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Publication Number
US-11903383-B2
Publication Date
2024-02-20
Expiration Date
Abstract
The present invention relates to methods and apparatus for the cryopreservation of biological samples involving a density assisted vitrification wherein a sample of biological material in a cryopreservation agent is cooled from its top surface, causing an ice layer to form thereon. As cooling continues the ice layer grows downwards through the sample to provide a cryoprotectant and biological material rich layer below the ice layer that undergoes vitrification as cooling continues to below the glass transition temperature.
Core Innovation
The invention is a method for cryopreservation of an initially liquid sample comprising a biological material in a cryopreservation medium, where the method cools the top surface of the sample to selectively form an ice layer at the top surface. Further cooling is applied to provide formation of ice from the ice layer at the top surface progressively downwardly toward a base of the sample whereat the biological material is substantially located or concentrated.
Cooling of the top surface is continued so that a layer of the sample below the ice layer solidifies as a glass. The method forms a vitrified, or amorphous solid, layer as a result of cooling below the glass transition temperature (Tg), with the cryopreservation medium being an aqueous solution comprising at least one cryoprotectant.
The approach is characterized by density-assisted/progressive directional vitrification, in which solute-free ice layer formation at the top drives cryoprotectant and biological material into a dense lower layer. The described approach supports cryopreservation of larger samples without extremely high cooling rates or pressure by using the progressively downward ice formation and subsequent vitrification below Tg.
Claims Coverage
The partial content provides one independent claim directed to the core cryopreservation method. The independent claim includes multiple inventive features, but no additional independent claims are provided in the received text.
Top-surface ice-layer formation and progressive downward ice formation
Cooling the top surface of the initially liquid sample to selectively form an ice layer at the top surface, and further cooling the sample to provide formation of ice from said ice layer at the top surface progressively downwardly toward a base of the sample whereat the biological material is substantially located or concentrated.
Vitrification below the glass transition temperature
Continuing the cooling applied to the top surface of the sample until a layer of the sample below the ice layer solidifies as a glass to form a vitrified, or amorphous solid, layer as a result of cooling below the glass transition temperature (Tg).
Aqueous cryopreservation medium comprising at least one cryoprotectant
Using a cryopreservation medium that is an aqueous solution comprising at least one cryoprotectant.
Across the claim coverage shown, the method centers on cooling the top surface to form an ice layer and drive progressive downward ice formation toward the sample base, followed by continued cooling until the region below the ice layer vitrifies as an amorphous solid below Tg using an aqueous cryopreservation medium comprising at least one cryoprotectant.
Stated Advantages
Enables cryopreservation of larger samples without extremely high cooling rates or pressure.
Documented Applications
Cryopreservation of biological material in an initially liquid sample, including biopsies, tissues, tissue constructs, proteins, antibodies, and cells.
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