Method of cryopreserving a suspension of biological material
AssigneeLifeCell Corporation
InventorsStephen A. Livesey, Anthony A. del Campo, Abhijit Nag
Life of this patent
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Abstract
This invention relates to methods, apparatus and solutions for cryopreserving microscopic biological materials for biologically extended periods of time. The method comprises treating a suspension of biological material, in an appropriate buffer, with a cryoprotectant or combination of cryoprotectants which raises the glass transition temperature range of the sample. One or more dry protectants may be added to the cryosolution. The cryosolution is then nebulized and rapidly cooled with novel apparatus, dried by molecular distillation, stored and then rehydrated in a buffer prior to its use. The solutions comprise novel mixtures of cryoprotectants.
Claims27
A method for preserving a suspension of biological material comprising:
- (a) preparing a cryosolution of a suspension of biological material, said cryosolution comprising a buffer, a cryoprotectant and a biological material;
- (b) nebulizing said cryosolution with a low frequency, ultrasonic nebulizer to form microdroplets;
- (c) cooling the microdroplets at a rate which results in the formation of amorphous phase ice and/or cubic phase ice with or without hexagonal phase ice in said cryosolution by spraying the microdroplets on a solid cryogenic surface;
- (d) continuously removing said cryosolution from said solid cryogenic surface after cooling to replenish said surface for further cooling of additional microdroplets;
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- (e) drying the cooled cryosolution to form a dried cryosolution.
A method for preserving a suspension of a microscopic biological material comprising:
- (a) preparing a cryosolution of a microscopic biological material, said cryosolution comprising a buffer, a cryoprotectant, and a biological material;
- (b) nebulizing said cryosolution With a low frequency, ultrasonic nebulizer to form discrete droplets, said droplets being less than about 200 .mu.m in diameter;
- (c) rapidly cooling said droplets resulting in the formation of amorphous phase ice, cubic phase ice and hexagonal phase ice by spraying the droplets on a solid cryogenic surface;
- (d) continuously removing the rapidly cooled cryosolution from said solid cryogenic surface after said ice has been formed to replenish said surface for cooling of additional droplets;
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- (e) drying said rapidly cooled cryosolution to remove said amorphous phase ice, cubic phase ice and hexagonal ice to form a dried cryosolution, said drying being completed to a final residual moisture content of less than about 5%.
Dependencies are inferred from the claim text.