这项工作得到了 NSF Grant （2152065） 和 NSF CAREER Award （1846857） 的支持。作者要感谢 Wachs 实验室的所有现任和前任成员为本协议做出贡献。 图 1 中的图表是在 Biorender 中制作的。

DRG 收获是按照内布拉斯加大学林肯分校的机构动物护理和使用委员会 （IACUC） 进行的。该研究使用 12 周龄 （~250 g） 的雌性 Sprague Dawley 大鼠。材料 表中列出了研究中使用的动物、试剂和设备的详细信息。
1. 多隔室器件制造和组装
<ol><li>多隔间设备的计算机辅助设计和 3D 打印 注意：MC 设备由三个隔室组成，用于分离 DRG 细胞体和神经突（图 2A）。MC 设备没有市售;但是，此处提供了 STL 文件（补充编码文件 1）以启用 3D 打印。打印 MC 需要一 （1） 天才能完成。</li>
	<li>使用兼容软件将 MC 的 STL 文件上传到 Resin 3D 打印机。 注意：理想的打印材料是高温树脂。使用高温树脂的一个关键优点是它具有生物相容性，并且可以高压灭菌和重复使用。数字光处理 （DLP） 3D 打印机或其他 3D 打印机也可用于打印设备。</li>
	<li>将打印的设备转移到异丙醇 （>96%） 的洗涤溶液中 30 分钟，以去除打印的 MC 设备表面的残留树脂。</li>
	<li>使用市售固化剂在 80 °C 下对设备后固化 120 分钟（参见 材料表）。固化剂通过暴露于温度和紫外线下来增强印刷 MC 器件的机械性能。</li>
	<li>将 MC 器件在 160 °C 的烘箱中热固化 180 分钟。</li>
	<li>使用高压灭菌器在重力循环中对设备消毒 45 分钟。</li>
</ol>2. 水凝胶制备
<ol><li>合成甲基丙烯酸透明质酸（MAHA，85%-115% 甲基丙烯酸化）并使用 Seidlits 等人描述的方案进行表征20。 注意：为了支持 DRG 的生长，通常使用由甲基丙烯酸透明质酸、I 型胶原蛋白和层粘连蛋白组成的三重水凝胶。研究结果表明，DRG 通常在 MAHA 中生长，范围为 1.25-2.5 mg/mL，胶原蛋白为 2.0-4.5 mg/mL。对于层粘连蛋白，发现 0.75 mg/mL 足以确保 DRG 的稳健生长。在此，详细介绍了用 1.25 mg/mL MAHA、4.5 mg/mL 胶原蛋白和 0.75 mg/mL 层粘连蛋白创建三重凝胶的方法。水凝胶制备应在层流柜中进行，以提供无菌工作空间。</li>
</ol>3. 光引发剂溶液制备
注意：在紫外线下交联 MAHA 需要光引发剂。它通常以 0.3% 到 0.6% 的百分比使用。
<ol><li>在 DRG 收获前 3 天准备光引发剂溶液。</li>
	<li>在制备光引发剂溶液之前，将超声处理水浴预热至 37 °C。</li>
	<li>在流动罩中使用无菌技术，通过将 NaHCO3 溶解在 5x Dulbecco 改良 Eagle 培养基 （DMEM）-HEPES 溶液中来制备 23.125 mg/mL 碳酸氢钠 （NaHCO3） 溶液。</li>
	<li>在玻璃样品瓶中，将 0.6% 重量/体积 （w/v） 光引发剂与 20% 体积/体积 1x 无菌磷酸盐缓冲盐水 （PBS） 和 80% 体积/体积 5X DMEM/HEPES 溶液混合。</li>
	<li>为避免光线，请用铝箔包裹玻璃瓶。但是，在将样品瓶转移到加热至 37 °C 的超声处理水浴中，超声处理 30-40 分钟之前，请确保去除铝箔。 注：光引发剂溶液必须避光，因为它在光照下会分解成自由基并开始聚合过程21.</li>
	<li>用 0.22 μm 注射器过滤器将溶液无菌过滤到新的无菌玻璃瓶中。</li>
</ol>4. 甲基丙烯酸酯透明质酸溶解
<ol><li>在制备光引发剂溶液的同一天（步骤 3）溶解甲基丙烯酸化透明质酸（在步骤 2 中制备）。</li>
	<li>将冷冻冻干的 MAHA 放入干燥器中 15 分钟以达到室温。</li>
	<li>在层流罩中工作并使用无菌称重技术，称量玻璃瓶中的冻干 MAHA。</li>
	<li>将必要体积的过滤光引发剂溶液添加到 MAHA 中，以达到所需的 MAHA 浓度。常见的最终凝胶浓度为 1.25 mg/mL MAHA 和 4.5 mg/mL 胶原蛋白。</li>
	<li>用实验室密封膜密封瓶盖，用铝箔包裹玻璃瓶，在室温下将其放在轨道摇床板上直至完全溶解（通常为 3 天）。偶尔，涡旋溶液以分解大块 MAHA，以确保 MAHA 均匀溶解。</li>
</ol>5. 设备组装
<ol><li>在 DRG 收获前一天将 MC 装置放入 48 孔板中。</li>
