Name: 作者聚焦：用于药物递送和免疫治疗的纳米颗粒技术的进展
Uploaded: 2023-11-10T12:50:00
Description: Watch this Scientific Journal Video about Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device at JoVE.com

资金来自 Fischell 生物工程系 （KM）、T32 GM080201 （MA）、Vogel 捐赠暑期奖学金 （MA）、LAM 基金会 （KM） 和美国肺脏协会 （KM）。作者要感谢米歇尔·卡卢津斯基（Michele Kaluzienski）在编辑方面的帮助。

该协议已获得UMD机构动物护理和使用委员会（IACUC）的批准。这些研究使用了 7 至 9 周龄雌性 C57BL/6J 小鼠的组织。这些动物是从商业来源获得的（见 材料表）。
1.手动解离
注意：此步骤改编自 Maisel K. 等人 7。
<ol><li>取出解剖的组织并将其置于含有5%胎牛血清（FBS）的冷细胞培养基中。</li>
	<li>使用锋利的解剖?...

用于单细胞分析的半自动机械酶组织解离

<ol>
	<li>Wiegleb, G., Reinhardt, S., Dahl, A., Posnien, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+dissociation+for+single-cell+and+single-nuclei+RNA+sequencing+for+low+amounts+of+input+material.">Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material.</a> Frontiers in Zoology. 19 (1), 27 (2022).</li><li>Weiskirchen, S., Tag, C. G., Sauer-Lehnen, S., Tacke, F., Weiskirchen, R., Ritti&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+murine+hepatic+stellate+cells.">Isolation and culture of primary murine hepatic stellate cells.</a> Fibrosis: Methods and Protocols. , 7113-7118 (2017).</li><li>Wang, 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=The+isolation+and+characterization+of+endothelial+cells+from+juvenile+nasopharyngeal+angiofibroma.">The isolation and characterization of endothelial cells from juvenile nasopharyngeal angiofibroma.</a> Acta Biochimica et Biophysica Sinica. 48 (9), 856-858 (2016).</li><li>Burja, 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=An+optimized+tissue+dissociation+protocol+for+single-cell+rna+sequencing+analysis+of+fresh+and+cultured+human+skin+biopsies.">An optimized tissue dissociation protocol for single-cell rna sequencing analysis of fresh and cultured human skin biopsies.</a> Frontiers in Cell and Developmental Biology. 10, 102022 (2022).</li><li>McBeth, C., Gutermuth, A., Ochs, J., Sharon, A., Sauer-Budge, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tissue+dissociation+for+rapid+extraction+of+viable+cells.">Automated tissue dissociation for rapid extraction of viable cells.</a> Procedia CIRP. 65, 88-92 (2017).</li><li>Welch, E. C., Yu, H., Barabino, G., Tapinos, N., Tripathi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric-field+facilitated+rapid+and+efficient+dissociation+of+tissues+into+viable+single+cells.">Electric-field facilitated rapid and efficient dissociation of tissues into viable single cells.</a> Scientific Reports. 12 (1), 10728 (2022).</li><li>Maisel, 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=Pro-lymphangiogenic+VEGFR-3+signaling+modulates+memory+T+cell+responses+in+allergic+airway+inflammation.">Pro-lymphangiogenic VEGFR-3 signaling modulates memory T cell responses in allergic airway inflammation.</a> Mucosal Immunology. 14 (1), 144-151 (2021).</li><li>Karmakar, 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=A+pilot+study+to+determine+the+utility+of+automated+tissue+dissociator+for+flowcytometry+based+evaluation+of+hematolymphoid+tumor+tissue+biopsies.">A pilot study to determine the utility of automated tissue dissociator for flowcytometry based evaluation of hematolymphoid tumor tissue biopsies.</a> Indian Journal of Hematology and Blood Transfusion. 38 (2), 403-410 (2022).</li><li>Montanari, 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=Automated-mechanical+procedure+compared+to+gentle+enzymatic+tissue+dissociation+in+cell+function+studies.">Automated-mechanical procedure compared to gentle enzymatic tissue dissociation in cell function studies.</a> Biomolecules. 12 (5), 701 (2022).</li><li>Genova, A., Dix, O., Saefan, A., Thakur, M., Hassan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carpal+tunnel+syndrome:+a+review+of+literature.">Carpal tunnel syndrome: a review of literature.</a> Cureus. 12 (3), e7333 (2020).</li></ol>

