Abstract
OBJECTIVES: There has been increasing interest in mesenchymal stem cells (MSCs) because of their potential use for regenerative therapy; however, there is no well-defined protocol for MSCs culture. This study compares techniques of conventional plate and microcarrier culturing of MSCs. METHODS AND RESULTS: Here, different conditions for isolation and expansion of rat MSCs have been examined and it was found that plating density and plating time in primary culture played important roles for culture of these rat MSCs. When plated at 10(8) /cm(2) density for 72 h, in primary culture, recycling stem cells (RS cells) predominated, and characteristics of rat MSCs (including morphology, growth rate, phenotype and differentiation potentials) remained stable during expansion until passage 14. For subculture of the cells, it was found that their growth rate when incubated at 33 °C was higher than those incubated at 37 °C, and maximal increase was 10- and 6-fold respectively. When cultured using microcarriers, at a density of 1 × 10(5) /mg beads, growth kinetics, phenotype and differentiation potentials also remained constant for cells between passage 2nd and 14th; their maximal number increased 16-fold. CONCLUSIONS: Compared to conventional plate culture, culture using gelatine porous microcarrier Cultispher-S was superior for large-scale production of rat MSCs.