	<li>使用 3 mL 注射器在 MC 设备下方的凹槽中涂抹一条细线硅凝胶（图 2B）。这将有助于设备牢固地连接到孔板上。</li>
	<li>在 #3 镊子的帮助下，将 MC 装置轻轻放入 48 孔板的孔中，如图 2C 所示。 注意：可以修改设备的大小以适应预期的应用，如果需要，可以使用不同尺寸的板。这些设备可以重复使用。要重复使用，必须轻轻地从所有隧道中洗掉水凝胶，并且装置下方的加载槽必须用 70% 乙醇轻轻搅拌。完成后，可以重新高压灭菌设备，以便在使用前进行灭菌。</li>
</ol>6. 动物准备
<ol><li>在收获开始前，用高压灭菌所有必要的解剖工具。</li>
	<li>获得一只 12-20 周龄的成年雄性或雌性 Sprague Dawley 大鼠。大鼠的性别不会影响 DRG 生长。</li>
	<li>根据批准的 IACUC 方案对大鼠实施安乐死，使用 CO2 过量作为安乐死的主要方法，根据美国兽医协会指南22 将双侧气胸穿刺作为安乐死的次要方法。</li>
	<li>将大鼠放在解剖台上的无菌垫上，腹侧朝下。用 70% 乙醇喷洒皮毛。</li>
</ol>7. 背根神经节 （DRG） 收获
<ol><li>准备含有 86% Neurobasal A 培养基、10% 胎牛血清 （FBS）、1% 青霉素-链霉素 （PS）、1% 谷氨酰胺和 2% B27 加 50x 的修剪培养基，然后转移到 24 孔板中。该培养基和孔板将用于在将收获的 DRG 嵌入水凝胶之前临时储存它们。</li>
	<li>穿上实验服、外科口罩、帽子、安全眼镜和手套。</li>
	<li>使用大钝鼻剪刀，切开颈椎底部的皮肤，然后沿着脊柱的长度向下切割。</li>
	<li>为了进一步将血液从椎管排出，请在肩胛骨下方切开并向下穿过肌肉以切断主要血管。</li>
	<li>使用大而锋利的剪刀将肌肉从脊柱上剪下来。尽可能多地清理肌肉以观察脊柱。</li>
	<li>去除椎骨的背段和外侧段。使用直的 Rongeur 和尖鼻剪刀去除椎骨的棘突和横突，从颈端开始，向腰端移动。</li>
	<li>切除脊髓。使用小尖鼻剪刀小心地剪断沿脊髓两侧连接到 DRG 的神经根。切开脊髓的颈端，小心地逐渐从椎管中抬出，切开与 DRG 相连的任何剩余神经根。 注意：精细运动技能对于确保 DRG 体在此过程中不被切断至关重要。</li>
	<li>要暴露 DRG，请使用弯曲的 Rongeur 去除椎骨的更多外侧部分。从中线向外拉，交替两侧。小心不要切开 DRG 体，因为它可能会对细胞体和轴突造成损害。</li>
	<li>使用 #3 镊子和直刃弹簧剪刀，去除椎骨每层之间的 DRG。从 DRG 中抓住脊神经根，小心地将它们从口袋中拉出，然后切开脊神经的另一端。小心不要直接抓住 DRG 主体。</li>
	<li>将 DRG 置于冰上的 24 孔板（包含修剪培养基，步骤 7.1）的孔中。</li>
	<li>收集完所有 DRG 后，清理工作区，将胴体放入高压灭菌袋中，并根据 IACUC 协议妥善存放/处理。</li>
</ol>8. DRG 修整和切割
<ol><li>在干净的工作台上用修剪培养基填充 60 mm 培养皿（步骤 7.1）。设置解剖镜和玻璃珠消毒器，并在镜旁放置一个带有修剪工具（#3 镊子和直刃弹簧剪刀）的高压灭菌工具箱。</li>
	<li>在解剖镜下，去除 DRG 周围的多余组织并修剪靠近 DRG 体的神经根（通常比周围的神经略暗）。</li>
	<li>用新鲜培养基填充第二个 60 mm 培养皿。在解剖镜下，将修剪的 DRG 切成约 0.5 毫米（或 0.5 毫米和 0.8 毫米之间）。在切割过程中，将尺子放在培养皿下方，以获得正确的切割 DRG 估计尺寸。</li>
	<li>将切割的 DRG 转移到新鲜的 24 孔板中，将培养基置于冰上。 注意：DRG 修整、切割和包埋应在层流柜中进行，以提供无菌工作空间。在没有层流柜的情况下，必须遵守无菌技术，建议使用最低限度的适当个人防护设备 （PPE）（实验室外套、口罩、护目镜和手套）并在整个过程中对所有工具进行消毒，以减少潜在的污染。</li>
</ol>9. 胶原蛋白中和
<ol><li>在修剪和切割 DRG 后，在 DRG 收获的同一天中和胶原蛋白。</li>
	<li>在冰上工作，将 ~8.16% 超纯水、~1.84% 1 M 氢氧化钠 （NaOH）、~10% 10x PBS 和溶液总体积的 ~80% 胶原蛋白移液到玻璃瓶中。</li>
	<li>使用无菌低吸附移液器吸头缓慢混匀，将胶原蛋白（通常最后添加）移液到混合物中。混合时确保移液器吸头保持在溶液中，以避免产生气泡。使用 pH 试纸检查中和胶原蛋白的 pH 值，确保其为 ~7。 注意：中和的胶原蛋白可以在冰上保持 2-3 小时而不胶凝;因此，一旦胶原蛋白被中和，就应迅速进行水凝胶制备。</li>
