Name: comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods
Uploaded: 2018-09-10T14:30:00
Description: Watch this Scientific Journal Video about Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods at JoVE.com

这项工作得到了国家自然科学基金 (81771617) 和广东省自然科学基金 (2016A030313178) 授予范的赠款的支持;深圳市基础研究基金 (JCYJ20170413165233512) 授予 x F 的补助金;和尤尼斯肯尼迪美国国立卫生研究院儿童健康 & 人类发展研究所授予编号 R01HD088549 (内容完全由作者负责, 不一定代表官方国家卫生研究院) 对 N.N. 的看法

所有鼠标实验严格遵循的协议 (SIAT-IRB-160520-YYS-FXJ-A0232) 批准的动物护理和使用委员会的深圳先进技术研究院, 中国科学院。
1. 胎盘硫酸软骨素 A 靶向脂质高分子纳米粒的合成
<ol><li>将甲氨喋呤-和微球负载的脂质高分子纳米粒子 (MNPs 和修订为期三) 和 plCSA-BP-共轭纳米粒子 (plCSA MNPs 和 plCSA 修订为期三) 进行了详细的描述, 其他18。</li>
</ol>2....

<ol>
	<li>Rodger, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Association+of+Factor+V+Leiden+and+Prothrombin+Gene+Mutation+and+Placenta-Mediated+Pregnancy+Complications:+A+Systematic+Review+and+Meta-analysis+of+Prospective+Cohort+Studies.">The Association of Factor V Leiden and Prothrombin Gene Mutation and Placenta-Mediated Pregnancy Complications: A Systematic Review and Meta-analysis of Prospective Cohort Studies.</a> PLOS Medicine. 7 (6), e1000292 (2010).</li><li>Rodger, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+thrombophilia+and+pregnancy+complications+revisited.">Inherited thrombophilia and pregnancy complications revisited.</a> Obstetrics &amp; Gynecology. 112 (2 Pt 1), 320-324 (2008).</li><li>Brenner, B., Aharon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombophilia+and+adverse+pregnancy+outcome.">Thrombophilia and adverse pregnancy outcome.</a> Clinics in Perinatology. 34 (4), 527-541 (2007).</li><li>Fisk, N. M., McKee, M., Atun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+and+absolute+addressability+of+global+disease+burden+in+maternal+and+perinatal+health+by+investment+in+R&amp;D.">Relative and absolute addressability of global disease burden in maternal and perinatal health by investment in R&amp;D.</a> Tropical Medicine &amp; International Health. 16 (6), 662-668 (2011).</li><li>Fisk, N. M., Atun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Market+failure+and+the+poverty+of+new+drugs+in+maternal+health.">Market failure and the poverty of new drugs in maternal health.</a> PLOS Medicine. 5 (1), e22 (2008).</li><li>Kaitu'u-Lino, T. u. 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=Targeted+nanoparticle+delivery+of+doxorubicin+into+placental+tissues+to+treat+ectopic+pregnancies.">Targeted nanoparticle delivery of doxorubicin into placental tissues to treat ectopic pregnancies.</a> Endocrinology. 154 (2), 911-919 (2013).</li><li>King, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-homing+peptides+as+tools+for+targeted+delivery+of+payloads+to+the+placenta.">Tumor-homing peptides as tools for targeted delivery of payloads to the placenta.</a> Science Advances. 2 (5), e1600349 (2016).</li><li>Beards, F., Jones, L. E., Charnock, J., Forbes, K., Harris, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+Homing+Peptide-microRNA+Inhibitor+Conjugates+for+Targeted+Enhancement+of+Intrinsic+Placental+Growth+Signaling.">Placental Homing Peptide-microRNA Inhibitor Conjugates for Targeted Enhancement of Intrinsic Placental Growth Signaling.</a> Theranostics. 7 (11), 2940-2955 (2017).</li><li>Cureton, N., 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=Selective+Targeting+of+a+Novel+Vasodilator+to+the+Uterine+Vasculature+to+Treat+Impaired+Uteroplacental+Perfusion+in+Pregnancy.">Selective Targeting of a Novel Vasodilator to the Uterine Vasculature to Treat Impaired Uteroplacental Perfusion in Pregnancy.