Culture of cells from mammalian tissue cryopreserved without cryoprotection

Abstract

Donor cells for nuclear transfer are usually prepared by the culture of fresh tissue. However, animal carcasses are sometimes frozen without cryoprotectants and if it were possible to obtain live cells from carcasses (tissue) preserved in this manner, it could be very beneficial in nuclear transfer cloning of trophy or extinct animals. This study tested the hypothesis that tissue samples of skin, muscle, and oral mucosa could be cryopreserved without cryoprotection. The tissue samples were taken from euthanized goats and placed into a -20°C freezer for varying lengths of time. The samples were thawed by two different methods. One method was in 37°C water bath and the other was on ice, thawing to room temperature from 1°C to 25°C. The samples were then processed and placed into an incubator to evaluate cell growth. Skin samples frozen for up to 34 days obtained cell growth to confluency and the cells were then cryopreserved with cryoprotectant. The cells were able to tolerate the potentially lethal effects of ice nucleation and dehydration brought about by ice formation and colligative factors. Although this method of cryopreservation has been shown to yield growth of cells that might be useful for nuclear transfer cloning, it is not the recommended method to cryopreserve tissues if cryoprotectants are available or if only short term storage is needed. These procedures would be especially useful when a precious animal dies unexpectedly and cryoprotectant is not available and the sample can not be processed before 10 days.