Este trabalho foi financiado pela Royal Society URF concessão UF051616, Reino Unido e do Conselho Europeu de Investigação StG concessão 243261 a BS. O trabalho em laboratório MZ é também apoiada por um Instituto de Medicina Regenerativa da Califórnia concessão RB1-01417.

1. Isolamento de células progenitoras neurais <ol><li> Dissecar cérebros inteiros de E14-16 camundongos e coloque em meio DMEM/F12 frio basal. Remova todas as meninges sob um microscópio anatômico e cérebros de transferência em uma placa de Petri 35 mm. </li><li> Use fórceps finos para dissociar mecanicamente cérebros em fragmentos de tecido e transferi-los para um tubo de 15 ml, em seguida, centrifugar amostras a 800 rpm durante 3 min para remover os resíduos. </li><li> Adicionar DMEM/F12 bFGF contendo e EGF e tritura-se com uma pipeta de 1 ml. </li><li> Passar a suspensão de células através de um filtro de células para obter uma suspensão de célula única. </li><li> Células placa em frascos de 2-5 x 10 4 células / ml, em seguida realizar um meio completo mudar a cada 3 dias e passagem das células a cada 6 dias. </li><li> Depois de pelo menos 5 passagens, digerir neuroesferas para células individuais utilizando tripsina e EDTA e crescer em câmaras electrotactic poly-D-lysine/laminin-coated (preparada tal como descrito abaixo em 2). Use o meio de cultura contendo N2 suplemento,bFGF e EGF em todos os momentos para manter as propriedades de NPCs. </li></ol> 2. Preparação da câmara de electrotactic <ol><li> Prepare 22 x 11 mm tiras de vidro dividindo autoclavados 22 x 22 mm de espessura lamínulas no.1 ao meio usando uma caneta de diamante. </li><li> Criar um copo free-standing bem de dimensões interiores 22 x 10 mm colando quatro verticalmente em pé 22 x 11 mm com tiras de alto vácuo, graxa de silicone. Permitir que os poços completamente secar e endurecer durante a noite. </li><li> No dia seguinte, anexar dois 22 x 11 tiras de vidro mm, paralelamente uns aos outros deixando um intervalo de 10 mm, para a base de um prato de cultura 100 milímetros usando massa de silicone. Vedar a região entre estas tiras pela colocação de um 22 x 22 lamínula mm em cada extremidade, fixada no fundo do prato com massa lubrificante em três lados, mas não que mais próximo do centro. </li><li> Coloque o vidro bem preparado na etapa 2,2 na tampa desliza de forma que as paredes interiores criam um espaço confinado para propagaçãoas células para o fundo da placa. Água à prova de todas as articulações com graxa de silicone. Casaco esta região confinada sequencialmente com poli-D-lisina, em seguida, laminina: adicionar 1 ml de poli-D-lisina para dentro da câmara e deixar durante 5 min para permitir que o poli-D-lisina para se ligar a parte inferior da placa e lavar a câmara com PBS estéril, duas vezes, depois dilui-se a laminina em PBS estéril para se obter 20 mg / ml e usar para cobrir o fundo do prato. Deixe em temperatura ambiente durante a noite. </li><li> No dia seguinte, as células de colheita e de preparar 1 ml de suspensão contendo 1 x 104 células. Remover laminina a partir do vidro bem, permitindo que a secar ao ar completamente, e substituir com 1 ml de suspensão de células. Colocar a cápsula na incubadora a 37 ° C durante um mínimo de 4 horas para permitir a fixação. </li><li> Uma vez que as células são suficientemente confluente remover todo o meio a partir da câmara. Retire cuidadosamente o vidro bem. Formar um telhado sobre as células com cuidado anexando, com graxa de silicone, um autoclave 22 x 22 mm de vidro lamelade ligação entre as duas