该设备设计用于在研究环境中轻松组装，以提供来自整个组织的单细胞悬浮液，用于随后的单细胞分析。这些功能虽然很基本，但足以满足学术环境及其他环境中的研究人员的需求。使用该装置的一个主要优点是它有可能通过减少变异性来改进单细胞悬浮液的制备。此外，同时处理 12+ 个样品的能力可以提高样品间的一致性，这可能会受到手动解离方案所需时间增加的影响。在研究机构和行业环?...

无论是流式细胞术、识别不同细胞类型、单细胞测序，还是用于识别细胞之间的基因组或转录组变异，单细胞分析都已迅速成为新的生物医学发现的关键1。从感兴趣的组织中进行这种细胞分离需要切碎解剖的组织并将它们推入细细胞过滤器，以从所需细胞中过滤掉结缔组织（图1A）。分离贴壁细胞类型，例如树突状细胞或巨噬细胞，或来自特定纤维组织的细胞，需要额外的机械或酶分离步骤2,3,4。该过程通常是手动完成的，因此在评估细胞产量和样品活力时非常耗时且容易出现用户差异。因此，引入可定制的自动组织解离选项至关重要。虽然已经对设计此类系统进行了一些尝试，但现有的选项并不总是容易获得，特别是在学术实验室和资源匮乏的环境中，这主要是由于这些设备的成本过高5.此外，这些设备并不总是可以根据研究小组的个性化需求进行定制 6.
在这里，设计了一种组织解离装置，用于在消化酶和机械破坏的帮助下将整个组织或组织块自动消化成单细胞悬浮液。该设备可以在实验室中轻松组装，放入加热或冷却室中进行温度调节，根据所需数量的解离组织进行定制，并使用所需的解离方案进行编程。该装置的广泛使用可以显着提高细胞提取方案的可重复性，并提供一种节省时间的手动解离替代方案。
该设计允许通过自动化过程同时消化多达 12 个组织。该设备由 12 个并联的独立电机组成，并通过带有可调电压刻度盘的 AC/DC 适配器由标准壁式插头供电，以控制电机的旋转/速度。电机转动一个紧贴 C 型管顶部的六角螺栓。C型管通过亚克力板上的向下张力固定到位，亚克力板在两侧锁定在固定电机的顶板上（图1B）。由于电机是并联接线的，因此它们在任何给定电压下的速度变化不大，但即使电压保持恒定，负载（安装在设备上的 C 管数量）也会影响速度。为了测量每分钟转数 （rpm），在其中一个电机轴上安装了使用霍尔效应传感器和固定磁铁的转速计（补充图 1）。用于构建电机阵列的 CAD 文件在 补充编码文件 1 中提供。还包括一个可编程开关，通过反转传递到电机的正/负电荷来反转旋转方向。所有这些功能都使用编码软件（Arduino IDE软件，请参阅 材料表）集成在Arduino Nano（补充编码文件2）上。使用连接的按钮和 LCD 面板（补充图 2），可以创建和运行保存的和自定义的协议，在协议的指定时间自动反转旋转方向，使用电压调整速度（补充图 3），并显示当前电机速度和完成编程协议的剩余时间（补充图 4）。
在本研究中，使用该装置的机械-酶促组织解离和手动酶促组织解离制备单细胞悬浮液，以确定为下游应用回收的细胞中的差异（如果有的话）。根据每个组织的总细胞产量和细胞活力百分比评估细胞制剂。流式细胞术用于比较表面标志物表达的潜在差异。使用图形和统计分析软件对数据进行分析。使用未配对的 Welch t 检验 来比较成对的样本或组，样本量 n > 4 只小鼠代表 2 个重复实验。有关制造和组装此设备的详细说明，请参阅 补充文件 1。该协议所需的材料列在 材料表中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#188; inch acrylic sheet&#160; 12&#34; x 24&#34;</td>
			<td>Acrylic Mega Store</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189; inch acrylic sheet 12&#34; x 12&#34;</td>
			<td>SimbaLux</td>
			<td>SL-AS13-12x12</td>
			<td></td>
		</tr>
		<tr>
			<td>12 G stainless steel wire (for tension arms)</td>
			<td>Everbilt</td>
			<td>1000847413</td>
			<td></td>
		</tr>
		<tr>
			<td>16 G electrical wire (stranded)</td>
			<td>Best Connections</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2 x 3 mm magnet&#160;&#160;</td>
			<td>SU-CRO0587</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2-channel relay board (to reverse polarity of current to motors)</td>
			<td>AEDIKO</td>
			<td>AE06233</td>
			<td></td>
		</tr>
		<tr>
			<td>37 mm Diameter DC Motors (12 V, 200 rpm) x 12</td>
			<td>Greartisan</td>
			<td>N/A</td>
			<td>Rated Torque: 2.2 Kg.cm 
			Reduction Ratio: 1:22 
			Rated Current: 0.1 A 
			D Shaped Output Shaft Size: 6 x 14mm (0.24&#34; x 0.55&#34;) (D x L) 
			Gearbox Size: 37 x 25 mm (1.46&#34; x 0.98&#34;) (D x L) 
			Motor Size: 36.2 x 33.3 mm (1.43&#34; x 1.31&#34;) (D x L) 
			Mounting Hole Size: M3 (not included)</td>
		</tr>
		<tr>
			<td>AC/C Power Adapter with variable voltage controller&#160;&#160; (5 Amps, 3-12 volts)</td>
