞，包括在无菌层流和显微镜下处理它们，以及在微生物反应器生产过程中使用正确的细胞数量/液体体积速率，这可能根据所使用的特定细胞类型而有所不同。根据我们的经验，强烈建议在培养物中使用试剂之前新鲜制备试剂（这对于5-氮杂-CR储备溶液绝对至关重要）。此外，介质刷新必须在立体显微镜下进行，因为3D球面类器官可能会在介质更换过程中丢失。
该方法的主要优点是没有转基因和/或病毒载体要求;不同哺乳动物物种的稳健性和再现性;低成本;对不同细胞类型具有高度的灵活性。另一方面，由于微型生物反应器体积小，获得的数据数量有限，可能的限制。此外，使用高细胞密度可能导致低氧转移速率和悬浮生长受限。为了克服这些问题，仍然需要进一步制定扩大和（或）缩小战略。
重要的是要强调，所有基于3D球体的应用共同的关键方面是可重复性，生产效率，类器官大小均匀性以及对细胞生理学的影响。这些特征与培养系统内产生的机械力严格相关，并根据所使用的不同方法而变化。例如，可以在固定系统中使用非粘附培养皿培养多细胞类器官。这种方法主要基于扩散受限的条件，通常会导致松散聚集集群的形成。悬挂-下降技术也显示出相同的局限性。事实上，细胞悬浮液的沉积下降到组织培养皿盖子的下侧，导致微重力环境的产生，该环境将细胞集中在自由液 - 空气界面处，诱导产生低聚集的多细胞球体。一种可能的替代方案是由微调烧瓶技术代表的。然而，这种方法非常昂贵，因为它需要增加培养基的数量。此外，当用于表征或进一步的体外测试时，需要将形成的类器官转移到固定的培养系统中。所有这些问题都可以通过使用液体大理石微生物反应器来克服。事实上，它们提供了一个非粘附的液体表面，结合了固定和旋转方法的优点，诱导快速细胞聚集和 产生致密的球体。同时，每个大理石的凹形底部，球形和内部液体流动允许细胞沉降到微生物反应器的底部，从而形成大小和形状均匀的类器官。使用液体弹珠的另一个显着优势是最佳的气体交换，由于其球形形状，可以在整个表面上发生。
总之，这里描述的方案允许有效和简单地产生哺乳动物多能细胞。由于该策略不含病毒载体，并且不涉及使用任何基因转染，因此对于转化医学应用非常有希望，可以被认为是患者特异性细胞治疗的一个进步。此外，使用3D微生物反应器培养系统可能代表干细胞类器官技术的显着突破，并可能为不同细胞类型（如ESC，iPSC和MSC）的长期培养提供有利的微环境。另一个优点是体积小，可以研究微生物反应器内建立的丰富环境的旁分泌/自分泌信号传导的影响。

