3D神経幹細胞培養におけるローカライズたんぱく質：ハイブリッド可視化手法

When grown in suspension, neural progenitor cells isolated from neurogenic niches of the brain proliferate to form neurospheres. Here we show the isolation and serial sectioning of free floating postnatal hippocampal derived neurospheres to locate protein expression within the three-dimensional neuros sphere. We will perform immuno staining for a protein of interest in this case, Conexion 29, followed by assembly and translation of two dimensional microscopy images into a three dimensional reconstruction of the neuros sphere.

Visualization of proteins within the context of their native three dimensional structures, such as a free floating neuros sphere or a tissue, allows for a better understanding of protein function based upon location within these structures, and also allows the inference of possible rules for proteins with known localization, but unknown functions. Hi, I'm Sophia Ambo from the Neural Regeneration Laboratory at the University of Ottawa. And I'm Nicholas Valenzuela from the Carlton Immersive Media Studio at Carleton University in Ottawa.

Today we'll be demonstrating how to isolate, expand and serially section hippocampal derived postnatal neural progenitor cells as nerve spheres. In order to localize protein expression via immuno staining, We'll then use two dimensional images of the serial neurospheres sections collected following immuno staining to illustrate a method of reconstructing cellular and protein location in the three dimensional structure and thereby assess spatial organization of protein expression throughout the neurospheres. So let's get started.

Once brains have been collected, prepared to place them onto the chuck by creating a flat surface for the brains to properly rest. This is called blocking. Block the cerebellum using a razor blade and forceps.

Next, put a drop of crazy glue onto the chuck and fix the first brain onto it, anterior side up and dorsal side facing you. Proceed like so with the other brains for the last brain, it is prudent to position the dorsal side away from you as cutting is sometimes incomplete in this direction. If incomplete cutting should occur, use a scalpel to slice through and collect the section.

Speed is of the essence for the gluing step as the brains can't be exposed to air for too long. Once all brains are glued in, the chuck is placed in the vibrato and the holder filled with ice cold. A CSF.

Set the vibrator for a frequency of 8.5 and a speed of 3.5 to 4.5. Bring the blade into contact with the A CSF and set the automatic cut. Now we will cut 500 micron slices.

Keep the slices which contain the hippocampus. Place them in a dish filled with ice cold A CSF and discard the rest of the slices. Once all slices are collected, proceed to the hippocampal dissection.

Using a stereo microscope, we only take the dorsal hippocampus approximately between bgma minus 1.65 millimeters and minus two point 10 millimeters. Place the hippo campi in an empty ice cold dish. When all the hippo campi have been collected from the slices, it's time to bring them into the flow hood for mechanical dissociation.

Using a scalpel, spray the scalpel blade with 70%ethanol and flame to sterilize. Cut the tissue with the scalpel until it appears liquidy. Now using dissociation media already containing pepane DNAs and neural protease, collect the tissue by pipetting and place into a 15 milliliter tube.

For enzymatic dissection, place the tube in a rotating oven set to 37 degrees and let digestion take place for 45 minutes to an hour. Once digestion is complete, add 10 milliliters of sterile tissue culture PBS to stop the enzymatic reaction and spin the cells down at 300 times G for five minutes. Remove the supra natant and resuspend the pellet.

In five milliliters of PBS, use a pasture pipette and tate the sample to further dissociate the tissue. Keep tating until the pieces of tissue no longer get smaller. At this point, pellet the cells again.

Remove the PBS and resuspend the pellet in three milliliters of maintenance medium. Once you have, well triturated the sample. Take a 15 microliter aliquot and mix it with an equal volume of trian blue.

Take two 10 microliter samples and load a hemo cytometer for cell counting. Count the cells from three fields in each section for a total of six fields. Average numbers in calculate your cell density.

