WP4 - Serum Free Culture of MSCs

The use of MSCs in the clinic will most likely involve more than the minimal manipulation of harvesting and infusion. Expansion and differentiation steps in MSC culture involve the use of bovine serum-containing media. Bovine serum is the most widely used growth supplement for in vitro culture because of its high levels of growth-stimulatory factors and low levels of growth-inhibitory factors. However, the use of animal derived products greatly enhances the risk of prion, viral or zoonose contamination. By eliminating our reliance on animal or human based products, the risk of infection is eliminated (Mannello 2007, Halme 2006).

The need for a rationally developed serum free medium for the expansion and differentiation of human MSCs is necessary for good quality control of experiments between laboratories as well as for the culture of cells intended for clinical use. The use of current serum-free conditions in culturing human MSCs selects for a subpopulation of cells that can survive serum deprivation and continue proliferating (Pochampally 2004). These cells contained longer telomeres than control cells and expressed genes associated with embryonic cells such as octomer-binding transcription factor 4. A new compliment of medium, growth factors and cytokines must therefore be developed specifically to complement the display of receptors expressed on the MSC cell surface ensuring uniform expansion of the MSC population.

Serum Free Experimental Design
In WP3, the receptome of the MSC, cultured in vitro, from WP1, or unmodified from its in vivo state, from WP2, will be elucidated. From this display of growth factor or cytokine receptors we can rationally deduce the combination of growth factors that the MSC requires.

One of the critical aspects of this WP will be the testing and titration of several combinations of growth factors. We will assess how the combinations of growth factors might affect proliferation, CFU formation, viability, plating efficiency, DNA analysis, telomere length, apoptosis, senescence and OCT 3/4 expression. Moreover cells will undergo detailed phenotypic analysis and differentiation.

Fig. 4 MSCs cultivated in different media a) 2% FCS media supplemented with EGF and PDGF (Soukup 2006), b) 10% FCS media supplemented with FGF-2

Fig. 5 - Design of WP4: One of the critical aspects of this WP will be the testing and titration of several combinations of growth factors (data selected from receptome study - WP3). We will assess how the combinations of growth factors might affect proliferation, CFU formation, viability, plating efficiency, DNA analysis etc. Moreover cells will undergo phenotypic analysis and differentiation.