在过去的几十年里，被广泛接受的单向进展到细胞承诺和分化的概念被完全修改。已经证明，细胞规格可以颠倒过来，并且使用不同的方法可以将终末分化的细胞推向不那么投入和更高的宽容状态。
在提出的几种方法中，最有前途的方法之一涉及使用化合物诱导细胞进入所谓的化学诱导多能性。这种方法中使用的小分子能够相互作用并修饰成年成熟细胞的表观遗传特征，从而避免了对任何转基因和/或病毒载体1，2，3，4，5，6，7，8，9，10的需求。.最近的许多研究表明，通过提供诱导高甲基化基因11，12，13，14，15再激活的特定生化和生物刺激，可以将细胞从一种表型切换到另一种表型。这些去甲基化事件允许将终末分化的细胞转化为原始祖细胞，多能或高可塑性/多能细胞1，2，3，4，5，6，7，8，9，10。
同时，许多研究最近都集中在理解机械检测相关线索上，更具体地说，是使用机械力直接影响细胞可塑性和/或分化的可能性16，17，18，19。事实上，已经清楚地表明，细胞外基质（ECM）在控制细胞命运中起着关键作用。特别是，ECM产生的生物力学和生物物理信号直接调节分子机制和信号通路，影响细胞行为和功能20，21。这些最近的数据为开发新型3D培养系统铺平了道路，这些系统更接近于模仿体内细胞微环境，复制驱动细胞行为的机械和物理刺激。
我们在这里描述了一个两步方案，它将化学表观遗传擦除与机械衰老相关线索相结合，以产生哺乳动物多能细胞。在第一步中，将细胞与去甲基化分子5-氮杂胞苷（5-氮杂-CR）一起孵育。该试剂能够通过直接的10-11易位2（TET2）介导的作用8，10 和间接抑制DNA甲基转移酶（DNMT）22，23的组合作用来诱导显着的全局DNA去甲基化。该步骤诱导表观遗传阻滞的去除，随后重新激活多能性相关基因表达，因此产生高可塑性细胞1，2，3，8，10，以下称为"表观遗传擦除细胞"。在第二步中，将细胞封装在3D培养系统中。为此，将非反应性疏水性合成化合物聚四氟乙烯（PTFE;粒径为1μm）用作微生物反应器，其允许创建通过使用传统2D培养系统无法实现的细胞微环境10。PTFE粉末颗粒粘附在液滴的表面，其中细胞被重新悬浮并将液核与支撑表面隔离开来，同时允许内部液体和周围环境24之间的气体交换。由此获得的"PTFE微生物反应器"，也称为"液体大理石"，鼓励细胞彼此自由相互作用，促进3D细胞重排25，26，27，并通过生物机械测量相关线索10延伸并稳定地维持获得的高可塑性状态。

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7522</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>5-Azacytidine</td>
			<td>Sigma-Aldrich</td>
			<td>A2385</td>
			<td>5-aza-CR, for fibroblast epigenetic erasing</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR</td>
			<td>Bio-Rad Laboratories</td>
			<td>NA</td>
			<td>Thermal cycler for quantitative PCR</td>
		</tr>
		<tr>
			<td>Cytidine</td>
			<td>Sigma-Aldrich</td>
			<td>C4654</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>41966052</td>
			<td>For fibroblast isolation and culture medium</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>31885023</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>PBS; for biopsy and cell wash and for solution preparation</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT Micro Purification Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>61021</td>
			<td>mRNA estraction</td>
		</tr>
		<tr>
			<td>ESGRO Recombinant Mouse LIF Protein</td>
			<td>Sigma-Aldrich</td>
			<td>ESG1106</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>FGF-Basic (AA10-155) Recombinant Human Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0024</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td>For dish coating</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 2700</td>
			<td>Applied Biosystems</td>
			<td>NA</td>
			<td>Thermal cycler for qualitative PCR</td>
		</tr>
		<tr>
			<td>Global DNA Methylation ELISA Kit</td>
			<td>CELL BIOLABS</td>
			<td>STA-380</td>
			<td>Methylation study</td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M7801</td>
			<td>Qualitative PCR</td>
		</tr>
		<tr>
			<td>Guanosine</td>
			<td>Sigma-Aldrich</td>
			<td>G6264</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-10 Nutrient Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>31550031</td>
			<td>For ESC medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>KOVA glasstic slide 10 with grids</td>
			<td>Hycor Biomedical</td>
			<td>87144</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>Leica MZ APO Stereo Microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td>For organoid observation</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>Component of fibroblast and ESC media</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td>Component of ESC medium</td>
		</tr>
		<tr>
			<td>Millex-GS 0.22 &#181;m pore filters</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>For solution sterilization</td>
		</tr>
		<tr>
			<td>M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant</td>
			<td>Promega</td>
			<td>M3681</td>
			<td>mRNA reverse transcription</td>
		</tr>
		<tr>
			<td>Multiskan FC Microplate Photometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>51119000</td>
			<td>For ELISA plate reading</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE300 Inverted Phase Contrast Microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>For cell observation</td>
		</tr>
		<tr>
			<td>Perkin Elmer Thermal Cycler 480</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Thermal cycler for reverse transcription</td>
		</tr>
		<tr>
			<td>Poly(tetrafluoroethylene) 1 &#956;m particle size</td>
			<td>Sigma-Aldrich</td>
			<td>430935</td>
			<td>For generating micro-bioreactor</td>
		</tr>
		<tr>
			<td>PureLink Genomic DNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K182001</td>
			<td>Genomic DNA estraction</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Cells-to-CT Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1728</td>
			<td>Quantitative PCR</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 100X20 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833902</td>
			<td>For fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Standard</td>
			<td>Sarstedt</td>
			<td>833900</td>
			<td>For Fibroblast isolation</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35X10 mm, Suspension</td>
			<td>Sarstedt</td>
			<td>833900500</td>
			<td>Bacteriology petri dish for liquid marble culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 Well,Standard,F</td>
			<td>Sarstedt</td>
			<td>833924005</td>
			<td>For liquid marble culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>For fibroblast dissociation</td>
		</tr>
		<tr>
			<td>Tube 15ml, 120x17mm, PS</td>
			<td>Sarstedt</td>
			<td>62553041</td>
			<td>For cell suspension centrifugation</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td>Component of nucleoside mix for ESC medium</td>
		</tr>
	</tbody>
</table>

