Culture of rat mesenchymal stem cell using peripheral blood-derived plasma as the culture medium supplement

Background and objectivesIn current protocol for isolation and expansion of mesenchymal stem cells (MSCs), the use of fetal calf serum (FCS) as a medium supplement is inevitable. FCS is immunogenic for human subjects and may transfer infection in the case of transplantation. In the search for an appropriate substitute for FCS, in the present study the effect of plasma prepared from peripheral blood on the growth of MSCs has been examined. Materials and MethodsMarrow cells obtained from rat long bones were cultivated both in the mediums containing FCS and in those with plasma prepared from rat peripheral blood for the primary culture and the three consequent passages. The cells present in all four passages were evaluated at each culture stage for population doubling number, viability, and the rate of proliferation by cell count, MTT assay, and plotting growth curve, respectively. The cells from primary culture were also examined with respect to their clonogenic activity. All experiments were replicated 10 times and the average values for each group were statistically compared. Furthermore, passage 3 cells from each group were examined in terms of bone and adipogenic differentiation by specific staining as well as RT-PCR. ResultsThe cultures from plasma group appeared morphologically more homogenous than those from FCS group. In general, the cells from FCS groups had a better status in terms of total population doubling number and MTT test but these differences were not significant in passage 3. Moreover, growth curve plotted for each group indicated that the proliferation of cells in the plasma group is somewhat slower than those in FCS groups. The culture of plasma groups showed more colon cells compared to FCS groups but the colons in the latter appeared larger than the former. The cells from both groups were readily differentiated into osteoblastic and adipogenic cells lineages; this was confirmed by alizarin red and oil red staining, the bone expression of osteopontin and osteocalcin, and the expression of PPAR-alfa, PPAR-gamma, and C/EBP-alpha for adipose cells. ConclusionsTaken together, plasma as a substitute for FCS can support proliferation of MSCs and maintains their viability in vitro. Although this support was somehow less strong than FCS, plasma could still be considered a safe substitute for FCS.