			<td>Mo-gu</td>
			<td>J19091-2-MG-US</td>
			<td></td>
		</tr>
		<tr>
			<td>AC-DC 5 V 1 A Precision buck converter step down transformer</td>
			<td>Walfront</td>
			<td>1A</td>
			<td>(power adapter for powering Arduino Nano)</td>
		</tr>
		<tr>
			<td>Arduino Nano&#160;&#160; (Lafvin)</td>
			<td>LAFVIN</td>
			<td>8541582500</td>
			<td></td>
		</tr>
		<tr>
			<td>Buttons&#160;</td>
			<td>Awpeye</td>
			<td>Push-button</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL6/J mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington</td>
			<td>CLS4 LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Double sided foam tape</td>
			<td>SANKA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Sided prototyping circuit board</td>
			<td>deyue</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma- Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical solder and soldering iron</td>
			<td>LDK</td>
			<td>1002P</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical Tape</td>
			<td>3M</td>
			<td>03429NA</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Gibco</td>
			<td>16140089</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td>GraphPad, La Jolla, CA</td>
			<td></td>
			<td>Graphing and statistical analysis software</td>
		</tr>
		<tr>
			<td>Hall effect sensor Dimensions : 0.79 x 0.79x 0.39 inches</td>
			<td>SunFounder</td>
			<td>43237-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hex Coupler 6 mm Bore Motor Brass x 2 x 12</td>
			<td>Uxcell</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hex head bolts (M4-.70 X 12 Hex Head Cap Screw) x 12</td>
			<td>FAS</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Jumper wires (for Arduino Nano)</td>
			<td>ELEGOO</td>
			<td>EL-CP-004</td>
			<td></td>
		</tr>
		<tr>
			<td>LCD screen</td>
			<td>JANSANE</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>M3 Hex Socket Head Cap Screws x 12</td>
			<td>Shenzhen 
			Baishichuangyou 
			Technology co.Ltd</td>
			<td>310luosditaozhuang</td>
			<td></td>
		</tr>
		<tr>
			<td>M3 Stainless SteelMachine screws Flat Head Hex Socket Cap Screws (30 mm) x 36</td>
			<td>Still Awake</td>
			<td>a52400001</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick disconnect terminal connectors</td>
			<td>IEUYO</td>
			<td>22010064</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>46232</td>
			<td></td>
		</tr>
		<tr>
			<td>Terminal adapter shield Expansion board for Arduino Nano&#160; 12&#34; x 24&#34;</td>
			<td>Shenzhen 
			Weiyapuhua 
			Technology</td>
			<td>60-026-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanical dissociation of tissues for single cell analysis using a motorized device