</ol>10. 水凝胶制造
<ol><li>在冰上工作，将层粘连蛋白（终浓度为 0.75 mg/mL）和 1x PBS 移液到玻璃瓶中。</li>
	<li>使用无菌低吸附移液器吸头，将中和的胶原蛋白和 MAHA 溶液移液到混合物中，以达到所需的浓度。</li>
	<li>缓慢混匀并检查所得水凝胶的 pH 值（应为 ~7）。</li>
</ol>11. DRG 嵌入
<ol><li>执行多区间嵌入。<ol><li>将 65.1 μL 和 52.8 μL 的预混合水凝胶分别移液到体室和神经突室中。 注意：这假设在 48 孔板中使用 MC 设备。可以根据所使用的 MC 设备的大小更改这些卷。</li>
		<li>轻轻地将切割的 DRG 嵌入胞室（图 2A），确保它们不会划伤孔板的底部。将 DRG 放在中间，以便神经突可以通过隧道长入相邻的隔室。</li>
		<li>在 37 °C 的培养箱中对水凝胶进行热交联 30 分钟。发生这种情况时，请打开 UV 灯进行预热。</li>
		<li>热交联后，UV 交联水凝胶 90 秒。</li>
		<li>将 DRG 生长培养基添加到水凝胶和 DRG 中，并在一小时后进行完整的培养基更换，以消除培养基中残留的未反应或残留的光引发剂痕迹。</li>
		<li>每 3 天更换半培养基，并通过在显微镜下观察来监测 DRG 的生长。</li>
		<li>~4 周后，当 DRG 神经突开始生长时（图 3A），使用荧光板成像仪捕获图像并使用 FIJI 量化神经突的长度 （ImageJ）。</li>
	</ol></li>
</ol>12. 控制 DRG 嵌入
<ol><li>在 48 孔板（无 MC）中移取 250 μL 水凝胶。</li>
	<li>轻轻地将切割的 DRG 放在孔的中间，然后一半向下放入水凝胶中，确保它不会划伤孔板的底部。</li>
	<li>在 37 °C 的培养箱中对水凝胶进行热交联 30 分钟。</li>
	<li>UV 交联水凝胶 90 秒。</li>
	<li>将 DRG 生长培养基添加到水凝胶和 DRG 中，并在一小时后进行完整的培养基更换。</li>
	<li>每 3 天更换一半培养基，并通过在显微镜下观察来监测 DRG 的生长。</li>
	<li>~4 周后，当 DRG 神经突开始生长时（图 3B），使用荧光板成像仪捕获图像。 注意：任何明场显微镜都可用于成像。基于板的自动化显微镜使这一过程更快。在没有该设备的情况下控制包埋在普通凝胶中，以比较 DRG 神经突在多区室中的生长。一旦用户验证了 DRG 在 MC 和对照凝胶中的生长相似，他们就不需要每次都重复对照凝胶。</li>
</ol>13. DRG 成像
<ol><li>使用荧光板成像仪在一系列焦距下以 4 倍放大倍率捕获明场图像，以可视化整个 DRG 和 MC 设备。</li>
	<li>调整图像的亮度和对比度，以便于查看神经突。</li>
	<li>将图像另存为 .tiff 文件。</li>
</ol>14. DRG 神经突定量
<ol><li>下载并安装斐济 （ImageJ）。</li>
	<li>打开 ImageJ。打开图像文件并确保文件为单色文件。</li>
	<li>增强对比度，以便可以看到最小的神经突。通过使用快捷键 Ctrl+Shift+C 打开亮度和对比度选项来执行此操作。</li>
	<li>根据需要调整控制器的滑块以可视化神经突，然后单击 应用 以获得所需的亮度和对比度。</li>
	<li>打开 Plugins 菜单，选择 Segmentation，然后单击 Simple Neurite Tracer，打开神经突示踪器。</li>
	<li>通过单击神经突的一端正对 DRG 主体来开始新的轨迹。</li>
	<li>单击神经突上的另一个点以添加轨迹，直到端到端跟踪神经突。点击 完成路径 追踪神经突后。</li>
	<li>将跟踪长度转换为实际单位，并通过将其复制并粘贴到 Excel 中来保存结果。</li>
	<li>使用 FIJI 中的直线工具绘制一条与比例尺长度相同的线（不要放开线的末端），并查看工具栏下可见的长度值。length 值用于转换。</li>
	<li>测量从 DRG 两侧延伸的六个长神经突的长度（图 3C、D）并计算这些神经突的平均长度，以给出 DRG 的代表性平均长度，如图 4 所示。 注：已在普通凝胶中的 DRG 外植体上进行了免疫染色测试，以显示非神经元支持细胞的存在7。将DRG在室温下固定在4%多聚甲醛（PFA）中，用1x PBS洗涤3次，每次15分钟，并在4°C下储存在1x PBS中直至免疫染色。</li>
	<li>对于 MC 设备中的外植体，移除 MC 设备并按照上述相同的过程进行操作7。</li>
</ol>

