Name: preparation of neuronal co-cultures with single cell precision
Uploaded: 2014-05-20T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

लेखकों SEM इमेजिंग के लिए SU-8 निर्माण और मारिया बेकर (ISAs) के लिए Ulrich Marggraf (ISAs) के लिए आभारी हैं. अनुसंधान आर्थिक रूप से ड्यूश Forschungsgemeinschaft (DFG WE3737/3-1), एक Bundesministerium फर Bildung und Forschung अनुदान (BMBF 0101-31P6541) द्वारा और Ministerium फर अभिनव, Wissenschaft und Forschung डेस लैंडेस Nordrhein-Westfalen द्वारा समर्थित किया गया. Heike Hardelauf धन्यवाद अंतर्राष्ट्रीय लाइबनिट्स ग्रेजुएट स्कूल &quot;सिस्टम्स बायोलॉजी लैब पर एक चिप&quot; वित्तीय सहायता के लिए.

प्रोटोकॉल neuroscientists के लिए इरादा कर रहे हैं, और चित्रा 2 में संक्षेप हैं. जैसे यह व्यावसायिक या संस्थागत सुविधाओं को आउटसोर्स है PDMS प्रतिकृति के लिए इस्तेमाल किया SU-8 स्वामी की उस microfabrication की सलाह देते है. ?...