</a> Theranostics. 7 (15), 3715-3731 (2017).</li><li>Paul, J. W., 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=Drug+delivery+to+the+human+and+mouse+uterus+using+immunoliposomes+targeted+to+the+oxytocin+receptor.">Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor.</a> American Journal of Obstetrics and Gynecology. 216 (3), e281-e283 (2017).</li><li>Zhang, B., 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=Placenta-specific+drug+delivery+by+trophoblast-targeted+nanoparticles+in+mice.">Placenta-specific drug delivery by trophoblast-targeted nanoparticles in mice.</a> Theranostics. 8 (10), 2765-2781 (2018).</li><li>Fan, 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=Noninvasive+monitoring+of+placenta-specific+transgene+expression+by+bioluminescence+imaging.">Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging.</a> PloS One. 6 (1), e16348 (2011).</li><li>Murata, M., Tahara, K., Takeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+surface-modified+liposomes+to+evaluate+their+behavior+after+pulmonary+administration.">Real-time in vivo imaging of surface-modified liposomes to evaluate their behavior after pulmonary administration.</a> European Journal of Pharmaceutics and Biopharmaceutics. 86 (1), 115-119 (2014).</li><li>Ito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+whole-body+multimodality+imaging+of+gastric+cancer+peritoneal+metastasis+combining+fluorescence+imaging+with+ICG-labeled+antibody+and+MRI+in+mice.">New whole-body multimodality imaging of gastric cancer peritoneal metastasis combining fluorescence imaging with ICG-labeled antibody and MRI in mice.</a> Gastric Cancer. 17 (3), 497-507 (2014).</li><li>Mazza, 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=Liposome-Indocyanine+Green+Nanoprobes+for+Optical+Labeling+and+Tracking+of+Human+Mesenchymal+Stem+Cells+Post-Transplantation+In+Vivo.">Liposome-Indocyanine Green Nanoprobes for Optical Labeling and Tracking of Human Mesenchymal Stem Cells Post-Transplantation In Vivo.</a> Advanced Healthcare Materials. 6 (21), (2017).</li><li>Greco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+ultrasound+for+in+vivo+pregnancy+diagnosis+and+staging+of+placental+and+fetal+development+in+mice.">High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice.</a> PloS One. 8 (10), e77205 (2013).</li><li>Spurney, C. F., Leatherbury, L., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasound+database+profiling+growth,+development,+and+cardiovascular+function+in+C57BL/6J+mouse+fetuses.">High-frequency ultrasound database profiling growth, development, and cardiovascular function in C57BL/6J mouse fetuses.</a> Journal of the American Society of Echocardiography. 17 (8), 893-900 (2004).</li><li>Zhang, B., 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=Synthesis+and+characterization+of+placental+chondroitin+sulfate+A+(plCSA)+-targeting+lipid-polymer+nanoparticles.">Synthesis and characterization of placental chondroitin sulfate A (plCSA) -targeting lipid-polymer nanoparticles.</a> Journal of Visualized Experiments. , (2018).</li><li>Devraj, K., Guerit, S., Macas, J., Reiss, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vivo+Blood-brain+Barrier+Permeability+Assay+in+Mice+Using+Fluorescently+Labeled+Tracers.">An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers.</a> Journal of Visualized Experiments. (132), (2018).