用于研究 DRG 培养中神经元超敏反应的高级体外模型

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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=Exploring+the+possibilities+of+using+in+vitro+model+for+neuropathic+pain+studies.">Exploring the possibilities of using in vitro model for neuropathic pain studies.</a> Neurosci Res Notes. 5, 144 (2022).</li><li>Park, S. 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=A+three-dimensional+in+vitro+model+of+the+peripheral+nervous+system.">A three-dimensional in vitro model of the peripheral nervous system.</a> NPG Asia Mater. 13 (1), 2 (2021).</li><li>Caparaso, S. M., Redwine, A. L., Wachs, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+multi-compartment+in+vitro+model+for+dorsal+root+ganglia+phenotypic+assessment.">Engineering a multi-compartment in vitro model for dorsal root ganglia phenotypic assessment.</a> J Biomed Mater Res B Appl Biomater. 111 (11), 1903-1920 (2023).</li><li>Krug, A. 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=Evaluation+of+a+human+neurite+growth+assay+as+specific+screen+for+developmental+neurotoxicants.">Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.</a> Arch Toxicol. 87 (12), 2215-2231 (2013).</li><li>F, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+stiffness+negatively+affects+axon+elongation,+growth+cone+area+and+F-actin+levels+in+a+collagen+type+I+3D+culture.">Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.</a> J Tissue Eng Regen Med. 16 (2), 151-162 (2021).</li><li>Spearman, B. S., 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=Tunable+methacrylated+hyaluronic+acid-based+hydrogels+as+scaffolds+for+soft+tissue+engineering+applications.">Tunable methacrylated hyaluronic acid-based hydrogels as scaffolds for soft tissue engineering applications.</a> J Biomed Mater Res A. 108 (2), 279-291 (2020).</li><li>Zhu, W., Oxford, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+of+neonatal+and+adult+DRG+neurons+correlates+with+the+differential+sensitization+of+TRPV1+responses+to+nerve+growth+factor.">Differential gene expression of neonatal and adult DRG neurons correlates with the differential sensitization of TRPV1 responses to nerve growth factor.</a> Neurosci Lett. 500 (3), 192-196 (2011).</li><li>Van De Wijdeven, R., 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=Structuring+a+multi-nodal+neural+network+in+vitro+within+a+novel+design+microfluidic+chip.">Structuring a multi-nodal neural network in vitro within a novel design microfluidic chip.</a> Biomed Microdevices. 20, 1-8 (2018).</li><li>Chan, J. R., 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=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43 (2), 183-191 (2004).</li><li>Ng, B. K., Chen, L., Mandemakers, W., Cosgaya, J. M., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterograde+transport+and+secretion+of+brain-derived+neurotrophic+factor+along+sensory+axons+promote+Schwann+cell+myelination.">Anterograde transport and secretion of brain-derived neurotrophic factor along sensory axons promote Schwann cell myelination.</a> J Neurosci. 27 (28), 7597-7603 (2007).</li><li>Xiao, 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=BDNF+exerts+contrasting+effects+on+peripheral+myelination+of+NGF-dependent+and+BDNF-dependent+drg+neurons.">BDNF exerts contrasting effects on peripheral myelination of NGF-dependent and BDNF-dependent drg neurons.</a> J Neurosci. 29 (13), 4016-4022 (2009).</li><li>Seidlits, S. 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=The+effects+of+hyaluronic+acid+hydrogels+with+tunable+mechanical+properties+on+neural+progenitor+cell+differentiation.">The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation.</a> Biomaterials. 31 (14), 3930-3940 (2010).</li><li>Tomal, W., Ortyl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-soluble+photoinitiators+in+biomedical+applications.">Water-soluble photoinitiators in biomedical applications.</a> Polymers. 12 (5), 1073 (2020).</li><li>Leary, S., 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> Avma guidelines for the euthanasia of animals: 2013 edition. , (2013).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Res Notes. 9 (1), 82 (2016).</li><li>Naveed, 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=Simple+methods+of+dissection+protocols+for+the+rapid+isolation+of+rat+dorsal+root+ganglia+under+the+non-microscopic+condition.">Simple methods of dissection protocols for the rapid isolation of rat dorsal root ganglia under the non-microscopic condition.</a> Neuroscience Research Notes. 6 (2), 212 (2023).</li><li>Perner, C., Sokol, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+and+culture+of+murine+dorsal+root+ganglia+neurons+to+study+neuropeptide+release.">Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.</a> STAR Protocols. 2 (1), 100333 (2021).</li><li>Bai, 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=A+high-resolution+anatomical+rat+atlas.">A high-resolution anatomical rat atlas.</a> J Anatomy. 209 (5), 707-708 (2006).</li><li>Bian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+hyaluronic+acid+hydrogel+crosslinking+density+and+macromolecular+diffusivity+on+human+MSC+chondrogenesis+and+hypertrophy.">The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy.</a> Biomaterials. 34 (2), 413-421 (2013).</li><li>Haberberger, R. V., Barry, C., Dominguez, N., Matusica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dorsal+root+ganglia.">Human dorsal root ganglia.</a> Front Cell Neurosci. 13, 271 (2019).</li><li>Schwaid, A. G., Krasowka-Zoladek, A., Chi, A., Cornella-Taracido, I. <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+the+rat+and+human+dorsal+root+ganglion+proteome.">Comparison of the rat and human dorsal root ganglion proteome.</a> Scientific Rep. 8 (1), 13469 (2018).</li></ol>