<ol>
	<li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+control+of+neurite+development+by+nerve+growth+factor.">Local control of neurite development by nerve growth factor.</a> Proc Natl Acad Sci USA. 74 (10), 4516-4519 (1997).</li><li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sympathetic+neurons+in+compartmentalized+cultures.+I.+Local+control+of+neurite+growth+by+nerve+growth+factor.">Development of sympathetic neurons in compartmentalized cultures. I. Local control of neurite growth by nerve growth factor.</a> Dev Biol. 93 (1), 1-12 (1982).</li><li>Taylor, 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=Microfluidic+multicompartment+device+for+neuroscience+research.">Microfluidic multicompartment device for neuroscience research.</a> Langmuir. 19, 1551-1556 (2003).</li><li>Taylor, 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=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2 (8), 559-565 (2005).</li><li>Kilinc, D., 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=Wallerian-like+degeneration+of+central+neurons+after+synchronized+and+geometrically+registered+mass+axotomy+in+a+three-compartmental+microfluidic+chip.">Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip.</a> Neurotox. Res. 19, 149-161 (2011).</li><li>Li, L., 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=Spatiotemporally+controlled+and+multifactor+involved+assay+of+neuronal+regeneration+after+chemical+injury+in+an+integrated+microfluidics.">Spatiotemporally controlled and multifactor involved assay of neuronal regeneration after chemical injury in an integrated microfluidics.</a> Anal. Chem. 84 (15), 6444-6453 (2012).</li><li>Kim, Y. T., Karthikeyan, K., Chirvi, S., Dave, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-optical+microfluidic+platform+to+study+injury+and+regeneration+of+single+axons.">Neuro-optical microfluidic platform to study injury and regeneration of single axons.</a> Lab Chip. 9, 2576-2581 (2009).</li><li>Hellman, A. N., 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=Examination+of+axonal+injury+and+regeneration+in+micropatterned+neuronal+culture+using+pulsed+laser+microbeam+dissection.">Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection.</a> Lab Chip. 10 (16), 2083-2092 (2010).</li><li>Kunze, A., 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=Co-pathological+connected+primary+neurons+in+a+microfluidic+device+for+Alzheimer+studies.">Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies.</a> Biotechnol. Bioeng. 108 (9), 2241-2245 (2011).</li><li>Liu, W. W., Goodhouse, J., Jeon, N. L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+chamber+for+analysis+of+neuron-to-cell+spread+and+axonal+transport+of+an+alpha-herpesvirus.">A microfluidic chamber for analysis of neuron-to-cell spread and axonal transport of an alpha-herpesvirus.</a> PLoS One. 3 (6), (2008).</li><li>Markus, A., 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=Varicella-Zoster+virus+(VZV)+infection+of+neurons+derived+from+human+embryonic+stem+cells:+Direct+demonstration+of+axonal+infection,+transport+of+VZV,+and+productive+neuronal+infection.">Varicella-Zoster virus (VZV) infection of neurons derived from human embryonic stem cells: Direct demonstration of axonal infection, transport of VZV, and productive neuronal infection.</a> J. Virol. 85 (13), 6220-6233 (2011).</li><li>Dinh, N. D., 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=Microfluidic+construction+of+minimalistic+neuronal+co-cultures.">Microfluidic construction of minimalistic neuronal co-cultures.</a> Lab Chip. 13 (7), 1402-1412 (2013).</li><li>Tan, W. H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trap-and-release+integrated+microfluidic+system+for+dynamic+microarray+applications.">A trap-and-release integrated microfluidic system for dynamic microarray applications.</a> Proc. Natl. Acad. Sci. USA. 104 (4), 1146-1151 (2007).</li><li>Frimat, J. -. P., 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=A+microfluidic+array+with+cellular+valving+for+single+cell+co-culture.">A microfluidic array with cellular valving for single cell co-culture.</a> Lab Chip. 11 (2), 231-237 (2011).</li><li>Di Carlo, D., Aghdam, N., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+enzyme+concentrations,+kinetics,+and+inhibition+analysis+using+high-density+hydrodynamic+cell+isolation+arrays.">Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays.</a> Anal. Chem. 78 (14), 4925-4930 (2006).</li><li>Frimat, J. P., 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=The+network+formation+assay:+A+spatially+standardized+neurite+outgrowth+analytical+display+for+neurotoxicity+screening.">The network formation assay: A spatially standardized neurite outgrowth analytical display for neurotoxicity screening.</a> Lab Chip. 10, 701-709 (2010).</li><li>Hardelauf, H., 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=High+fidelity+neuronal+networks+formed+by+plasma+masking+with+a+bilayer+membrane:+analysis+of+neurodegenerative+and+neuroprotective+processes.">High fidelity neuronal networks formed by plasma masking with a bilayer membrane: analysis of neurodegenerative and neuroprotective processes.</a> Lab Chip. 11 (16), 2763-2771 (2011).</li><li>Whitesides, G. 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=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annu. Rev. Biomed. Eng. 3, 335-373 (2001).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Frimat, J. P., 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=Plasma+stencilling+methods+for+cell+patterning.">Plasma stencilling methods for cell patterning.</a> Anal. Bioanal. Chem. 395 (3), 601-609 (2009).</li><li>Huang, N. P., 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=Poly(L-lysine)-g-poly(ethylene+glycol)+layers+on+metal+oxide+surfaces:+surface-analytical+characterization+and+resistance+to+serum+and+fibrinogen+adsorption.">Poly(L-lysine)-g-poly(ethylene glycol) layers on metal oxide surfaces: surface-analytical characterization and resistance to serum and fibrinogen adsorption.</a> Langmuir. 17, 489-498 (2001).</li><li>Li, N., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+topographical+and+biochemical+cues+by+axons+during+growth+on+microfabricated+3-D+substrates.">Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates.</a> Exp. Cell Res. 311 (2), 307-316 (2005).</li><li>Huang, L. R., Cox, E. C., Austin, R. H., Sturm, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+particle+separation+through+deterministic+lateral+displacement.">Continuous particle separation through deterministic lateral displacement.</a> Science. 304 (5673), 987-990 (2004).</li><li>Yang, Z., 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=A+review+of+nanoparticle+functionality+and+toxicity+on+the+central+nervous+system.">A review of nanoparticle functionality and toxicity on the central nervous system.</a> J. R. Soc. Interface. 7, (2010).</li><li>Brenneman, K. A., 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=Direct+olfactory+transport+of+inhaled+manganese+((MnCl2)-Mn-54)+to+the+rat+brain:+Toxicokinetic+investigations+in+a+unilateral+nasal+occlusion+model.">Direct olfactory transport of inhaled manganese ((MnCl2)-Mn-54) to the rat brain: Toxicokinetic investigations in a unilateral nasal occlusion model.</a> Toxicol. Appl. Pharm. 169 (3), 238-248 (2000).</li><li>Magal&#227;es, A. C., 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=Uptake+and+neuritic+transport+of+scrapie+prion+protein+coincident+with+infection+of+neuronal+cells.">Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells.</a> J. Neurosci. 25 (21), 5207-5216 (2005).</li><li>Diogenes, M. J., 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=Extracellular+alpha-synuclein+oligomers+modulate+synaptic+transmission+and+impair+LTP+via+NMDA-receptor+activation.">Extracellular alpha-synuclein oligomers modulate synaptic transmission and impair LTP via NMDA-receptor activation.</a> J Neurosci. 32 (34), 11750-11762 (2012).</li><li>Egger, B., 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=In+vitro+imaging+of+primary+neural+cell+culture+from+Drosophila.">In vitro imaging of primary neural cell culture from Drosophila.</a> Nat. Protoc. 8 (5), 958-965 (2013).</li></ol>