能够分离和制备用于分析组织样本的单细胞对于新的生物医学发现和研究迅速变得至关重要。单细胞分离的手动方案非常耗时，并且容易出现用户差异。自动化机械方案能够减少处理时间和样品变异性，但在资源匮乏的研究环境中不容易获得或具有成本效益。这里描述的设备是为使用市售材料进行半自动组织解离而设计的，作为学术实验室的低成本替代品。提供了制造、组装和操作设备设计的说明。该解离方案可靠地产生单细胞悬浮液，其细胞产量和样品活力与跨多个小鼠组织的手动制备相当。该方案能够为每台设备同时处理多达 12 个组织样本，使需要大样本量的研究更易于管理。随附的软件还允许自定义设备协议，以适应不同的组织和实验限制。

Single-cell analysis has rapidly become crucial for new biomedical discoveries, whether for applications such as flow cytometry, identifying different cell types, single-cell sequencing, or for identifying genomic or transcriptomic variations between cells1. Performing such cell isolations from tissues of interest&#160;requires mincing dissected tissues and pushing them through a fine cell strainer to filter out connective tissue from the desired cells (Figure 1A). Isolating adherent cell types, such as dendritic cells or macrophages, or cells from particularly fibrous tissues, requires additional mechanical or enzymatic separation steps2,3,4. This process is generally done manually, making it highly time-consuming and prone to user variability when assessing cell yields and sample viability. Therefore, it is crucial to introduce customizable options for automated tissue dissociation. While some attempts have been made to design such systems, the existing options are not always readily accessible, particularly in academic labs and lower-resource settings, largely due to the cost-prohibitive nature of these devices5. Furthermore, these devices are not always customizable to the individual needs of a research group6.
Here, a tissue dissociator device was designed to automate the digestion of whole tissues or tissue pieces into single-cell suspensions with the aid of digestive enzymes and mechanical disruption. This device can be easily assembled in the lab, placed into heating or cooling chambers for temperature regulation, customized for the required number of tissues to dissociate, and programmed with the desired dissociation protocols. The broad use of this device could significantly improve the reproducibility of cell extraction protocols and provide a time-saving alternative to manual dissociation.
The design allows for the simultaneous digestion of up to 12 tissues through an automated process. The device is composed of 12 individual motors wired in parallel and powered by a standard wall plug through an AC/DC adapter with an adjustable voltage dial to control the rotation/speed of the motors. The motors turn a hex bolt that fits snugly into the top of the C-tubes. The C-tubes are held in place by downward tension on an acrylic plate that latches on either side to the top plate where the motors are secured (Figure 1B). Because the motors are wired in parallel, their speed at any given voltage should not vary much, but the load (the number of C-tubes mounted on the device) will affect speed even when the voltage is kept constant. To measure rotations per minute (rpm), a tachometer has been incorporated using a hall effect sensor and a fixed magnet on one of the motor shafts (Supplementary Figure 1). The CAD files for building motor arrays are provided in Supplementary Coding File 1. Also included is a programmable switch to reverse the direction of rotation by reversing the positive/negative charges delivered to the motors. All of these features are integrated using coded software (Arduino IDE software, see Table of Materials) on an Arduino Nano (Supplementary Coding File 2). Using connected buttons and an LCD panel (Supplementary Figure 2), it is possible to create and run saved and custom protocols, automatically reverse the rotational direction at specified times of a protocol, adjust speed using the voltage (Supplementary Figure 3), and display the current motor speed and time left to complete a programmed protocol (Supplementary Figure 4).
For the present study, single-cell suspensions were prepared using both mechanical-enzymatic tissue dissociation with this device and manual-enzymatic tissue dissociation to determine differences, if any, in cells recovered for downstream applications. The cell preparations were evaluated based on total cell yields per tissue and&#160;percent cell viability. Flow&#160;cytometry was used to compare potential differences in surface marker expression. Data were analyzed using graphing and statistical analysis software. Unpaired Welch t-tests were used to compare pairs of samples or groups, with sample sizes n &#62; 4 mice representing 2 replicate experiments. Detailed instructions for the fabrication and assembly of this device can be found in Supplementary File 1. Materials needed for this protocol are listed in the Table of Materials.

