This work was supported by the Royal Society URF grant UF051616, UK and the European Research Council StG grant 243261 to BS. The work in MZ lab is also supported by a California Institute of Regenerative Medicine grant RB1-01417.

1. Neural progenitor cell isolation
<ol>
 <li>Dissect whole brains from E14-16 mice and place in cold DMEM/F12 basal medium. Remove all meninges under an anatomical microscope and transfer brains into a 35 mm Petri dish.</li>
 <li>Use fine forceps to mechanically dissociate brains into tissue fragments and transfer them to a 15 ml tube, then centrifuge samples at 800 rpm for 3 min to remove debris.</li>
 <li>Add DMEM/F12 containing bFGF and EGF and triturate with a 1 ml pipette.</li>
 <li>Pass the cell suspension through a cell strainer to obtain a single cell suspension.</li>
 <li>Plate cells into flasks at 2-5 x 104 cells/ml, then perform a full medium change every 3 days and passage the cells every 6 days.</li>
 <li>After at least 5 passages, digest neurospheres to single cells using trypsin and EDTA and grow on poly-D-lysine/laminin-coated electrotactic chambers (prepared as described below in 2). Use the growing medium containing N2-supplement, bFGF and EGF at all times to maintain properties of NPCs.</li>
</ol>
2. Preparation of the electrotactic chamber
<ol>
 <li>Prepare 22 x 11 mm strips of glass by dividing autoclaved 22 x 22 mm no.1 thickness coverslips in half using a diamond pen.</li>
 <li>Create a free-standing glass well of interior dimensions 22 x 10 mm by gluing together four vertically standing 22 x 11 mm strips with high-vacuum silicone grease. Allow the wells to completely dry and harden overnight.</li>
 <li>The following day, attach two 22 x 11 mm glass strips, parallel to each other leaving a gap of 10 mm, to the base of a 100 mm culture dish using silicone grease. Seal off the region between these strips by placing a 22 x 22 mm cover slip at each end, attached to the bottom of the dish with grease on three sides, but not that closest to the centre.</li>
 <li>Place the glass well prepared in step 2.2 onto the cover slips so that the interior walls create a confined space for seeding the cells onto the bottom of the plate. Water-proof all joints with silicone grease. Coat this confined region sequentially with poly-D-lysine then laminin: add 1 ml poly-D-lysine into the chamber and leave for 5 min to allow the poly-D-lysine to bind to the bottom of the plate; wash the chamber with sterile PBS twice, then dilute laminin in sterile PBS to yield 20 &mu;g/ml and use to cover the bottom of the plate. Leave at room temperature over night.</li>
 <li>The following day, harvest cells and prepare 1 ml of suspension containing 1 x 104 cells. Remove laminin from the glass well, allowing it to air dry completely, and replace with 1 ml of cell suspension. Place the dish in the incubator at 37&deg; C for a minimum of 4 hours to allow for attachment. </li>
 <li>Once the cells are sufficiently confluent remove all medium from the chamber. Carefully remove the glass well. Form a roof over the cells by carefully attaching, with silicone grease, an autoclaved 22 x 22 mm glass coverslip bridging between the two 22 x 11 mm strips. Cover the cells with a few drops of medium to avoid drying out.</li>
 <li>Form an isolated medium reservoir at each end of the chamber by creating two watertight silicone grease barriers that run from one edge of the dish to the other, over the chamber roof. Fill the chamber with fresh medium, ensuring a flow-through from one medium reservoir to the other. Return the dish to the incubator for 12 hours to allow for cell recovery.</li>
</ol>
3. Application of an electric field to the electrotactic chamber
<ol>
 <li>Prepare a lid to cover the dish by drilling two holes, one positioned over each reservoir of the migration chamber.</li>
 <li>Replace all medium in the chamber with culture medium containing 25 mM HEPES buffer and transfer the dish to the temperature-controlled imaging system. Set up the experimental parameters for time-lapse and multi-position recording. Align the chamber so that the cathode and anode are on the left and right respectively, to ensure that the EF vector runs horizontally as viewed down the microscope and recorded in the imaging system.</li>
 <li>Fill two beakers with Steinberg's solution (58 mM NaCl, 0.67 mM KCl, 0.44 mM Ca(NO3)2.4H20, 1.3 mM MgSO4.7H20 and 4.6 mM Trizma base, pH 7.4). Connect a separate beaker to each medium reservoir using pre-prepared glass bridges (glass tubes ~13 cm long and ~3 mm in diameter, and bent into a U-shape by heating in a Bunsen flame), filled with 2 % (w/v) Steinberg's-agar solution, passing through the holes in the lid. Complete the electric circuit by placing Ag/AgCl electrodes connected to a DC power supply into each beaker of Steinberg's solution.</li>
 <li>Set the voltage dial on the power supply to 0 and switch on. Measure the voltage across the electrotactic chamber while turning up the voltage dial, using a voltage meter, and adjust to suit the experimental requirements.</li>
 <li>Start time-lapse recording. Perform a medium change and readjust the voltage, as required, every hour. Fresh medium, drugs, or chemical agents may be added to the reservoirs as required. When perform medium change, two options can be considered as below:
 <ol>
 <li>Option No.1 - To pause the time-lapse recording temporarily, carefully remove glass bridges from chamber to avoid perturbing cover lid, use a sterilized Pasteur pipette gently replace all medium with fresh medium and put the glass bridges back, then resume the recording.</li>
 <li>Option No.2 - Alternatively, custom made cover lid with 4 holes (two positioned over each reservoir of the migration chamber) can also be used for medium change purpose. Two for glass bridges connection, the other two for changing medium. Option 2 allows continuous recording without interference during medium change.</li>
 </ol>
 </li>
</ol>
4. Preparation of the organotypic spinal cord slice
<ol>
 <li>Dissect lumbar spinal cords of 2 week old C57 BL/6 mice.</li>
 <li>Slice spinal cords into 500 &mu;m thick sections with a McIlwain tissue chopper.</li>
 <li>Separate slices under an anatomical microscope and select slices with intact sagittal/axial spinal cord structure.</li>
 <li>Plate slices in a 35 mm Petri dish containing 30 &mu;l Matrigel and place them as close to the centre as possible and maintain them in a 5 % CO2 incubator at 37 &deg;C for at least 30 min until the Matrigel proteins self-assemble to produce a thin film that covers the surface of the spinal cord slice. It is very important to make sure that Matrigel has been assembled completely, incubate the Petri dish at 37 &deg;C for another 30 min if necessary.</li>
 <li>Add 4-6 ml DMEM/F-12 medium containing 25 mM HEPES buffer and 15-20% fetal calf serum very gently so as to avoid medium flow directly onto the slice. Ensure that the slice is not entirely submerged in medium, leaving the surface of explants well exposed to the air. Change medium twice weekly.</li>
</ol>
5. Injection of Hoechst 33342 labelled NPCs into the organotypic spinal cord slice
<ol>
 <li>Prepare the NPC suspension at 1 &times; 106 cells/&mu;l.</li>
 <li>Pre-incubate the cell suspension in medium with 5 &mu;M Hoechst 33342 for 30 min.</li>
 <li>Use capillary glass tube to microinject 2 &mu;l of suspension into the spinal cord slice slowly under a microscope. Make sure capillary glass tube passes through the matrigel (pink part under microscope) and reach to the inside of spinal cord slice (grey tissue under microscope), remain inside the spinal cord slice for at least 30 seconds to avoid cell suspension run over. Put the petri dish containing spinal cord slice into incubator (37&deg;C 5%CO2) and leave it over night.</li>
 <li>Next day, apply an EF of 500 mV/mm to the spinal cord slice containing Hoechst 33342-labelled NPCs in the electrotactic chamber (using the methods described in 3).</li>
</ol>
6. Representative Results
When NPCs were exposed to a range of physiological EFs they showed highly directed cell migration towards the cathode (Figure 1). The same observation was also made at a single cell level on the organotypic spinal cord slice ex vivo model, a 3D environment mimicking in vivo conditions (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> 
Figure 1. NPCs show directed migration in EFs. PCs showed highly directed migration towards the cathode when exposed to EFs, red lines and blue arrows represent trajectories and direction of cell movement (A). B shows migration paths of NPCs. Bar: 50 &mu;m.
<img alt="Figure 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> 
Figure 2. Transplanted NPCs show directed migration towards the cathode in the organotypic spinal cord slice. (A) NPCs labeled with Hoechst 33342 were transplanted into the organotypic spinal cord slice at the starting point of the EF treatment. NPCs migrated directionally towards the cathode for 2.5 hours, at which point the EF polarity was reversed (B). Altering EF polarity triggered a sharp reversal of electrotaxis towards the new cathode (C). (D) Image of transplanted NPCs within the spinal cord slice at the end of the time-lapse recording. (E) A 3D reconstruction of transplanted NPCs within the spinal cord slice. 3D scanning sections were 300 &mu;m in thickness, starting from the middle and ending at the bottom of the slice. Dotted lines indicate the relative positions of the same population of transplanted cells at the beginning, reversal, and end points of the EF treatment (A - C, respectively). Arrow heads indicate the same population of Hoechst 33342 labelled NPCs. Bar: 50 &mu;m.