a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells

细胞表型可以用不同的方法逆转或修饰，具有每种技术特有的优点和局限性。在这里，我们描述了一种新策略，该策略将化学表观遗传擦除与机械检测相关线索相结合，以产生哺乳动物多能细胞。需要两个主要步骤。在第一步中，成体成熟（终末分化）细胞暴露于表观遗传橡皮擦5-氮杂胞苷以驱动它们进入多能状态。该方案的这一部分是基于对控制细胞命运和分化的表观遗传机制的日益了解而开发的，并且涉及使用表观遗传修饰剂来消除细胞分化状态，然后驱动进入瞬时高可塑性窗口。
在第二步中，将擦除的细胞封装在聚四氟乙烯（PTFE）微生物反应器（也称为Liquid Marbles）中，以促进3D细胞重排以扩展并稳定地保持获得的高可塑性。PTFE是一种非反应性疏水性合成化合物，其使用允许产生细胞微环境，这在传统的2D培养系统中无法实现。该系统通过生物机械检测相关线索鼓励和促进多能性的维持。
这里描述的技术程序是允许在成人体细胞中诱导和维持高可塑性状态的简单策略。该协议允许在所有测试的哺乳动物物种中衍生出高可塑性细胞。由于它不涉及基因转染的使用，并且没有病毒载体，因此它可能代表了转化医学应用的显着技术进步。此外，微生物反应器系统通过体外重新创建特定的微环境，允许长期培养高可塑性细胞，即ESC，iPSCs，表观遗传擦除细胞和MSC，从而为干细胞类器官技术提供了显着的进步。

