I resuscitate human primary fibroblasts(P5) and culture in DMEM(Gibco, 11965) plus 10% FBS(Gibco) and 100 U/mL penicillin, 100 μg/mL streptomycin, but these cells proliferate very slow and differentiate into myofibroblasts almost 100% at about 7 to 10 days. What is the problem?
Hi Xiaolin,
While it is true that cytokines are important the main reason your Fibs differentiate into MyoFibs is that you presumably are growing them on plastic, a rigid substrate. Any fibroblast (or mesenchyme/parenchymal cell) will organise their cytoskeleton to generate tension on a rigid substrate. This is mostly mediated via cell adhesion receptors (integrins). The fibronectin, vitronectin and glycosaminglycans coat the plastic and mediate cell attachment. The stronger the attachment/affinity the greater the development of tension, aka myodifferentiation. Of course as attachment is cation-dependent, you can modulate the strength of adhesion by controlling the availability of divalent cations and theoretically also differentiation. Dermal fibroblasts are pre-programmed to contract as part of normal response to dermal trauma/wounding. It is part of early wound healing responses aimed at restoring tissue homeostasis (i.e close the wound).
So is there other roles for serum? Sure!! What is serum? It is clotted plasma, or if you like, post-thrombosis plasma. In other words it is what one would expect to find in a wound! It is a trauma signal - so off course any fibroblast would respond by contracting and transdifferentiating into a myofibroblast. If you prefer this is the scarring response.
How to prevent? Do not use serum! ...go serum free! And do not use trauma-associated attachment factors. Only then might you obtain a near-normal mesenchyme!
The differentiation state of these cells depends on the presence of cytokines and growth factors. Try a different brand or lot of FBS.
I agree with christian but would strongly suggest a serum free medium. We culture our (lung)fibroblasts in Quantum 333 (PAA Austria) with good success and stability. Only for some experiments we use DMEM+FBS.
good luck
Gernot
Hi Xiaojin. Do you mean DMEM instead DMSO? And what kind of tissue fibroblast come?
I think you should use 10% FBS in DMEM , because differentiation state of these cells depends on the presence of cytokines and growth factors,
Sorry, I made a mistake. I mean DMEM and I had change it. Fibroblasts I cultured came from human dermal.
Fibroblasts are a highly heterogeneous cells. I isolate CAFs from different patients and the proliferation rate is very different among fibroblasts. Don't worry about the proliferation rate of your fibroblasts. I use both DMEMF12 10% FBS and Quantum333. Quantum333 has also serum, but only selected components. When fibroblasts seems to be in a senescent state, try to culture them w/o FBS and very very important, when subculturing them use only 1:2 dilution....no more
Fibroblasts plated at low density will differeniate into myofibroblasts at confluence. What is the density of human dermal fibroblasts that you are plating? How are you defining myofibroblasts?
Hi Xiaolin,
While it is true that cytokines are important the main reason your Fibs differentiate into MyoFibs is that you presumably are growing them on plastic, a rigid substrate. Any fibroblast (or mesenchyme/parenchymal cell) will organise their cytoskeleton to generate tension on a rigid substrate. This is mostly mediated via cell adhesion receptors (integrins). The fibronectin, vitronectin and glycosaminglycans coat the plastic and mediate cell attachment. The stronger the attachment/affinity the greater the development of tension, aka myodifferentiation. Of course as attachment is cation-dependent, you can modulate the strength of adhesion by controlling the availability of divalent cations and theoretically also differentiation. Dermal fibroblasts are pre-programmed to contract as part of normal response to dermal trauma/wounding. It is part of early wound healing responses aimed at restoring tissue homeostasis (i.e close the wound).
So is there other roles for serum? Sure!! What is serum? It is clotted plasma, or if you like, post-thrombosis plasma. In other words it is what one would expect to find in a wound! It is a trauma signal - so off course any fibroblast would respond by contracting and transdifferentiating into a myofibroblast. If you prefer this is the scarring response.
How to prevent? Do not use serum! ...go serum free! And do not use trauma-associated attachment factors. Only then might you obtain a near-normal mesenchyme!
Dear David Mollevi and David Leavesley, thanks very much for your response and clear explanations. What you said indeed deepen my understanding about fibroblasts. I think culture my fibroblasts in serum- free medium should be a good solution for my problem, because I used to culture my fibroblasts in serum- free medium, and they grew well without obvious morphological change.
If this don't work, I think maybe there were some errors during my cells freezing.
Concept, Eric?
It ain't a concept mate! Its fact. You just need to join the dots (and read a lot outside your own discipline).
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology