Method of designing pre-freezing protocol in cryopreservation of biological materials

Procedures of determining the pre-freezing protocol in cryopreservation of biological materials are discussed. In order to cryopreserve biological materials, especially tissues with large sizes, the concentration of cryoprotective agents (CPAs) should be high enough after pre-freezing process, because a high concentration CPA solution can be vitrified a rather low cooling rate. In the pre-freezing process, the concentration of CPA is raised gradually to avoid osmotic stress. In this paper, a conventional method of designing the pre-freezing protocol is proposed. The rate of increase of the concentration of CPA can be calculated on the basis of Kedem-Katchalsky equations using the membrane permeabilities, Lp, w and s. Optimal protocol can be determined by calculating a process in which the normalized volume of the cell is kept constant whole through the process.