本方案描述了从成体细胞生成和稳定维持哺乳动物多能细胞的所有步骤。这种方法已经成功地从不同的哺乳动物物种（即小鼠，猪和人类）中分离出成纤维细胞。这里报道的代表性结果是从所有细胞系获得的，无论来源物种如何。
形态学分析表明，在与去甲基化剂5-氮杂-CR孵育18小时后，成纤维细胞封装在PTFE微生物反应器（3D Post 5-aza-CR）聚集体中并形成3D球形结构，显示出均匀的尺寸几何形状，在所有三个物种中都考虑。（图2A-C，3D Post 5-aza-CR）。86.31±4.13%的包封细胞显着修饰其表型，显示出通常与高可塑性表型相关的特征8。相比之下，培养到2D标准条件下的5-aza-CR细胞后，尽管用圆形或椭圆形取代了典型的成纤维细胞细长形状，但尺寸要小得多，具有较大的颗粒状细胞核，保留了单层分布（图2）。形态学变化伴随着3D和2D后5-氮杂-CR细胞中多能性相关基因表达的开始。还观察到POU 5类同源盒1（OCT4），Nanog同源盒（NANOG），ZFP42锌指蛋白（REX1）和性别决定区Y-box 2（SOX2）的转录，在未处理的成纤维细胞（T0）中不存在（图3， 图4， 图5）。此外，定量PCR分析表明，与在2D标准塑料培养皿（2D Post 5-aza-CR）中培养的细胞相比，上述基因以及10-11易位-2（TET2），上皮细胞粘附分子（EPCAM）和钙粘蛋白1（CDH1）基因在3D后5-aza-CR细胞（图3， 图4， 图5，蓝条）中显着上调（图3， 图 4、 图 5、橙色条）。同时，在3D和2D后5-氮杂-CR细胞中可以清楚地检测到成纤维细胞特异性标记Thy-1细胞表面抗原（THY1）的显着下调（图3， 图4， 图5）。
通过对DNA全局甲基化的ELISA分析也证实了高可塑性状态的实现，该分析表明3D和2D后5-氮杂-CR细胞中的甲基化水平显着降低（图6A-C）。此外，与基因表达结果一致，与2D Post 5-aza-CR细胞相比，3D Post 5-aza-CR细胞（图6A-C，橙色条）的DNA甲基化水平显着降低（
更有趣的是，3D Post 5-aza-CR细胞在所有后续培养期间保持获得3D球形结构（图2A，3D 28d）并保持多能性相关基因的高表达水平（图3，图4和图5蓝条）以及低DNA甲基化水平（图6A-C，蓝条），直到28天， 当文化被逮捕时。相比之下，尽管2D Post 5-aza-CR细胞在用去甲基化剂处理后转录相同的多能性基因，但它们在第7天就拒绝了这种表达（图3，图4，图5，nd）。同样，甲基化水平的下降在最初的72小时内保持不变;然后甲基化缓慢增加，通过培养的第7天（图6A-C，橙色条）返回与未处理的成纤维细胞（图6 A-C，T0，白条）相当。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61655/61655fig01.jpg"> 图1:PTFE微生物反应器中的细胞封装和类器官回收。 （A）细菌培养皿中装满PTFE以制备粉末床。（B）将含有细胞的单个培养基液滴分配在PTFE床的顶部。（C）培养皿以圆周运动轻轻旋转以涂覆液滴并产生微生物反应器。（D）在末端切割1000μL移液器吸头（红色箭头）和（E）用于收集微生物反应器。（F）将液体大理石转移到干净的培养皿中以稳定它，（G）放入96孔板（一个大理石/孔）中，（H）漂浮在介质上。（I）为了收集新形成的类器官，用针刺穿微生物反应器，（J）在立体显微镜下回收获得的细胞聚集体。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61655/61655fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61655/61655fig02v2.jpg"> 图2:封装在PTFE微生物反应器中的哺乳动物表观遗传擦除细胞形成3D球形结构。 用PTFE封装并用5-aza-CR形式的3D球形结构（3D Post 5-aza-CR）处理的小鼠（A），猪（B）和人（C）细胞，在所有随后的培养期间（3D 28d）中稳定地维持;比例尺，100 μm）。相比之下，将小鼠（A），猪（B）和人（C）细胞接种到塑料培养皿上并用5-aza-CR处理，将典型的成纤维细胞细长形状（T0）替换为圆形上皮样表型并保留单层分布（2D Post 5-aza-CR）。到培养的第7天，2D细胞恢复到其原始的细长形状，该形状在随后的培养期间稳定地保持（2D 28 d;比例尺，100 μm）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61655/61655fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61655/61655fig03.jpg"> 图3:包封在PTFE微生物反应器中的小鼠表观遗传擦除细胞显示出多能性相关基因表达的高水平和长期维持。 Oct4，Nanog，Rex1，Sox2，Tet2，Epcam，Cdh1和Thy1基因在小鼠未经处理的成纤维细胞（T0，白条），暴露于5-aza-CR（Post 5-aza-CR）的成纤维细胞中的转录水平，以及在PTFE封装（蓝条）和标准塑料培养皿（橙色条）培养细胞的不同培养点。报告基因表达值时，最高表达值设置为1，所有其他表达值均与此相关。不同的上标表示显著差异（P < 0.05）。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61655/61655fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61655/61655fig04.jpg"> 图4:包封在PTFE微生物反应器中的猪表观遗传擦除细胞显示出多能性相关基因表达的高水平和长期维持。 