Abstract: The invention discloses a multi-layer cell freezing vial and method of ohtention thereof. The method of obtention is a cell freezing and thawing process that avoids the need of incorporating new fresh medium to the thawed cells for the dilution of the cryoprotective agent. This is achieved by the previous freezing of an extra layer of culture medium in addition to the frozen ceil solution containing said cryoprotective agent. At the time of thawing, the culture medium and the cell solution mix themselves, turning the concentration of said cryoprotective agent to a non-toxic dilution and achieving the effective exclusion of said cryoprotective agent from the living cells.