Hi, I'm Steve. I work in Dr.Flanagan's lab in the Department of Pathology at uc, Irvine. Today I'm going to show you how to split human neural precursor cells.
This technique involves coding a new flask with a solution of human fibronectin, lifting the cells off with cell dissociation buffer, then neutralizing the cells dissociation buffer with a 10%serum solution, centrifusion the cell suspension and resus suspending the cells in fresh, medium, and transferring cell suspension into a new flask. In our lab, we culture human neural precursor cells by changing half of their media every other day, and we passage them about once a week by splitting one to two. When I passage cells, I may count them if I want to trade them for a immuno chemistry experiment, or if I want to do a transfection with a nuclear effector.
It's important to use a cell dissociation buffer when packaging cells because first, it's non enzymatic, unlike trypsin, and It's much gentler on cells. Hi, we're in the tissue culture room Now, and before I show you what a confluent flask of human precursor cells looks like, first I'm going to prepare the tissue culture. First, I'm going to turn off the UV light and turn on the light and the airflow.
Next, I'm going to disinfect the hood with 70%ethanol by spraying a paper towel and wiping down the hood. It's important to spray a paper towel with 70%ethanol and not the inside of the hood directly because spraying the hood with 70%ethanol might damage the HEPA filter, which is really expensive. Next, I'm gonna spray all the reagents and instruments.
And finally, I'm going to spray the vacuum hoses, and we are all set. Let's have a look at these cells. So these cells look about 80%confluent.
These are human neural precursor cells isolated from human fetuses, and we grow them in T 25 flasks. We split them about every week, one to two. I'm ready to passage my cells now.
And first I'm going to bring a new T 25 flask that I coated for four hours with human fibronectin from BD biosciences. So I removed the fibronectin solution. I'm going to rinse this flask with BBS before I'm going to move the cells into the flask.
And now I'm going to get the cells out of the 37 degree incubator. There they are. Now I'm going to remove the PBS wash solution from the new flask that was coated with human fibronectin.
And I'm going to add two milliliters of old conditioned media into the new flask. So now I need to rinse my cells with PBS before I can take him off the surface. So first, I'm going to remove the remaining media, add about two mils of PBS right away without wetting the cells dry out.
I'm going to wait about two minutes before removing PBS and putting on sale dissociation buffer in order to take the sales off the surface of the flak. Now I'm going to remove PBS, and again, I'm gonna try to add cell dissociation buffer right away so that the cells don't dry out. So I'm going to add 1.5 mils of cell dissociation buffer.
And unlike PBS, I'm going to add it directly onto the cells and I'm going to try to cover as much surface as possible. So I'm moving the flask from side to side as I slowly add this buffer onto the cells. And I'm going to wait now about five minutes for the cells to start coming off the surface.
And I'm going to leave the flask at room temperature in the hood. As you can see here, cells first start rounding up and then they start coming off the surface and you can actually see some, some cells floating already. And if you tap the flask widely on the side, that's gonna help them come off.
So it's been five Minutes, and as you can see, the solution has become really dense with cells that have come off the surface. And now I'm going to neutralize the effect of cell dissociation buffer by adding 4.5 mils of serum containing medium. So I'm going to use 10%heat inactivated fetal bovine serum in D-M-E-M-F 12 media, and I'm going to add it directly on onto the surface of the flask to make sure there are no cells remaining attached.
And I'm going to, and I'm going to gently pipette up and down to dislodge any, any remaining cells. And then I'm going to transfer the cell solution into a 15 mil conical tube. And I'm going to spin it at a thousand RPM for one minute.
So the cells are done spinning and we have here a beautiful cell pallet. I'm going to remove the serum media very, very carefully without trying to disturb the pallet. And then I'm going to resuspend the pallet in four mils of, of the media.
So I'm going to try to resuspend the pellet so that there are, are, there are no cell clumps left so that the solution becomes homogeneous and contains, contains no clumps. And since I'm doing a one to two split, I'm going to take two of the four mills and transfer them into the new flask that already contains two mil of the condition media. And finally, I'm going to label the flask with the cell type, the passage number, and the date.
This is a look at at the cells that we passaged half an hour ago. And as you can see, they, most of them have attached down to the surface and have started sending out processes. That's all Folks.
And now I'm ready to count.
