# US 2013/0157362 A1 · Method of liquid nitrogen surface vitrification > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0157362 A1 - Document ID: US20130157362A1 - Status: Published application - Kind: A1 - Application number: 13/818,072 - Filed: 2011-08-17 - Published: 2013-06-20 - Earliest claimed: 2010-08-20 - Assignees: Inguran, LLC - Inventors: Fuliang Du; Jie Xu; Juan Moreno - Web page: https://patentgrep.com/patent/US20130157362A1 ## CPC - A01N 1/10: Preservation of living parts - A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators - A01N 1/126: Physiologically active agents, e.g. antioxidants or nutrients - A01N 1/14: Mechanical aspects of preservation; Apparatus or containers therefor - A01N 1/16: Physical preservation processes - A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time - C12N 5/0604: {Whole embryos; Culture medium therefor} - C12N 5/0609: {Oocytes, oogonia (fertilised oocytes C12N5/0604)} - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A method of liquid nitrogen surface vitrification requiring an embryo washed in a rinsing medium, then incubated in a base medium and incubated in a hold medium before being washed in a vitrification medium and produced into a vitrification droplet (270). For forming the droplet, vitrification medium (210), an intermediary fluid such as air, followed by vitrification medium containing at least one embryo (250) are aspirated into the channel. The vitrification droplet consequently can contain an air bubble (220). The vitrification droplet can be produced from an instrument with a channel and dropped directly into liquid phase nitrogen producing a vitrified droplet. The vitrified droplet can be stored in cryo-vessels, and warmed for revitalization of biological function of vitrified biological cell mass or tissues, such as oocytes and/or embryos. ## Claims ### Claim 1 A method of forming a vitrification droplet comprising the step of: a) aspirating vitrification medium into a channel; b) aspirating an intermediary fluid into the channel; c) aspirating vitrification medium containing at least one embryo or at least one oocyte into the channel; and d) expelling the vitrification medium, the air, and the vitrification medium containing the at least one embryo or at least one oocyte from the channel to form a vitrification droplet at the end of the channel. ### Claim 2 (depends on 1) The method according to claim 1, wherein the vitrification medium and the vitrification medium containing at least one embryo are separated by the intermediary fluid in the channel prior to the step of expelling. ### Claim 3 (depends on 1) The method according to claim 1, wherein the step of expelling vitrification medium, the intermediary fluid, and the vitrification medium containing the at least one embryo or at least one oocyte from the channel results in a vitrification droplet formed at the end of the channel entraining the at least one embryo or at least one oocyte. ### Claim 4 (depends on 1) The method according to claim 1, wherein the intermediary fluid comprises air or a gas. ### Claim 5 (depends on 4) The method according to claim 4, wherein the vitrification droplet contains at least one air bubble and at least one embryo or oocyte. ### Claim 6 (depends on 1) The method of claim 1, further comprising the step of dropping the vitrification droplet into liquid nitrogen. ### Claim 7 (depends on 5) The method according to claim 5, wherein the vitrification droplet is dropped from between about 5 cm and about 60 cm into the liquid nitrogen. ### Claim 8 (depends on 5) The method of claim 5, wherein the vitrification droplet is dropped into the liquid nitrogen from a distance between about 0.1 cm and 5 cm. ### Claim 9 (depends on 1) The method according to claim 1, further comprising the step of incubating the vitrification medium at between about 37° C. and 42° C. prior to the steps of aspirating. ### Claim 10 (depends on 1) The method according to claim 1, wherein the vitrification medium includes a coloring dye. ### Claim 11 (depends on 1) The method according to claim 1, wherein the at least one embryo or at least one oocyte comprises an embryo which has been produced from sex selected sperm. ### Claim 12 A method of liquid nitrogen surface vitrification of embryos or oocytes comprising the steps of: a. obtaining at least one embryo or at least one oocyte; b. washing the at least one embryo or at least one oocyte with a vitrification medium; c. forming a droplet containing at least one embryo or at least one oocyte; and d. dropping the droplet into liquid nitrogen wherein the at least one embryo or at least one oocyte becomes vitrified upon contact with the liquid nitrogen. ### Claim 13 (depends on 12) The