High throughput system for production and vitrification of biomaterials in cryoprotectant droplets

Patent metadata

Field	Value
Publication number	US-12458016-B2
Application number	US-202017431361-A
Country	US
Kind code	B2
Filing date	Feb 25, 2020
Priority date	Feb 28, 2019
Publication date	Nov 4, 2025
Grant date	Nov 4, 2025

Abstract

Methods and vitrification systems for biological samples are provided. The vitrification system has a rotatable cryowheel ( 210 ) with a facing surface ( 220 ). Droplets of a composition that includes a biological sample are released onto the facing surface. Droplets are rapidly pulled beneath the surface of the cryogenic coolant ( 160 ) to generate vitrified samples ( 180 ). Methods and cryopreservation devices are also provided that incorporate the vitrification systems.

First claim

What is claimed is:

1 . A vitrification system comprising:
a rotatable cryowheel comprising a porous facing surface, wherein the rotatable cryowheel is in a cryogenic container, wherein the facing surface of the cryowheel lightly adheres droplets when the droplets are released onto the facing surface of the cryowheel as the cryowheel emerges above the level of a cryogenic coolant in the cryogenic container, wherein the droplets comprise a biological sample and wherein rotation of the cryowheel submerges the droplets of the biological sample on the facing surface into the cryogenic coolant.

2 . The system of claim 1 , wherein a material for the facing surface of the cryowheel is selected from the group consisting of balsa wood, polystyrene foam, and ethylene vinyl acetate foam.

3 . The system of claim 1 , further comprising an apparatus for driving the rotation of the cryowheel.

4 . The system of claim 1 , further comprising a scraper for detaching the biological sample from the facing surface in the cryogenic coolant and forming a vitrified biological sample.

5 . The system of claim 4 , further comprising a tray, wherein the tray is configured to receive the vitrified biological sample.

6 . The system of claim 1 , wherein the droplet further comprises cryoprotectants and laser absorbers.

7 . The system of claim 1 , wherein the cryowheel comprises a notched edge for engaging a driving system for rotation.

8 . The system of claim 1 , wherein the biological sample is selected from the group consisting of zebrafish embryos, pancreatic islets, Xenopus oocytes, C. elegans, germplasm, coral germplasm, coral larvae, mammalian tissue, mammalian germplasm, bacteria or protozoans.

9 . A cryopreservation device comprising:
at least a first input port and a second input port; a mixing system comprising tubing with an inlet end and an outlet end, wherein the inlet end is operably connected to the first port and the second port, wherein the tubing is configured to mix compositions entering from the at least first inlet port and second input port and as the compositions traverse the tubing from the inlet end to the outlet end; a droplet production head comprising a nozzle with a tip and an actuator, the nozzle operably attached to the outlet end of the tubing in the mixing system and the actuator configured to tap the side of the nozzle at set intervals to dislodge a consistent sized droplet of the combined composition from the tip of the nozzle; and a rotatable cryowheel comprising a porous facing surface, wherein the rotatable cryowheel is in a cryogenic container, wherein the facing surface of the cryowheel lightly adheres the droplets of the composition when released onto the facing surface of the cryowheel as the cryowheel emerges above the level of a cryogenic coolant in the cryogenic container, wherein rotation of the cryowheel submerges the droplets of the composition into the cryogenic coolant, wherein the droplets comprise a biological sample.

10 . The device of claim 9 , wherein the mixing system comprises a serpentine mixing system comprising at least three vertical posts configured to retain the tubing in a serpentine configuration, promoting the thorough mixing of the compositions introduced into the mixing system.

11 . The device of claim 10 , wherein the serpentine mixing system is configured to dispose the tubing on a level plane and to prevent biological samples of density different from the density of surrounding composition from accumulating in low or high points of the tubing.

12 . The device of claim 9 , wherein the actuator is a solenoid actuator.

Assignees

• Univ Minnesota
• Smithsonian Inst

Inventors

• Zuchowicz Nikolas
• Hagedorn Mary
• Daly Jonathan
• Zhan Li
• Khosla Kanav
• Bischof John

Classifications

• F25D3/11

  with conveyors carrying articles to be cooled through the cooling space · CPC title

• A01N1/145 Primary

  Stationary or portable vessels generating cryogenic temperatures, e.g. liquid nitrogen baths · CPC title