Keeping in mind the sample is diluted. In trian blue plate the cells in low adherence dishes at a density of 2.5 times 10th to the five cells per dish in a final volume of five milliliters of maintenance medium. If low adherence dishes aren't available, you can use petri dishes.

In this case, you'll have to tap the neurospheres in the morning and afternoon every day for the first six days so the neurospheres don't adhere to the bottom of the dish. Add EG, F and FGF before placing the dishes in the incubator. These growth factors will need to be replenished every two days for the entire length of the culture On day in vitro 14, fix the neurospheres using formaldehyde to a final concentration of about 4%by adding it directly to the culture and incubating at room temperature for 20 minutes.

With rocking pellet the cells by centrifuging at 300 times G for five minutes. Wash the cells twice with PBS and resuspend in a 15 to 20%sucrose solution made up in 10 millimolar phosphate buffer. Leave to cryo protect at four degrees for at least 24 hours prior to sectioning on a cryostat.

Remove the cryo protected neuro spheres from the refrigerator and place them in a 60 millimeter dish. For serial sectioning where a collection of every single section is crucial, I found it best to select neurospheres of good size and shape using a stereo microscope using a pipetter place, a neuros sphere into a cryo mold filled with OCT compound. Mark the spot where the neurospheres located onto the cryo mold.

This step will help find the neuros sphere during sectioning. Once five to 10 spheres have been placed into the mold rapidly, freeze them using CO2. Using the indications on the cryo mold mark where the neurospheres are located directly into the frozen OTC block.

Remove the OCT block from the mold and place it onto the cryostat chuck. Fix the mold to the chuck using CO2 and place it in the cryostat. Allow the samples to equilibrate for at least 30 minutes.

Once this is done, cut quickly to the marks made on the mold, then start collecting every section from then on. Checking to see where the neuros sphere slices begin using a light microscope Once sectioning is complete, store the slides in a freezer until you're ready to perform immunochemistry. For this step, take out the slides containing one of the serially cut neurospheres and include the slides with five previous and five subsequent sections.

This will ensure you image the entire neuros sphere. Using a nuclear counter stain will be a must in order to find all the sections of your neuros sphere. So keep this in mind as you plan out your immuno staining while the slides by applying PBS and allowing to warm to room temperature for at least five minutes, we use antibody buffer that contains 0.3%Triton X 100 for permeation and 3%bovine serum albumin for blocking.

Dilute your primary antibody appropriately in this buffer and incubate in the human chamber overnight at four degrees. Use PBS to float the paraform off the neurospheres and carefully remove it using forceps. This is the first of four five minute PBS washes.

Apply the secondary antibody to slides and incubate them at room temperature for one hour. As before, float the paraform off the slide using PBS and remove it using forceps. Again, this is the first of four five minute PBS washes.

In the third wash, we include HOS 3 3 2, 5 8 as a nuclear counter stain, which is diluted to 0.5 micrograms per milliliter. In PBS, there is one more five minute wash following the hos wash. Once all washes are complete, apply a generous amount of mounting medium to the bottom of the slide and cover with a glass cover slip.

Secure the corners with nail polish. Once all slides are cover slip, secure the rest of the cover slip edges. Image the slide right away or stir them at minus 20 until you're ready to do so.

When imaging, finding the cells on the slide is the hardest part, it will be easy to detect cells belonging to the sphere if you look for them using the nuclear counter stain. Once you find the beginning of the neuros sphere, which may comprise only a few cells, image every section from then on. Taking shots of the nuclear stain, your protein or proteins of interest and a phase contrast image.

This phase contrast image will be very important in the modeling stages. Images with resident scale bars of 10 micrometers and 50 micrometers established in open lab 3.56 software are imported into Photoshop. CS four using a standard canvas size of 3, 300 by 2, 550 pixels.

All serial section images are saved as TIFF files maintaining the standard canvas size. Open the first and second phase, contrast images of the neurospheres serial sections. Increase the transparency of the second section under the image tab, open image rotation and choose arbitrary.

