Sperm processing method, apparatus and related media compositions

Patent metadata

Field	Value
Publication number	US-11279913-B2
Application number	US-201815875679-A
Country	US
Kind code	B2
Filing date	Jan 19, 2018
Priority date	Jan 20, 2017
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

Abstract

Embodiments of the present invention relate generally to processes, systems, and compositions useful in manipulating a ratio of viable X chromosome bearing sperm to viable Y chromosome bearing sperm in at least one sperm population and useful for preserving the resulting manipulated sperm population. In some embodiments a cryoprotectant may be incorporated into various medias used in manipulating the sperm sample, such as in a staining media, a sheath fluid, and a collection media.

First claim

We claim:

1. A method of increasing resolution while analyzing sperm comprising:
staining a sperm sample having viable X-chromosome bearing sperm and viable Y-chromosome bearing sperm with a staining media;
contacting the stained sperm sample with a sheath fluid in a flow path; and
manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population;
the sheath fluid comprising cryoprotectant comprising a polyalcohol, the polyalcohol at a vol./vol. or wt./vol. concentration between about 0.1% and about 6% in the sheath fluid thereby increasing resolution.

2. The method of claim 1 further comprising the step of collecting the manipulated sperm population in a collection media.

3. The method of claim 2 , wherein the collection media comprises an amount of cryoprotectant.

4. The method of claim 2 , wherein each of the staining media, the sheath fluid and the collection media include an amount of cryoprotectant.

5. The method of claim 1 , wherein the step of manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population further comprises either selecting sperm for separation and collection or for photo-damage and collection.

6. The method of claim 1 further comprising the step of freezing the manipulated sperm sample.

7. The method of claim 1 , further comprising one or more of the following processing steps: holding; transporting; buffering; chilling; warming; diluting; concentrating; exciting with a laser; charging; deflecting; ablating; collecting; shaking; oscillating; magnetically separating; oxygenating; labeling; precipitating; centrifuging; resuspending; mixing; dialyzing; cryostabilizing; microchip processing; and flow cytometry processing.

8. The method of claim 1 , wherein the polyalcohol is selected from the group consisting of: ethylene glycol; glycerol; erythritol; threitol; arabitol; ribitol; xylitol; sorbitol; galactitol; iditol; volemitol; fucitol; inositol; a glycylglycitol, and combinations thereof.

9. The method of claim 1 , wherein the polyalcohol is selected from the group consisting of: propylene glycol, butane triol and combinations thereof.

10. The method of claim 1 , wherein at least one of the sheath fluid and the staining media further comprises an antioxidant.

11. The method of claim 10 , wherein the antioxidant comprises an antioxidant selected from the group consisting of: pyruvate, vitamin B12; vitamin B12 vitamers; vitamin E; vitamin E vitamers; tocopherol; tocotrienol; α-tocophery; alpha ketoglutarate; derivatives thereof and combinations thereof.

12. The method of claim 1 , wherein the sheath fluid comprises the cryoprotectant at a vol./vol. or wt./vol. concentration between about 0.1% and about 2%; between about 2% and about 4%; between about 4% and about 6%; between about 1% and about 2%; between about 2% and about 3%; between about 3% and about 4%; between about 4% and about 5%; between about 5% and about 6%; between about 2% and about 6%; or between about 3% and about 5%.

13. The method of claim 1 , wherein the sheath fluid comprises a saline buffered with phosphates, TRIS citrate, or HEPES.

14. The method of claim 1 , wherein cryoprotectant is added to the staining media in a first amount and added to the sheath fluid in a second amount, and wherein the second amount is greater than the first amount.

15. The method of claim 1 , wherein increasing resolution comprises increasing a peak to valley ratio formed by the viable X-chromosome bearing sperm and the viable Y-chromosome bearing sperm.

16. The method of claim 1 , wherein the step of manipulating comprises sorting the sperm sample at an event rate of at least 20,000 events per second.

17. A method of processing sperm comprising:
staining a sperm sample with a staining media having a DNA selective dye;
injecting the stained sperm sample into a flow of sheath fluid;
exposing the stained sperm sample in the flow of sheath fluid to an electromagnetic radiation source which causes a detectable response in the DNA selective dye;
detecting the response of the DNA selective dye to the electromagnetic radiation exposure;
manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population; and
collecting the at least one manipulated sperm population in one or more collection vessels having collection medias therein;
the staining media, the sheath fluid and the collection medias comprising a cryoprotectant, the cryoprotectant comprising a polyalcohol.

18. The method of claim 17 , wherein the staining media comprises a first amount of the cryoprotectant, the sheath fluid comprises a second amount of the cryoprotectant, and the collection media comprises a third amount of the cryoprotectant.

19. The method of claim 18 , wherein the first amount of the cryoprotectant is less than the second amount of the cryoprotectant and the third amount of the cryoprotectant.

20. The method of claim 18 , wherein the third amount of the cryoprotectant is greater than the first amount of the cryoprotectant and the second amount of the cryoprotectant.

21. The method of claim 17 , wherein the polyalcohol is selected from the group consisting of: ethylene glycol; glycerol; erythritol; threitol; arabitol; ribitol; xylitol; sorbitol; galactitol; iditol; volemitol; fucitol; inositol; a glycylglycitol, and combinations thereof.

22. The method of claim 17 , wherein the polyalcohol is selected from the group consisting of: propylene glycol, butane triol and combinations thereof.

23. The method of claim 17 , wherein at least one of the sheath fluid, the staining media and the collection media further comprises an antioxidant.

24. The method of claim 23 , wherein the antioxidant comprises an antioxidant selected from the group consisting of: vitamin B12; vitamin B12 vitamers; vitamin E; vitamin E vitamers; tocopherol; tocotrienol; α-tocophery; alpha ketoglutarate; derivatives thereof and combinations thereof.

25. The method of claim 17 , wherein at least one of the sheath fluid, the staining media, and the collection media further comprises a citrate, citric acid, citric acid monohydrate, or sodium citrate.

26. The method of claim 17 , wherein the sheath fluid comprises the cryoprotectant at a vol./vol. or wt./vol. concentration between about 0.1% and about 6%.

27. The method of claim 17 , wherein the sheath fluid comprises the cryoprotectant at a vol./vol. or wt./vol. concentration between about 0.1% and about 2%; between about 2% and about 4%; between about 4% and about 6%; between about 1% and about 2%; between about 2% and about 3%; between about 3% and about 4%; between about 4% and about 5%; between about 5% and about 6%; between about 2% and about 6%; or between about 3% and about 5%.

28. The method of claim 17 , wherein the staining media comprises the cryoprotectant at a vol./vol. or wt./vol. concentration between about 0.1% and about 1%; between about 1% and about 2%; between about 2% and about 3%; between about 3% and about 4%; between about 2% and about 4%; or between about 1.5% and about 3%.

29. The method of claim 17 , wherein the collection media comprises the cryoprotectant at a vol./vol. or wt./vol. concentration between about 1% and abou

Assignees

• Inguran Llc

Inventors

• Gilligan Thomas B
• Gonzalez-Marin Clara
• Vishwanath Ramakrishnan

Classifications

• G01N1/30

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title

• A01N1/126

  Physiologically active agents, e.g. antioxidants or nutrients · CPC title

• A01N1/125

  Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators · CPC title

• A01N1/124

  Disinfecting agents, e.g. antimicrobials · CPC title

• A01N1/12

  Chemical aspects of preservation · CPC title