Systems and methods for cryopreservation of cells

Inventors

Woods, Erik John • Critser, John K.

Assignees

COOK REGENTEC LLC • Sexton Biotechnologies Inc

Abstract

An auto-nucleating device includes a hollow tube containing a crystalline cholesterol matrix therein. The ends of the tube are closed by a membrane that is impermeable to the cholesterol but permeable to liquids contained in a cryopreservation vessel. The auto-nucleating device is disposed within the vessel and provides a site for ice nucleation during freezing of the liquid within the vessel. One such cryopreservation vessel is a flexible vial having a closed port at one adapted to be pierced by a needle to withdraw the liquid within. The opposite end of the vial is initially open to receive the liquid. The end is then sealed to form a closed system for cryopreservation. Another cryopreservation vessel includes an adaptor mounted to a port of a liquid container. The adaptor includes one tubular branch that is closed by a needle septum while another tubular branch includes a barbed fitting for engaging a flexible tube. The flexible term terminates in a needle septum. The container is initially at below-atmospheric pressure. In use, the liquid sample is injected by needle through the septum in the second branch. Once the container is filled with the sample, the flexible tube is heat sealed and severed just above the barbed fitting. The sealed container may then be subject to a cryopreservation protocol. After thawing, the sample liquid may be withdrawn by a needle puncturing the septum in the first branch.

Core Innovation

The invention relates to an auto-nucleating device for introduction into a cryopreservation vessel prior to freezing of a liquid contained therein. The device includes an elongated hollow tube sized for introduction into the cryopreservation vessel, and an ice-nucleating composition disposed within the hollow tube. The hollow tube is sealed at both ends, where at least one end is sealed with a membrane that is impermeable to the ice-nucleating composition but permeable to the liquid contained within the cryopreservation vessel.

The disclosed auto-nucleating device is configured so that a crystalline cholesterol (sterol) ice-nucleating matrix can be retained within the elongated hollow tube while cryopreservation liquid permeates through the membrane-sealed ends. The ice-nucleating composition can be provided as a coating on the interior of the hollow tube or as a solid matrix disposed within the tube. The device is introduced into the cryopreservation vessel prior to freezing to reduce supercooling and promote extracellular ice nucleation in relation to freezing.

Cryopreservation vessel configurations are also disclosed for use with the auto-nucleating device. A flexible tubular body can be used to introduce a liquid sample and can define a closed port adapted to be pierced by a needle for withdrawal of the liquid sample, with the auto-nucleating device disposed within the tubular body. The disclosure includes vessel formats intended to be compatible with practical banking formats and tests showing improved freeze-thaw outcomes and reduced temperature change during fusion for cryopreservation solutions.

Claims Coverage

The independent claims cover three main areas: an auto-nucleating device inserted into a cryopreservation vessel prior to freezing, a second auto-nucleating device variant with membrane-sealed ends, and a cryopreservation vessel that integrates the auto-nucleating device and includes a needle-pierceable closed port. Across these claims, there are three inventive feature clusters.

Overall, the claim set focuses on an auto-nucleating device that retains an ice-nucleating composition inside an elongated hollow tube using membranes that are impermeable to the ice-nucleating composition but permeable to the cryopreservation liquid, and on cryopreservation vessel formats that incorporate this device while providing a needle-pierceable closed port for withdrawal.

Stated Advantages

Documented Applications