Name: multi-stream perfusion bioreactor integrated with outlet fractionation for dynamic cell culture
Uploaded: 2022-07-20T13:45:00
Description: Watch this Scientific Journal Video about Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture at JoVE.com

这项研究是在拨款号的支持下进行的。R01EB012521、R01EB028782 和 T32 GM008339 来自美国国立卫生研究院。

1. 准备孔板灌注零件
<ol><li>准备管材<ol>
<li>为每个要灌注的细胞培养物切割两个长度的硅胶管（内径1.6 mm）。确保用作上游管路的片段足够长，可以从注射器泵到达培养箱内的细胞培养物，并且下游片段可以从细胞培养物到达馏分收集器的最远延伸位置。</li>
		<li>在每根管子的两端都有一个独特的标签，并贴上胶带。</li>
	</ol>
</li>
	<li>为孔板准备塞子<ol>
<li>为要灌注的孔板的每个孔...

<ol>
	<li>Welsh, D. K., Yoo, S. H., Liu, A. C., Takahashi, J. S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+individual+fibroblasts+reveals+persistent,+independently+phased+circadian+rhythms+of+clock+gene+expression.">Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.</a> Current Biology. 14 (24), 2289-2295 (2004).</li><li>Talaei, 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=A+mathematical+model+of+the+dynamics+of+cytokine+expression+and+human+immune+cell+activation+in+response+to+the+pathogen+Staphylococcus+aureus.">A mathematical model of the dynamics of cytokine expression and human immune cell activation in response to the pathogen Staphylococcus aureus.</a> Frontiers in Cellular and Infection Microbiology. 11, 711153 (2021).</li><li>Kemas, A. M., Youhanna, S., Zandi Shafagh, R., Lauschke, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-dependent+glucose+consumption+dynamics+in+3D+primary+human+liver+cultures+measured+by+a+sensitive+and+specific+glucose+sensor+with+nanoliter+input+volume.">Insulin-dependent glucose consumption dynamics in 3D primary human liver cultures measured by a sensitive and specific glucose sensor with nanoliter input volume.</a> FASEB Journal. 35 (3), 21305 (2021).</li><li>Muzzey, D., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+time-lapse+fluorescence+microscopy+in+single+cells.">Quantitative time-lapse fluorescence microscopy in single cells.</a> Annual Review of Cell and Developmental Biology. 25, 301-327 (2009).</li><li>Calligaro, H., Kinane, C., Bennis, M., Coutanson, C., Dkhissi-Benyahya, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+method+to+assess+the+endogenous+activity+and+the+light-response+of+the+retinal+clock+in+mammals.">A standardized method to assess the endogenous activity and the light-response of the retinal clock in mammals.</a> Molecular Vision. 26, 106-116 (2020).</li><li>Battat, S., Weitz, D. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+outlook+on+microfluidics:+the+promise+and+the+challenge.">An outlook on microfluidics: the promise and the challenge.</a> Lab on a Chip. 22 (3), 530-536 (2022).</li><li>Petrenko, V., Saini, C., Perrin, L., Dibner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+measurement+of+circadian+clock+gene+expression+and+hormone+secretion+in+human+primary+cell+cultures.">Parallel measurement of circadian clock gene expression and hormone secretion in human primary cell cultures.</a> Journal of Visualized Experiments. (117), e54673 (2016).</li><li>Yamagishi, K., Enomoto, T., Ohmiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-culture-based+secreted+bioluminescence+reporter+assay+in+living+cells.">Perfusion-culture-based secreted bioluminescence reporter assay in living cells.</a> Analytical Biochemistry. 354 (1), 15-21 (2006).</li><li>Watanabe, 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=Multichannel+perfusion+culture+bioluminescence+reporter+system+for+long-term+detection+in+living+cells.">Multichannel perfusion culture bioluminescence reporter system for long-term detection in living cells.</a> Analytical Biochemistry. 402 (1), 107-109 (2010).</li><li>Murakami, N., Nakamura, H., Nishi, R., Marumoto, N., Nasu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+circadian+oscillation+of+melatonin+release+in+pineal+cells+of+house+sparrow,+pigeon+and+Japanese+quail,+using+cell+perfusion+systems.">Comparison of circadian oscillation of melatonin release in pineal cells of house sparrow, pigeon and Japanese quail, using cell perfusion systems.