Ice-free preservation of large volume tissue samples for viable, functional tissue banking

Inventors

BROCKBANK, Kelvin GM • Campbell, Lia H.

Assignees

Lifenet Health

Abstract

Large volume cellular material may be preserved by combining the cellular material with a cryoprotectant formulation/medium/solution containing at least one sugar and then subjecting the cellular material to a vitrification preservation protocol.

Core Innovation

The invention provides methods for preserving living large volume cellular material by exposing the cellular material to a cryoprotectant formulation containing from 0.5 M to 2 M of at least one sugar selected from trehalose and/or sucrose. The cellular material is obtained from intestine, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins and lymph nodes, and has a volume greater than 4 mL. The methods obtain cryopreserved cellular material while avoiding ice-induced damage.

The preservation protocol includes a cooling protocol, a storage phase, and a warming protocol in which ice-induced damage does not occur. During the cooling protocol, a continuous controlled rate of about -20.1 to about -22° C. per minute is used to cool the cellular material from a point of initiation temperature to -80° C. or lower, where the point of initiation temperature is from about +4 to about +30° C. After completion of the preservation protocol, the methods obtain the cryopreserved cellular material.

A related aspect increases production yield of viable cryopreserved cellular material by exposing a large volume cellular material to a cryoprotectant containing 0.5 M to 2 M of at least one sugar selected from trehalose and/or sucrose, for a predetermined amount of time to form a cryopreservation formulation. The cryopreservation formulation is subjected to a preservation protocol comprising cryopreservation at about -80° C. or lower using the cooling protocol, storage phase, and warming protocol, followed by recovering the cryopreserved cellular material.

Another aspect specifies preservation where the cellular material is a heart valve. In this method, the cryoprotectant formulation contains from 0.5 M to 2 M of at least one sugar selected from trehalose and/or sucrose, and the preservation protocol again includes cooling with a continuous controlled rate from a point of initiation temperature in a range from about +4 to about +30° C. to -80° C. or lower, followed by obtaining cryopreserved cellular material. In specific embodiments, the heart valve is obtained from a mammalian source and is immersed in the sugar-containing cryoprotectant formulation, without contacting the heart valve via perfusion with a cryoprotectant-containing solution before, during, or after exposure.

Claims Coverage

The document contains three independent claims. Across them, the main inventive features involve sugar-containing (trehalose and/or sucrose) cryoprotectant exposure for large-volume tissues, an ice-damage-avoiding preservation protocol with defined cooling, storage, and warming phases, and defined success metrics including cell viability, with further specialization for heart valves.

Across the independent claims, the coverage centers on preserving living large volume cellular material and obtaining cryopreserved cellular material without ice-induced damage by exposing the material to a cryoprotectant formulation with 0.5 M to 2 M trehalose and/or sucrose, and applying a preservation protocol with cooling, storage, and warming. One independent claim further requires increased production yield with recovered cell viability at least 60%, and another specializes the cellular material to a heart valve.

Stated Advantages

Documented Applications