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).

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इस वीडियो लेख के प्रकाशन कॉर्निंग द्वारा प्रायोजित है।

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.

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&gt;। 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।

<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.

डोनट आकार के लिए ठोस रेशम स्पंज precut तंत्रिका ऊतक (2A चित्रा) जैसी एक बंटे वास्तुकला प्राप्त करने के लिए एक अद्वितीय और सरल विचार प्रतिनिधित्व करते हैं। एक छोटी मात्रा के भीतर बोने और उच्च घनत्व सेल के विकास की अनुमति के असाधारण उच्च सतह क्षेत्र में लगभग 500 माइक्रोन परिणामों के छेद के आकार के साथ पाड़ की अत्यधिक झरझरा संरचना (2x10 7 / एमएल) (चित्रा 2 बी)। इसके अतिरिक्त, पाड़ के उच्च porosity बढ़ाया समय तख्ते पर घने सेल संस्कृतियों के बेहतर व्यवहार्यता में जिसके परिणामस्वरूप पोषक तत्वों और कचरे के अप्रतिबंधित प्रसार के लिए अनुमति देता है। न्यूरॉन्स बोने पर तेजी से और समान पाड़ छिद्रों की सतह के लिए देते हैं। सेल लगाव पूरा हो गया है और कोशिकाओं रेशम सब्सट्रेट पर equilibrated हैं, के बाद स्पंज पाड़ axonal नेटवर्क के गठन (चित्रा 3 के लिए एक 3 डी वातावरण प्रदान करने के लिए मुलायम कोलेजन मैट्रिक्स से भर जाता है </strong&gt;)। न्यूरॉन्स केवल 2 डी सतहों (चित्रा 3 बी) के साथ मिल गया के रूप में मिश्रित नेटवर्क में जिसके परिणामस्वरूप छिद्रों की सतह पर एक्सटेंशन के रूप में विकसित कोलेजन मैट्रिक्स के अभाव में एक्सोन और सेल शरीर के compartmentalization संभव नहीं है। इसके विपरीत, कोलेजन जेल neuronal सेल निकायों रेशम पाड़ (चित्रा 3 सी) में जुड़े रहते हैं, जबकि 3 डी में व्यापक axonal परिणाम का समर्थन करता है जो एक meshwork प्रदान करता है। इस विकास पैटर्न सेल निकायों और शुद्ध axonal नेटवर्क मस्तिष्क में कॉर्टेक्स के ग्रे और सफेद पदार्थ जो जैसा दिखता है (चित्रा 3 डी), के compartmentalization की ओर जाता है। इसके अलावा माइक्रोस्कोपी से, निर्माणों प्रयोग के पीछे के सवाल पर निर्भर करता है, अन्य तरीकों 18 की एक किस्म के साथ मूल्यांकन किया जा सकता है। उदाहरण के लिए, निर्माणों से डीएनए, आरएनए और प्रोटीन के अलगाव आसानी से हो वाणिज्यिक किट (चित्रा 4) का उपयोग किया जा सकता है। न्यूक्लिक एसिड और प्रोटीन पे की उपजआर निर्माण शुरू में रेशम मचानों पर वरीयता प्राप्त कोशिकाओं की संख्या पर निर्भर करता है। अधिक कोशिकाओं वरीयता प्राप्त डीएनए, आरएनए और प्रोटीन की उपज अधिक है। <img alt="चित्र 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> रेशम स्पंज पाड़ तैयारी के लिए इस्तेमाल चित्रा 1. PTFE मोल्ड आयाम:।। 10 सेमी व्यास, 2 सेमी ऊंचाई <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">यह आंकड़ा का एक बड़ा संस्करण देखने के लिए यहां क्लिक करें।</a> <img alt="चित्र 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> चित्रा 2. 3 डी मस्तिष्क की तरह ऊतक मॉडल। (ए) झरझरा रेशम स्पंज पाड़। इससे पहले कोलेजन (हरा-जीवित कोशिकाओं, लाल मृत कोशिकाओं) embedding रेशम पाड़ पर बोने पर 1 दिन में न्यूरॉन्स की (बी) लाइव / मृत धुंधला हो जाना। सही पक्ष पर पैनल मा दिखानेलाल फ्रेम में कब्जा क्षेत्र के gnification। <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">यह आंकड़ा का एक बड़ा संस्करण देखने के लिए यहां क्लिक करें।</a> <img alt="चित्र तीन" src="/files/ftp_upload/52970/52970fig3.jpg" /> चित्रा 3. Neuronal 3D मस्तिष्क की तरह ऊतक मॉडल में बंटे परिणाम (ए) पाड़ डिब्बों:। लाल सेल शरीर डिब्बे, ब्लू-axonal डिब्बे। (बी) कोलेजन एम्बेडिंग से पहले बोने पर 1 दिन में neuronal विकास पैटर्न। (सी)। सेल शरीर के डिब्बे में बोने पर दिन 7 में neuronal परिणाम। (डी) axonal डिब्बे में बोने पर दिन 7 में neuronal परिणाम। ग्रीन -। ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">इस का एक बड़ा संस्करण देखने के लिए यहां क्लिक करें</a>आंकड़ा। <img alt="चित्रा 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> (ए) आरएनए और डीएनए, और पाड़ प्रति वरीयता प्राप्त कोशिकाओं की राशि के संबंध में निर्माण प्रति (बी) प्रोटीन की चित्रा 4. उम्मीद उपज (एसडी ± n = 5)। <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">यह आंकड़ा का एक बड़ा संस्करण देखने के लिए यहां क्लिक करें ।</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.

फूट डालना तंत्रिका ऊतक के इंजीनियर 3 डी सिल्क कोलेजन आधारित मॉडल

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).

इलेक्ट्रिक फील्ड नियंत्रित 2 डी और 3 डी वातावरण में तंत्रिका कोशिकाओं पूर्वज निर्देशित प्रवासन

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

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).

ऊतक के मूल्यांकन के लिए चुंबकीय अनुनाद Elastography पद्धति इंजीनियर निर्माण विकास

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.

सप्ताह में तीन पत्रक पॉलिमर वाल्व के सापेक्ष द्रव्यगतिकी आकलन के लिए प्रोटोकॉल

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.

एक मानव 3 डी के ऊतक इंजीनियरिंग<em&gt; इन विट्रो</em&gt; ट्यूमर परीक्षा प्रणाली

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.

एक माउस मॉडल में ऊतक इंजीनियर वेसल्स का सृजन और कलम बांधने का काम

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.

एक 3 डी ऊतक इंजीनियर मानव इसोफेजियल mucosal मॉडल का उत्पादन, लक्षण और क्षमता का उपयोग करता

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.

एक संयुक्त 3 डी ऊतक इंजीनियर<em&gt; इन विट्रो</em&gt; /<em&gt; सिलिको</emविशिष्ट mutational पृष्ठभूमि में ड्रग प्रभावशीलता की भविष्यवाणी के लिए&gt; फेफड़े ट्यूमर मॉडल

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.

उपास्थि और त्वचा ऊतक Bioink Precellularization के लिए एक उपंयास निष्क्रिय मिश्रण इकाई तकनीक के उपयोग के अनुरूप का मुद्रण

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.

एक माउस मॉडल में एक जैव सिंथेटिक ऊतक इंजीनियर Tracheal भ्रष्टाचार के बोने और आरोपण

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.

चूहों में परिचारक एसोफेगल पुनर्निर्माण के लिए ऊतक-इंजीनियर भ्रष्टाचार

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