High aspect ratio devices and methods for vitrification of biological samples by rapid cooling
Methods of bulk cryopreservation of C. parvum oocysts by vitrification using high aspect ratio cryopreservation devices are disclosed. Cryopreserved oocysts exhibit high viability, maintain infectivity in vitro, and are infectious to interferon-γ knockout mice. The course of the infection is comparable to that observed with unfrozen oocysts.
1 . 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.
2 . The method of claim 1 , wherein the dehydrating agent is a salt, a sugar, or a combination thereof.
3 . The method of claim 2 , wherein the dehydrating agent is a sugar comprising sucrose, trehalose, raffinose, stachyose, dextran, or a combination of any two or more thereof.
4 . The method of claim 1 , wherein the one or more cryoprotective agents are selected from the group consisting of glycerol, ethylene glycol, 1,2-propanediol, and dimethyl sulfoxide (DMSO).
5 . The method of claim 1 , wherein step (c) comprises increasing the total concentration of the one or more cryoprotective agents in the vitrification solution by at least 15%.
6 . The method of claim 1 , wherein the cells in the vitrification solution are cooled at a rate equal to or greater than 100,000 to 250,000° C./minute.
7 . The method of claim 1 , further comprising, prior to step (a), exposing the cells to a permeabilization solution comprising a permeabilizing agent for a time and under conditions sufficient to increase uptake of the cryoprotective agent by the cells compared to uptake of the cryoprotective agent by untreated cells.
8 . The method of claim 7 , wherein the permeabilizing agent is hypochlorite.
9 . The method of claim 1 , wherein a level of infectivity or viability, or both, of the cells after vitrification is at least 50% of the level of infectivity or viability, or both, of untreated cells.
10 . The method of claim 1 , wherein the cells are oocytes.
11 . The method of claim 1 , wherein the one or more CPAs comprise DMSO.
12 . The method of claim 1 , wherein the dehydrating agent comprises trehalose and the one or more CPAs comprise DMSO.
13 . The method of claim 1 , wherein in step (c) the concentration of the one or more CPAs in the vitrification solution is increased to a total concentration of up to 50%.
14 . The method of claim 13 , wherein the dehydrating agent is a salt, a sugar, or a combination thereof.
15 . The method of claim 14 , wherein the dehydrating agent is a sugar comprising sucrose, trehalose, raffinose, stachyose, dextran, or a combination of any one or more thereof.
16 . The method of claim 13 , wherein the one or more CPAs are selected from the group consisting of glycerol, ethylene glycol, 1,2-propanediol, and dimethyl sulfoxide (DMSO).
17 . The method of claim 16 , wherein the one or more CPAs comprise DMSO.
18 . The method of claim 16 , wherein the dehydrating agent comprises trehalose and the one or more CPAs comprise DMSO.