未经处理的成纤维细胞（T0，白条），暴露于5-aza-CR（5-aza-CR后）的成纤维细胞中的OCT4，NANOG，REX1，SOX2，TET2，EPCAM，CDH1和THY1基因的转录水平，以及在PTFE封装（蓝条）和标准塑料培养皿（橙色条）培养细胞的不同培养点。报告基因表达值时，最高表达值设置为1，所有其他表达值均与此相关。不同的上标表示显著差异（P < 0.05）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61655/61655fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61655/61655fig05.jpg"> 图5:包封在PTFE微生物反应器中的人类表观遗传擦除细胞显示出多能性相关基因表达的高水平和长期维持。 人未处理的成纤维细胞（T0，白条），暴露于5-aza-CR（5-aza-CR后）的成纤维细胞中的OCT4，NANOG，REX1，SOX2，TET2，EPCAM，CDH1和THY1基因的转录水平，以及在PTFE封装（蓝条）和标准塑料培养皿（橙色条）培养细胞的不同培养点。报告基因表达值时，最高表达值设置为1，所有其他表达值均与此相关。不同的上标表示显著差异（P < 0.05）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61655/61655fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61655/61655fig06v2.jpg"> 图 6:PTFE 微生物反应器增强 5-氮杂-CR 去甲基化效果，并在哺乳动物表观遗传学擦除的成纤维细胞中维持长期 DNA 低甲基化。 将封闭在PTFE微生物反应器（蓝条）中的小鼠（A），猪（B）和人（C）细胞的全局DNA甲基化水平或电镀在暴露于5-aza-CR（Post 5-aza-CR）的标准塑料（橙色条）上并在ESC培养基中培养28天。未经处理的成纤维细胞（T0;白条）。结果表示具有5个独立生物重复的3个独立实验的平均±SD。不同的上标表示显著差异（P < 0.05）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61655/61655fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">成纤维细胞分离培养基 （100 mL）</td>
		</tr>
<tr>
<td>DMEM，高糖，丙酮酸盐</td>
			<td>77 毫升</td>
		</tr>
<tr>
<td>胎牛精华素，合格，热灭活</td>
			<td>20 毫升</td>
		</tr>
<tr>
<td>L-谷氨酰胺溶液</td>
			<td>1 毫升</td>
		</tr>
<tr>
<td>抗生素抗真菌剂溶液 （100×）</td>
			<td>2 毫升</td>
		</tr>
<tr>
<td colspan="2"></td>
		</tr>
<tr>
<td colspan="2">成纤维细胞培养基 （100 mL）</td>
		</tr>
<tr>
<td>DMEM，高糖，丙酮酸盐</td>
			<td>88 毫升</td>
		</tr>
<tr>
<td>胎牛精华素，合格，热灭活</td>
			<td>10 毫升</td>
		</tr>
<tr>
<td>L-谷氨酰胺溶液</td>
			<td>1 毫升</td>
		</tr>
<tr>
<td>抗生素抗真菌剂溶液 （100×）</td>
			<td>1 毫升</td>
		</tr>
<tr>
<td colspan="2"></td>
		</tr>
<tr>
<td colspan="2">ESC 培养基 （10 mL）</td>
		</tr>
<tr>
<td>火腿的F-10营养混合物</td>
			<td>3，99 毫升</td>
		</tr>
<tr>
<td>DMEM，低葡萄糖，丙酮酸盐</td>
			<td>3，99 毫升</td>
		</tr>
<tr>
<td>敲除血清替代</td>
			<td>1 毫升</td>
		</tr>
<tr>
<td>胎牛精华素，合格，热灭活</td>
			<td>500 微升</td>
		</tr>
<tr>
<td>抗生素抗真菌剂溶液 （100×）</td>
			<td>100 微升</td>
		</tr>
<tr>
<td>L-谷氨酰胺溶液</td>
			<td>100 微升</td>
		</tr>
<tr>
<td>MEM 非必需氨基酸溶液 （100X）</td>
			<td>100 微升</td>
		</tr>
<tr>
<td>2-巯基乙醇（10毫安）</td>
			<td>100 微升 （0.1 毫微米）</td>
		</tr>
<tr>
<td>核苷混合物（0.3 M 鸟苷，0.3 M 腺苷，0.3 M 胞苷，0.3 M 尿苷和 0.1 M 胆苷）</td>
			<td>100 μL（3 mM 鸟苷、3 mM 腺苷、3 mM 胞苷、3 mM 尿苷和 1 mM 胆苷）</td>
		</tr>
<tr>
<td>ESGRO重组小鼠LIF蛋白（1000单位/毫升）</td>
			<td>10 μL （1 单位/毫升）</td>
		</tr>
<tr>
<td>重组人碱性FGF （bFGF） （5 μg/mL）</td>
			<td>10 微升 （5 纳克/毫升）</td>
		</tr>
</tbody></table>表1:成纤维细胞分离培养基、成纤维细胞培养基和ESC培养基的组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>目标</td>