本研究的作者声明他们没有利益冲突。

该协议概述了一种收获成年 Sprague Dawley DRG 并在 3D 天然水凝胶中培养它们的方法。与这种方法相反，从小鼠和大鼠中收集 DRG 的其他方法包括分离脊柱。切除的脊柱减半，并切除脊髓以露出 DRG 23,24,25。脊髓损伤会限制血液供应，从而影响 DRG 和内部神经元26。这降低了 DRG 的活性，使手术方法更合适。此外，还...

慢性疼痛是全球残疾和失业的主要原因1。慢性疼痛影响着全球约 20% 的成年人，并造成了巨大的社会和经济负担2，在美国，每年的总成本估计在 560 至 6350 亿美元之间3。
慢性疼痛患者表现出的主要外周特征是神经刺激阈值降低，这导致神经系统对刺激更敏感 4,5。降低的刺激阈值会导致对先前非痛苦刺激的痛苦反应（痛觉异常）或对痛苦刺激的反应增强（痛觉过敏）6。目前的慢性疼痛治疗疗效有限，由于疼痛表现的机制差异，在动物模型中成功的治疗往往在人体试验中失败7。能够更准确地模拟外周敏化机制的体外模型有可能增加新疗法的翻译 8,9。此外，通过对培养系统中致敏神经的关键方面进行建模，研究人员可以更深入地了解驱动降低阈值的机制，并确定逆转阈值的新治疗靶点10。
理想的 体外 平台或微生理系统将结合远端神经突和背根神经节 （DRG） 细胞体的物理分离、三维 （3D） 细胞环境以及天然支持细胞的存在，以密切模拟 体内 条件。然而，Caparaso 等人最近的一篇论文 11 表明，当前的 DRG 培养平台缺乏这些关键特征中的一个或多个，这使得 它们在体内复制 条件方面不足。尽管这些平台易于设置，但它们并不模拟外周致敏的生物学基础，因此可能无法转化为 体内 疗效。为了解决这一限制，已经开发了一种生理学相关的 体外 模型，用于在具有三个隔离隔室的水凝胶基质中培养背根神经节 （DRG），以允许神经突和 DRG 细胞体的时间流体分离11。该模型提供了生理学和解剖学相关性，有可能在 体外研究神经元的外周敏化。
人们对在 3D 培养中使用 DRG 外植体的兴趣日益浓厚，是因为它们能够促进稳健的神经突生长，这是 DRG 活力的间接指标12。虽然原代新生儿或胚胎 DRG 外植体主要用于当前的体外培养平台13,14，但使用成年啮齿动物的外植体提供了更好的成熟神经元生理学模型，与新生儿或胚胎啮齿动物的外植体相比，它与人类 DRG 生理学非常相似15。外植体 DRG 是指主要通过维持天然非神经元支持细胞来保存天然 DRG 组织的细胞和分子组织。在此，该协议描述了在多室 （MC） 装置中从成年 Sprague Dawley 大鼠收获和培养 DRG 外植体的方法（图 1）。
在从颈椎、胸椎和腰椎培养 DRG 中已显示出有效性，神经突生长没有明显的差异。对于此应用，目标是诱使神经突生长到设备的外室中;因此，本文没有区分 DRG 水平。但是，如果特定实验需要，则可以定制 DRG 水平以满足实验者的需求。目前还有其他用于 DRGs16 3D 培养的区室化培养模型，但是，这些装置不包含保存的天然非神经元支持细胞，这可能会限制翻译。保留收获的 DRG 的天然结构很重要，因为它可以确保非神经元支持细胞的保留，这些支持细胞与 DRG 神经元的相互作用对于维持这些神经元的功能特性至关重要。几项研究将解离的 DRG 与非天然神经元细胞（如雪旺细胞）共培养，以促进神经元的髓鞘形成 17,18,19。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>For trimming and cutting DRG</td>
		</tr>
		<tr>
			<td>10x DMEM</td>
			<td>MilliporeSigma</td>
			<td>D2429</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS (autoclaved)</td>
			<td>Prepared in lab</td>
			<td></td>
			<td>7.3 - 7.5 pH</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>VWR</td>
			<td>82050-892</td>
			<td>To temporarily store harvested and cut DRGs</td>
		</tr>
		<tr>
			<td>3 mL Syringe sterile, single use</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>48 well plates</td>
			<td>Greiner Bio-One</td>
			<td>677180</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td>To hold media for trimming and cutting</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Fisherbrand</td>
			<td>01-213-104</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Plus 50x</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Collagen type I</td>
			<td>Ibidi</td>
			<td>50205</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved cup Friedman Pearson Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16221-14</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Dumont #3 forceps</td>
			<td>Fine Science Tools</td>
			<td>11293-00</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Form cure</td>
			<td>Form Labs</td>
			<td></td>
			<td>curing agent</td>
		</tr>
		<tr>
			<td>Form wash</td>
			<td>Form Labs</td>
			<td></td>
			<td>To wash excess resins off MC</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials (8 mL)</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>224724</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>ThermoFisher</td>
			<td>35050-061</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Millipore Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>High temp V2 resin</td>
			<td>FormLabs</td>
