फूट डालना तंत्रिका ऊतक के इंजीनियर 3 डी सिल्क कोलेजन आधारित मॉडल
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
केंद्रीय तंत्रिका तंत्र (सीएनएस), संरचनात्मक कार्यात्मक, संक्रामक या अपक्षयी नाड़ी से जुड़े विकारों की एक किस्म से प्रभावित हो सकते हैं। एक अनुमान के अनुसार 68 लाख लोग एक दुनिया भर में एक से बढ़ सामाजिक आर्थिक बोझ का प्रतिनिधित्व करता है जो मस्तिष्क संबंधी बीमारियों का एक परिणाम के रूप में हर साल मर जाते हैं। हालांकि, विकारों के केवल कुछ उपचार उपलब्ध है। इसलिए, मस्तिष्क संबंधी बीमारियों से पीड़ित रोगियों के लिए अभिनव चिकित्सकीय रणनीति के लिए एक महत्वपूर्ण आवश्यकता है। दुर्भाग्य से, कई सीएनएस-लक्षित चिकित्सा विज्ञान की वजह से physiologically प्रासंगिक कार्यात्मक readouts के साथ तीव्र और जीर्ण प्रभावों के मूल्यांकन की अनुमति नहीं है, जो अपर्याप्त पूर्व नैदानिक अनुसंधान मॉडल, का उपयोग करने के लिए भाग में, क्लिनिकल परीक्षण के दौरान विफल हो।
पिछले दशकों में महत्वपूर्ण अनुसंधान के प्रयासों के बावजूद, सीएनएस की संरचना और समारोह के बारे में अज्ञात एक विशाल राशि है। अधिक ज्ञान प्राप्त करने के लिए, पशु मॉडल अक्सर हम कर रहे हैंएड विशेष रूप से पूर्व नैदानिक अध्ययन में, इस तरह के घाव मस्तिष्क चोट (TBI) या पागलपन के रूप में रोग राज्यों, मॉडल करने के लिए। हालांकि, जानवरों से मनुष्यों में दोनों सीएनएस की शारीरिक रचना में, साथ ही समारोह, जीन अभिव्यक्ति और चयापचय 2-4 में काफी भिन्न होते हैं। दूसरी ओर, 2 डी में इन विट्रो संस्कृतियों कोशिका जीव विज्ञान की जांच करने के लिए आम तरीका कर रहे हैं और नियमित रूप से दवाओं की खोज के लिए उपयोग किया जाता है। हालांकि, 2 डी सेल संस्कृतियों जटिलता और मानव मस्तिष्क 5-7 की तुलना में शारीरिक प्रासंगिकता की कमी है। कम लागत और सादगी 2 डी सेल संस्कृति के अध्ययन की या पशु मॉडल द्वारा प्रदान की जटिलता के लिए कोई विकल्प नहीं है, वहीं 3 डी ऊतक इंजीनियरिंग विट्रो और इन विवो दृष्टिकोण में 2 डी के बीच मौजूद है कि अंतर को बंद करने के लिए सुधार अनुसंधान मॉडल उत्पन्न कर सकता है। 3 डी ऊतक इंजीनियरिंग 3 डी सेल सेल बातचीत और biomaterial मचानों द्वारा प्रदान की बाह्य संकेतों द्वारा हासिल की और physiologically प्रासंगिक प्रयोगात्मक शर्तों प्रदान करता है। सैर3 डी संस्कृतियों के मूल्य के पीछे महत्वपूर्ण सबूत ite, वहाँ इस तरह के स्टेम सेल व्युत्पन्न organoid संस्कृतियों 8-10 के रूप में केवल कुछ ही 3 डी सीएनएस ऊतक मॉडल, 11 neurospheroids वर्तमान में कर रहे हैं और हाइड्रोजेल संस्कृतियों 12,13 छितरी हुई है। बहुपरत लिथोग्राफी 14, और 3 डी प्रिंटिंग 15 सहित उन्नत तकनीकी विधियों फेफड़े, जिगर और गुर्दे ऊतक के अध्ययन के लिए उपयोग किया गया है। हालांकि, इस तरह cortical वास्तुकला और जीव विज्ञान की नकल उतार रूप में बंटे न्यूरोनल विकास के लिए अनुमति है कि 3 डी सीएनएस मॉडल, की कमी नहीं है। Neuronal सेल निकायों से neurites के अलग विकास पहले से अक्षतंतु पथ ट्रेसिंग, कैल्शियम बाढ़, नेटवर्क वास्तुकला और गतिविधियों के अध्ययन की अनुमति microfabrication 16,17 का उपयोग करके 2 डी संस्कृतियों में प्रदर्शन किया गया है। यह विचार सेल निकायों और axonal इलाकों मस्तिष्क 18 की स्तरीय वास्तुकला नकल उतार अलग डिब्बों में स्थित हैं, जहां एक 3 डी ध्रुवीकृत तंत्रिका ऊतक विकसित करने के लिए हमें प्रेरित </sup>। हमारा दृष्टिकोण एक सीमित मात्रा में कोशिकाओं के उच्च घनत्व accommodates और एक कोलेजन जेल में घने axonal नेटवर्क के परिणाम की अनुमति देता है जो अद्वितीय रेशम पाड़ डिजाइन के उपयोग पर आधारित है। यहाँ हम पाड़ निर्माण और neuronal संस्कृति सहित मस्तिष्क की तरह ऊतक से भरा विधानसभा प्रक्रिया प्रदर्शित करता है।
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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