Amnion-Derived MSC Separation via Single-Step Enzyme Treatment

The patent divides the amnion into distinct layers (epithelial cell layer and extracellular matrix layer) and applies different enzyme treatments to each layer. The epithelial cell layer is treated with trypsin to remove epithelial cells, while the extracellular matrix layer is treated with collagenase to release mesenchymal stromal cells. This segmented approach allows selective removal of contaminants while preserving the target cells, thereby improving both purity and recovery rate.

The patent extracts and removes specific contaminating components (epithelial cells) from the amnion tissue before extracting the target mesenchymal stromal cells. By first removing the epithelial cell layer through trypsin treatment, the subsequent collagenase treatment can more effectively isolate mesenchymal stromal cells without contamination, thus improving recovery rate while maintaining high purity.

The patent modifies the chemical composition parameters of the cryopreservation solution by reducing DMSO concentration from conventional high levels (e.g., 10%) to lower levels (e.g., 5% or less) and replacing it with alternative cryoprotectants such as hydroxyethyl starch or dextran. This parameter change maintains the cryopreservation effectiveness during storage while eliminating the harmful cytotoxic effects of high DMSO concentrations during thawing, thus improving post-thaw cell survival.

The patent introduces alternative cryoprotectants (hydroxyethyl starch or dextran) as intermediary substances that fulfill the cryopreservation function without the harmful effects of high DMSO. These alternative agents act as mediators that protect cells during storage while being less harmful during thawing, thus resolving the contradiction between storage reliability and post-thaw survival.