tiras de 22 x 11 mm. Cobrir as células com algumas gotas de meio para evitar a secagem. </li><li> Formar um reservatório de meio isolado em cada extremidade da câmara de criando duas barreiras estanques massa de silicone que são executados a partir de uma aresta do prato para o outro, sobre o telhado de câmara. Encher a câmara com meio fresco, assegurando um fluxo através de um reservatório de meio para o outro. Retornar o prato para a incubadora durante 12 horas para permitir a recuperação de células. </li></ol> 3. Aplicação de um campo elétrico para a câmara de electrotactic <ol><li> Prepara-se uma tampa para cobrir o prato por perfuração de dois furos, um posicionado sobre cada reservatório da câmara de migração. </li><li> Substituir todo o meio na câmara com meio de cultura contendo 25 mM de tampão HEPES e transferir o prato para o sistema de imagem com temperatura controlada. Configure os parâmetros experimentais para time-lapse e multi-posição de gravação. Alinhar a câmara de modo a que o cátodo eo ânodo sãodo lado esquerdo e direito, respectivamente, para assegurar que o vector de EF corre horizontalmente para baixo como visto ao microscópio e registada no sistema de imagem. </li><li> Encher com a solução de duas provetas de Steinberg (58 mM de NaCl, 0,67 mM de KCl, 0,44 mM de Ca (NO3) 2 0,4 H 2 0, 1,3 mM MgSO4 0,7 H 2 0 e 4,6 mM de Trizma base, pH 7,4). Ligue um copo separado para cada reservatório média usando pontes pré-preparadas de vidro (tubos de vidro ~ 13 cm de comprimento e ~ 3 mm de diâmetro, e dobrado em forma de U por aquecimento em uma chama de Bunsen), cheios com 2% (w / v) de solução Steinberg&#39;s-ágar, passando através dos orifícios na tampa. Completar o circuito eléctrico através da colocação de Ag / AgCl eléctrodos ligados a uma fonte de alimentação DC em cada copo de solução de Steinberg. </li><li> Coloque o selector de tensão no fornecimento de energia para 0 e ligar. Medir a tensão através da câmara de electrotactic ao ligar-se o selector de tensão, utilizando um medidor de tensão, e ajustar para se adequar aos requisitos experimentais. </li><li> Iniciar lapso de tempo de gravação. Realizar uma troca de meio e reajustar a tensão, se necessário, a cada hora. Meio fresco, drogas ou agentes químicos podem ser adicionados para os reservatórios, conforme necessário. Quando realizar troca de meio, duas opções pode ser considerada como a seguir: <ol><li> Opção n º 1 - Para pausar a gravação de lapso de tempo temporariamente, remova cuidadosamente pontes de vidro da câmara para evitar perturbar a tampa de cobertura, use uma pipeta Pasteur esterilizada suavemente substituir todos meio com meio fresco e colocar as pontes de vidro de volta, em seguida, retomar a gravação. </li><li> Opção No.2 - Alternativamente, feito tampa de cobertura com 4 furos (dois posicionado sobre cada reservatório da câmara de migração) também pode ser utilizado para efeitos de troca de meio. Dois para o vidro conexão pontes, os outros dois para mudar médio. Opção 2 permite gravação contínua, sem interferências durante a mudança de meio. </li></ol></li></ol> 4. Preparação da fatia de medula espinhal organotípico <ol><li> Dissecar Lumbcabos de ar da coluna de 2 semanas de idade C57 BL / 6 ratos. </li><li> Corte medula espinhal em 500 mm de espessura com um triturador de tecido McIlwain. </li><li> Fatias separadas de um microscópio anatômico e selecionar fatias com estrutura intacta cabo sagital / axial da coluna vertebral. </li><li> Fatias placa numa placa de Petri contendo 35 milímetros 30 uL Matrigel