作者聚焦：用于药物递送和免疫治疗的纳米颗粒技术的进展

使用电动设备对组织进行机械解离以进行单细胞分析

Our lab develops nanoparticles as tools to study biological barriers and to overcome these barriers at mucosal surfaces to improve drug delivery and immunotherapy. One of the most notable developments is the COVID-19 vaccines by Pfizer, BioNTech, and Moderna that use lipid nanoparticle formulations to deliver mRNA for vaccination. There are a lot of ongoing efforts to improve immunotherapies like vaccines and cancer treatments using nanoparticles, as these formulations could improve target delivery to immune cells and reduce the systemic side effects of immunotherapies.Single cell analysis is an essential component of immunology in biomedical research, one of the most common techniques being flow cytometry. Performing such cell isolations from tissues of interest requires reliable tissue dissociation. Making laboratory research more accessible without compromising quality and accuracy can be challenging.Automating processes makes technical and labor intensive steps easier to learn and allows trainees to gain experience doing more sophisticated analyses without affecting the reliability of results. The main advantage of this protocol and the accompanying device design is its customizability and cost effectiveness. The ability to process up to 12 tissues simultaneously reduces processing times.This low cost alternative makes this technology more accessible in lower resourced research settings.

这种半自动机械方案可以复制手动处理细胞的实验结果。使用该装置和通过手动解离制备的细胞悬液在小鼠肺、肾和心脏组织中显示出相当的细胞产量和样品活力（图 2A、B）。免疫细胞群，如T细胞和树突状细胞，不受分离方案差异的显着影响（图2C）。同样，对这些免疫细胞群表面标记物表达的分析显示，在手动和设备分离之间，分离的T...

Watch this Scientific Journal Video about Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device at JoVE.com

提供了组织结合酶法和半自动机械解离以产生用于下游分析（例如流式细胞术）的单细胞悬浮液的通用方案。包括为该协议开发的低成本机械装置的制造、组装和操作说明。

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. ...

我们的实验室开发纳米颗粒作为研究生物屏障的工具，并克服粘膜表面的这些屏障，以改善药物递送和免疫治疗。最显着的发展之一是辉瑞、BioNTech 和 Moderna 的 COVID-19 疫苗，它们使用脂质纳米颗粒配方递送 mRNA 用于疫苗接种。目前有很多正在进行的努力来改进免疫疗法，如使用纳米颗粒的疫苗和癌症治疗，因为这些制剂可以改善对免疫细胞的靶向递送并减少免疫疗法的全身副作用。单细胞分析是生物医学研究中免疫学的重要组成部分，最常见的技术之一是流式细胞术。从目标组织中进行此类细胞分离需要可靠的组织解离。在不影响质量和准确性的情况下使实验室研究更容易进行可能具有挑战性。自动化流程使技术和劳动密集型步骤更容易学习，并允许受训者在不影响结果可靠性的情况下获得进行更复杂分析的经验。该协议和随附设备设计的主要优点是其可定制性和成本效益。能够同时处理多达 12 个组织，从而缩短了处理时间。这种低成本的替代方案使这项技术在资源匮乏的研究环境中更容易获得。

