استنادا الحرير الكولاجين المهندسة 3D نموذج الاستقطاب العصبية الأنسجة
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
الجهاز العصبي المركزي (CNS) يمكن أن تتأثر مجموعة متنوعة من الاضطرابات التي تنطوي على الأوعية الدموية، الهيكلية والوظيفية، المعدية أو التنكسية. ما يقدر بنحو 6.8 مليون شخص يموتون سنويا نتيجة للاضطرابات العصبية، وهو ما يمثل عبئا الاجتماعي والاقتصادي المتزايد في جميع أنحاء العالم 1. ومع ذلك، سوى عدد قليل من اضطرابات الحصول على العلاجات المتاحة. ولذلك، هناك حاجة ماسة لوضع استراتيجيات علاجية مبتكرة للمرضى الذين يعانون من الاضطرابات العصبية. للأسف، فشلت العديد من العلاجات CNS المستهدفة خلال التجارب السريرية، ويرجع ذلك جزئيا إلى الاستفادة من عدم كفاية نماذج الأبحاث ما قبل السريرية، التي لا تسمح بتقييم الآثار الحادة والمزمنة مع قراءات من الناحية الفسيولوجية الوظيفية ذات الصلة.
على الرغم من الجهود البحثية الهامة على مدى العقود الماضية، هناك كمية هائلة مجهولة حول بنية ووظيفة الجهاز العصبي المركزي. من أجل كسب المزيد من المعرفة، نماذج حيوانية هي في كثير من الأحيان لناإد لنموذج الحالات المرضية، مثل إصابات الدماغ (TBI) أو الخرف، وخاصة في الدراسات ما قبل السريرية. ومع ذلك، الحيوانات تختلف اختلافا كبيرا من البشر على حد سواء في تشريح الجهاز العصبي المركزي، وكذلك في وظيفة، التعبير الجيني والأيض 2-4. من ناحية أخرى، 2D الثقافات في المختبر هي طريقة شائعة للتحقيق في بيولوجيا الخلية وتستخدم بشكل روتيني لاكتشاف الأدوية. ومع ذلك، مزارع الخلايا 2D تفتقر إلى التعقيد والأهمية الفسيولوجية بالمقارنة مع الدماغ البشري 5-7. في حين لا يوجد بديل عن التكلفة المنخفضة وبساطة 2D دراسات ثقافة الخلية أو تعقيد المقدمة من النماذج الحيوانية، يمكن أن هندسة الأنسجة 3D توليد نماذج محسنة البحوث من أجل سد الفجوة القائمة بين 2D في التجارب المختبرية والنهج الجسم الحي. تقدم هندسة الأنسجة 3D الظروف التجريبية أكثر ذات الصلة من الناحية الفسيولوجية التي كتبها 3D التفاعلات خلية خلية والعظة خارج الخلية التي تقدمها السقالات بيولوجية تحقيقها. Despالفنار الأدلة كبير وراء قيمة الثقافات 3D، وهناك حاليا سوى عدد قليل 3D CNS نماذج الأنسجة مثل الجذعية المشتقة من خلية ثقافات عضوي الشكل 8-10، neurospheroids 11 ومشتتة الثقافات هيدروجيل 12،13. وقد تم استخدام الأساليب الفنية المتطورة بما في ذلك متعددة الطبقات الحجرية 14، و3D الطباعة 15 لدراسة الرئة والكبد وأنسجة الكلى. ومع ذلك، هناك عدم وجود نماذج 3D CNS التي تسمح للنمو الخلايا العصبية مجزأة، مثل محاكاة العمارة القشرية وعلم الأحياء. وقد تم فصل النمو من neurites التي من أجسام الخلايا العصبية سابقا أظهر في الثقافات 2D باستخدام التصنيع الدقيق 16،17 السماح للدراسة تتبع محور عصبي المسالك، تدفق الكالسيوم وهندسة الشبكات والأنشطة. ألهمتنا هذه الفكرة لتطوير الأنسجة العصبية الاستقطاب 3D حيث توجد أجسام الخلايا ومساحات محور عصبي في أماكن مختلفة تحاكي العمارة الطبقات من الدماغ 18 </sup>. ويستند نهجنا على استخدام فريدة من نوعها الحرير تصميم سقالة التي تستوعب كثافة عالية من الخلايا في حجم المحصورة ويسمح ثمرة من شبكة كثيفة محور عصبي إلى هلام الكولاجين. نحن هنا لشرح إجراءات التجميع الكامل لمثل الدماغ الأنسجة بما في ذلك تصنيع السقالة وثقافة العصبية.
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.
Divulgations
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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