e colocá-los tão perto do centro quanto possível e mantê-las em um 5% de CO 2 incubadora a 37 ° C durante pelo menos 30 min, até que as proteínas de Matrigel auto-montagem para produzir uma fina película que cobre a superfície da fatia da medula espinhal. É muito importante ter a certeza de que Matrigel foi montado completamente, incubar a placa de Petri a 37 ° C durante mais 30 min, se necessário. </li><li> Adicionar 4-6 ml DMEM/F-12 meio contendo 25 mM de tampão HEPES e 15-20% de soro fetal de vitelo muito suavemente, de modo a evitar o fluxo médio directamente sobre a fatia. Assegurar que a fatia não é inteiramente submerso no meio, deixando a superfície de explantes bem expostos ao ar. Troca de meio duas vezes por semana. </li></ol> 5. Injeção de Hoechst 33342 NPCs marcados na fatia cordão espinhal organotípico <ol><li> Preparar a suspensão NPC em 1 × 10 6 células / uL. </li><li> Pré-incubar a suspensão de células em meio com 5 uM Hoechst 33342 durante 30 min. </li><li> Use tubo capilar de vidro para microinject 2 ul de suspensão para a fatia da medula espinal lentamente sob um microscópio. Assegure-se tubo de vidro capilar passa através do matrigel (parte rosa sob microscópio) e atingem para o interior da fatia da medula espinal (tecido cinza sob microscópio), permanecem dentro da fatia da medula espinal por pelo menos 30 segundos para evitar a suspensão de células atropelado. Colocar a placa de Petri contendo fatia da medula espinal em incubadora (37 ° C a 5% de CO 2) e deixá-la durante a noite. </li><li> No dia seguinte, aplicar um EF de 500 mV / mm para a fatia da medula espinal contendo Hoechst 33342 marcadas NPCs na câmara de electrotactic (usando os métodos descritos no3). </li></ol> 6. Os resultados representativos Quando NPCs foram expostas a uma gama de EFs fisiológicas que mostraram a migração de células altamente dirigido para o cátodo (Figura 1). A mesma observação foi também feita a um nível única célula na medula espinal organotípico fatia modelo ex vivo, um mimético ambiente 3D em condições in vivo (Figura 2). <img alt="A Figura 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> Figura 1. NPCs mostrar migração dirigida na FE. PCs apresentaram migração altamente dirigido para o cátodo quando expostos a FE, linhas vermelhas e flechas azuis representam trajetórias e direção do movimento das células (A). B mostra os caminhos migratórios de NPCs. Bar: 50 um. <img alt="A Figura 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> Figura 2. NPCs transplantados mostram a migração dirigida para o cátodo na fatia cordão espinal organotípico </strong&gt;. (A) NPCs marcadas com Hoechst 33342 foram transplantadas para a fatia cordão espinal organotípico no ponto de partida do tratamento EF. NPCs migrado direccionalmente no sentido do cátodo durante 2,5 horas, altura em que a polaridade EF foi invertida (B). Alterando a polaridade EF desencadeou uma forte reversão de electrotaxis em direção ao cátodo nova (C). (D) da imagem de NPCs transplantados no interior da fatia da medula espinal no final da gravação de lapso de tempo. (E) A reconstrução 3D de NPCs transplantadas dentro da fatia da medula espinhal. 3D secções de digitalização eram 300 mm de espessura, a partir do meio e que termina na parte inferior da fatia. As linhas pontilhadas indicam as posições relativas da mesma população de células transplantadas, no início, a inversão, e os pontos finais do tratamento EF (A - C, respectivamente). Cabeças de seta indicam a mesma população de Hoechst 33342 NPCs marcados. Bar: 50 um.

