We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

脑组织分离方案经塔夫茨大学机构动物护理和使用委员会，并符合卫生指南实验动物的护理和使用（机构动物护理和使用委员会B2011-45）全国学院。 1.丝绸脚手架准备<ol><li>如先前19,20描述了从家蚕蚕茧丝绸溶液的制备。 <ol><li>横切茧成用剪刀8等份。使用约11茧为5克支离破碎的蚕茧。 （15分钟） </li><li>准备2升的0.02M 的 Na 2 CO 3溶液，并把它用热板煮沸。 （15分钟） </li><li>称取5克零散蚕茧和煮沸中的 Na 2 CO 3溶液中30分钟。搅拌煮沸丝每隔几分钟用锅铲。这一步，​​也被称为脱胶，净化丝素蛋白由亲水性蛋白质，sericins。 （30分钟） </li><lI&gt;拧丝心蛋白通过手和冲洗在蒸馏水至少三次洗出任何残留的丝胶和化学品。 （5分钟） </li><li>将湿纤蛋白上的培养皿中，并干燥，在流罩O / N的丝心蛋白提取物。 </li><li>第二天称量干纤蛋白质量和放置丝心蛋白在玻璃烧杯中。 （15分钟） </li><li>为了通过4乘以干素蛋白的质量溶于9.3中号溴化锂溴化锂溶液，计算所需体积（毫升）的素慢慢倒入溴化锂溶液中的丝素蛋白，用抹刀沉浸所有的丝素纤维。覆盖烧杯以防止蒸发，并将其放置在60℃下4小时，以使纤维溶解。 （15分钟） </li><li>使用注射器，收集烧杯丝心蛋白溶液和其装载到截留分子量3500的透析盒。执行透析蒸馏水48小时。换水每隔几个小时。 </li><li>使用注射器，收集丝心蛋白溶液卡匣到50ml锥形管中并离心两次以9,000rpm（〜12700×g离心）在4℃下20分钟。倾上清液到新的管中的每个离心步骤后，弃去沉淀。 （40分） </li><li>通过估计干重测量丝心蛋白的浓度。倒入1毫升丝溶液成权衡船。干燥在烤箱的样品在60℃下2小时。称量干燥丝纤蛋白和由100的丝溶液的预期浓度所获得的权重乘法计算的丝纤蛋白溶液的浓度为6-9％（重量/体积）。 </li><li>通过将其在蒸馏水稀释调整丝浓度至6％（重量/体积）。 停止点:液体丝纤蛋白可存放于4℃最多一个月在密闭容器中。 </li></ol></li><li>制取丝溶液多孔支架。 <ol><li>筛粒状的NaCl以分离颗粒尺寸500-600微米，这将在后面的步骤中使用。丢弃颗粒小号尺寸低于500微米以上600微米。 （15分钟） </li><li>倾的30ml 6％丝溶液到聚四氟乙烯（PTFE）的模具（ 图1）。轻轻地撒60克过筛氯化钠过的丝绸。敲击容器以获得盐的均匀层。孵育48小时，在室温聚合丝。 </li><li>将含有支架的PTFE模具在烘箱在60℃下1小时以完成聚合和蒸发任何剩余的液体。 </li><li>放置的PTFE模具的含量在含有2升蒸馏水48小时浸出的盐的烧杯中。换水，每天2-3次。除去从模具海绵支架时盐完全浸出停止点 :该海绵可以存储浸没在水中，在4℃的密闭容器中，以防止支架从脱水。 </li><li>准备就绪后，切出直径5毫米的穿刺打孔的支架。预切支架达到约2mm的高度。浦NCH ​​脱离与2毫米直径活检穿孔（图2A）的支架的中心。 （1小时） </li><li>高压釜浸入水中支架消毒他们（湿循环，121℃，20分钟）停止点 :该海绵可以存储浸没在水中，在4℃的密闭容器中，以防止支架从脱水。 </li><li>计划细胞接种之前，浸泡在无菌0.1毫克/毫升聚D-赖氨酸（PDL）溶液支架。孵育1小时，在37℃。 </li><li>洗支架用磷酸盐缓冲盐水（PBS）3倍以除去未结合的PDL。 （30分钟） </li></ol></li></ol> 2.隔离大鼠皮层神经元<ol><li>解剖皮层从胚胎第18天（E18）Sprague-Dawley大鼠通过Pacifici和佩鲁齐27获得批准动物协议之后如前所述。 （2小时） </li><li>为20分钟，在3孵育10皮层在5毫升的0.25％胰蛋白酶的0.3％DNA酶I（来自牛胰腺）7℃。 </li><li>通过加入1mg / ml的大豆蛋白质的等体积灭活胰蛋白酶。 </li><li>磨碎移液器向上用10ml巴斯德吸管将皮质和向下20次，直到产生单细胞悬浮液。要轻柔，避免气泡形成。 （10分钟） </li><li>离心细胞悬浮液在127×g离心5分钟。 </li><li>重悬在10ml培养基的细胞沉淀（Neurobasal培养基，1×B27添加剂，1×Glutamax的，1％青霉素/链霉素）。