Bu çalışma Royal Society URF hibe UF051616, Birleşik Krallık ve BS Avrupa Araştırma Konseyi StG hibe 243.261 tarafından desteklenmiştir. MZ laboratuvar çalışmaları da Rejeneratif Tıp hibe RB1-01.417 bir California Institute tarafından desteklenmektedir.

1. Sinir progenitör hücre izolasyonu <ol><li> Soğuk DMEM/F12 bazal orta E14-16 fareler ve yerden bütün beyinleri parçalara ayır. 35 mm petri içine anatomik bir mikroskop ve transfer beyinleri altındaki tüm meninksler çıkarın. </li><li> Mekanik doku parçaları içine beyinleri ayrışır ve 15 ml tüp aktarmak için ince pens kullanın, sonra enkaz kaldırmak için 3 dk süreyle 800 rpm&#39;de örnekleri santrifüj. </li><li> DMEM/F12 içeren bFGF ve EGF ekleyin ve 1 ml pipet ile çiğnemek. </li><li> Tek bir hücre süspansiyonu elde etmek için bir hücre süzgecinden hücre süspansiyonu geçmektedir. </li><li> 2-5 de ölçülü balona Plakalı hücreleri x 10 4 hücre / ml, sonra tam bir orta 3 gün ve geçiş hücreleri her 6 günde bir değiştirmek gerçekleştirmek. </li><li> En az 5 pasajlar sonra, özet neurospheres tek hücrelerin tripsin ve EDTA kullanılarak ve poly-D-lysine/laminin-coated electrotactic odalarına (2, aşağıda tarif edildiği gibi hazırlanmıştır) üzerinde yetişebilir. , N2-takviyesi içeren büyüyen orta kullanınbFGF ve EGF her zaman NPCs özellikleri sağlamak için. </li></ol> 2. Electrotactic haznesi hazırlanması <ol><li> Bir elmas kalem kullanarak yarısında 22 x 22 mm kalınlık no.1 lamelleri otoklava Bölünen ile camı 22 x 11 mm şeritler hazırlayın. </li><li> Yapıştırılarak de iç boyutları 22 x 10 mm ayaklı cam oluşturun birlikte dört dikey yüksek vakum silikon gres ile 22 x 11 mm şeritler ayakta. Tamamen kuruması için kuyu ve sertleşmesine gecede izin verin. </li><li> Ertesi gün, silikon yağı kullanılarak bir 100 mm kültürü çanağı baz, 10 mm arasında bir boşluk bırakarak birbirlerine paralel olarak, iki adet 22 x 11 mm ebadındaki bir cam şeritler takın. Merkeze yakın bir 22 x 22 mm kapak üç taraftan gres tabağın tabanına bağlı her iki ucunda kayma, fakat yerleştirerek bu şeritler arasındaki bölgede kapatmak. </li><li> Iyi kapak üzerine adım 2.2 hazırlanmış cam yerleştirin iç duvarları tohumlama için kapalı bir alan yaratmak böylece sığarplakanın alt üzerine hücreleri. Silikon gres Su geçirmez, tüm eklemleri. Coat poli-D-lizin sonra da laminin ile sırayla bu dar bir bölgeyi: haznesine 1 ml poli-D-lizin ekleyin ve poli-D-lizin plakanın alt bağlamak için izin verecek şekilde 5 dakika boyunca terk; haznesi yıkayıp Steril PBS ile iki kez, daha sonra 20 ug / ml verim ve plakanın alt kapsayacak şekilde kullanmak için steril PBS içinde laminin seyreltik. Gece boyunca oda sıcaklığında bırakın. </li><li> Ertesi gün, hasat hücreleri ve 1 x 104 hücre içeren 1 ml süspansiyon hazırlamak. Tamamen kuru hava ve hücre süspansiyonu 1 ml ile değiştirmek için izin de cam laminin çıkarın. Tutturulması için izin vermek üzere 4 saat arasında en az 37 ° C&#39;de inkübatörde çanağı yerleştirin. </li><li> Hücreleri yeterince konfluent olduğunuzda odasından tüm orta kaldırın. Dikkatle de camı kaldırın. Silikon yağ, bir otoklavlanmış 22 x 22 mm cam coverslip ile dikkatlice takarak hücreleri üzerinde bir çatı Formuiki adet 22 x 11 mm şeritler arasındaki köprü. Kurumasını önlemek için orta birkaç damla hücreleri örtün. </li><li> Odanın çatı üzerinde tepsinin bir kenarından diğerine koşmak iki su geçirmez silikon gres bariyerler oluşturarak odasının her sonunda izole bir ortamda rezervuar oluşturur. Bir akış yoluyla bir orta rezervuar diğerine sağlanması, taze aracı madde ile haznesi doldurun. Hücre geri kazanımı için izin vermek için 12 saat için inkübatöre geri dönmek çanağı. </li></ol> 3. Electrotactic odasına bir elektrik alanı uygulanması <ol><li> Iki delik, göç odasının her rezervuar üzerine yerleştirilmiş bir delerek çanak kapsayacak bir kapak hazırlayınız. </li><li> 25 mM HEPES tampon içeren kültür ortamı ile haznesi tüm orta yerini ve sıcaklık kontrollü bir görüntüleme sistemi çanağı transfer. Zaman ayarlı, çok-pozisyon kayıt için deney parametrelerini. Katod ve anot böylece haznesi hizalamakSağ sırasıyla sol ve üzerinde, EF vektör çalışmasını sağlamak için yatay olarak mikroskop aşağı incelendi ve görüntüleme sistemi kaydedildi. </li><li> Steinberg&#39;in çözeltisi (58 mM NaCl, 0.67 mM KCI, 0.44 mM Ca (NO 3) 2 ,4 H 2 0, 1,3 mM MgSO 4 0,7 H 2 0 ve 4.6 mM Trizma baz, pH 7,4) ile iki beher doldurun. % 2 (w / ile doldurulmuş önceden hazırlanmış cam köprüler (~ 13 cm uzunluğunda ve ~ 3 mm çaplı ve bir Bunsen alev içinde ısıtmak suretiyle U-biçimli bir bükülebilmektedir cam tüpler), her biri kullanılarak, orta rezervuara ayrı bir behere Connect Kapağın deliklerden geçerek v) Steinberg&#39;s-agar çözeltisi,. Steinberg çözümü