</a> Brain Research. 651 (1-2), 209-214 (1994).</li><li>Erickson, P., Houwayek, T., Burr, A., Teryek, M., Parekkadan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+flow+cell+culture+system+for+precision+cell+stimulation+and+time-resolved+profiling+of+cell+secretion.">A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion.</a> Analytical Biochemistry. 625, 114213 (2021).</li><li>Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drug Delivery: Engineering Principles for Drug Therapy. , (2001).</li><li>Fogler, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Elements of Chemical Reaction Engineering. 4th edn. , (2006).</li><li>Conesa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Reactor Design: Mathematical Modeling and Applications. , (2019).</li><li>Toson, P., Doshi, P., Jajcevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explicit+residence+time+distribution+of+a+generalised+cascade+of+continuous+stirred+tank+reactors+for+a+description+of+short+recirculation+time+(bypassing).">Explicit residence time distribution of a generalised cascade of continuous stirred tank reactors for a description of short recirculation time (bypassing).</a> Processes. 7 (9), 615 (2019).</li><li>Tamayo, A. G., Shukor, S., Burr, A., Erickson, P., Parekkadan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+leukemic+T-cell+transcriptional+dynamics+in+vivo+with+a+blood-based+reporter+assay.">Tracking leukemic T-cell transcriptional dynamics in vivo with a blood-based reporter assay.</a> FEBS Open Biology. 10 (9), 1868-1879 (2020).</li><li>Newell, B., Bailey, J., Islam, A., Hopkins, L., Lant, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterising+bioreactor+mixing+with+residence+time+distribution+(RTD)+tests.">Characterising bioreactor mixing with residence time distribution (RTD) tests.</a> Water Science and Technology. 37 (12), 43-47 (1998).</li><li>Dubois, J., Tremblay, L., Lepage, M., Vermette, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dynamics+within+a+bioreactor+for+tissue+engineering+by+residence+time+distribution+analysis+combined+with+fluorescence+and+magnetic+resonance+imaging+to+investigate+forced+permeability+and+apparent+diffusion+coefficient+in+a+perfusion+cell+culture+chamber.">Flow dynamics within a bioreactor for tissue engineering by residence time distribution analysis combined with fluorescence and magnetic resonance imaging to investigate forced permeability and apparent diffusion coefficient in a perfusion cell culture chamber.</a> Biotechnology and Bioengineering. 108 (10), 2488-2498 (2011).</li><li>Gaida, L. 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=Liquid+and+gas+residence+time+distribution+in+a+two-stage+bioreactor+with+cell+recycle.">Liquid and gas residence time distribution in a two-stage bioreactor with cell recycle.</a> HAL Open Science. , (2008).</li><li>Rodrigues, M. E., Costa, A. R., Henriques, M., Azeredo, J., Oliveira, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wave+characterization+for+mammalian+cell+culture:+residence+time+distribution.">Wave characterization for mammalian cell culture: residence time distribution.</a> New Biotechnology. 29 (3), 402-408 (2012).</li><li>Olivet, D., Valls, J., Gordillo, M. A., Freix&#243;, A., S&#225;nchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+residence+time+distribution+technique+to+the+study+of+the+hydrodynamic+behaviour+of+a+full-scale+wastewater+treatment+plant+plug-flow+bioreactor.">Application of residence time distribution technique to the study of the hydrodynamic behaviour of a full-scale wastewater treatment plant plug-flow bioreactor.</a> Journal of Chemical Technology and Biotechnology. 80 (4), 425-432 (2005).</li></ol>