计数细胞。预期的细胞浓度是约2×10 7 /毫升。 （20分钟） </li></ol> 3.建设大会与文化<ol><li>支架接种用的细胞。 <ol><li>将无菌支架和所有必要的用具的细胞培养罩内。使用无菌镊子放置在96孔细胞培养板中的支架分配每孔1的支架。 （10分钟） </li><li>沉浸在细胞培养基中的支架以平衡它们之前细胞种子ING。孵育1小时，在37℃。 （10分钟） </li><li>吸出多余的支架介质。 </li><li>适用于100微升细胞悬浮液/支架。 （10分钟） </li><li>孵育细胞在37℃CO / N，以使细胞附着到所述支架。 </li><li>第二天上午吸出非附着的细胞，并应用200μl/孔的新鲜培养基。 （10分钟） </li></ol></li><li>脚手架胶原基质中嵌入。 （2小时） <ol><li>放置10×PBS中，水，1N NaOH和大鼠尾I型胶原在冰上。制备胶原的工作溶液按照制造商的说明。保持在冰上，直至细胞接种构建体准备（高达1小时）。 </li><li>从培养箱中取出细胞接种丝构建体和吸出多余的培养基。 </li><li>使用无菌镊子支架转交到空孔的孔板和浸入每个支架在100μl的3毫克/毫升的胶原溶液。放置组织培养板背面在孵化30分钟，以允许胶原的聚合。 </li><li>申请将100μl预热的细胞培养基/孔。培养所述构建体一周每天改变介质通过更换培养基的一半的体积。 </li></ol></li></ol> 4.显微镜分析<ol><li>活/死染色（1小时） <ol><li>制备碘化丙啶（PI）为1mg / ml（1.5毫摩尔）的蒸馏水的贮液。 </li><li>制备的荧光素二乙酸酯（FDA）的2毫克/毫升的丙酮储备液。 </li><li>制备含有10μM的PI和0.15μM的FDA的在PBS中稀释的工作溶液。预温水浴中的溶液至37℃。 </li><li>用预热的PBS洗涤细胞以除去血清酯酶。吸出PBS。 </li><li>应用细胞上的预热工作溶液2分钟，用PBS洗涤。 </li><li>使用啶显微镜图像细胞（PI前λ= 490纳米，EMλ= 570纳米; FDA前λ= 490ñ男，EMλ= 514纳米）。污渍保持稳定40分钟。延长染色会导致从由细胞PI摄取和细胞内共定位的PI / FDA的所得非特异性染色。 </li></ol></li><li>免疫染色<ol><li>在培养所需的时间点固定细胞，用4％多聚甲醛（PFA），用于在室温30分钟。由于煤灰的毒性油烟定影步骤应在通风橱中进行。 </li><li>洗支架与3×PBS 停止点 :该PFA固定构建体可以在进行进一步的步骤之前被存储在PBS在4℃下数天。 </li><li>孵育在PBS含第一抗体的O / N的0.2％的Triton，0.25％牛血清白蛋白（BSA）的支架在4℃。 </li><li>翌日丢弃染色液并用PBS洗支架3×30分钟，以除去未结合的第一抗体。 </li><li>孵育样品与0.2％的Triton稀释二级抗体，0.25％BSA的PBS进行2小时，在室温。 </li><li>除去染色液并用PBS洗支架3×30分钟，以除去未结合的次级抗体。 </li><li>图像使用共聚焦显微镜20X物镜通过取样品为100μm的z切片用1微米步骤支架。以可视化的薄突起图像的分辨率应该是最低1,024×1,024像素。 </li></ol></li><li> RNA，DNA和蛋白质分离 注意:RNA，DNA和蛋白质的分离使用根据制造商的说明书一商业AllPrep的DNA / RNA /蛋白质Mini试剂盒进行。 <ol><li>使用无菌镊子转移支架出培养板的一个2毫升无菌试管中。将每管1支架。 </li><li>加入200μl的RLT缓冲液到每个管中。 </li><li>用无菌microscissors分段来扰乱结构。 </li><li>以均化破碎的组织，该管的内容传送到离心柱。在离心旋转2分钟全速。将裂解物均化，因为它通过旋转柱。 </li><li>收集通过流动，并立即用它来分离RNA，DNA和蛋白质，按照制造商的协议。 </li><li>量化的核酸的产量的任何其他兼容方法（例如，的NanoDrop）。 </li><li>量化，根据制造商的说明书用BCA蛋白质测定蛋白质产率。使用酶标仪在波长562nm处测量试验的结果。 注意:核酸和相对于细胞密度每构建体蛋白的预期产量示于图4。 </li></ol></li></ol>