<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>

Não temos nada a divulgar.

Os protocolos que usamos são baseadas em estudos anteriores 5,11. Usando esses métodos de cultura, estável e elétrico condições atuais pode ser mantida enquanto a aplicação de um EF através de pontes de ágar, solução Steinberg e Ag / AgCl, para células ou fatias cultivadas em câmaras electrotactic custom-concebidas de dimensões padronizadas e precisas. A profundidade das câmaras pode ser ajustada para acomodar diferentes espessuras amostra 11, e, no caso de células, o tamanho da c?...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> FGF-básico Humano Recombinante </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / mL </td></tr><tr><td> EGF humano recombinante </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / mL </td></tr><tr><td> N2 suplemento líquido (100X) </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12 médio (glucose elevada) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poli-D-Lisina </td><td> Millipore </td><td> Um-003-E </td><td></td></tr><tr><td> Natural do mouse laminina </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Fator de crescimento reduzido da membrana basal Matrix (Matrigel) </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> Tampão HEPES </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> McIlwain tissue chopper </td><td> O Laboratório de Engenharia de Mickle Co Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> A Dow Corning massa de silicone de alto-vácuo </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

Endógenos campos eléctricos (EFS) ocorrem naturalmente in vivo e desempenham um papel crítico durante tecido / órgão de desenvolvimento e regeneração, incluindo a do 1,2 sistema nervoso central. Estes EFs endógenos são gerados por regulação celular de transporte iónico combinada com a resistência eléctrica de células e tecidos. Tem sido relatado que aplicada EF tratamento pode promover a reparação funcional de lesões da medula espinal em animais e humanos 3,4. Em particular, EF-dirigida a migração celular tem sido demonstrada em uma ampla variedade de tipos de células 5,6, incluindo as células progenitoras neurais (NPCs) 7,8. Aplicação de corrente direta (DC) FE não é uma técnica comumente disponíveis na maioria dos laboratórios. Nós descrevemos os protocolos de execução de FE DC para celular e culturas de tecidos previamente 5,11. Aqui apresentamos um vídeo de demonstração de métodos padronizados com base em uma intensidade de campo calculada para configurar uma 2Dd ambientes 3D para NPCs, e investigar respostas celulares a EF estimulação em ambas as condições unicelulares de crescimento em 2D, ea fatia de medula espinhal organotípico em 3D. O cordslice vertebral é um tecido destinatário ideal para estudar comportamentos NPC ex vivo, pós-transplante, porque a organização do tecido citoarquitetônicos está bem preservada dentro dessas culturas 9,10. Além disso, este modelo ex vivo também permite que os procedimentos que não são tecnicamente viável para controlar as células in vivo utilizando lapso de tempo de gravação ao nível da célula individual. É extremamente essencial para avaliar comportamentos de células em não só um ambiente 2D, mas também numa condição 3D organotípico que imita o ambiente in vivo. Este sistema permitirá que imagens de alta resolução usando tampa de vidro pratos à base de tecido ou cultura de órgão com rastreamento 3D da migração única célula in vitro e ex vivo e pode ser uma etapa intermediária antes de passar para a viparadigmas vo.

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

Este protocolo demonstra métodos utilizados para estabelecer ambientes 2D e 3D em câmaras de design personalizado electrotactic, que pode acompanhar células<em&gt; In vivo / ex vivo</em&gt; Usando lapso de tempo de gravação no nível da célula única, a fim de investigar galvanotaxis / electrotaxis e outras respostas celulares ao dirigir atuais campos (DC) elétricos (EFS).

Campo elétrico controlado de migração dirigida de células progenitoras neurais em ambientes 2D e 3D

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 visualizações.  Cardiff University. Este protocolo demonstra métodos utilizados para estabelecer ambientes 2D e 3D em câmaras de design personalizado electrotactic, que pode acompanhar células In vivo / ex vivo Usando lapso de tempo de gravação no nível da célula única, a fim de investigar galvanotaxis / electrotaxis e outras respostas celulares ao dirigir atuais campos (DC) elétricos (EFS).

Vídeo: 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.

Adaptação de um robô Haptic em um fMRI 3T

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

Bioengenharia

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.

Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem

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.

Ordenando células individuais e único embriões em 3D Confinamento: Um novo dispositivo de alta Screening conteúdo

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.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

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.

Electrotaxis Estudos de pulmão células de câncer usando um Multichannel Dual-elétrico-campo Microfluidic Chip

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.

Melhorar 2D e 3D Pele<em&gt; In Vitro</em&gt; Models Usando 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 ...

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.

Matrizes 2D e 3D para estudar formação e atividade de Invadosome Linear

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.

Caracterizando a migração celular no tridimensional em Vitro ferida ambientes

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.

Melhorado o hidrogel 3D culturas de células gliais primárias para In Vitro modelagem de Neuroinflammation

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.

Manipulação de células-tronco neurais únicas e neurônios em fatias cerebrais usando microinjeção robótica

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