			<td colspan="2">PCR 引物套装</td>
			<td>物种</td>
		</tr><tr><td rowspan="2">Cdh1</td>
			<td colspan="2">前锋: 嘎嘎</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向: GTTGACCGTCCCTTCACAGT</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">埃普卡姆</td>
			<td colspan="2">前锋: TGGCAACAAGTTGCTCTCTG</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向:CTTGTCGGTTCTTCGACTC</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">纳诺格</td>
			<td colspan="2">Forward: AGGCTGATTTGGTTGGTGTC</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向:CCAGGAAGACCCACACTCAT</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">10月4日</td>
			<td colspan="2">转发: ACACCTGGCTTCAGACTTCG</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">Reverse: AGTTGCTTTCCACTCGTGCT</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">雷克斯1</td>
			<td colspan="2">前锋: CAGGTTCTGGAAGCGAGTTC</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向: GACAAGCATGTGCTTCCTCA</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">袜子2</td>
			<td colspan="2">前锋: AGAACCCCAAGATGCACAAC</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向:CTCCGGGAAGCGTGTACTTA</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">Tet2</td>
			<td colspan="2">前锋: GCACAGGGAGCAAGAGATTC</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向:ATGTTGACATTGCCAGTGGA</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">你的1</td>
			<td colspan="2">前锋: AACTCTTGGCACCATGAACC</td>
			<td>鼠</td>
		</tr><tr><td colspan="2">反向:GCACGTGTGTCCTCTTCTCT</td>
			<td>鼠</td>
		</tr><tr><td rowspan="2">高密度氢呋喃1</td>
			<td colspan="2">前锋: GAATGACAATGGCCCCATAC</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">Reverse: AGGTGGTCACCTCTCTCTCTG</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">断续器</td>
			<td colspan="2">前锋: GCTTGGTCAGTGCCAGTGTA</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">反向:CTTCTGACCCCAGCAGTGTT</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">纳诺格</td>
			<td colspan="2">前锋: ATCCAGCTTGTCCCCAAAG</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">Reverse: ATTTCATTCGCTGGTTCTGG</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">华侨城4</td>
			<td colspan="2">前锋: GTTCAGCCAAACGACCATCT</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">反向:CTCCAGGTTTCTCTCTC</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">雷克斯1</td>
			<td colspan="2">前锋: CTTCAAGGAGAGCGCAAAAC</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">反向:TGTCCCCAATCAAAAATGCT</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">断续器</td>
