Dear Scientists of Researchgate,
After a lot of trial and error, we managed to grow and maintain L929 fibroblasts in-house. We managed to grow a few monolayers that looked.. okay.
So far, what worked best for me was to plate 100,000-200,000 cells in 2mL of EMEM with 10% FBS for 24-48 hours, then replace the medium layer with pure EMEM (no Fetal bovine serum added). The cells differentiated relatively well.
Our team doesn't have any prior experience working with adherent mammalian cell lines, so I was wondering - does anyone have any tips on making sure L929 cells differentiate into fibroblasts with a higher percentage? And if anyone has experience working with these cells - could you please evaluate the quality of the monolayers I have attached pictures of? Any feedback would be greatly appreciated.
-Tim S
ATCC images of L929 cells look like this, but I think they are too stellate/not bipolar enough to resemble connective tissue fibroblasts, more like melanocytes or fetal/embryonic fibroblasts. The following paper available on ResearchGate has L929 that look a little more like fibroblasts. Check their culture/differentiation conditions:
Article Novel materials to enhance corneal epithelial cell migration...
Here is another link to a ResearchGate paper with L929 cells that look quite like fibroblasts, although there are some unusual cytoplasmic inclusions. Check their culture/differentiation conditions:
Article Alveolar blood clots and platelet-rich fibrin induce in vitr...
You can try: stiff substrates, TGF-beta treatment, fibronectin-coated plates are examples of microenvironments that stimulate fibroblast-myofibroblast differentiation. Good luck.
As shown in the image, L929 cells seems to be starved and it could be due to their culture in serum-free EMEM. Under starved conditions, cell attachment, elongation, stretching and adhesion to the culture flask are drastically reduced. For more detail on the culture of fibroblast cell lines, see our published paper "Modulation of a5b1 Integrin Functions by the Phospholipid and Cholesterol Contents of Cell Membranes. Journal of Cellular Biochemistry 77:517–528
(2000).
Article Modulation of α5β1 integrin functions by the phospholipid an...
I would suggest you to supplement 10% FBS in DMEM or EMEM medium that provides all growth factors, nutrients and cell cycle proteins required for growth, development, cell adhesion and spreading of cultured cells in the flask. You can use the fibronectin-coated flask for better cell adhesion, spreading and differentiation.
Good luck
Thank you Melville! I will look through the papers to see what I can add to promote differentiation into fibroblasts.
Shail, thank you for your response as well. Out of curiosity - what properties of the cell line in the image shows that its starved/suboptimal?
I attached another image of a monolayer I have - before I introduced experimental conditions, so the cells were only in pure MEM for a day. Please let me know if you can if this monolayer has better health.
Thank you all,
-Tim S
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line