microfluidic arraying तकनीक न्यूनतर संस्कृतियों की स्थापना के लिए सटीक एक न्यूरॉन से निपटने में सक्षम बनाता है कि अपनी तरह का पहला है. Microfluidic संरचनाओं के साथ सेल पैटर्न के लिए पंक्ति में सीटू biomaterial patterning विधि में, ?...

<html> Neuronal ऊतक अत्यधिक जटिल है; स्थानिक सेल संपर्क और विशेष रूप से अक्षतंतु और dendrite outgrowths के माध्यम से के माध्यम से परिभाषित परतों और डिब्बों के भीतर और प्लास्टिक कनेक्टिविटी के साथ आदेश दिया है कि एक विषम सेलुलर मिश्रण. नई तकनीक गहरी अंतर्दृष्टि और रोग, विकास, और स्वस्थ समारोह के लिए केंद्रीय सुलझाना तंत्र हासिल करने के लिए अधिक से अधिक प्रयोगात्मक स्वतंत्रता प्रदान करने के लिए आवश्यक हैं. Campenot कक्ष 1,2 और अधिक हाल ही में microfabricated embodiments 3,4 चुनिंदा विभिन्न दैहिक आबादी उपद्रव और भी उनके neurite outgrowths करने की क्षमता के साथ नेटवर्क neuronal सह संस्कृतियों के पूर्व vivo तैयारी के लिए इस्तेमाल किया जा सकता है. ये microfluidic उपकरणों उदाहरण के लिए रासायनिक 5,6 या लेजर axotomy 6-8, tauopathy 9, वायरल प्रसार 10,11, और axons 4 में mRNA स्थानीयकरण निम्नलिखित अक्षतंतु अध: पतन और उत्थान अध्ययन करने के लिए इस्तेमाल किया गया है. ईएनटी &quot;&gt; Neurobiologists की पहुंच का विस्तार, तकनीकी विकास न्यूनतर neuronal सह संस्कृतियों तैयार करने के लिए आवश्यक हैं. इस एकल कक्ष और उप सेलुलर परिशुद्धता के साथ प्रणाली की जांच के लिए neuronal नेटवर्क के सुलझाव सक्षम बनाता है. न्यूनतम सेल नंबर के लिए आवश्यकता पार्किंसंस रोग, कान से सर्पिल ganglia, परिधीय न्यूरॉन्स के लिए प्रासंगिक डोपामिनर्जिक substantia nigra कोशिकाओं सहित दुर्लभ प्रकार की कोशिकाओं, विश्लेषण, और स्टेम कोशिकाओं के लिए संभावना को खोलता है. इस के अलावा, सेलुलर अर्थव्यवस्था 3Rs पहल के लिए प्रासंगिक है. इन microfluidic प्लेटफार्मों, बड़े पैमाने पर विषाक्तता स्क्रीन या अन्य उच्च throughput का प्रयोग, पशु न्यूरॉन्स की आवश्यकता डेटा अमीर प्रयोगात्मक श्रृंखला अब माना जा सकता है. इस पत्र में एक microfluidic डिवाइस के निर्माण और उपयोग के लिए प्रोटोकॉल में वर्णित हैं. सीटू biomateri में एक साथ संयोजन में microfluidic arrayingअल patterning विधि न्यूनतम सेल नंबर का उपयोग अत्यधिक परस्पर neuronal सह संस्कृतियों के पंजीकरण के लिए इस्तेमाल किया जा सकता है. Microfluidic arraying एक अंतर प्रवाह दृष्टिकोण microstructured जाल एक fluidic सर्किट (चित्र 1 में SEM छवियों के साथ सचित्र) के भीतर तैनात हैं जिससे 12-15, पर आधारित है. neurite परिणाम चैनलों को inlets - पथ 0 → 1 microstructured apertures की एक रैखिक सरणी के लिए न्यूरॉन्स के परिवहन के लिए कम fluidic प्रतिरोध (नि. 2&gt; आर 1) है. एक एकल कोशिका से जाल की रिहाइश स्थानीय स्तर पर पड़ोसी जाल में बाद में कोशिकाओं को फँसाने के लिए सुव्यवस्थित हटाने के प्रवाह में बाधा उत्पन्न करती. सरणी में जाल की पूरी अधिभोग अतिरिक्त न्यूरॉन्स को हटाने के लिए ऑपरेशन का एक बाईपास मोड के उत्पादन के लिए चक्करदार पथ (0 → 2) में सुव्यवस्थित हटाने की fluidic अनुपात (आर 1&gt; आर 2) स्विच. चित्रा 1. Microfluidic सर्किट. Neurite परिणाम चैनलों से जुड़े संस्कृति कक्षों flanking साथ एक न्यूरॉन arraying के लिए ए) अंतर प्रतिरोध fluidic सर्किट,. बी, meniscus लगाए micropillars साथ bilayer बंटे न्यूरॉन सह संस्कृति सरणी के सी) SEM छवियों. इस डिजाइन के साथ, त्रिशूल के आकार का न्यूरॉन फँसाने संरचनाओं neurite outgrowths का स्फुरण बढ़ावा देने के लिए इस्तेमाल किया गया. चित्रा और रसायन विज्ञान की रॉयल सोसायटी (आरएससी) की अनुमति के साथ reproduced कथा 12. <a href="https://www.jove.com/files/ftp_upload/51389/51389fig1highres.jpg" target="_blank">बड़ी छवि को देखने के लिए यहां क्लिक करें.</a> तलीय substrates पर micropatterned neuronal नेटवर्क की तैयारी आसानी exampl के लिए (प्राप्त किया जा सकता है<html> हमारे समूह से तों, Frimat एट अल. 16 और Heike एट अल. 17) देखें. हालांकि, PDMS उपकरणों के भीतर और microfluidic चैनलों के लिए इनमें से सुक्ष्ममापी पैमाने संरेखण की आवश्यकता के साथ bioactive सामग्री पैटर्न encapsulating एक प्रमुख तकनीकी चुनौती बन गया है. खंड 3.1 में के लिए एक प्रोटोकॉल में चिप, या बगल में, biomaterial पैटर्न की तैयारी में प्रस्तुत किया है. इन नमूनों लंबा संस्कृति timescales दौरान न्यूरॉन पंजीकरण सक्षम और डिब्बों के बीच outgrowths को बढ़ावा देने. Meniscus-लगाए microstructures न्यूरॉन साइटों और neurite परिणाम चैनलों arraying साथ एक तथाकथित पानी मुखौटा संरेखित