			<td>FLHTAM02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronic Acid Sodium Salt</td>
			<td>MilliporeSigma</td>
			<td>53747</td>
			<td>Used to make MAHA</td>
		</tr>
		<tr>
			<td>Irgacure</td>
			<td>MilliporeSigma</td>
			<td>410896</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>R&#38;D Systems</td>
			<td>344600501</td>
			<td></td>
		</tr>
		<tr>
			<td>Large blunt-nose scissors</td>
			<td>Militex</td>
			<td>EG5-26</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Large forceps (serrated tips)</td>
			<td>Militex</td>
			<td>9538797</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Large sharp-nosed scissors</td>
			<td>Fine Science Tools</td>
			<td>14010-15</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Low Retention pipette tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-017</td>
			<td>For pipetting collagen and MAHA</td>
		</tr>
		<tr>
			<td>Methacrylated hyaluronic acid (MAHA)</td>
			<td>Prepared in lab</td>
			<td>N/A</td>
			<td>85 - 115 % methacrylation</td>
		</tr>
		<tr>
			<td>Nerve Growth Factor (NGF)</td>
			<td>R&#38;D Systems</td>
			<td>556-NG-100</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Neurobasal A Media</td>
			<td>ThermoFisher</td>
			<td>10888022</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS)</td>
			<td>EMD Millipore</td>
			<td>516106</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>pH test strips</td>
			<td>VWR International</td>
			<td>BDH35309.606</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#181;L)</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td></td>
		</tr>
		<tr>
			<td>Preform 3.23.1 software</td>
			<td>Formslab</td>
			<td></td>
			<td>To upload STL file</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Charles River</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resin 3D printer</td>
			<td>Form Labs</td>
			<td>Form 3L</td>
			<td>3D printing MC device</td>
		</tr>
		<tr>
			<td>Small sharp-nosed scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>MilliporeSigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight cup rongeur</td>
			<td>Fine Science Tools</td>
			<td>16004-16</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Straight edge spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Surgical Scaplel blade (No. 10)</td>
			<td>Fisher Scientific</td>
			<td>22-079-690</td>
			<td></td>
		</tr>
		<tr>
			<td>Syring filters, PES (0.22 &#181;m)</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiny spring scissors</td>
			<td>World Precision Instruments</td>
			<td>14003</td>
			<td>For trimming and cutting DRG</td>
		</tr>
		<tr>
			<td>UV lamp</td>
			<td>Analytik Jena US</td>
			<td></td>
			<td>To photocrosslink hydrogel (15 - 18 mW/cm2)</td>
		</tr>
	</tbody>
</table>

harvesting, embedding, and culturing dorsal root ganglia in multi-compartment devices to study peripheral neuronal features

疼痛最常见的周围神经元特征是刺激阈值降低或背根神经节 （DRG） 终末神经超敏反应。这种超敏反应的一个拟议原因与外周组织中的免疫细胞和神经元之间的相互作用有关。 体外 模型为理解这些机制如何导致伤害感受器超敏反应提供了基础知识。然而， 体外 模型面临着将疗效转化为人类的挑战。为了应对这一挑战，已经开发了一种生理学和解剖学相关的 体外 模型，用于在 48 孔板的三个孤立隔室中培养完整的背根神经节 （DRG）。初级 DRG 是在人道安乐死后从成年 Sprague Dawley 大鼠身上收获的。修剪多余的神经根，并将 DRG 切成合适的大小进行培养。然后，DRG 在天然水凝胶中生长，从而在所有隔室中实现稳健生长。这种多室系统提供了从神经突、生理相关细胞类型和机械特性中分离 DRG 细胞体的解剖学相关分离，以研究神经细胞和免疫细胞之间的相互作用。因此，该培养平台为研究治疗隔离策略提供了有价值的工具，最终导致一种改进的预测疼痛的筛查方法。