<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>

We have nothing to disclose.

The protocols we use are based on previous studies5,11. Using these methods, stable culture and electric current conditions can be maintained while applying an EF via agar bridges, Steinberg's solution, and Ag/AgCl electrodes, to cells or slices cultured in custom-designed electrotactic chambers of standardised and precise dimensions. The depth of chambers can be adjusted to accommodate for different sample thicknesses11, and in the case of cells, chamber size can be modified to accommodate however ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>FGF-basic Recombinant Human</td>
 <td>Invitrogen</td>
 <td>PHG0026</td>
 <td>20 ng/mL</td>
 </tr>
 <tr>
 <td>EGF Recombinant Human</td>
 <td>Invitrogen</td>
 <td>PHG0311</td>
 <td>20 ng/mL</td>
 </tr>
 <tr>
 <td>N2-Supplement (100X) liquid</td>
 <td>Invitrogen</td>
 <td>02048</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM/F12 medium (high glucose)</td>
 <td>Invitrogen</td>
 <td>31330-095</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-D-Lysine</td>
 <td>Millipore</td>
 <td>A-003-E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Natural mouse Laminin</td>
 <td>Invitrogen</td>
 <td>23017-015</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Growth factor reduced Basement Membrane Matrix (Matrigel)</td>
 <td>B.D. Biosciences</td>
 <td>354230</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES buffer</td>
 <td>Gibco</td>
 <td>15630</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>McIlwain tissue chopper</td>
 <td>The Mickle Laboratory Engineering Co Ltd</td>
 <td>TC752-PD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dow Corning high-vacuum silicone grease</td>
 <td>Sigma-Aldrich</td>
 <td>Z273554</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

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

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

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

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11.6K Views.  Cardiff University. This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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