करने के लिए उपयोग किया जाता है. सतहों उजागर biomaterial स्वरूप को परिभाषित करने के लिए विघटित कर रहे हैं, जबकि पानी का मुखौटा, प्लाज्मा उपचार के दौरान आसंजन अणु कोटिंग्स की रक्षा करता है. इसके अलावा, प्रोटोकॉल सेल संस्कृति के लिए और विभिन्न सह संस्कृति डिब्बों के चुनिंदा उपचार के लिए आवश्यक fluidic अलगाव के लिए प्रदान की जाती हैं. ve_content &quot;&gt; प्रोटोकॉल इसी तरह, सीटू biomaterial patterning में वाष्पीकरण और सतह तनाव घटना का शोषण, सीधा है. पाली की प्रतिकृति (dimethylsiloxane) (PDMS) microfluidic उपकरणों 18 के लिए नरम लिथोग्राफी के उपयोगकर्ता के अनुकूल सिद्धांतों का लाभ उठाने के लिए तैयार है, और कर रहे हैं केवल एक सस्ती हाथ से आयोजित प्लाज्मा स्रोत की आवश्यकता होती है. microfluidic सर्किट इन आपरेशनों बनाने को प्रभावी ढंग से कार्यक्रमों सेल लदान और डिब्बे विशिष्ट उपचार बस सही नीचे पोर्ट में सामग्री वितरण और ऊपर से aspirating की बात है. इस तरीके में, यह neurobiologists देने का इरादा है उनकी अपनी प्रयोगशालाओं में microfluidic उपकरणों को तैयार करने और उपयोग करने के लिए स्वतंत्रता.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PDMS Sylgard 184</td>
			<td style="width:163px;">Dow Corning</td>
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			<td height="21" style="height:21px;">PDMS Elastosil RT 601</td>
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			<td height="21" style="height:21px;width:235px;">Coverslips</td>
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			<td>130-160 mm thick</td>
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			<td height="21" style="height:21px;width:235px;">3 mm Biopsy Punches</td>
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			<td height="21" style="height:21px;">Tygon Tubing</td>
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			<td>1.65 mm ID; 3.35 mm OD</td>
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			<td height="21" style="height:21px;">1 mL Syringe (Inkjekt and Omnifix)</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:235px;">4-Way Tubing Connector</td>
			<td style="width:163px;">VWR or Fisher Scientific</td>
			<td style="width:143px;"></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Flow Regulator</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:143px;">722645</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:235px;">0.5 mm Pins</td>
			<td style="width:163px;">Dressmaking Departments</td>
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			<td height="42" style="height:42px;width:235px;">PLL-g-PEG</td>
			<td style="width:163px;">SuSoS</td>
			<td style="width:143px;">PLL(20)-g[3.5]-PEG(5)</td>
			<td>Stability in storage can be an issue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6407</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-lysine-FITC</td>
			<td style="width:163px;">Sigma-Aldrich</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-ornithine</td>
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			<td height="21" style="height:21px;width:235px;">fibronectin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">laminin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">L2020</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Inverted Fluorescent Microscope</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Example Aspiration Pump</td>
			<td style="width:163px;">KNF Neuberger, Laboport</td>
			<td style="width:143px;">N811KVP</td>
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		</tr>
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			<td height="42" style="height:42px;width:235px;">Hand Held Corona Discharge Device</td>
			<td style="width:163px;">Leybold-Heraeus, USA</td>
			<td style="width:143px;">VP23</td>
			<td>May not comply with your country&#39;s safety standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Femto Plasma Oven</td>
			<td style="width:163px;">Diener Electronic</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Vacuum Dessicator or Centrifuge</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of neuronal co-cultures with single cell precision

