极化神经组织工程三维蚕丝胶原蛋白为基础的模式
Summary
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.
Abstract
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.
Introduction
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>。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。
Protocol
Representative Results
Discussion
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.
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Na2CO3 | Sigma-Aldrich | 223530 | – |
| LiBr | Sigma-Aldrich | 213225 | – |
| MWCO 3500 dialysis cassettes | Thermo Fisher | 66110 | – |
| Heating plate | Fisher Scientific | Isotemp | – |
| Centrifuge 5804 R | Eppendorf | – | – |
| Sieve | Fisher Scientific | – | No. 270, No. 35, No. 30 |
| PTFE mold | – | – | made in house (Figure 1) 10 cm diameter, 2 cm height |
| NaCl | Sigma-Aldrich | 71382 | – |
| Biopsy Punch 5mm | World Precision Instrument | 501909 | – |
| Biopsy Punch 2mm | World Precision Instrument | 501908 | – |
| poly-D-lysine | Sigma-Aldrich | P6407-5MG | final concentration 10 ug/ml, dissolved in water |
| PBS | Sigma-Aldrich | D1283-500ML | – |
| Trypsin | Sigma-Aldrich | T4049-500ML | – |
| DNase | Roche | 10104159001 | final concentration 0.3% |
| Soybean protein | Sigma-Aldrich | T6522-100MG | final concentration 1 mg/ml |
| Neurobasal medium | Gibco | 21103049 | warm up to 37°C before use |
| B27 supplement 50x | Gibco | 17504-044 | – |
| Glutamax | Gibco | 35050-061 | – |
| Penicilin Streptomycin | Corning | 30-002-CI | – |
| Collagen I, rat tail, 100 mg | Corning | 354236 | final concentration 3 mg/ml |
| NaOH | Sigma-Aldrich | S2770 | corrosive |
| Propidium Iodide | Sigma-Aldrich | P4170-10MG | toxic |
| Fluorescein Diacetate | Sigma-Aldrich | F7378-5G | – |
| Olympus MVX10 | Olympus | – | – |
| Paraformaldehyde | Sigma-Aldrich | P6148 | toxic, final concentration 4% |
| Bovine Serum Albumin | Sigma-Aldrich | A7906-10G | – |
| anti-βIIITubulin antibody | Sigma-Aldrich | T2200 | rabbit 1:500 |
| anti-rabbit Alexa-546 | Molecular Probes | A11010 | goat 1:250 |
| goat serum | Sigma-Aldrich | G9023-5ML | – |
| Leica SP2 confocal microscope | Leica | – | objective 20x |
| QIAshredder | Qiagen | 79654 | – |
| AllPrep DNA/RNA/Protein Mini Kit | Qiagen | 80004 | – |
| Ethyl alcohol, Pure | Sigma-Aldrich | E7023 | – |
| 2-Mercaptoethanol | Sigma-Aldrich | 63689 | |
| NanoDrop 2000c/2000 UV-Vis Spectrophotometer | Thermo Scientific | – | – |
| BCA protein assay | Thermo Scientific | 23225 | – |
| Plate reader Spectramax M2 | Molecular Devices | – | absorbance 562 nm |
| Micro Scissors, Economy, Vannas-type | TedPella | 1346 | – |
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