这项工作描述了具有多个培养基来源的灌注细胞培养系统的组装和操作，并展示了一个具体的例子，其中测量了NF-κB驱动的基因表达响应于TNF-α瞬时脉冲的动态。对灌注系统组分的RTD进行测量和建模，并使用信号卷积来预测细胞暴露于TNF-α脉冲和TNF-α在收集的流出介质中的分布。将细胞暴露于脉冲并收集分数40小时，之后在分数中测量GLuc并与预测的TNF-α信号一起绘制，以揭示刺激 - 反应动力学。<...

许多重要的生物过程发生在细胞和组织培养物中，时间尺度为几分钟到几小时1，2，3。虽然可以使用延时显微镜4，生物发光1或其他方法以自动方式观察和记录其中一些现象，但涉及收集用于化学分析的培养物上清液样品的实验通常在静态细胞培养物中手动进行。由于频繁或下班后采样时间点的不便，手动采样限制了某些研究的可行性。静态培养方法的进一步缺点包括涉及受控的瞬时暴露于化学刺激的实验。在静态培养物中，必须手动添加和去除刺激，并且刺激曲线仅限于随时间的步进变化，而培养基的变化也会添加和去除其他培养基成分，这可能会以不受控制的方式影响细胞5。流体系统可以克服这些挑战，但现有设备带来了其他挑战。微流体装置伴随着专业设备和培训的生产和使用成本过高，需要微分析方法来处理样品，并且灌注后细胞难以从装置中回收6。对于这里描述的实验类型，很少有大流体系统7，8，9，10，并且它们由内部制造的多个定制部件组成，需要多个泵或馏分收集器。此外，作者不知道除了用于悬浮培养的搅拌罐生物反应器之外，任何市售的大流体灌注细胞培养系统，这些系统对生物制造有用，尽管不是为建模和研究生理学而设计的。
作者之前报告了低成本灌注生物反应器系统的设计，该系统几乎完全由市售部件11组成。该系统的基本版本使孔板中的多种培养物能够保存在CO2 培养箱中，并连续灌注来自注射器泵的培养基，而来自培养物的流出培养基流随着时间的推移使用具有定制修改的馏分收集器自动分馏到样品中。因此，该系统能够自动取样培养基上清液，并随着时间的推移连续地将溶质输入到培养物中。该系统是大流体和模块化的，可以很容易地修改，以满足新型实验设计的需要。
这里介绍的方法的总体目标是构建，表征和使用灌注细胞培养系统，该系统能够进行实验，其中测量细胞随时间推移的物质分泌或吸收速率，和/或细胞暴露于精确的瞬态溶质信号。本视频文章介绍了如何组装碱基设置，该设置能够使用单个注射器泵和改进的馏分收集器同时灌注多达六种细胞培养物。还介绍了基础系统上的两个有用变体，它们利用额外的泵和部件来允许将细胞暴露于瞬态溶质浓度信号的实验，包括短暂的脉冲和药代动力学样曲线12， 如图1所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63935/63935fig01.jpg"> 图1：灌注系统设计的三种变化 （顶部）基本灌注系统。（中）带有旋塞阀的灌注系统，用于多种介质来源。（底部）灌注系统带有搅拌罐，以模拟混合良好的分布体积。 <a href="https://www.jove.com/files/ftp_upload/63935/63935fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
由于流动中的色散和扩散，溶质信号在通过流动系统时会失真或“模糊”。这种失真可以通过使用停留时间分布（RTD）来量化13。本文介绍了如何对灌注系统的组件进行示踪剂实验（图 2），并提供 MATLAB 脚本来根据测量数据生成 RTD。关于这一分析的详细解释可以在作者之前的论文11中找到。附加的MATLAB脚本将适当的函数拟合到RTD并提取物理参数，并使用RTD执行信号卷积，以预测用户输入的溶质信号将如何通过灌注系统14传播和扭曲。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63935/63935fig02.jpg"> 图 2：停留时间分布。 流量系统组件的RTD，例如这个长度的管道，是通过向系统输入示踪剂脉冲并测量它在退出收集的馏分时如何“涂抹”来测量的。这个数字是从埃里克森等人11修改而来的。 <a href="https://www.jove.com/files/ftp_upload/63935/63935fig02large.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>18 Gauge 1 1/2- in Disposable Probe Needle For Use With Syringes and Dispensing Machines</td>
			<td>Grainger</td>
			<td>5FVK2</td>
			<td></td>
		</tr>
		<tr>
			<td>293T Cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>HEK 293T cells used in the Representative Results experiment.</td>
		</tr>
		<tr>
			<td>96-Well Clear Bottom Plates, Corning</td>
			<td>VWR</td>
			<td>89091-010</td>
			<td>Plates for measuring dye concentrations in RTD experiments and GLuc in representative results experiment.</td>
		</tr>
		<tr>
			<td>BD&#160;Disposable Syringes with Luer-Lok Tips, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFrac Fraction Collector&#160;</td>
			<td>Bio-Rad</td>
			<td>7410002</td>
			<td>Fraction collector that can be used for a single stream, or modified using our method to enable collection from multiple streams.</td>
		</tr>
		<tr>
			<td>Clear High-Strength UV-Resistant Acrylic 12&#34; x 12&#34; x 1/8&#34;</td>
			<td>McMaster-Carr</td>
			<td>4615T93</td>
			<td>This sheet is cut using a laser cutter according to the DXF file in the supplemental materials to produce the multi-head dispenser that can be attached to the BioFrac fraction collector.</td>
		</tr>
		<tr>
			<td>Coelenterazine native</td>
			<td>NanoLight Technology</td>
			<td>303</td>
			<td>Substrate used in Gaussia luciferase bioluminescence assay in representative results.</td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated Multiple Well Plates, size 48&#160;wells, polystyrene plate, flat bottom wells</td>
			<td>Millipore Sigma</td>
			<td>CLS3548</td>
			<td>Used to grow and perfuse 293T cells in representative results.</td>
		</tr>
		<tr>
			<td>Corning&#160;Costar Flat Bottom Cell Culture Plates, size 12 wells</td>
			<td>Fisher Scientific</td>
			<td>720081</td>
			<td>Can be plugged and used as a stirred tank to produce pharmacokinetic profiles in perfusion. Can also contain cells for perfusion.</td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>11965126</td>
			<td></td>
		</tr>
		<tr>
			<td>Epilog Zing 24 Laser</td>
			<td>Cutting Edge Systems</td>
			<td>Epilog Zing 24</td>