her behere bir DC güç kaynağına bağlı Ag / AgCl elektrot yerleştirerek elektrik devresi tamamlayın. </li><li> 0 ve açmak için güç kaynağı gerilimi kadranını. Bir gerilim ölçer kullanarak, gerilim çevirmeli açarken electrotactic odasının arasındaki gerilimi ölçün ve deneysel gereksinimlerine uygun şekilde ayarlamak. </li><li> Time-lapse kayıt başlatın. Orta değişiklik yapın ve her saat, gerektiği gibi, gerilimi tekrar ayarlayın. Gerekli olan taze aracı madde, ilaç ya da kimyasal ajanlara rezervuar ilave edilebilir. Orta değişiklik yaparken, iki seçenek aşağıdaki gibi düşünülebilir: <ol><li> Opsiyon No.1 - geçici duraklama time-lapse kayıt için dikkatli kapak kapak rahatsızlık vermesini önlemek için odasından cam köprüleri kaldırmak, taze ortam ile tüm orta yerine hafifçe steril bir Pastör pipeti kullanın ve cam köprüler geri koymak, sonra kayda devam. </li><li> Seçenek No.2 - Alternatif olarak, aynı zamanda özel bir orta değişim amaçlı kullanılabilir 4 delikler (iki geçiş bölmesinin üzerine yerleştirilmiş her bir rezervuar) ile kapak kapak yapmıştır. Cam köprü bağlantısı için iki, diğer iki orta değiştirmek için. Seçenek 2 orta boy değişimi sırasında müdahalesi olmadan sürekli kayıt sağlar. </li></ol></li></ol> 4. Organotipik omurilik dilim hazırlanması <ol><li> Lumb teşrih2 haftalık C57 BL / 6 farelerde ar omurilik. </li><li> Bir McIlwain doku kıyıcı ile 500 mikron kalınlığında kesitler halinde omurilik dilimleyin. </li><li> Ayrı anatomik bir mikroskop altında dilimleri ve sağlam aksiyel / sagital omurilik yapısıyla dilim seçin. </li><li> Matrigel proteinler üretmek için kendini araya kadar 30 ul Matrigel ve koyun içeren bir 35 mm Petri kabındaki Plaka dilimler mümkün olduğunca merkeze yakın ve en az 30 dakika 37 ° C&#39;de% 5 CO2 inkübatör içinde onları korumak spinal kord dilim yüzeyine ince bir tabaka içerir. Tamamen, bir 30 dakika gerekirse 37 ° C&#39;de Petri inkübe Matrigel monte edilmiş emin olmak çok önemlidir. </li><li> Dilim doğrudan orta akışını önlemek için 25 mM HEPES tampon ve çok yavaşça olarak% 15-20 oranında fetal sığır serumu içeren 4-6 ml DMEM/F-12 orta ekleyin. Kesit tamamen de maruz eksplantlarının yüzeyi bırakarak, orta sokulmasına emin olmakhava. Haftada iki kez orta değiştirin. </li></ol> 5.. Organotipik omurilik dilim içine Hoechst 33342 etiketli NPCs Enjeksiyon <ol><li> 1 NPC süspansiyon × 10 6 hücre / ml hazırlayın. </li><li> 30 dakika boyunca 5 uM Hoechst 33.342 olan bir ortamda hücre süspansiyonu ön-kuluçkaya. </li><li> Mikroskop altında yavaş yavaş omurilik dilim içine süspansiyon 2 ul microinject kapiler cam tüp kullanın. Kapiller cam tüp matrigel (mikroskop altında pembe kısmı) geçer ve omurilik dilim (mikroskop altında gri doku) içine ulaşmak emin olun, üzerinde çalıştırmak hücre süspansiyonu önlemek için en az 30 saniye boyunca omurilik dilim içinde kalır. Inkübatöre omurilik dilim içeren petri (37 ° C&#39;de% 5 CO 2) koyun ve gece boyunca bekletin. </li><li> Ertesi gün, (açıklanan yöntemler kullanılarak electrotactic odacık içinde 33.342-etiketli NPCs Hoechst içeren spinal kord dilimlenmesi 500 mV / mm arasında bir EF uygulamak3). </li></ol> 6. Temsilcisi Sonuçlar NPC onlar katot doğru yönlendirilmiş yüksek hücre migrasyonu (Şekil 1) gösterdi fizyolojik EF&#39;in bir dizi ortaya çıktı. Aynı gözlem organotipik omurilik dilim ex vivo modelde, in vivo koşullarda bir 3D ortamı taklit eden (Şekil 2) tek bir hücre düzeyinde yapıldı. <img alt="Şekil 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> Şekil 1.. NPC EF&#39;in yönlendirilmiş göç göstermektedir. EF&#39;in maruz kaldığında PC&#39;ler katot yönelik yüksek yönettiği göç gösterdi, kırmızı çizgiler ve mavi oklar hücre hareket yörüngeleri ve yön (A) temsil eder. B NPC göç yollarını gösterir. Bar: 50 mikron. <img alt="Şekil 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> Şekil 2. Nakledilen NPC organotipik omurilik dilim katot yönelik göç göstermek </strong&gt;. (A) Hoechst 33342 ile etiketlenmiş NPC&#39;ler EF tedavinin başlangıç ​​noktasında organotipik omurilik dilim içine transplante edildi. NPC EF polarite (B) tersine edildiği noktada, 2,5 saat süreyle katoda doğru yönde göç ettiler. EF kutup değiştirme, yeni katot (C) doğru electrotaxis keskin bir geri tetikledi. Time-lapse kayıt sonunda omurilik dilim içinde nakledilen NPC (D) Görüntü. Omurilik dilim içinde nakledilen NPC (E) A 3D rekonstrüksiyon. 3D tarama bölümleri dilim altındaki ortasında başlayan ve biten, kalınlığı 300 mikron idi. Noktalı çizgiler, başlangıçta, ters ve EF tedavi uç noktaları (- C, sırasıyla A) ile nakledilen hücrelerin aynı popülasyon parçasının göreli konumlarını gösteren. Ok uçları Hoechst 33342 etiketli NPC aynı nüfusa hitap etmektedir. Bar: 50 mikron.