			<td colspan="2">前锋: GCCCTGCAGTACAACTCCAT</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">反向: GCTGATCATGTCCCGTAGGT</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">断续器2</td>
			<td colspan="2">前锋: 卡加阿卡阿卡特加加</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">Reverse: GAATGGCTCGGTCTGAAG</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">THY1</td>
			<td colspan="2">Forward: GGCATCGCTCTCTTGCTAAC</td>
			<td>猪的</td>
		</tr><tr><td colspan="2">Reverse: GCTGGAGAAGTTGGTTCGAG</td>
			<td>猪的</td>
		</tr><tr><td rowspan="2">高密度氢呋喃1</td>
			<td colspan="2">前锋: TGCCCAGAAAATGAAAAG</td>
			<td>人</td>
		</tr><tr><td colspan="2">反向:GTGTATGTGGCAATGCGTTC</td>
			<td>人</td>
		</tr><tr><td rowspan="2">断续器</td>
			<td colspan="2">前锋: GCTGGTGTGTGAACACTGCT</td>
			<td>人</td>
		</tr><tr><td colspan="2">反向:ACGCGTTGTGATCTCCTTCT</td>
			<td>人</td>
		</tr><tr><td rowspan="2">纳诺格</td>
			<td colspan="2">前锋: AGAAAAACAACTGGCCGAAG</td>
			<td>人</td>
		</tr><tr><td colspan="2">反向:TGCTCCAGGACTGGATGTTC</td>
			<td>人</td>
		</tr><tr><td rowspan="2">华侨城4</td>
			<td colspan="2">前锋: AATTTGCCAAGCTCCTGAAG</td>
			<td>人</td>
		</tr><tr><td colspan="2">反向:GTTGCCTCTCTCCT</td>
			<td>人</td>
		</tr><tr><td rowspan="2">雷克斯1</td>
			<td colspan="2">前锋: ACGTTTCGTGTGTGTCCCTTTC</td>
			<td>人</td>
		</tr><tr><td colspan="2">Reverse: TATAACCGCTTTTGGGGTTG</td>
			<td>人</td>
		</tr><tr><td rowspan="2">断续器</td>
			<td colspan="2">前锋: ACACCAATCCCATCCACACT</td>
			<td>人</td>
		</tr><tr><td colspan="2">反向:GCAAACTCTGCAAAGCTC</td>
			<td>人</td>
		</tr><tr><td rowspan="2">断续器2</td>
			<td colspan="2">Forward: CCCACTTACCTGCGTTTCAT</td>
			<td>人</td>
		</tr><tr><td colspan="2">反向:ACTGTGACCTTTCCCCACTG</td>
			<td>人</td>
		</tr><tr><td rowspan="2">THY1</td>
			<td colspan="2">前锋: AGATCCCAGAACCATGAACC</td>
			<td>人</td>
		</tr><tr><td colspan="2">Reverse: GCACGTGTGTTTCTTTGTCTCA</td>
			<td>人</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td>目标</td>
			<td colspan="2">塔克曼化验目录号</td>
			<td>物种</td>
		</tr><tr><td>阿克布</td>
			<td colspan="2">Mm02619580_g1</td>
			<td>鼠</td>
		</tr><tr><td>Cdh1</td>
			<td colspan="2">Mm01247357_m1</td>
			<td>鼠</td>
		</tr><tr><td>埃普卡姆</td>
			<td colspan="2">Mm00493214_m1</td>
			<td>鼠</td>
		</tr><tr><td>加普德</td>
			<td colspan="2">Mm99999915_g1</td>
			<td>鼠</td>
		</tr><tr><td>纳诺格</td>
			<td colspan="2">Mm02019550_s1</td>
			<td>鼠</td>
		</tr><tr><td>10月4日</td>
			<td colspan="2">Mm03053917_g1</td>
			<td>鼠</td>
		</tr><tr><td>雷克斯1</td>
			<td colspan="2">Mm03053975_g1</td>