			<td>Laser cutter used to produce multi-head dispenser from acrylic sheet. Other laser cutters may be used.</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Syringes for Single Use, Luer-Lock, 20 mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-460</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Syringes for Single Use, Luer-Lock, 60 mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-461</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>Microcentrifuge tubes for collecting fractions.</td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Round Bottom Disposable Borosilicate Glass Tubes with Plain End</td>
			<td>Fisher Scientific</td>
			<td>14-961-26</td>
			<td>Glass tubes for collecting fractions.</td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;SureOne Micropoint Pipette Tips, Universal Fit, Non-Filtered</td>
			<td>Fisher Scientific</td>
			<td>2707410</td>
			<td>300 ul pipette tips that best fit the multi-head dispenser and tubing to act as dispensing tips.</td>
		</tr>
		<tr>
			<td>Gibco&#160;DPBS, powder, no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>21600010</td>
			<td>Phosphate buffered saline.</td>
		</tr>
		<tr>
			<td>Labline 4625 Titer Shaker</td>
			<td>Marshall Scientific</td>
			<td>Labline 4625 Titer Shaker</td>
			<td>Orbital shaker used to keep stirred tanks mixed.</td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-Sterile; 10/PK</td>
			<td>Cole-Parmer</td>
			<td>EW-30600-04</td>
			<td>Used to join multiple inlet streams for RTD experiments and cell culture experiments.</td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polycarbonate, Straight, Female Luer x Cap; 25/PK</td>
			<td>Masterflex</td>
			<td>UX-45501-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polypropylene, Straight, Female Luer to Hosebarb Adapters, 1/16&#34;</td>
			<td>Cole-Parmer</td>
			<td>EW-45508-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polypropylene, Straight, Male Luer Lock to Hosebarb Adapter, 1/16&#34; ID</td>
			<td>Cole-Parmer</td>
			<td>EW-45518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polypropylene, Straight, Male Luer Lock to Plug Adapter; 25/PK</td>
			<td>Masterflex</td>
			<td>EW-30800-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Precision Pump Tubing, Platinum-Cured Silicone, L/S 14; 25 ft</td>
			<td>Masterflex</td>
			<td>EW-96410-14</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2019b</td>
			<td>Version R2019b. Newer versions may also be used. Some older versions may work.</td>
		</tr>
		<tr>
			<td>NE-1600 Six Channel Programmable Syringe Pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Rack Set F1</td>
			<td>Bio-Rad</td>
			<td>7410010</td>
			<td>Racks to hold collecting tubes in the fraction collector.</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha (HEK293-expressed) Protein, CF</td>
			<td>Bio-Techne</td>
			<td>10291-TA-020</td>
			<td>Cytokine used to stimulate 293T cells in representative results.</td>
		</tr>
		<tr>
			<td>Saint Gobain Solid Stoppers, Versilic Silicone, Size: 00, Bottom 10.5mm</td>
			<td>Saint Gobain</td>
			<td>DX263015-50</td>
			<td>Fits 48-well plates.</td>
		</tr>
		<tr>
			<td>Saint Gobain Solid Stoppers, Versilic Silicone, Size: 4 Bottom 21mm</td>
			<td>Saint Gobain</td>
			<td>DX263027-10</td>
			<td>Fits 12-well plates.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, 10.0 N Aqueous Solution APHA; 1 L</td>
			<td>Spectrum Chemicals</td>
			<td>S-395-1LT</td>
			<td></td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault Systems</td>
			<td>SolidWorks</td>
			<td>CAD software used to create the multi-head dispenser DXF file.</td>
		</tr>
		<tr>
			<td>Varioskan LUX multimode microplate reader</td>
			<td>ThermoFisher Scientific</td>
			<td>VL0000D0</td>
			<td>Plate reader.</td>
		</tr>
		<tr>
			<td>Wilton Color Right Performance Color System Base Refill, Blue</td>
			<td>Michaels</td>
			<td>10404779</td>
			<td>Blue food dye containing Brilliant Blue FCF, used as a tracer in RTD experiments. Absorbance spectrum peaks at 628 nm.</td>
		</tr>
	</tbody>
</table>

