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.

Staggered Herringbone Microfluid Device for the Manufacturing of Chitosan/TPP Nanoparticles: Systematic Optimization and Preliminary Biological Evaluation

“…Cell nuclei were stained by Hoechst33258 (Sigma Aldrich, St Luis, MO, USA). Cell membrane was observed highlighting the CD44 expression on the hMSCs surface by immunocytochemistry assay as previously described [52]. Cells were observed by confocal microscopy (obj mag 63×) and all confocal images were elaborated by using ImageJ software to evaluate the CS/TPP NPs uptake in hMSCs.…”

Untitled

“…Feasible presence of human ovarian follicle-derived MSCs was published formerly, and these research findings illuminate previous inquiries (Kossowska-Tomaszczuk et al 2009b;Riva et al 2014). Vimentin, which is a specific protein of cytoskeleton intermediate filaments of mesenchymal cells, was chosen to determine cell populations isolated from human FF (Riva et al 2014). Immunofluorescence showed an intense cytoplasmic staining for vimentin in MSC at different times of culture.…”

“…Our observations indicated that this fluid, despite the presence of granulosa and thecal cells (Honda et al 2007;Kossowska-Tomaszczuk et al 2009b), consist of heterogeneous cell populations involving a cell collection exchanging fibroblast-like morphology and other characteristics of MSCs (Kucia et al 2005). Feasible presence of human ovarian follicle-derived MSCs was published formerly, and these research findings illuminate previous inquiries (Kossowska-Tomaszczuk et al 2009b;Riva et al 2014). Vimentin, which is a specific protein of cytoskeleton intermediate filaments of mesenchymal cells, was chosen to determine cell populations isolated from human FF (Riva et al 2014).…”

“…The human ovarian follicular fluids (~10-15mL) was obtained from 78 infertile patients treated with controlled ovarian hyperstimulation for in vitro fertilization (IVF)from the center of reproductive medicine of AJUMS general hospital. Briefly, the samples were gathered from women under IVF therapy during oocytes retrieval through ultrasound-guided aspiration needle (Lai et al 2015;Riva et al 2014). To get better quality control, oocyte retrieval process was conducted by two operators.…”

“…Then, PBS was used to wash it, and 4% ice-cold paraformaldehyde was applied to fix it for 10 to 15 minutes. When it was re-washed three times with PBS, the permeabilization of the cells was done by 0.1% Triton X-100 at room temperature for 10 minutes (Riva et al 2014). Afterwards, the blocking solution (1% BSA,1x PBS) was used to block non-specific binding of the antibodies for 30-45 min, and incubation was done with anti-vimentin (rabbit antihuman at a ratio of 1:200; Santa Cruz), anti-OCT4 (rabbit antihuman, 1:200; Santa Cruz), anti-NANOG (rabbit antihuman, 1:200; Santa Cruz), anti-ZP2 and anti-ZP3 (mouse monoclonal,1:100; Santa Cruz) antibody at 4℃ overnight.…”