			<td>鼠</td>
		</tr><tr><td>袜子2</td>
			<td colspan="2">Mm03053810_s1</td>
			<td>鼠</td>
		</tr><tr><td>Tet2</td>
			<td colspan="2">Mm00524395_m1</td>
			<td>鼠</td>
		</tr><tr><td>你的1</td>
			<td colspan="2">Mm00493681_m1</td>
			<td>鼠</td>
		</tr><tr><td>阿克特布</td>
			<td colspan="2">Ss03376563_uH</td>
			<td>猪的</td>
		</tr><tr><td>高密度氢呋喃1</td>
			<td colspan="2">Ss06942341_m1</td>
			<td>猪的</td>
		</tr><tr><td>断续器</td>
			<td colspan="2">Ss03384752_u1</td>
			<td>猪的</td>
		</tr><tr><td>加普德</td>
			<td colspan="2">Ss03375629_u1</td>
			<td>猪的</td>
		</tr><tr><td>纳诺格</td>
			<td colspan="2">Ss04245375_s1</td>
			<td>猪的</td>
		</tr><tr><td>华侨城4</td>
			<td colspan="2">Ss03389800_m1</td>
			<td>猪的</td>
		</tr><tr><td>雷克斯1</td>
			<td colspan="2">Ss03373622_g1</td>
			<td>猪的</td>
		</tr><tr><td>断续器</td>
			<td colspan="2">Ss03388002_u1</td>
			<td>猪的</td>
		</tr><tr><td>断续器2</td>
			<td colspan="2">Ss06880359_m1</td>
			<td>猪的</td>
		</tr><tr><td>THY1</td>
			<td colspan="2">Ss03376963_u1</td>
			<td>猪的</td>
		</tr><tr><td>阿克特布</td>
			<td colspan="2">Hs01060665_g1</td>
			<td>人</td>
		</tr><tr><td>高密度氢呋喃1</td>
			<td colspan="2">Hs01023895_m1</td>
			<td>人</td>
		</tr><tr><td>断续器</td>
			<td colspan="2">Hs00901885_m1</td>
			<td>人</td>
		</tr><tr><td>加普德</td>
			<td colspan="2">Hs02786624_g1</td>
			<td>人</td>
		</tr><tr><td>纳诺格</td>
			<td colspan="2">Hs02387400_g1</td>
			<td>人</td>
		</tr><tr><td>华侨城4</td>
			<td colspan="2">Hs00999632_g1</td>
			<td>人</td>
		</tr><tr><td>雷克斯1</td>
			<td colspan="2">Hs00399279_m1</td>
			<td>人</td>
		</tr><tr><td>断续器</td>
			<td colspan="2">Hs01053049_s1</td>
			<td>人</td>
		</tr><tr><td>断续器2</td>
			<td colspan="2">Hs00325999_m1</td>
			<td>人</td>
		</tr><tr><td>THY1</td>
			<td colspan="2">Hs00174816_m1</td>
			<td>人</td>
		</tr></tbody></table>表 2:引物信息。

Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

我们在这里提出了一种将化学表观遗传擦除与机械检测相关线索相结合的方法，以有效地产生哺乳动物多能细胞，而无需基因转染或逆转录病毒载体。因此，这种策略对转化医学很有希望，并且代表了干细胞类器官技术的显着进步。

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

a-two-step-strategy-that-combines-epigenetic-modification

Nanyang Technological University

Research

JoVE Journal

Biology

2.2K 次观看.  Università degli Studi di Milano. 我们在这里提出了一种将化学表观遗传擦除与机械检测相关线索相结合的方法，以有效地产生哺乳动物多能细胞，而无需基因转染或逆转录病毒载体。因此，这种策略对转化医学很有希望，并且代表了干细胞类器官技术的显着进步。

视频: A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-他的净化:一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

自动化芯片起实验产生上万HeLa细胞表观遗传配置文件

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...