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<li>Hopkins, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silk+hydrogels+as+soft+substrates+for+neural+tissue+engineering%5BTitle%5D)+AND+%22Adv+Funct+Mater%22%5BJournal%5D)">Silk hydrogels as soft substrates for neural tissue engineering.</a> Adv Funct Mater. 23 (41), 5140-5149 (2013).</li>
<li>Pacifici, M., Peruzzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+rat+embryonic+neural+cells%3A+A+quick+protocol%5BTitle%5D)+AND+%22Vis.+Exp%22%5BJournal%5D)">Isolation and culture of rat embryonic neural cells: A quick protocol.</a> Vis. Exp. (63), e3965(2012).</li>
</ol>

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Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

中枢神经系统（CNS）可受到各种涉及血管，结构，功能，感染或变性病症。估计有680万人，每年死于神经障碍的结果，它代表全世界日益社会经济负担1。然而，只有少数的病症有可用的治疗。因此，存在对用于从患者的神经系统疾病的患者的创新的治疗策略迫切需要。不幸的是，许多中枢神经系统靶向治疗在临床试验中由于不充分的临床前研究模型，不允许与生理有关职能读数急性和慢性影响评价的利用率失败，部分原因。 尽管在过去几十年显著的研究工作，还有大量未知的有关中枢神经系统的结构和功能。为了获得更多的知识，动物模型往往我们编模型的病理状态，例如创伤性脑损伤（TBI）或痴呆，特别是在临床前研究。然而，动物从人都在中枢神经系统的解剖结构，以及在功能上，基因表达和代谢2-4显著不同。另一方面，2D 体外培养物来研究细胞生物学的常规方法和常规用于药物发现。然而，二维细胞培养物缺乏的复杂性和生理相关性相比，人类的大脑5-7。虽然没有替代品的成本二维细胞培养研究低和简单或动物模型所提供的复杂性，3D组织工程可能会产生更好的研究模型关闭了2D之间存在体外和体内的方法的差距。三维组织工程提供了通过三维细胞 - 细胞相互作用和由生物材料支架提供胞外线索实现多种生理相关的实验条件。 DESP伊特3D背后的文化价值的显著的证据，目前只有少数的3D中枢神经系统组织模型，比如干细胞衍生的类器官培养8-10，neurospheroids 11和分散的水凝胶文化12,13。先进的技术方法，包括多层光刻14，和3D打印15已被用于研究肺，肝，肾和组织。然而，缺乏中枢神经系统的3D模型，允许条块分割的神经元生长，如模仿皮质结构和生物学。轴突从神经元细胞体中分离生长先前已经在2D培养通过使用微加工16,17允许轴突道追踪，钙内流，网络体系结构和活动的研究证实。这个想法激发了我们能够开发出3D偏光神经组织，是细胞体和轴突束分布在不同的车厢模仿大脑18的分层体系结构</sup&gt;。我们的方法是基于使用独特的蚕丝支架设计容纳细胞高密度在密闭体积，并允许稠密轴突网络的过度生长成胶原凝胶。在这里，我们表现出脑状组织，包括支架制造和神经元文化的全面组装过程。