Chronic pain is the leading cause of disability and loss of work globally1. Chronic pain affects about 20% of adults globally and imposes a significant societal and economic burden2, with total costs estimated between $560 and $635 billion every year in the United States3.
The main peripheral feature exhibited by chronic pain patients is a lowered stimulation threshold of nerves, which leads to the nervous system being more responsive to stimuli4,5. The lowered stimulation threshold can result in a painful response to a previously non-painful stimulus (allodynia) or a heightened response to a painful stimulus (hyperalgesia)6. Current chronic pain treatments have limited efficacy, and treatments that succeed in animal models often fail in human trials due to mechanistic differences in pain manifestation7. In vitro models that can more accurately mimic peripheral sensitization mechanisms have the potential to increase the translation of new therapeutics8,9. Further, by modeling key aspects of sensitized nerves in a culture system, researchers could develop a deeper understanding of the mechanisms that drive lowered thresholds and identify novel therapeutic targets that reverse them10.
The ideal in vitro platforms or microphysiological systems would incorporate the physical separation of distal neurites and dorsal root ganglia (DRG) cell body, a three-dimensional (3D) cellular environment, and the presence of native support cells to closely mimic in vivo conditions. However, a recent paper by Caparaso et al.11 shows that current DRG culture platforms lack one or more of these key features, making them insufficient in replicating in vivo conditions. Even though these platforms are easy to set up, they do not mimic the biological basis of peripheral sensitization and thus may not translate to in vivo efficacy. To address this limitation, a physiologically relevant in vitro model has been developed for the culture of dorsal root ganglia (DRG) within a hydrogel matrix with three isolated compartments to allow temporal fluidic isolation of neurites and DRG cell bodies11. This model offers both physiological and anatomical relevance, which has the potential to study peripheral sensitization of neurons in vitro.
The growing interest in the use of DRG explants in a 3D culture is due to their ability to facilitate robust neurite growth, which serves as an indirect indicator of DRG viability12. While primary neonatal or embryonic DRG explants are predominantly used in current in vitro culture platforms13,14, using explants from adult rodents provides a better model of mature neuronal physiology, which closely mimics human DRG physiology compared to explants from neonatal or embryonic rodents15. Explant DRGs refer to the preservation of the cellular and molecular tissue of native DRG tissue, primarily by maintaining native non-neuronal support cells.Herein, this protocol describes the methodology to harvest and culture DRG explants from adult Sprague Dawley rats in a multi-compartment (MC) device (Figure 1).
Efficacy has been shown in culturing DRGs from the cervical, thoracic, and lumbar spine with no observable differences in neurite growth. For this application, the objective was to elicit neurite growth into the outer compartments of the device; therefore, this article did not discriminate among DRG levels. However, if needed for a specific experiment, the DRG level can be tailored to meet experimenters&#39; needs. There are currently other compartmentalized culture models for the 3D culture of DRGs16, however, these devices do not contain preserved native non-neuronal support cells, which can limit translation. Preserving the native structure of harvested DRGs is important because it ensures the retention of non-neuronal support cells, whose interactions with DRG neurons are essential for maintaining the functional properties of these neurons. Several studies co-cultured dissociated DRGs with non-native neuronal cells such as Schwann cells to promote myelination of neurons17,18,19.

作者聚焦：提高慢性疼痛治疗效果的体外和体内模型

在多室装置中收获、包埋和培养背根神经节以研究外周神经元特征

Our research seeks to understand some of the underlying mechanisms that contribute to chronic musculoskeletal pain, so we can develop novel targeted treatments. Thus, we have developed in vitro and in vivo models of chronic low back pain and neuronal sensitization. Current therapeutics that work in vivo in animal models often fail clinical trials due to mechanistic differences between animal models and human subject chronic pain phenotype.Individual test by that model, nociceptor hypersensitivity present in chronic pain have the potential to identify more promising treatments for chronic pain in humans. Our protocol clearly demonstrates how to isolate primary dorsal root ganglia, and culture them in a multi compartment device. By keeping intact primary dorsal root ganglia, we maintain native support cells and culture them in a multi compartment device, allowing anatomically relevant isolation between neuronal somas and peripheral neurites.This tool will help to culture primary dorsal root ganglia in a more physiologically and anatomically relevant way, thereby increasing translational efficacy of findings. Specifically, we are excited to use this system to identify novel treatment for nociceptor hypersensitivity present in chronic pain.

本方案描述了一种在多隔室 （MC） 装置中从成年 Sprague Dawley 大鼠收获和培养 DRG 的技术。如图 1 所示， 从成年大鼠身上收获的 DRG 被修剪并切成 ~0.5 mm。然后将修剪和切割的 DRG 嵌入 MC 装置胞体区域的水凝胶中（图 2）并在神经突定量前培养 27 天。将 DRG 在普通凝胶中培养作为对照。用于本实验的水凝胶制剂浓度为 4.5/1.25 mg/mL 胶原蛋白：MA...

