The use of discontinuous density gradients in stem cell research and application.

Discontinuous density gradients have been shown to be very efficient in sperm enrichment in assisted reproductive technologies (ART). Percoll [polyvinylpyrrolidone (PVP)-coated silica] which is a colloidal agent commonly used in such gradients is now confined to only research purposes in ART because its PVP component was considered toxic for clinical applications. The PVP component was replaced with silane instead and silane-coated colloidal silica marketed as Puresperm is currently being used for clinical sperm enhancement procedures in ART. The efficiency of these two agents has not been tested for human embryonic stem cell (hESC) and cancer cell research and clinical application. Herein we describe discontinuous density gradient protocols using PureSperm and Percoll for the separation and enrichment of hESCs and hepatocarcinoma cells from a mixed cell population. Trypan blue cell viability and CellTracker green cell counts are used to accurately quantify the extent of cell separation. We show that Puresperm provides good cell separation and enrichment with higher viable cell counts and greater number of fractions compared to Percoll. PureSperm being superior and safer compared to Percoll would be a better alternative for cell separation and enrichment in stem and cancer cell research and clinical applications.