Name: engineered 3d silk-collagen-based model of polarized neural tissue
Uploaded: 2015-10-23T14:00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

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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J., Park, J., Kim, H. J., Wada, M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+aqueous-derived+biomaterial+scaffolds+from+silk+fibroin.">Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin.</a> Biomaterials. 26 (15), 2775-2785 (2005).</li><li>Gil, E. S., Mandal, B. B., Park, S. H., Marchant, J. K., Omentetto, F. G., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicoidal+multi-lamellar+features+of+RGD-functionalized+silk+biomaterials+for+corneal+tissue+engineering.">Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering.</a> Biomaterials. 31 (34), 8953-8963 (2010).</li><li>Hopkins, A. 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=Silk+hydrogels+as+soft+substrates+for+neural+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+embryonic+neural+cells:+A+quick+protocol.">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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</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&#160;</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 (Figure 1) 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 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</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 ug/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 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;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 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 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-&#946;IIITubulin antibody&#160;</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&#160;</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

Research

JoVE Journal

Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...