该协议提供了一种在多隔室 （MC） 装置中从成年 Sprague Dawley 大鼠收获和培养外植背根神经节 （DRG） 的技术。

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

harvesting-embedding-culturing-dorsal-root-ganglia-multi-compartment

Research

JoVE Journal

Bioengineering

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

443 次观看.  University of Nebraska-Lincoln. 我们的研究旨在了解导致慢性肌肉骨骼疼痛的一些潜在机制，以便我们可以开发新的靶向治疗方法。因此，我们开发了慢性腰痛和神经元敏化的体外和体内模型。由于动物模型和人类受试者慢性疼痛表型之间的机制差异，目前在动物模型中起作用的体内疗法经常无法通过临床试验。通过该模型的单独测试，慢性疼痛中存在的伤害感受器超敏反应...

视频: 作者聚焦：提高慢性疼痛治疗效果的体外和体内模型

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

科研

生物工程

Assembly of Multi-Compartment Device and Harvesting of Dorsal Root Ganglia (DRG) from Adult Sprague Dawley Rats

To begin, use a three milliliter syringe to apply a thin line of silicone gel into the groove beneath the MC device. Using number three forceps gently position the MC device into the well of a 48 well plate. Place the device into a 48 well plate one day before harvesting the dorsal root ganglion.Place the euthanized rat on the dissection platform. Using large blunt nose scissors, make an incision through the skin at the base of the cervical spine and extend it down the length of the spine. To further drain blood from the spinal canal, cut beneath the shoulder blades and through the muscle to sever major blood vessels.Using large sharp nose scissors, cut the muscle away from the spine, then remove the dorsal segments of the vertebrae. Use straight rongeur and sharp nose scissors to remove the spinous and transverse processes of the vertebrae starting at the cervical end and moving towards the lumbar end. Afterward, remove the spinal cord.Using small sharp nose scissors, carefully sever the nerve roots connected to the dorsal root ganglia along both sides of the spinal cord. Now cut the cervical end of the spinal cord and gradually lift it out of the spinal canal, severing any remaining nerve roots connected to the dorsal root ganglia. To expose the dorsal root ganglia, use a curved rongeur to remove additional lateral parts of the vertebrae.Using number three forceps and straight edge spring scissors, extract the dorsal root ganglia between each vertebra level. Grip the spinal nerve roots from the dorsal root ganglia. Gently pull them out of the pocket and cut through the other end of the spinal nerve.Lastly, place the dorsal root ganglia into wells of a 24 well plate on ice.

多室装置的组装和成年 Sprague Dawley 大鼠背根神经节 （DRG） 的收获

Trimming, Cutting, and Embedding the Dorsal Root Ganglion for Culturing in a Multi-Compartment (MC) Device

To begin, take the harvested dorsal root ganglia. On a clean bench, fill a 60-millimeter Petri dish with trimming media. Arrange the dissecting scope and glass bead sterilizer, and place an autoclave toolbox with trimming tools next to the scope.Using the dissecting scope, remove any excess tissue around the dorsal root ganglion, and trim the nerve roots close to the dorsal root ganglion body. Now, fill a second 60-millimeter Petri dish with fresh media. Using the dissecting scope, cut the trimmed dorsal root ganglia to approximately 0.5 millimeters.Transfer the cut dorsal root ganglia into a fresh 24-well plate containing media on ice. Then, pipette 65.1 and 52.8 microliters of the premixed hydrogel into the soma and neurite compartments, respectively. Carefully embed the cut dorsal root ganglia into the soma compartment.Thermally cross-link the hydrogel in the incubator at 37 degrees Celsius for 30 minutes. Then, UV cross-link the hydrogel for 90 seconds. Afterward, add dorsal root ganglia growth media to the hydrogel and dorsal root ganglia.Perform a complete media change after one hour to remove any unreactive or residual photo-initiator in the media.

修剪、切割和包埋背根神经节，以便在多室 （MC） 设备中培养

Quantification of Dorsal Root Ganglion Neurite Growth in a Multi-Compartment (MC) Device Using Fiji

After imaging the dorsal root ganglion cultured in a multi-compartment device, launch Image J and open the image file. To enhance the contrast and make the smallest neurites visible using the shortcuts Control with Shift and C, open the Brightness and Contrast option. Adjust the brightness and contrast sliders to visualize the neurites.Then click on Apply. Now, navigate to Plugins, select Segmentation, and click Simple Neurite Tracer to open the neurite tracer. Then, click on one end of the neurite at the dorsal root ganglion body to initiate a new trace, and continue adding points along the neurite until it is fully traced.Then click on Complete Path. Using the straight line tool in Fiji, draw a line of the same length as the scale bar without releasing the end of the line. And note the length value under the toolbar.Set the scale to convert the neurite length to real units and copy the results to Excel. Then, convert the lengths to real units in Excel. Then, measure the length of six long neurites extending from both sides of the dorsal root ganglion, and calculate the average length and standard deviation of the dorsal root ganglion.On days 27 and 21, there was robust growth of neurites in the multi compartment and plain gels, respectively. The average length of neurites in the multi compartment device was comparable to neurite length in control plane gels.