Sperm processing method, apparatus and related media compositions
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.
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 about 2%; between about 2% and about 4%; between about 4% and about 6%; between about 3% and about 5%; or between about 3.5% and about 5.5%; or at about 4.5%.
30. The method of claim 17 , wherein the sheath fluid comprises a saline buffered with phosphates, TRIS citrate, or HEPES.