High aspect ratio devices and methods for vitrification of biological samples by rapid cooling

A method for high-volume cryopreservation of a plurality of cells by vitrification, the method comprising:

• (a) incubating the cells in a vitrification solution comprising a dehydrating agent for a time and under conditions sufficient to dehydrate the cells;
• (b) adding one or more cryoprotective agents (CPAs) to the vitrification solution at a total CPA concentration of up to 30% and incubating the cells in the vitrification solution for a time and under conditions sufficient for uptake of the one or more CPAs by the cells;
• (c) after step (b), adding an additional amount of the one or more CPAs to the vitrification solution to increase a total concentration of the one or more CPAs in the vitrification solution by at least an additional 10% and incubating the cells for up to one minute;
• (d) loading the cells in the vitrification solution into a vitrification cassette, wherein the vitrification cassette comprises:
• a planar bottom layer;
• a planar top layer;
• a planar intermediate layer disposed between the bottom layer and the top layer and enclosing a chamber within the intermediate layer, wherein the chamber has a height of about 50 to about 150 μm, a volume of at least 200 μL to 500 μL, and outer dimensions of the planar top layer and planar bottom layer of about 25 to about 100 mm by about 15 to about 80 mm;
• and
• at least one port fluidly connected to the chamber,
• wherein the bottom, the intermediate, and the top layers are laminated together or held together mechanically;
• and
• (e) cooling the cells in the vitrification solution in the vitrification cassette to a temperature less than or equal to a glass transition temperature of the vitrification solution at a rate equal to or greater than 30,000° C./minute, wherein the cooling causes vitrification of the plurality of cells.