mechanical-dissociation-tissues-for-single-cell-analysis-using

Research

JoVE Journal

Bioengineering

Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. Manual protocols for single-cell isolations are highly time-consuming and prone to user variability. Automated mechanical protocols are able to reduce processing time and sample variability but aren't easily accessible or cost-effective in lower-resourced research settings. The device described here was designed for semi-automated tissue dissociation using commercially available materials as a low-cost alternative for academic laboratories. Instructions to fabricate, assemble, and operate the device design have been provided. The dissociation protocol reliably produces single-cell suspensions with comparable cell yields and sample viability to manual preparations across multiple mouse tissues. The protocol provides the ability to process up to 12 tissue samples simultaneously per device, making studies requiring large sample sizes more manageable. The accompanying software also allows for customization of the device protocol to accommodate varying tissues and experimental constraints.

A general protocol for the combined enzymatic and semi-automated mechanical dissociation of tissues to generate single-cell suspensions for downstream analyses, such as flow cytometry, is provided. Instructions for the fabrication, assembly, and operation of the low-cost mechanical device developed for this protocol are included.

科研

生物工程

Semi-Automated Mechanical Dissociation of Mouse Tissues to Generate Single-Cell Suspensions

To begin, place the dissected mouse tissue in a cold cell culture medium containing 5%FBS. Using sharp dissection scissors, chop the tissue until approximately five millimeters fragments are obtained. Transfer the chopped fragments to a C-tube and add one milliliter of cell culture media.Load the C-tube onto the motorized device and fit the tube holder plate over the D-shaped tube bottoms. Latch the tension arms into the acrylic plate to secure the tubes to the motor plate. To set a custom program on the motorized device from the main menu, press the blue button to choose custom mode.In the first custom mode menu, press the green button to specify the duration of forward rotation to 30 seconds. Then press the blue button to select the on screen value. In the second custom mode menu, press the green button to specify the duration of reverse rotation to 10 seconds.Then press the blue button to select the on screen value. In the third custom mode menu, press the red button to specify the selection loops to four times. Then press the blue button to select the on screen value.Adjust the voltage control dial to 200 RPM and start the custom program. Next, add digestion enzyme in four milliliters of cold culture media containing dissected tissues. Again, load the C-tube onto the device and repeat the custom program as demonstrated previously.After the run, transfer the device with the loaded tubes to a 37 degree Celsius incubator for 45 minutes. Next, load the C-tube onto the device and set the custom program at 50 RPM with forward rotation to 270 seconds. Reverse rotation to 30 seconds and looping nine times.Add EDTA to a five millimolar final concentration to the sample. Set the custom program at 100 RPM with forward rotation to 30 seconds. Reverse rotation to 10 seconds and looping twice.Next, pass the cell suspension through a 70 micrometer cell strainer and collect the filtrate in a 50 or 15 milliliter tube. Centrifuge the collected cell suspension at 300 G for five minutes at four degrees Celsius and discard the supernatant. Re-suspend the pellet in one milliliter of red blood cell lysis buffer, and incubate for one minute.Neutralize the lysis buffer with nine milliliters of cold PBS. Again, centrifuge the cells and re-suspend the pellet in the desired buffer or media. Cell suspension prepared with motorized device and manual dissociation exhibited comparable cell viability and yields across mouse lung, kidney, and heart tissues.Immune cell populations like T-cells and dendritic cells were not significantly affected by the isolation protocol. Surface marker expression also showed similar frequencies of isolated T-cells in mean fluorescence intensity for the antigen presentation marker MHC II in dendritic cells between manual and device isolation.