<table><tbody><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223530</td><td></td></tr><tr><td>LiBr</td><td>Sigma-Aldrich</td><td>213225</td><td></td></tr><tr><td>MWCO 3500 dialysis cassettes&amp;nbsp;</td><td>Thermo Fisher</td><td>66110</td><td></td></tr><tr><td>Heating plate</td><td>Fisher Scientific</td><td>Isotemp</td><td></td></tr><tr><td>Centrifuge 5804 R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Sieve</td><td>Fisher Scientific</td><td></td><td>No. 270, No. 35, No. 30</td></tr><tr><td>PTFE mold</td><td></td><td></td><td>made in house (&lt;strong&gt;Figure 1&lt;/strong&gt;) 10 cm diameter, 2 cm height</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71382</td><td></td></tr><tr><td>Biopsy Punch 5&amp;#160;mm</td><td>World Precision Instrument</td><td>501909</td><td></td></tr><tr><td>Biopsy Punch 2&amp;#160;mm</td><td>World Precision Instrument</td><td>501908</td><td></td></tr><tr><td>poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td>final concentration 10&amp;#160;&amp;#956;g/ml, dissolved in water</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D1283-500ML</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T4049-500ML</td><td></td></tr><tr><td>DNase</td><td>Roche</td><td>10104159001</td><td>final concentration 0.3%</td></tr><tr><td>Soybean protein</td><td>Sigma-Aldrich</td><td>T6522-100MG</td><td>final concentration 1&amp;#160;mg/ml</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>warm up to 37&amp;#160;&amp;deg;C before use</td></tr><tr><td>B27 supplement 50x</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicilin Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Collagen I, rat tail, 100&amp;#160;mg</td><td>Corning</td><td>354236</td><td>final concentration 3&amp;#160;mg/ml</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S2770</td><td>corrosive</td></tr><tr><td>Propidium Iodide</td><td>Sigma-Aldrich</td><td>P4170-10MG</td><td>toxic</td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378-5G</td><td></td></tr><tr><td>Olympus MVX10</td><td>Olympus</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>toxic, final concentration 4%</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906-10G</td><td></td></tr><tr><td>anti-&amp;beta;IIITubulin antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T2200</td><td>rabbit 1:500</td></tr><tr><td>anti-rabbit Alexa-546</td><td>Molecular Probes</td><td>A11010</td><td>goat 1:250</td></tr><tr><td>goat serum</td><td>Sigma-Aldrich</td><td>G9023-5ML</td><td></td></tr><tr><td>Leica SP2 confocal microscope</td><td>Leica</td><td></td><td>objective 20X</td></tr><tr><td>QIAshredder</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>AllPrep DNA/RNA/Protein Mini Kit</td><td>Qiagen</td><td>80004</td><td></td></tr><tr><td>Ethyl alcohol, Pure</td><td>Sigma-Aldrich</td><td>E7023&amp;nbsp;</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>63689</td><td></td></tr><tr><td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>BCA protein assay</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Plate reader Spectramax M2</td><td>Molecular Devices</td><td></td><td>absorbance 562 nm</td></tr><tr><td>Micro Scissors, Economy, Vannas-type</td><td>TedPella</td><td>1346</td><td></td></tr></tbody></table>

engineered 3d silk-collagen-based model of polarized neural tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

