Isolation, expansion and differentiation of rMSC
Five New Zealand female rabbits were included in the study under guidelines determined by the Local Ethical Committee.
Five mL samples of bone marrow aspirates from femur were drawn and treated to induce haemolysis. The remaining nucleated cell suspensions were centrifuged at 500 g for 10 minutes and the pellets were resuspended in low glucose DMEM supplemented with 10% fetal calf serum (FCS), 10 U/mL penicillin G, 10 μg/mL streptomycin, 2 mM L-glutamine.
Cells were counted and resuspended in standard culture medium (SCM): α-MEM, 20% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine. Plating concentration was 105cells/cm2 in 25 cm2 tissue culture flasks. After 24 hours the non adherent cells were removed by washing with PBS. Fresh SCM was added twice a week up to 90% confluence (passage 0, P0); at P0, CFU-F were counted by visual examination to evaluate the rMSC number in the primary culture. Cells were then harvested for further expansion and re-plated at 5000 cells/cm2. At the end of each passage (90% confluence) cells were counted. Cell Doubling (CD), Cumulative Population Doublings (CPD) and Doubling Time (DT) values were calculated following established formulae.
Osteogenic, chondrogenic and adipogenic differentiation were performed following standard protocols.
Determination of influence of culture passage and plating density on cell proliferation and clonogenic potential
Aliquots of rMSC from different culture passages (P1, P2 and P3) were assayed for cell proliferation (fold increase), and clonogenic ability (CFU-F assay).
Cells were plated at 10 cells/cm2, 100 cells/cm2 and 1000 cells/cm2 in SCM in 12-well tissue culture plates in duplicate. Every day for a week, cells from each culture density (in triplicate) were detached and counted in a haemocytometer. Viability was assessed by 0.4% Trypan Blue exclusion test. Fold increase was calculated dividing the number of harvested cells at 90% confluence by the number of plated cells.
To evaluate the clonogenic potential the CFU-F assay was performed as follows: rMSC were seeded in SCM, at 10 cells/cm2, 100 cells/cm2 and 1000 cells/cm2 in 6-well tissue culture plates. Colonies were counted on day 7 and 10. Cells were then stained with Crystal Violet (0.5%) in methanol at RT for 10 minutes, washed twice with PBS and visually counted.
Selection of different culture conditions and media for rMSC expansion and clonogenic ability
P3 cells were plated at 10 cells/cm2 and 100 cells/cm2 either in α-MEM, or DMEM, or Medium199 (M199), or Mesenchymal Stem Cells Basal Medium (MSCBM). FCS 20%, Penicillin 100 U/mL, Streptomycin 0.1 mg/mL and L-Glutamine 2 mM were added to α-MEM, DMEM and M199. To evaluate the effects of growth factor supplementation, 10 ng/mL human Epidermal Growth Factor (hEGF) or 10 ng/mL fibroblast growth factor (bFGF) or insulin 5 ug/mL were added to all media, with the exception of MSCBM.
To assess the effects of different serum supplements, α-MEM, DMEM and M199 containing Penicillin 100 U/mL, Streptomycin 0.1 mg/mL and L-Glutamine 2 mM, were supplemented either with 20% FCS or 10% FCS – 10% Horse Serum (HS).
After expansion for 8 days cell were counted. Fold increase and CFU-F number were calculated.
Epitope analysis of rMSC: FACS and immunochemical analyses
FACS analyses were performed on P3 cells by using anti-MHC I, anti-MHC II, anti-CD14, anti-CD45, anti-CD44, anti-β-1-integrin and anti-CD90 mouse monoclonal antibodies. As secondary antibody a FITC goat anti mouse IgG was used. P3 cells were used for immunohistochemistry. As primary antibodies anti-β-1-integrin, anti-CD90, anti-MHC I, anti-MHC II, anti-vimentin, anti-α-smooth-actin, anti-cytokeratin, anti-desmin were used. Reactivity was visualized by streptavidine-peroxidase method.