Rotate the second section until you are able to identify points of geometric and structural continuity between images. Repeat for all serial sections, saving each section as a separate layer. Ensure that each image layer corresponding to phase and protein expression are links so that all rotations for each channel are performed at the same time.

Create a plane in Maya that is of the same proportions as the Photoshop file from the status line of Maya's user interface. Change the menu bar from its default animation setting to surfaces. Use a primitive subdivision sphere to create the cell body with the sphere selected.

Begin manipulating its vertices by right clicking the right mouse button on the object and selecting vertex from the marking menu. Further, elaborate the surface of the sphere by changing the display level from the marking menu from the subdivision surfaces tab of the main menu. Open the collapse hierarchy options box and set the number of levels to two.

Collapse the sphere. Change the menu bar from surfaces to polygons With the sphere selected. Open the sculpt geometry tool from the mesh tab of the main menu.

Refine the surface of the cell to its final form. Employing the sculpt geometry tool set from the tool settings tab. Repeat this process for each cell of your typology to simulate the variety of progenitor cells present in the neurospheres.

In this demonstration, 10 differing progenitor cells will form the basis of the neuros sphere. Select the cell typology and freeze transformations with the cells of your typology. Established two surface shaders will be designed to represent both cells expressing the protein connects in 29 and cells where the protein is absent.

Open the hyper shade window from the windows rendering editor's tab of the main menu. Choose a ramp shader from the surface materials tab. Open the attribute editor and set the shader color incandescent ambient color bump, map and specular color attributes.

Assign a Brownian 3D texture to the ambient color node and a 2D fractal texture to the bump mapping node. Select the resultant shader input and output connections and export the selected network. Select the cell typology and open the export selection options box.

Choose the file type Maya A-S-C-I-I and uncheck the include these inputs box export selection. Open the aligned serial sections file in Adobe Photoshop. Establish a key of pencil strokes to mark the location of each progenitor cell in the serial sections.

In this demonstration, a key of three strokes. A solid square. A square with a dashed line and a square with a cross will be used to indicate whether a cell is located in one, two or three sections.

This key of strokes is further defined by color red representing cells expressing conexion 29 and white representing cells not expressing Conexion 29. Turn the phase layer on and begin marking the center of each progenitor cell with a white pencil stroke. Use a separate layer within the section folder with which to build your maps.

Toggle between sections to locate the cell's position, ensuring that cells on more than one section are properly denoted. With the CX 29 layer turned on. Select the cells that fall in the regions where CX 29 is positively expressed.

Choose the fill command from the edit tab of Photoshop's main menu bar and change the white strokes to red with the maps completed. Save each section as a separate JPEG image in the source images folder of the Maya project. To do this, a new Maya project will first have to be created from the status line of Maya's user interface.

Change the menu bar from its default animation setting to polygons import the planes. Do ma and scale bar. ma files from the projects seen folder with the plain selected.

Assign a Lambert shader by clicking the right mouse button on the object and opening the assign new material tab in the attribute editor. Click the checkered box to the right of the materials color attribute from the pop-up menu. Choose file from the 2D texture section under the file attribute section in the attribute editor.

Click the file icon to the right of the image name attribute. Choose the first section from the source images folder. Switch the camera of the workspace window from the perspective view to the default orthographic top view by opening that panels tab.

Select the create polygon tool from the mesh tab of the main menu and trace the outline of the section. Choose the first map from the source images folder and assign it to the newly created polygon plane. Following the steps established to create the polygon plane with the object selected, choose UV from the marking menu by clicking the right mouse button over the plane.

Select all the planes UV points and open the UV texture editor from the windows tab of the main menu, scale the UVS proportionally to fit the section plane. Repeat this process for each section of the neuros sphere, ensuring that the spaces between each section correspond to the height of the scale bar from the status line of Maya's user interface. Change the menu bar from its default animation setting to dynamics, leaving only the first section of the neuros sphere visible.