multi-stream perfusion bioreactor integrated with outlet fractionation for dynamic cell culture

某些细胞和组织功能在几分钟到几小时的动态时间尺度内运行，而传统培养系统无法很好地解决这些功能。这项工作开发了一种低成本的灌注生物反应器系统，该系统允许将培养基连续灌注到细胞培养模块中，并在下游模块中分馏以测量这种规模的动态。该系统几乎完全由市售部件构成，可以并行化以同时在常规多孔细胞培养板中进行独立实验。本视频文章演示了如何组装基础设置，该设置只需要一个多通道注射器泵和一个改进的馏分收集器即可并行灌注多达六种培养物。还提出了模块化设计的有用变体，允许受控的刺激动力学，例如溶质脉冲或药代动力学样曲线。重要的是，当溶质信号通过系统时，它们会因溶质色散而失真。此外，还描述了一种使用MATLAB用示踪剂测量灌注装置组件的停留时间分布（RTD）的方法。RTD可用于计算多室系统中的流动如何使溶质信号失真。该系统非常坚固且可重复，因此基础研究人员可以轻松采用它，而无需专门的制造设施。

使用来自方案第5部分的多个培养基来源的灌注系统来测量由活化B细胞（NF-κB）转录因子的核因子kappa-轻链增强子驱动的报告基因的表达动力学，以响应肿瘤坏死因子α（TNF-α）的1.5 h脉冲。HEK293细胞使用含有高西亚荧光素酶（GLuc）的基因构建体的慢病毒载体稳定地转导，由含有NF-κB响应元件的称为NFKB的启动子驱动，以产生NFKB-GLuc HEK293细胞， 其通过 荧光激活细胞分选（FACS）分选以分离转?...

Watch this Scientific Journal Video about Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture at JoVE.com

