In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

Abstract

To characterize different cell populations in the human ovary, morphological and functional characteristics of cell populations collected during routine IVF procedures were studied. Cells obtained from follicular fluid grew in vitro under minimal medium conditions, without growth factor, including leukaemia-inhibiting factor. Morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial-like and also neuron-like features. Morpho-functional characteristics of fibroblast-like cells were similar to mesenchymal stem cells, and, in particular, were positive for mesenchymal stemness markers, including CD90, CD44, CD105, CD73, but negative for epithelial proteins, such as cytokeratins, CD34 and CD45 antigens. Cell proliferation activity at different times and colony-forming unit capability were evaluated, and multipotency of a subset of granulosa cells was established by in-vitro differentiation studies (e.g. osteogenic, chondrogenic and adipogenic differentiation). This study suggests that cells provided by mesenchymal plasticity can be easily isolated by waste follicular fluid, avoiding scraping of human ovaries, and cultivated in minimal conditions. Successful growth of such progenitor cells on three-dimensional cryogel scaffold provides the basis for future developments in tissue engineering. This culture system may be regarded as an experimental model in which biological behaviour is not influenced by specific growth factors.

Keywords: 3D scaffold; granulosa cells; human follicular fluid; in-vitro differentiation; leukemia-inhibiting factor; mesenchymal stem cells.