The overall goal of the following experiment is to efficiently passage the neurospheres so that cell to cell contact is maintained throughout the lifespan of the cultured cells. This technique creates expansion conditions required for human neural progenitor cells. This is achieved by combining up to two T 1 75 flasks of the same cells, and transferring to a conical tube, allowing for the transfer of the spheres to a Petri dish as a second step condensed to suspended neurospheres into a manageable mass on the Petri dish, which allows the blade to cut the H NPCs into small clusters.
Next, the passaged or chopped neurospheres are resuspended in order to remove the cells from the Petri dish and quat into the new flasks. Results are obtained that show the chopping technique provides a long-term growth strategy based on the expansion curves demonstrating long-term logarithmic growth rates compared to cells passaged via the standard enzymatic approach. Visual demonstration of this technique is critical as a sphere.
Condensation steps are difficult to describe in writing. There are also several tricks to aid in the process. If excess media is not fully removed from the Petri dish, the spheres will simply be pushed around in the media as opposed to split into smaller clusters.
Begin this procedure by addressing the media color of the flask of H NPCs. Scan through the flask under a microscope to observe the cells. If possible, use a radical to examine the size of several spheres.
If many spheres have a diameter of 300 micrometers or greater, proceed with the chopping process and chop the cells every seven to 10 days. Then place the flask back into the incubator and set up the chopper. Turn on the power switch of the chopper.
In the BSC, set the chopping thickness to 200 micrometers and the blade force to 180 degrees or the nine o'clock position. Next, confirm that the automatic speed knob is rotated as far counterclockwise as possible. Then move the table release knob as far to the right as possible, and confirm the plate holder is stable.
Rotate the manual arm manipulator clockwise to raise the arm to its maximum level. After that aseptically transfer a sterile double-edged chopping blade onto the chopper arm bolt. Using a pair of forceps, place the clasp over the blade using the forceps.
The curved portion of the clasp must be over the top edge of the arm. Next, hold the clasp onto the arm and secure the nut onto the bolt. With the sterile nut driver leaving the nut a quarter turn loose.
In this procedure, transfer the suggested amount of maintenance media into the new flask. After that aseptically transfer the cells from the incubator to the BSC. Lean the flask on a tube rack and allow the spheres to settle in the flask.
One settled. Aspired up to 12 milliliters of supernatant. With a 10 milliliter serological pipette, rinse all loosely adherent spheres from the surface of the flask.
Repeat as necessary and allow the spheres to settle between Rens. Next, transfer the suggested volume of conditioned media into the new flask. Then transfer all the remaining conditioned media and spheres into a new 15 milliliter conical tube.
Allow the spheres to settle and discard the used flask. After that, slowly transfer the cells from the 15 milliliter conical tube onto the 60 millimeter Petri dish or shim disc in the lowest feasible volume. Keep the remaining conditioned media as it will be used to rinse the cells from the dish post chop.
Try to minimize the surface area covered by the media and spheres on the dish. Next, begin condensing the sphere pool by transferring the super natin back into the 15 milliliter conical tube. Remove as much media as possible from the top of the media cell pool with an aerosol barrier tipped micro pipetter.
Until it is not possible to remove media without spheres, then spread the pool out. Using the side of the pipette tip to increase surface area, tip the Petri dish slightly and use the side of the pipette tip to gently slide all of the spheres to one side of the pool. After all the cells have been relocated, slowly tip the Petri dish to the opposite direction to allow the media to separate from the spheres.
Afterward, transfer the cleared media back into the 15 milliliter conical tube. Use the side of the pipette tip to gently slide all the spheres back to the center of the Petri dish. So the pool has a diameter of 0.5 to 2.4 centimeters.
It is important to keep the depth of the sphere pool shallow. If the pool is too deep, the spheres will be pushed aside during chopping. Now, transfer the Petri dish onto the plate holder and secure it under the plate holder hooks.
Use the table release knob to slide the plate holder to the left so the blade is clear of the spheres and locked into gear. Next, lower the chopper arm by rotating the manual manipulator knob clockwise until the blade snaps down flat onto the petri dish. Press down on the arm mount while tightening the nut.
Then push the reset button. Once steady the Petri dish with one hand while turning the automatic arm manipulator knob clockwise to the 90 degrees or 12 o'clock position and start chopping when all the spheres have been chopped. Rotate the plate holder 90 degrees.
Then loosen the bolt, lower the arm and re-tighten the nut. Repeat the chopping procedure. After that aseptically, transfer the Petri dish from the plate holder onto a working space in the BSC.
Transfer one milliliter of conditioned media onto the chopped spheres. Then gently resuspend and transfer the chopped spheres into a new 15 milliliter conical tube. Try to avoid forming bubbles and repeat as many times as necessary to collect the chopped spheres.
Measure the volume of the conditioned media and chopped spheres. Add the appropriate amount of maintenance media to achieve the suggested volume in table two column E.After that, Eloqua the sphere suspension into each new flask only transfer the sphere suspension into one flask at a time and resuspend the spheres between transfers. The final volume in each flask should equal the suggested volume in table two Column F.Place the chopped h NPCs in the incubator.
This figure shows the projected cell numbers of H NPCs frozen at passage 19, then thawed and expanded as an adherent monolayer using enzymatic dissociation compared to neurospheres passaged via the chopping method. Here are the representative images of pre-chopped and post chopped spheres at 10 x magnification. This image shows the thaw passage 29 H NPCs that have been plated for one day and stained for nest and expression, whereas this image shows the thaw passage 29 H NPCs that have been plated for seven days in the absence of growth factors and stained for GFAP and beta three tubulin.
After watching this video, you should have a good understanding of how to use the automated chopping technique to passage HBCs, which maintains cell to cell contact throughout the life of the cells and culture. This procedure can be adapted to a multitude of cell types, such as neuro stem cells derived from pluripotent cells or cardio spheres.