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

本文提出了一种构建和操作低成本、多通道灌注细胞培养系统的方法，用于测量细胞过程中溶质分泌和吸收速率的动态。该系统还可以使细胞暴露于动态刺激曲线中。

多流灌注生物反应器集成了出口分馏功能，用于动态细胞培养

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

这种方法有助于了解分泌速率如何随时间变化以及细胞如何响应时变的固体信号。该技术的主要优点是成本低，需要很少的培训，并且丰富的系统设置可以适应新的实验。首先用背景溶液填充一个注射器，然后将其装入第一个注射泵中。用示踪剂溶液填充另一个注射器，并将其装入第二个注射器泵中。使用鲁尔连接器将两个注射器连接到四路旋塞阀的三个端口中的两个。将旋塞阀关闭到后台溶液中，并将示踪剂溶液泵入旋塞阀，直到其从打开的端口滴出。停止泵，不要进一步调整注射器。确保将注射器泵的移动杆向上推到注射器柱塞上，以便在泵启动时立即开始流动。关闭示踪剂溶液的旋塞阀，并将背景溶液泵入旋塞阀，直到所有残留的示踪剂溶液都从打开的端口中冲洗出来。停止泵，不要进一步调整注射器。设置 RTD 分析所需的流量系统组件。将末端插入移液器吸头分配器中，并将背景溶液泵入组件，直到其完全充满。将示踪剂溶液的泵设置为所需的流速。将旋塞阀关闭到后台溶液并启动示踪剂溶液的流动。同时，启动分数收集器。在短时间内继续示踪剂溶液的流动，以近似示踪剂的脉冲输入。对于 RTD，建议以每小时 1 毫升的流速进行脉冲持续时间为 10 分钟。在示踪剂溶液脉冲周期结束时停止示踪剂溶液泵。快速关闭示踪剂溶液的旋塞阀，并以相同的流速启动背景溶液的流动。允许背景溶液流动并收集馏分，直到所有示踪剂通过系统并进入收集的馏分。在无菌条件下，将带有针头的塞子插入孔板培养物中，并将针头拉起。塞子就位后，将针头降低到所需的灌注高度，因为出口针的高度决定了稳定的液位。用公头鲁尔帽盖住针头，并将整个孔板保持在37摄氏度的培养箱中直至使用。准备用于灌注的两种培养基，将首先分配的培养基标记为一种，另一种培养基为两种。对于要灌注的每种培养物，用培养基填充一个注射器，以持续其分配的整个持续时间以及足够的体积以最初填充灌注系统。用中等的两个注射器填充第二个注射器，以持续其分配的整个持续时间。将两个注射器连接到四向旋塞阀的三个端口中的两个。可能需要一定长度的管子将注射器连接到旋塞阀。通过关闭介质 1 的旋塞阀并将介质 2 分配到旋塞阀中，直到它刚开始从打开的端口滴出来准备旋塞阀。然后关闭介质 2 的旋塞阀，并将介质 1 分配到旋塞阀中，直到所有残留的介质 2 从打开的端口中冲洗出来。使用母头到倒钩型鲁尔连接器将上游管连接到开放式旋塞阀端口。在管子的另一端插入一个公头到倒钩鲁尔连接器。从注射器中分配介质，直到上游管充满介质，并继续制备下游管，如前所述。将公头到倒钩鲁尔连接器插入下游管道的一端，并用母头鲁尔帽盖住。小心地将所有准备好的管子，注射器和孔板带到将用于灌注的培养箱中。将注射泵和馏分收集器放置在培养箱附近的所需位置。将注射泵放在培养箱的顶部或附近，并将馏分收集器放在靠近端口的培养箱旁边。将注射器装入泵中。将所有上游和下游管的封端捆绑在一起，并通过端口将它们从培养箱外部推到内部。将下游管的开口端插入馏分收集器多头分液器的分液器吸头中。在培养箱内，将尽可能多的上游管的松弛拉入培养箱，以最大限度地延长管道的长度，流动介质可以通过该管道接收热量和二氧化碳。对于每个堵塞的井，快速拔下该井的针头以及上游和下游管的盖子，然后用它们的鲁尔连接器将它们连接在一起。连接所有部件后，以相对较高的速度短暂运行注射泵，以确保所有流都正常流动。此时，如果需要开始对充满介质的下游管子进行实验，请继续运行泵，直到全部装满，否则，请停止泵。设置介质一的注射器泵流量和馏分收集频率，并同时启动两台机器开始实验。当需要更换介质源时，请迅速停止注射泵以获得中等源。将旋塞阀关闭至中等温度，然后启动用于中等温度 2 的注射泵。如果以后需要，请以类似的方式将源切换回中等源。收集所需实验持续时间的分数。测量两个RTD实验的馏分中的示踪剂浓度，并从数据MATLAB脚本输入RTD以产生两个RTD.单管和多块串联的管子通过单个轴向分散模型很好地拟合，并且在线添加灌注的48孔板导致偏差可以忽略不计，允许一米管RTD和整个系统的RTD都适合轴向色散函数。使用拟合到一米管RTD数据的轴向色散模型演示了模型拟合不良的示例，并与数据一起绘制。更改了初始参数猜测以产生良好的模型拟合，其中tau近似等于灌注系统体积除以其体积流速。90分钟的TNF-α脉冲被定义为系统的输入信号，并与一米管RTD卷积以确定孔板入口处的TNF-α信号。输入信号还与整个系统的RTD卷积，以确定收集的分数中系统出口处的TNF-alpha信号。HEK 293细胞被设计成由含有NF-kappaB应答元件的启动子驱动的GLuc。实验显示，在TNF-α暴露后，由NF-kappaB驱动的GLuc表达显着增加和缓慢降低。随附的论文中描述了该系统的变化，包括不需要输入信号和将细胞暴露于pharma-hu连接溶质谱的实验的更简单设置。

Research

JoVE Journal

Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

人卵巢组织与工程化内皮细胞联合移植: 一种以细胞为基础的快速灌注与直接分泌的融合策略

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

成像引导的生物反应器，用于生成生物工程气道组织

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

在定制灌注生物反应器中获取和灌注-脱细胞猪血管化皮瓣

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

使用 离体 灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...