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

Biz ifşa hiçbir şey yok.

Kullandığımız protokolleri önceki çalışmalarda 5,11 dayanmaktadır. Standart ve hassas boyutlarda özel tasarlanmış electrotactic odaları kültüre hücreleri veya dilimleri için agar köprüler, Steinberg çözümü ve Ag / AgCl elektrot aracılığı ile bir EF uygularken bu yöntemleri kullanarak, istikrarlı bir kültür ve elektrik mevcut koşullar sağlanabilir. Odacık derinliği farklı örnek 11 kalınlığı için uygun şekilde ayarlanabilir, ve hücrelerin durumda, oda boyut...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> FGF-temel Rekombinant İnsan </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / ml </td></tr><tr><td> EGF Rekombinant İnsan </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / ml </td></tr><tr><td> N2-Supplement (100X) sıvı </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12 orta (yüksek glukoz) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poli-D-Lizin </td><td> Millipore </td><td> A-003-E </td><td></td></tr><tr><td> Doğal fare Laminin </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Büyüme faktörü Basement Membran Matrix (Matrigel) azaltılmış </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> HEPES tampon </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> McIlwain doku kıyıcı </td><td> Mickle Laboratuvarı Mühendislik Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> Dow Corning, yüksek vakum silikon gres </td><td> Sigma-Aldrich </td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