Campenot चैंबर के microfluidic embodiments तंत्रिका विज्ञान समुदाय से महान ब्याज को आकर्षित किया है. इन परस्पर सह संस्कृति प्लेटफार्मों संक्रमण और बीमारी प्रचार करने के लिए विकास और कार्यात्मक तंत्रिका जीव विज्ञान फैले, सवालों की एक किस्म की जांच करने के लिए इस्तेमाल किया जा सकता है. हालांकि, पारंपरिक प्रणालियों महत्वपूर्ण सेलुलर ऐसे प्राथमिक डोपामिनर्जिक substantia nigra, सर्पिल ganglia, और Drosophilia मेलानोगास्टर न्यूरॉन्स के रूप में कम बहुतायत कोशिकाओं के अध्ययन के लिए अपर्याप्त आदानों (डिब्बे प्रति कई हजारों),,, और उच्च throughput प्रयोग के लिए अव्यावहारिक आवश्यकता होती है. घने संस्कृतियों भी अत्यधिक स्थानीय स्तर पर दो संस्कृतियों के परस्पर कुछ outgrowths (&lt;10%) के साथ, उलझ रहे हैं. (मैं) एक microfluidic न्यूरॉन विधि arraying, और प्लाज्मा patterning biomaterial कोटिंग्स regi करने के लिए (द्वितीय) एक पानी मास्किंग विधि: इस पत्र में सीधा microfluidic और patterning प्रोटोकॉल इन चुनौतियों से निपटने के जो वर्णन किया गया हैंन्यूरॉन्स ster और डिब्बों के बीच परिणाम को बढ़ावा देने. न्यूनतर neuronal सह संस्कृतियों उच्च स्तर (&gt; 85%) intercompartment कनेक्टिविटी के साथ तैयार थे और एकल कक्ष परिशुद्धता के साथ उच्च throughput तंत्रिका जीव विज्ञान के प्रयोगों के लिए इस्तेमाल किया जा सकता है.

