Methods for bulk cryopreservation encapsulated islets

Abstract

A method for bulk cryopreservation of biological material includes the steps of providing a flexible container, such as a freezer bag, containing biological material that is treated with a cryoprotectant and freezing the biological material to below -100.degree. C. and preferably below -196.degree. C. for deep freeze long-term storage. In the preferred embodiment, the bag is placed in a holder that maintains the cross-sectional area of the bag essentially constant and small enough (e.g., about 5 mm width to facilitate uniform heat transfer to and from all regions the bag. This facilitates uniform nucleation of the biological material after supercooling which enables controlled and uniform slow cooling through from about -7.5.degree. C. to a temperature in the range of about -40.degree. C. to -80.degree. C., thereby maintaining the viability of the cells. The single freezer bag method facilitates cryopreservation of large amounts of material, such as entire preparations of isolated islets, as compared to conventional multiple tube cryopreservation protocols and yields similar or improved recovery. In addition, the single freezer bag method reduces the risk of cross-contamination, a problem when adding and withdrawing material form multiple tubes before, during and after the cryopreservation freeze-thaw cycle. The bag construction also reduces the risk of freezing medium (e.g., liquid nitrogen) seeping into the bag and causing the bag to burst when thawing the material. The present invention is directed, in particular, to the cryopreservation of encapsulated islets in a flexible container wherein the container thickness during freezing is maintained around 5 mm. The freezing may also be accomplished by supercooling the encapsulated islets and inducing ice nucleation.