Abstract
Background aimsCurrent procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. MethodsWe compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. ResultshAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. ConclusionsThis study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.
Highlights
The current effort in regenerative medicine is the use of human stem cells that are easy to collect, high proliferating, with large plasticity and without ethical issues
Amniotic fluid cell cultures were harvested by trypsinization, and subjected to c-Kit immunoselection by MACS® technology (Miltenyi Biotec, Germany). Human amniotic fluid stem cells (hAFSCs) were subcultured routinely at 1:3 dilution and not allowed to expand beyond the 70% of confluence. hAFSCs were grown in culture medium (AmnioChrome II) including an enriched basal medium and growth supplement, since it is a medium used for the culture of human amniotic fluid cells obtained from amniocentesis
The clonogenic capacity of C and D samples are shown in Fig. 1 C,D where the first graph displays the number of colony forming unit (CFU) obtained after 10 days depending on the cell seeded density: where the initial cell concentration was 80 cells/cm2 the number of colonies is comparable, while at 160 cells/cm2, the D samples produce a higher colony-forming unit (CFU) number
Summary
The current effort in regenerative medicine is the use of human stem cells that are easy to collect, high proliferating, with large plasticity and without ethical issues. HAFSCs samples were cultured in flask in cytogenetic laboratories for two weeks, can be devoted to research purpose, instead of being discarded This long time in culture, before the step of freezing and selection, may change the properties of the stem cells sub-population. In this study we verify if a direct freezing of the amniotic fluid, taken at the time of amniocentesis, is able to maintain and / or improve the potential of the sub-population of stem cells For this purpose, the proliferative rate during in vitro passages, before and after the selection process (based on c-kit antigen expression), has been analyzed by population doubling time calculation. HAFSCs, deriving from the two storage methods (samples C and D), were induced to differentiate towards mesenchymal, endodermal and ectodermal lineages, in order to verify if the differentiation potential is comparable
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
