Sperm suspensions for sorting into X or Y chromosome-bearing enriched populations

• US 20050244805A1
• Filed: 03/29/2005
• Published: 11/03/2005
• Est. Priority Date: 03/29/2004
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Crowley, Kathleen S., Ludwig, Cindy L., Graves, Charles N.
• Granted Patent: US 7,838,210 B2
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: A61K 35/52 Sperm; Prostate; Seminal fluid; Leydig cells of testes
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Abstract

Sperm cell suspensions comprising a motility inhibitor are disclosed. The cells contained in such suspensions tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into gender enriched populations, thereby resulting in post-sort compositions with an increased number of viable or motile sperm. Processes for forming such cell suspensions, as well as processes for staining sperm cells, are also disclosed.

Claims

1. A sperm cell suspension comprising viable spermatozoa and a composition which down-regulates carbohydrate uptake by the spermatozoa, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml.

2. A sperm cell suspension comprising viable, immotile sperm, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml.

12. The sperm suspension of claim 2, wherein the suspension comprises a source of carbonate.

13. The sperm suspension of claim 2, wherein the suspension comprises NaHCO₃, KHCO₃, and C₆H₈O₇.H₂O.

14. The sperm suspension of claim 2, wherein the suspension is formed from a buffer comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water.

15. The sperm suspension of claim 2, wherein the concentration of spermatozoa in the suspension is at least 1.25×

10⁸ spermatozoa per ml.

16. The sperm suspension of claim 2, wherein the concentration of spermatozoa in the suspension is at least 1.5×

10⁸ spermatozoa per ml.

17. The sperm suspension of claim 2, wherein the concentration of spermatozoa in the suspension is at least 1.75×

10⁸ spermatozoa per ml.

18. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 9.0×

10⁵ spermatozoa per ml.

19. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 7×

10⁵ spermatozoa per ml.

20. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 5×

10⁵ spermatozoa per ml.

21. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 2×

10⁵ spermatozoa per ml.

22. The suspension of claim 2, wherein the concentration of spermatozoa in the suspension is less than about 1×

10⁵ spermatozoa per ml.

27. The suspension of claim 13, further comprising a DNA-selective dye.

3. A sperm cell suspension comprising viable sperm, potassium and optionally sodium, the concentration of spermatozoa in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1;

1, respectively.

4. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.25:

1, respectively.

5. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.5:

1, respectively.

6. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 1.75:

1, respectively.

7. The suspension of claim 3 wherein the molar ratio of potassium to sodium is greater than 2:

1, respectively.

8. A sperm cell suspension comprising viable, immotile sperm and a DNA-selective dye.

9. The suspension of claim 8, wherein the dye is a DNA selective fluorescent dye.

10. The suspension of claim 8, wherein the dye is a UV excitable or a visible light excitable dye.

11. The suspension of claim 8, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®

conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′

H-2,5′

-bibenzimidazol-2′

-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide.

23. A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a motility inhibiting amount of potassium, and a DNA selective dye.

24. The process of claim 23 wherein the staining mixture is under an atmosphere having an enriched partial pressure of CO₂ relative to air.

25. A process of forming a sperm cell suspension for use in cell sorting, the process comprising combining a sperm cell source with a composition which inhibits the motility of sperm cells to form a sperm cell suspension, the concentration of sperm cells in the suspension being less than about 1×

10⁶ or at least 1×

10⁸ sperm cells per milliliter.

26. The process of claim 25, wherein the cell suspension comprises a DNA-selective dye.