जल मास्किंग कारनामे तनाव सतह. हवा तरल इंटरफेस पर दबाव सहिष्णुता वक्रता [की त्रिज्या के साथ inversely तराजू <img fo:content-width="1in" src="files/ftp_upload/51389/51389eq1.jpg" width="113" /> , Microfluidic आयामों पर अत्यधिक स्थिर इंटरफेस का निर्माण किया. micropillars प्रभाव?...

Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

Preparation of Neuronal Co-cultures with Single Cell Precision

एक न्यूरॉन microfluidic arraying और biomaterial कोटिंग्स में चिप प्लाज्मा patterning के लिए पानी मास्किंग के लिए प्रोटोकॉल में वर्णित हैं. अत्यधिक परस्पर सह संस्कृतियों न्यूनतम सेल आदानों का उपयोग कर तैयार किया जा सकता है.

सिंगल सेल प्रेसिजन साथ Neuronal सह संस्कृति की तैयारी

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

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Neuroscience

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

युगपत वोल्टेज दबाना और Amperometry साथ dopamine जारी की एकल कोशिका मापन

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

एकल कोशिका और एकल कील संकल्प के साथ suprathreshold तंत्रिका गतिविधि के ऑप्टिकल रिकॉर्डिंग

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

एक साथ पूर्व और बाद synaptic electrophysiological रिकॉर्डिंग से<em&gt; Xenopus</em&gt; तंत्रिका पेशी सह संस्कृतियों

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

पशु व्यवहार जाग में एकल इकाई रिकॉर्डिंग के लिए microwires की सरणियों का आयोजन दाखिल के लिए एक प्रक्रिया

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Neuronal फायरिंग दरों के हेरफेर से astrocytic रिसेप्टर्स की Plasticity उत्प्रेरण

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

मल्टी इलेक्ट्रोड सारणियों पर हो Organotypic स्पाइनल कॉर्ड सह संस्कृतियों में कार्यात्मक उत्थान की जांच कर

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Neuronal गतिविधि के बाह्य रिकॉर्डिंग जाग चूहों में neuroactive पदार्थों के Microiontophoretic आवेदन के साथ संयुक्त

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

गर्भाशय में Electroporation Neuronal उप-जनसंख्या के excitability और एकल कोशिका कनेक्टिविटी अध्ययन के लिए दृष्टिकोण

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

न्यूरॉन-मैक्रोफेज सह संस्कृतियों सक्रिय करने के लिए मैक्रोफेज Neurite के साथ आणविक कारकों स्रावित गतिविधि

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

रक्त-व्युत्पन्न न्यूरोनल कोशिकाओं की जीएम-मुक्त पीढ़ी

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...