Hide all other sections by changing the display settings from the display tab of the main menu with the CV curve tools options box open, select one linear from the CV curve settings. Viewing the scene from the top view camera begin assigning points to the cell maps creating one curve for the CX 29 negative cells and the CX 29 positive cells. The resultant curves are flat when viewed from the side view camera.

Approximate the thickness of the sections by shifting the points of the curve in the Y axis. Using the scale bar as a guide. Repeat this process for each section of the neuros sphere.

Import the cell typology. ma file from the scenes folder of the project and duplicate the cell typology for both the CX 29 negative and CX 29 positive.Progenitors. Open the hyper shade window from the windows rendering editors tab of the main menu.

Import the previously built shaders assigning them to the cells of the typology from the outliner window. Remove the file name from the beginning of the imported objects. This will become important in later stages of the modeling process.

Select the particles tab from the main menu and create a particle emitter for the first CV curve. Open the particle shape one tab and under emission attributes, change the max count from negative one to the number of points on your first curve. In the render attributes tab, change the particle render type to blobby surface.

Click on the current render type box below and change the radius of the particles to 0.01. Attach the particles to the vertices of the curve by first selecting the particles. Then shift selecting the curve.

Open the goal options from within the particles tab of the main menu and set the goal weight to one. Click create. Select the cells from number one to 10 in order in the outliner window and open the in instant replacement options box from the particles tab of the main menu.

In the instance objects box, all 10 cells should be listed in order. Choose the correct particle shape name from the dropdown list of the particle object to instance tab. Click create By default only the first cell that was selected will replace the particles along the curve in order to get all 10 to appear and cycle randomly among the particles an expression is needed.

A second expression will ensure that the cells emit randomly in the same way every time. The range slider is repositioned with the emitter selected. Open the attribute editor in the particle shaped tab under add dynamic attribute.

Click on the general box under long name. Write random underscore index in attribute type switch from scaler to per particle array and hit add in the per particle array attributes tab. A random underscore index box has been added.

Clicking the right mouse button over the empty space select create expression from the dropdown box. Type the following text in the expression box to complete the expression. Open the instant search geometry replacement tab in the attribute editor and in the general options object index.

Select random underscore index from the dropdown list. Bring the time slider to the first frame and hit play. The cell types are now randomly distributed among the particles.

Repeat this process for each section of the neuros sphere. Ensure that each new emitter instance and random index is differentiated by name and organized appropriately in the outliner window in the render. Using section of the render settings window, change the render from Maya software to mental ray.

Open the quality tab and under ray tracing change the reflection setting to zero. Open the frame buffer tab and change the color clip from raw to alpha and uncheck. Pre multiply.

Open the channel box layer editor and change the layer editor settings from display to render. Select the imported cells of the typology and click the create new layer and assign selected objects icon. Rename the layer ambient occlusion.

Clicking the right mouse button over the newly created layer, select attributes from the menu to the right of the render layer box. Click on the presets button and select occlusion from the dropdown list. Select the MIB underscore AMB underscore occlusion one tab and change the samples from 16 to 2 56.

Reopen the channel box layer editor and under the options tab, open the render all layers options box, select composite and keep layers with the ambient occlusion layer selected. Change it from normal to multiply from the dropdown box just above the layer list, render two passes of the neuros sphere once with the ambient occlusion layer turned on and once with the master layer turned on. Save the images as IFF files in the projects images folder.

Open both images in Adobe Photoshop. Place the ambient occlusion layer on top of the master layer and set it to multiply. Create a background and flatten the image.

We've just shown you how to culture. Hippocampal derive neurogen cells as neurospheres How to serially section neuros sphere culture is using a cryostat and how to perform immuno staining for a protein of interest. We have also shown you how to model the spatial location of that protein expression in three dimensions using a combination of cell culture registration and three dimensional modeling software.

We hope this helps you and good luck in your experiments.