Systems and methods for cryopreservation of cells
Inventors
Woods, Erik John • Critser, John K.
Assignees
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Publication Number
US-8222027-B2
Publication Date
2012-07-17
Expiration Date
Abstract
An auto-nucleating device includes a tube containing a crystalline cholesterol matrix. 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 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, and an opposite end that is initially open to receive the liquid. Another vessel includes an adaptor mounted to liquid container with a tubular branch closed by a needle septum and another tubular branch provided with a barbed fitting for engaging a flexible tube that terminates in a needle septum. In another embodiment, the vessel includes an inlet and vent branch at the top of the container and an outlet septum at a bottom opening.
Core Innovation
The invention relates to an auto-nucleating cryopreservation system that includes an ice-nucleating solid-state cholesterol matrix held inside an elongated hollow tube. The hollow tube contains an ice-nucleating composition disposed within the tube and is sealed at both ends, with at least one end sealed with a membrane that is impermeable to the ice-nucleating composition but permeable to the liquid contained within the cryopreservation vessel, enabling extracellular ice nucleation with reduced supercooling.
The system is configured so the ice-nucleating composition is restrained by membrane impermeability while liquid can permeate the membrane, supporting minimal intracellular ice. The auto-nucleating device is disposed within a container for receiving and storing a liquid sample, with the device introduced through an inlet fitting into the container. The described configuration supports cryopreservation using a nucleating device inside the vessel to control freezing behavior rather than relying on manual seeding.
The disclosure provides multiple cryopreservation vessel embodiments compatible with the device, including a flexible closed vial with a needle-pierceable port and an arrangement positioned so the nucleating device is not needle-accessible, an adaptor-based container having a needle-pierceable septum and a tube-engaging fitting, and an enclosed container with inlet/vent tubing using a sterile micro-filter vent and a bottom needle septum for post-thaw withdrawal. The described embodiments include sealing and closure structures such as heat-sealed tube ends, and one embodiment is optionally configured under below-atmospheric initial pressure.
Claims Coverage
The independent claim covers a cryopreservation device with an internal auto-nucleating device including an elongated hollow tube holding an ice-nucleating composition, sealed by membranes that separate the composition from the liquid sample while allowing liquid permeability. The independent claim further covers specific inlet/adaptor, septum, tube engagement, and closure features, with multiple dependent claims refining fitting, septum piercing, tube orientation, heat-sealed opposite end closure, and container pressure below atmospheric.
Membrane-sealed internal ice-nucleating hollow tube for extracellular nucleation
An auto-nucleating device disposed within the container, the auto-nucleating device including an elongated hollow tube sized for introduction through the inlet fitting, an ice-nucleating composition disposed within the hollow tube, and a seal at both ends of the tube, at least one end sealed with a membrane that is impermeable to the ice-nucleating composition but permeable to the liquid contained within the cryopreservation vessel.
Adaptor and inlet/tube arrangement with needle-pierceable septum and sealed opposite end
An adaptor mounted to the fitting, the adaptor having a first tubular branch and a second tubular branch, the second tubular branch terminating in a tube engaging fitting; a septum closing the first tubular branch, adapted to be pierced by a needle; a tube engaged at one end to the tube engaging fitting on the second tubular branch; and a closure at the opposite end of the tube.
Below-atmospheric container pressure configuration
The container is configured to be kept at a pressure below atmospheric pressure.
Overall, claim coverage emphasizes an elongated hollow tube containing an ice-nucleating composition sealed with membranes impermeable to the composition but permeable to the vessel liquid, and a defined inlet/adaptor/tube arrangement including a needle-pierceable septum and a tube with a sealed opposite end. Dependent refinements add specific fitting/septum and closure implementations, including tube heat-sealing at the opposite end and, in one refinement, keeping the container pressure below atmospheric.
Stated Advantages
Provides extracellular ice nucleation with reduced supercooling.
Supports minimal intracellular ice.
Human semen studies indicate higher post-thaw motility with the auto-nucleating device versus manual seeding and versus unseeded controls.
Bound cholesterol shows no significant cytotoxicity over incubation.
Documented Applications
Cryopreservation of cells, including human semen, using an auto-nucleating device versus manual seeding and unseeded controls.
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