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+from+the+central+nervous+system+of+rats+with+EAE.">Isolation of mononuclear cells from the central nervous system of rats with EAE.</a> Journal of Visualized Experiments. (10), 527 (2007).</li><li>Watson, E. D., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+structures+and+transport+functions+in+the+mouse+placenta.">Development of structures and transport functions in the mouse placenta.</a> Physiology. 20 (3), 180-193 (2005).</li><li>Frangioni, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Current Opinion in Chemical Biology. 7 (5), 626-634 (2003).</li><li>Flores, L. E., Hildebrandt, T. B., Kuhl, A. A., Drews, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+staging+of+spontaneous+embryo+resorption+by+ultrasound+biomicroscopy+in+murine+pregnancy.">Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.</a> Reproductive Biology and Endocrinology. 12, 38 (2014).</li><li>Khankin, E. V., Hacker, M. R., Zelop, C. M., Karumanchi, S. A., Rana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+high-frequency+ultrasonography+to+evaluate+cardiovascular+and+uteroplacental+blood+flow+in+mouse+pregnancy.">Intravital high-frequency ultrasonography to evaluate cardiovascular and uteroplacental blood flow in mouse pregnancy.</a> Pregnancy Hypertension. 2 (2), 84-92 (2012).</li><li>Phoon, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tools+for+the+developmental+biologist:+ultrasound+biomicroscopy+of+mouse+embryonic+development.">Imaging tools for the developmental biologist: ultrasound biomicroscopy of mouse embryonic development.</a> Pediatric Research. 60 (1), 14-21 (2006).</li><li>Pallares, P., Gonzalez-Bulnes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+ultrasonographic+characterization+of+phenotypic+changes+during+embryo+development+in+non-anesthetized+mice+of+different+genotypes.">Non-invasive ultrasonographic characterization of phenotypic changes during embryo development in non-anesthetized mice of different genotypes.</a> Theriogenology. 70 (1), 44-52 (2008).</li><li>Parvani, J. G., Gujrati, M. D., Mack, M. A., Schiemann, W. P., Lu, Z. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+&#946;3+integrin+by+targeted+ECO/siRNA+nanoparticles+inhibits+EMT+and+metastasis+of+triple-negative+breast+cancer.">Silencing &#946;3 integrin by targeted ECO/siRNA nanoparticles inhibits EMT and metastasis of triple-negative breast cancer.</a> Cancer Research. 75 (11), 2316-2325 (2015).</li><li>Zhang, B., 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=Targeted+delivery+of+doxorubicin+by+CSA-binding+nanoparticles+for+choriocarcinoma+treatment.">Targeted delivery of doxorubicin by CSA-binding nanoparticles for choriocarcinoma treatment.</a> Drug Delivery. 25 (1), 461-471 (2018).</li><li>Jenkins, D. E., 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=Bioluminescent+imaging+(BLI)+to+improve+and+refine+traditional+murine+models+of+tumor+growth+and+metastasis.">Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis.</a> Clinical &amp; Experimental Metastasis. 20 (8), 733-744 (2003).</li><li>Keelan, J. A., Leong, J. W., Ho, D., Iyer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+and+safety+considerations+of+nanoparticle-mediated+drug+delivery+in+pregnancy.">Therapeutic and safety considerations of nanoparticle-mediated drug delivery in pregnancy.</a> Nanomedicine. 10 (14), 2229-2247 (2015).</li></ol>