固体丝海绵预切成甜甜圈形状代表一个独特的和简单的想法实现条块架构类似的神经组织（图2A）。具有大约500微米的结果特别高的表面积，允许一个小体积内的接种和高细胞密度生长的孔径高度多孔支架的结构（2×10 7 / ml）的（图2B）。此外，该支架的高孔隙率允许营养物和废物产生稠密的细胞培养物在扩展的时间帧优越生存能力无限制扩散。在播种神经元迅速而均匀地附着到所述支架的孔的表面上。 后细胞附着完成并将细胞平衡的蚕丝基板上，海绵支架填充有柔软的胶原基质，以便提供3D环境轴突网络形成（图3 </strong&gt;）。在不存在胶原基质的轴突和细胞体的条块是不可能的，因为神经元仅在导致混合网络，与2D表面（ 图3B）中发现的孔的表面生长的扩展。与此相反，在胶原凝胶提供支持在三维广泛轴突生长，而神经元细胞体保持附着在蚕丝支架（图3C）一个小梁。这种增长模式导致的细胞体和纯轴突网络（ 图3D），这类似于在脑皮层的灰质和白质的条块分割。 除了 ​​显微镜，所述构建体可以用各种其他方法18来评估，根据实验背后的问题。例如，DNA，RNA和蛋白质从构建的隔离可以容易地使用商业试剂盒（图4）执行的。核酸和蛋白质的pe屈服ř构建取决于细胞初始接种于丝支架的数量。越细胞接种的DNA，RNA和蛋白质的产率越高。 <img alt="图1" src="/files/ftp_upload/52970/52970fig1.jpg" /> 用于丝绸海绵支架制备图1. PTFE模具的尺寸:10厘米直径的2厘米高<a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52970/52970fig2.jpg" /> 图2. 3D脑状组织模式。（A）多孔丝海绵支架。 （ 二）现场/死亡的神经元，在1天播种后染色丝绸支架前胶原嵌入（绿活细胞，红死细胞）。右侧面板显示的马红色帧拍摄区gnification。 <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52970/52970fig3.jpg" /> 图3.神经元在大脑的三维状组织模式条块分割的产物（A）脚手架车厢:红细胞体室，蓝轴突间室。 （ 二）在胶原蛋白嵌入1日前日向接种神经元的生长方式。 （C）。在接种在细胞体内室神经元向外生长7天。 （ 四）播种后神经生长在第7天，在轴突间室。绿色- βIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">请点击此处查看这是一个更大的版本</a>数字。 <img alt="图4" src="/files/ftp_upload/52970/52970fig4.jpg" /> （一）RNA和DNA，和（B）与细胞每支架播种量每结构蛋白图4.预期收益率（±SD，N = 5）。 <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.5K 次观看.  Tufts University. Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

视频: Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

科研

癌症研究

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

神经科学

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

微凝胶-细胞外基质复合材料支持人类神经结构的嵌入式3D打印

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

iPSC 来源的神经元和星形胶质细胞的 3D 生物打印共培养物

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

作者聚焦:推进 3D 细胞建模 – 一种用于神经共培养的高通量方法 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

3D 打印在微型神经元细胞培养设备中的应用

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

作者聚焦:用于分析大脑功能的模块化神经元网络

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

微尺度神经元细胞培养装置的双光子聚合3D打印

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

摘要

探索更多视频

使用胶原基支架的神经母细胞瘤三维体外仿生模型

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

接口3D工程神经细胞微电极阵列:一种创新

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

微凝胶-细胞外基质复合材料支持人类神经结构的嵌入式3D打印

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

微尺度神经元细胞培养装置的双光子聚合3D打印

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

摘要

探索更多视频

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

接口3D工程神经细胞微电极阵列:一种创新

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

微凝胶-细胞外基质复合材料支持人类神经结构的嵌入式3D打印

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

微尺度神经元细胞培养装置的双光子聚合3D打印

使用胶原基支架的神经母细胞瘤三维体外仿生模型