Hi everyone,
I am currently trying to differentiate Neural progenitor cells (purchased from a company that differentiated iPSCs to NPC) into cortical neurons as well as hippocampal neurons. I found significant protocols online however I have no experience with culturing NPCs. They have been behaving very weird like they wouldn't centrifuge sometimes ( i.e no pellet) or sometimes they wouldn't stick to laminin coated dishes or to say no attachment. I have come to find that the starting cell density is important however I am still struggling with the numbers. Does anyone have experience working with these cells? Is a particular passage number better for NPCs? What cell densities are ideal for them? All culturing tips are helpful!
Thank you!
-Sushmita
The company that supplied these cells to you should be able to help you with this. You may provide here the culture method that you followed. Then, it is easier for readers to give you any corrections or suggestions. No pellet and not attachment indicate that the cells might have died. Check for viability of the cells.
Hi Chandra
Thank you so much for replying. The company provided culturing tips and I am following it to the T, however, the cells are behaving a bit weird and we are both trying to figure out whats happening.
So throwing light on my process, these NPCs were P32. I thawed them and fed them with DMEM/F12 + FGF every other day.So far they looked healthy and great. I passaged them with accutase (5mL accutase in a T75), centrifuged and got a pellet. However, the subsequent passages (P34 or P35) either the cells wouldn't pellet (P34) or sometimes when it did in another lot they never attached. I have a feeling that the passage numbers are too high which is the reason for this variability but would appreciate if someone confirms this.
I also seeded them on glass coverslips (coated with PLO and laminin) as well as nanofiber scaffolds for differentiation. However, I am not getting the cell density right. I seeded at low density (50,000 cells/well) which looked okay and and at 100,000 cells/well which looked really bad. I am attaching images for both. Could someone help confirm this? Any insights are appreciated.
-Sushmita
Hi Chandra
Are those spores? The media didn't change color so I didn' think they were contaminated at that time. Do you have information on what's a good passage number for differentiations? And ideal seeding densities?
Thanks!
-Sushmita
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