在这篇手稿中, 我们概述了一个三方法系统, 以确定是否 plCSA-BP 制导纳米粒子是一个有效的工具, 以提供药物到胎盘。利用活体成像技术监测红外荧光干扰信号, 证实了 plCSA 的胎盘靶向特异性. 采用 HFUS 和 HPLC 法, 证明了 plCSA-bp 共轭纳米粒子能有效地将氨蝶呤输送到胎盘细胞, 而不是胎儿。
在活体荧光成像实验中, 孕鼠的妊娠年龄是重要的。胎盘在 E9.5<sup class="xref"...

胎盘介导的妊娠并发症, 包括先兆子痫、妊娠丢失、胎盘早剥和小胎龄 (SGA), 是常见的, 导致大量的胎儿和产妇发病率和死亡率1,2, 3, 很少有药物被证明是有效的治疗妊娠疾病4,5。在怀孕期间, 制定更有选择性和更安全的胎盘靶向药物的策略在现代药物治疗中仍然具有挑战性。
近年来, 几份报告着重于以胎盘为靶的工具, 用多肽或抗体包衣纳米微粒, 将药物子宫胎盘组织定向交付。这些包括抗表皮生长因子受体 (EGFR)6抗体, 肿瘤自导肽 (CGKRK 和 iRGD)7, 胎盘靶向肽8, 胎盘血管靶向肽9和抗体对催产素受体10。
在这里, 我们证明, 合成胎盘硫酸软骨素 a 结合肽 (plCSA BP) 可用于靶向交付纳米微粒及其药物有效载荷的胎盘11。plCSA-BP 制导纳米粒子与报告的子宫胎盘靶向方法是互补的, 因为它们靶向胎盘滋养层。
作为一种非侵入性的方法,在体内成像已被用来监测12小鼠胎盘特异基因的表达, 吲哚菁绿 () 已被广泛应用于用荧光成像系统跟踪纳米粒子13, 14,15。因此, 我们静脉注射注射 plCSA-BP-共轭纳米粒子负载的 plCSA-修订为期三), 以可视化的 plCSA, 在怀孕小鼠与荧光成像仪的分布。然后, 我们静脉注射注射甲氨蝶呤 (MTX) 加载 plCSA 核动力源到怀孕的小鼠。高频超声 (HFUS), 另一种无创, 实时成像工具16,17被用来监测胎儿和胎盘发育的小鼠。最后, 我们用高效液相色谱 (HPLC) 定量分析了甲氨蝶呤在胎盘和胎儿中的分布情况。
在本议定书中, 我们详细描述了用 plCSA-BP 引导 nanocarriers 评估胎盘靶向药物传递效率的三方法系统。

<table border="0" cellpadding="0" cellspacing="0" style="width:785px;" width="786">
	<tbody>
		<tr>
			<td>CD-1 mice</td>
			<td>Beijing Vital River</td>
			<td>201</td>
			<td>Female (8-12 week)</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328421</td>
			<td>for IV injection</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Soybean lecithin</td>
			<td>Avanti Polar Lipids</td>
			<td>441601</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>DSPE-PEG-COOH</td>
			<td>Avanti Polar Lipids</td>
			<td>880125</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>PLGA</td>
			<td>Sigma-Aldrich</td>
			<td>719897</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Ultrasonic processor</td>
			<td>Sonics</td>
			<td>VCX130</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Methotrexate (MTX)</td>
			<td>Sigma-Aldrich</td>
			<td>V900324</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Indocyanine green (ICG)</td>
			<td>Sigma-Aldrich</td>
			<td>1340009</td>
			<td>for in vivo imaging</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS spectrum instrument</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>for in vivo imaging</td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel</td>
			<td>Guanggong</td>
			<td>ZC4252418</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Lunan Pharmaceutical</td>
			<td>I0040</td>
			<td>for maintain the anesthesia</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td>TMG001</td>
			<td>for removing fur</td>
		</tr>
		<tr>
			<td>40 MHz transducer</td>
			<td>VisualSonics</td>
			<td>MS550S</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>High-frequency ultrasound imaging system</td>
			<td>VisualSonics</td>
			<td>Vevo2100</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>Avertin</td>
			<td>Sigma-Aldrich</td>
			<td>T48402</td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Mindray</td>
			<td>SK-500III</td>
			<td>forcardiac perfusion</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Meilunbio</td>
			<td>MA0083</td>
			<td>forcardiac perfusion</td>
		</tr>
		<tr>
			<td>1.5 mL Polypropylene tubes</td>
			<td>AXYGEN</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>88600V</td>
			<td></td>
		</tr>
		<tr>
			<td>Centriguge</td>
			<td>Cence</td>
			<td>H1650R</td>
			<td></td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311421</td>
			<td>for precipitating protein</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>SCIENTZ</td>
			<td>SCIENTZ-48</td>
			<td>for homogenizing tissue</td>
		</tr>
		<tr>
			<td>Syringe filter (0.45 &#956;m)</td>
			<td>Millipore</td>
			<td>SLHV033RS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sinopharm Chemical</td>
			<td>10019763</td>
			<td>for solving MTX</td>
		</tr>
		<tr>
			<td>HPLC vials</td>
			<td>Waters</td>
			<td>670650620</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sinopharm Chemical</td>
			<td>20032117</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>JKchemical</td>
			<td>932537</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>C18 column</td>
			<td>Waters</td>
			<td>186003966</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Shimadzu</td>
			<td></td>
			<td>for HPLC</td>
		</tr>
	</tbody>
</table>

comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods

目前尚无与胎盘相关的妊娠并发症的有效治疗方法, 并制定了靶向胎盘提供药物的战略, 同时尽量减少胎儿和产妇的副作用仍然具有挑战性。靶向纳米微粒载体为治疗胎盘疾病提供了新的机会。我们最近证明, 合成胎盘硫酸软骨素 a 结合肽 (plCSA BP) 可以用来指导纳米粒子向胎盘提供药物。在本议定书中, 我们详细描述了一个系统, 以评估药物交付胎盘由 plCSA BP, 采用三种不同的方法结合使用:在体内成像, 高频超声 (HFUS), 和高性能液相色谱法 (HPLC)。plCSA-bp 制导纳米粒子在活体动物胎盘中的应用, HFUS 和 HPLC 表明, plCSA-bp-共轭纳米粒子有效地将甲氨蝶呤送到胎盘。因此, 这些方法的组合可以作为一个有效的工具, 为靶向胎盘提供药物和制定新的治疗策略, 一些妊娠并发症。

在这篇手稿中, plCSA-BP-共轭纳米粒子负载甲氨蝶呤 (plCSA MNPs) 或一组 (plCSA 修订为期三) 被静脉注射到怀孕小鼠。体外成像显示, 在 plCSA 注射30分钟后, 子宫区域内有较强的干扰信号。修订为期三主要定位于肝脾区 (图 1A)。在 plCSA 注射后48小时, 怀孕小鼠被牺牲, 只在胎盘中暴露了干扰信号, 而胎儿没有信号可检测 (图 1B)。...

Watch this Scientific Journal Video about Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods at JoVE.com

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

我们描述一个系统, 利用三方法来评估胎盘靶向药物的安全性和有效性:在体内成像监测纳米微粒积累, 高频超声监测胎盘和胎儿发育, 并以 HPLC 量化药物对组织的传递。