method according to claim 12, between steps a and b further comprising the steps of: a. washing the at least one embryo or at least one oocyte in rinse medium; b. incubating the at least one embryo or at least one oocyte in a base medium; and c. incubating the at least one embryo or at least one oocyte in hold medium. ### Claim 14 (depends on 12) The method according to claim 12, wherein the volume of the droplet with at least one embryo or at least one oocyte is coordinated with the density of the vitrification medium in order to ensure vitrification occurs at a sufficient rate when the droplet contacts the water to prevent intracellular ice formation. ### Claim 15 (depends on 14) The method according to claim 14, wherein the temperature change occurs in the droplet at rate greater than 1×103 C/min, at rate greater than 1×104 C/min, or at rate greater than 1×105 C/min. ### Claim 16 (canceled) ### Claim 17 (canceled) ### Claim 18 (depends on 12) The method according to claim 12, wherein the at least one embryo or at least one oocyte comprises a first volume in the droplet V1 and wherein the droplet has a total volume V2. ### Claim 19 (depends on 18) The method according to claim 18, wherein the ratio of V1/V2 is less than 0.75, less than 0.5, less than 0.25, less than 0.1, less than 0.05 or less than 0.01. ### Claim 20 (canceled) ### Claim 21 (canceled) ### Claim 22 (canceled) ### Claim 23 (canceled) ### Claim 24 (canceled) ### Claim 25 (depends on 12) The method according to claim 12, wherein the droplet comprises about 0.2 μL to about 12 μL in volume. ### Claim 26 (depends on 12) The method according to claim 12, wherein the step of forming a droplet comprises the steps of: a. aspirating vitrification medium into a channel; b. aspirating air into the channel the channel; c. aspirating vitrification medium containing at least one embryo or at least one oocyte into the channel; and d. expelling the vitrification medium, the air, and the vitrification medium containing the at least one embryo or at least one oocyte from the channel. ### Claim 27 (depends on 26) The method of claim 26, wherein the air separates vitrification medium from vitrification medium containing the at least one embryo or at least one oocyte within the channel. ### Claim 28 (depends on 26) The method of claim 26, wherein expelling the air and fluids from the channel forms a drop at the end of the channel having an air bubble and at least one embryo or at least one oocyte. ### Claim 29 (canceled) ### Claim 30 (depends on 12) The method according to claim 12, wherein the step of obtaining at least one embryo or at least one oocyte further comprises the step of obtaining at least one embryo selected from the group consisting of: zygotes, cleaved embryos, morulaes, early blastocysts, regular blastocysts, expanding blastocysts, hatching/hatched blastocysts. ### Claim 31 (canceled) ### Claim 32 (depends on 30) The method according to claim 30, wherein the blastocysts comprise blastocysts selected from the group consisting of: blastocysts in day 5, blastocysts in day 6, blastocysts in day 7, and blastocysts in day 8. ### Claim 33 (canceled) ### Claim 34 (depends on 12) The method according to claim 12, wherein the size of the at least one embryo or at least one oocyte is between about 100 μm to about 500 μm. ### Claim 35 (depends on 12) The method according to claim 12, wherein the droplet is dropped into the liquid nitrogen from between about 1 cm to about 60 cm. ### Claim 36 (depends on 35) The method according to claim 35, wherein the droplet is dropped into the liquid nitrogen from between about 10 cm to about 20 cm. ### Claim 37 (depends on 12) The method according to claim 12, wherein the droplet is formed from by a pipette and/or by a tip. ### Claim 38 (depends on 13) The method according to claim 13, wherein the step of rinsing the at least one embryo or the at least one oocyte further comprises rinsing in a solution containing trypsin. ### Claim 39 (depends on 13) The method according to claim 13, wherein the step of incubating the at least one or at least one oocyte in a hold medium further comprises incubating the at least one at least one embryo or oocyte for about 1 minute to about 12 minutes. ### Claim 40 (depends on 12) The method according to claim 12, wherein the step of washing the at least one embryo or the at least one oocyte in vitrification medium further comprises washing in about a 10 μL to about a 20 μL vitrification droplet. ### Claim 41 (canceled) ### Claim 42 (canceled) ### Claim 43 (depends on 12) The method according to claim 12, wherein the at least one embryo or at least one oocyte comprises an embryo produced with sex selected sperm. ### Claim at position 44 44-73. (canceled)