Endojen elektrik alanlar (EF&#39;in) in vivo doğal olarak bulunur ve merkezi sinir sistemi 1,2 de dahil olmak üzere doku / organ gelişimi ve yenilenmesi sırasında kritik bir rol oynar. Bu endojen EF&#39;in hücre ve dokuların elektriksel direnci ile birlikte iyonik ulaşım hücresel regülasyon tarafından oluşturulur. Bu uygulamalı EF tedavi hayvanlarda ve insanlarda 3,4 omurilik yaralanmalarında fonksiyonel onarım teşvik ettiği bildirilmiştir. Özellikle, EF-yönlendirmeli hücre göçü sinir progenitör hücreleri (NPCs) 7,8 dahil olmak üzere hücre tipleri 5,6 geniş bir yelpazede, kanıtlanmıştır. Doğru akım (DC) EF&#39;in Uygulama en laboratuarlarında yaygın olarak mevcut bir teknik değildir. Biz hücre ve doku kültürleri, daha önce 5,11 DC EF&#39;in uygulanması için ayrıntılı protokoller tanımlamışlardır. Burada 2D bir kurmak için hesaplanan alan gücüne dayalı standart yöntemlerin bir video gösterisi sunmakNPC d 3D ortamlarda, ve 2D hem de tek hücre büyümesi koşullarında EF stimülasyon ve 3D organotipik omurilik dilim hücresel yanıtları incelemektir. Spinal cordslice cytoarchitectonic doku organizasyon iyi bu kültürlerin 9,10 içinde korunmuş çünkü NPC ex vivo davranışları, transplantasyon sonrasında, eğitim için ideal bir alıcı dokudur. Ayrıca, bu ex vivo model de tek hücre seviyesinde time-lapse kayıt kullanılarak in vivo olarak hücreleri izlemek için teknik olarak mümkün değildir prosedürleri sağlar. Bu eleştirel bir 2D ortamda sadece hücre davranışlarını değerlendirmek için gerekli değil, aynı zamanda in vivo ortamda taklit edebilmektedir 3D organotipik durumdadır. Bu sistem sağlayacak in vitro ve ex vivo olarak tek hücre göç 3D izleme ile doku veya organ kültüründe kapak cam tabanlı yemekleri kullanarak ve vi geçmeden önce bir ara adım olabilir yüksek çözünürlüklü görüntülemevo paradigmalar.

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Bu protokol hücreleri izleyebilirsiniz özel tasarımlı electrotactic odalarında 2D ve 3D ortamlar oluşturmak için kullanılan yöntemler, gösteriyor<em&gt; In vivo / ex vivo</em&gt; Akım (DC) elektrik alanlar (EF&#39;in) yönlendirmek için galvanotaksis / electrotaxis ve diğer hücresel yanıtları incelemek amacıyla, tek hücre seviyesinde time-lapse kayıt kullanarak.

2D ve 3D Ortamlarda Sinir Progenitör Hücreleri Elektrik Alanı kontrollü Yönetmen Göç

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

electric-field-controlled-directed-migration-neural-progenitor-cells

Research

JoVE Journal

Bioengineering

11.6K Görüntüleme.  Cardiff University. Bu protokol hücreleri izleyebilirsiniz özel tasarımlı electrotactic odalarında 2D ve 3D ortamlar oluşturmak için kullanılan yöntemler, gösteriyor In vivo / ex vivo Akım (DC) elektrik alanlar (EF'in) yönlendirmek için galvanotaksis / electrotaxis ve diğer hücresel yanıtları incelemek amacıyla, tek hücre seviyesinde time-lapse kayıt kullanarak.

Video: Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

3T fMRI Haptik Robot Adaptasyon

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Biyomühendislik

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 boyutlu montaj Self-Peptid Hidrojel İnsan Sinir Progenitör Hücreler Yetiştiriciliği

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Tek Hücreleri ve 3D Sınırlaması Tek Embriyolar Sipariş: Yüksek İçerik Tarama için Yeni Bir Cihaz

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

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.

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.

Polarize Sinir Doku Engineered 3D İpek-kolajen bazlı Modeli

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 ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Bir Çok Kanallı Çift elektrik alan mikroakışkan Chip kullanarak Akciğer Kanseri Hücreleri Electrotaxis Çalışmaları

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

Geliştirilmesi 2D ve 3D Cilt<em&gt; İn Vitro</emMakromoleküler Dışlamasını Kullanma&gt; Modeller

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Doğrusal Invadozom Oluşumu ve Aktivitesi Çalışmak İçin 2D ve 3D Matrisler

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

İçinde üç boyutlu Vitro hücre göç karakterize ortamlar yara

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Geliştirilmiş 3D hidrojel Neuroinflammation Vitro modelleme için birincil gliyal hücreler kültürleri

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

Robotik Mikroenjeksiyon Kullanarak Beyin Dilimlerinde Tek Nöral Kök Hücre ve Nöronların Manipülasyonu

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...