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

This method can help answer key questions in the nanomedicine field about the effectiveness and the safety of drug loaded on nanoparticle delivered to the mother and the fetus. The main advantage of this technique is that it facilitates an accurate biochemical measurement of the amount of drug delivered to the placenta. The implications of this technique extend toward the therapy of placenta-mediated pregnancy complications because it's important to ensure precise targeting of the drug delivery to the placenta.Visual demonstration of this method is critical, as high-frequency ultrasound evaluation of embryonic development requires patience and experience to master. For intravenous nanoparticle injection, transfer an Embryonic Day 14.5 pregnant mouse with heat-dilated tail veins into a restrainer, and clean the tail with an alcohol swab. Then insert the needle tip of a 28-gauge insulin syringe into a dilated tail vein, and slowly depress the plunger over five to 10 seconds with even pressure to deliver the nanoparticles into the tail vein.30 minutes after the injection, place the anesthetized pregnant mouse into the imaging chamber of an in vivo fluorescence imaging system in the supine position. Select 2D fluorescence and the appropriate photographic parameters for imaging the indocyanine green fluorescence signals, and set the exposure to auto and the excitation and emission wavelengths to 710 and 820 nanometers, respectively. Then image the animal, and return the pregnant mouse to its cage.48 hours after the injection, use Graefe forceps, Graefe tissue forceps, and dissecting scissors to collect the fetuses and placentas, and image the harvested tissues as just demonstrated for the whole animal. For high-frequency ultrasound evaluation of embryonic development, 24 hours after nanoparticle injection, secure the anesthetized pregnant dam onto a 37 to 42-degree Celsius preheated ultrasound imaging platform in the supine position, and place the 40-megahertz transducer into the mechanical arm. Then adjust the transducer position to obtain longitudinal images of the fetus and placenta with the region of interest within the focal zone.For B-mode imaging and analysis, select the B-mode and lower the transducer over the abdomen until the fetus and placenta come into view. Click Scan/Freeze to initiate and stop the imaging, Cine Store to store the cine loop, and Frame Store to store the frame images. Then click Measure to analyze the gestational sac length, fetal crown rump length, biparietal diameter, abdominal circumference, placental diameter, and placental thickness.For pulse wave Doppler imaging and analysis, using the same scan projection, first click Color, then select Pulse Wave, and place the sampling volume box in the center of the umbilical artery. Click Scan/Freeze to initiate imaging and Cine Store to collect the umbilical artery images. Then click Measure to calculate the umbilical artery peak velocity.For color Doppler mode imaging and analysis, using the same scan projection, click Color, and adjust the transducer position to obtain images of the fetal heart as demonstrated. Then click Measure to calculate the fetal heart rate of the stored cine image loop. For high-pressure liquid chromatography, or HPLC, analysis, first inject an Embryonic Day 14.5 pregnant mouse with a single dose of nanoparticles as demonstrated.After 24 hours, perfuse the heart of the pregnant animal with 50 milliliters of ice-cold 0.9%saline for 10 minutes to remove any unbound nanoparticles, and collect the fetuses and placentas for minus 80-degree Celsius storage. On the day of the analysis, add 500 microliters of freshly prepared homogenization solution to each 200-milligram tissue sample, and homogenize the samples two times at full speed for 30 seconds per dissociation. After the second homogenization, centrifuge the samples and filter the supernatants through a 0.45-micrometer syringe filter into an HPLC vial.Place the sample vials into an autosampler tray for injection, and turn on the HPLC degasser to remove air from the system. Next, turn on the flow to equilibrate the column with the mobile phase for 30 minutes to reduce baseline noise, and set the temperature of the column to 25 degrees Celsius. Then inject 20-microliter sample volumes at a one milliliter per minute flow rate within 30 minutes, and click Run Method to begin the analysis.When the runs are complete, manually change the mobile phase to HPLC-grade acetonitrile, and run the acetonitrile for approximately 15 minutes to protect the system. In vivo imaging reveals strong indocyanine green signals in the uterine tissue 30 minutes after placental chondroitin sulfate-bound fluorescence-conjugated nanoparticle injection, while the unbound nanoparticles mainly localize to the liver and splenic regions. 48 hours after injection, fluorescent signal is observed in the placenta of placental chondroitin sulfate-bound nanoparticle injected animals, with no signals detected within the fetus.High-frequency ultrasound monitoring of placental chondroitin sulfate-bound fluorescence-conjugated nanoparticle injected animals reveals a significant decrease in key fetal and placental developmental landmarks. Interestingly, treatment with methotrexate-bound nanoparticles also slightly impairs fetal and placental development, indicating that the nanoparticles may improve the delivery of methotrexate to the placenta via the enhanced permeability and retention effect. The measurement of methotrexate concentrations by HPLC indicates a retention time of seven minutes within the placentas of methotrexate-bound nanoparticle-injected mice.24 hours after injection, the placental methotrexate levels in the methotrexate-bound nanoparticle-injected group are significantly lower than that measured in the placental chondroitin sulfate and methotrexate-bound nanoparticle group, with no methotrexate detected in the fetuses of the latter. Methotrexate can still be detected in the placenta 48 hours after placental chondroitin sulfate and methotrexate-bound nanoparticle injection, demonstrating that placental chondroitin sulfate and methotrexate-bound nanoparticles cannot cross the placenta, thus minimizing potential adverse effects on the fetus. While attempting this procedure, it's important to remember that the placenta begin to form around Embryonic Day 9 1/2, and therefore the in vivo imaging experiment is better performed on Embryonic Day 10 1/2 or later.Following this procedure, other methods like immunofluorescent staining and analysis can be performed to answer additional questions about localization of the nanoparticles within the placenta. After its development, this technique paved the way for researcher in the field of reproductive medicine to explore the possibility of placenta-targeting treatment in pregnancy complications.

comprehensive-evaluation-effectiveness-safety-placenta-targeted-drug

Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

科研

生物工程

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...