Micromanipulation and storage apparatus and methods
A method of cryopreservation of biological material includes the steps of providing an apparatus including a vessel having a reservoir wherein the vessel has a channel formed in a portion of the reservoir, the channel including an intermediate restriction dimensioned to resist passage of the biological material but allow passage of liquid treatment solutions, and the channel including sub-reservoirs at either end of the restriction; introducing the biological material into a reservoir region of the apparatus for capture in a channel region of the vessel; irrigating and draining the biological material with a series of vitrification solutions introduced into a first of the sub-reservoirs and removed from a second of the sub-reservoirs; final draining the irrigation solutions from the vessel; and vitrifying the vessel and captive biological material.
1. A method of cryopreservation of biological material using an apparatus including a vessel having a reservoir, wherein said vessel has a channel formed in a portion of said reservoir, said channel including an intermediate restriction dimensioned to resist passage of said biological material but allow passage of liquid treatment solutions, and said channel including a first sub-reservoir at a first end of said restriction and a second sub-reservoir at a second end of said restriction, the method comprisin the steps of:
introducing said biological material into the first sub-reservoir of said vessel;
irrigating said biological material with a series of vitrification solutions introduced into the first sub-reservoir and removed from the second sub-reservoir;
removing the vitrification solutions from the second sub-reservoir, leaving a residual volume of liquid surrounding the biological material that is sufficiently small for vitrification; and
vitrifying the biological material and the residual volume of liquid surrounding the biological material within the vessel.
2. A method according to claim 1 , wherein said vitrification solutions include non-permeating and permeating cryoprotectants selected from the group consisting of Ethylene Glycol (EG), Dimethyl Sulphoxide (DMSO), Glycerol, and disaccharides solutions.
3. A method of cryopreservation of biological material using an apparatus including a vessel having a reservoir, wherein said vessel has a channel formed in a portion of said reservoir, said channel including an intermediate restriction dimensioned to resist passage of said biological material but allow passage of liquid treatment solutions, and said channel including a first sub-reservoir at a first end of said restriction and a second sub-reservoir at a second end of said restriction, the method comprising the steps of:
introducing said biological material into the first sub-reservoir of said vessel;
irrigating said biological material with a single vitrification solution gradually introduced into the first sub-reservoir;
removing the vitrification solution from the second sub-reservoir, leaving a residual volume of liquid surrounding the biological material that is sufficiently small for vitrification;
vitrifying the biological material and the residual volume of liquid surrounding the biological material within the vessel.
4. A method according to claim 3 , wherein said vitrification solution includes non-permeating and permeating cryoprotectants selected from the group consisting of Ethylene Glycol (EG), Dimethyl Sulphoxide (DMSO), Glycerol, and disaccharides solutions.
5. A method according to claim 3 , wherein said vitrification solution is gradually introduced at a rate to enhance sinking of said biological material to a bottom of the first sub-reservoir.
6. A method for pre-vitrification treatment of a biological material, using an apparatus including a vessel having a reservoir wherein said vessel has a channel formed in a portion of said reservoir, said channel including an intermediate restriction dimensioned to resist passage of said biological material but allow passage of liquid treatment solutions and said channel including a first sub-reservoir at a first end of said restriction and a second sub-reservoir at a second end of said restriction, the method comprising the steps of:
introducing said biological material into the first sub-reservoir of said vessel; and
irrigating said biological material with pre-vitrification solutions introduced into the first sub-reservoir and removed from the second sub-reservoir.
7. A method of thawing a vitrified biological material in an apparatus including a vessel having a reservoir wherein said vessel has a channel formed in a portion of said reservoir, said channel including an intermediate restriction dimensioned to resist passage of said biological material but allow passage of liquid treatment solutions, and said channel including a first sub-reservoir at a first end of said restriction and a second sub-reservoir at a second end of said restriction, the method comprising the steps of:
heating the vessel;
irrigating the biological material with a series of thawing solutions introduced into the first sub-reservoir and removed from the second sub-reservoir; and
recovering the thawed biological material from said vessel.
8. A method according to claim 1 , wherein said vitrification solutions are gradually introduced at a rate to enhance sinking of said biological material to a bottom of the first sub-reservoir.
9. A method according to claim 1 , wherein said vitrification solutions include non-permeating cryoprotectants selected from disaccharides solutions.
10. A method according to claim 3 , wherein said vitrification solution includes non-permeating cryoprotectants selected from disaccharides solutions.
11. A method according to claim 1 , wherein said vitrification solutions include permeating cryoprotectants selected from Ethylene Glycol (EG), Dimethyl Sulphoxide (DMSO), and Glycerol.
12. A method according to claim 3 , wherein said vitrification solution includes permeating cryoprotectants selected from Ethylene Glycol (EG), Dimethyl Sulphoxide (DMSO), and Glycerol.