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

• US 7,838,210 B2
• Filed: 03/29/2005
• Issued: 11/23/2010
• Est. Priority Date: 03/29/2004
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Inguran LLC
• Inventors: Graves, Charles N., Crowley, Kathleen S., Ludwig, Cindy L.
• Primary Examiner(s): Gitomer; Ralph
• Assistant Examiner(s): GOUGH, TIFFANY MAUREEN
• Application Number:
  US11/092,313
• Publication Number:
  US 20050244805A1
• Time in Patent Office: 2,065 Days
• Field of Search: 435/40.5, 435/325, 435/2
• 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 method of making a gender enriched sperm cell suspension comprising mixing a sperm cell suspension with a composition which inhibits motility to form a chemically immotile sperm cell suspension, staining said chemically immotile sperm cell suspension with a DNA-selective dye and sorting the stained chemically immotile sperm cell suspension to form a viable, gender enriched insemination population of X chromosome bearing or Y chromosome bearing sperm cells having a DNA-selected dye associated with their DNA.

2. The method of claim 1, 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.

3. The method of claim 1, wherein the chemically immotile sperm cell suspension is rendered chemically immotile during the sorting process by a composition which inhibits motility comprising a source of carbonate.

4. The method of claim 3, wherein said chemically immotile sperm cell suspension is rendered chemically immotile during the sorting process by a composition which inhibits motility comprising NaHCO₃, KHCO₃, and C₆H₈O₇·

H₂O.

5. The method of claim 4, wherein said chemically immotile sperm cell suspension is rendered chemically immotile during the sorting process by a composition which inhibits motility comprising 0.097 moles/L of NaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇·

H₂O in water.

6. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is at least 1.25×

10⁸ spermatozoa per ml.

7. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is at least 1.5×

10⁸ spermatozoa per ml.

8. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is at least 1.75×

10⁸ spermatozoa per ml.

9. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is less than about 9.0×

10⁵ spermatozoa per ml.

10. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is less than about 7×

10⁵ spermatozoa per ml.

11. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is less than about 5×

10⁵ spermatozoa per ml.

12. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is less than about 2×

10⁵ spermatozoa per ml.

13. The method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells sperm in the suspension is less than about 1×

10⁵ spermatozoa per ml.

14. The method of claim 1, wherein said viable spermatozoa suspension comprises viable frozen spermatozoa sperm.

15. The, method of claim 1, wherein a concentration of said viable, gender enriched insemination population of X chromosome bearing sperm cells or Y chromosome bearing sperm cells is sperm in the suspension being less than about 1×

10⁶ or at least about 1×

10⁸ sperm per ml.

16. The method of claim 1, wherein the chemically immotile sperm cell suspension is rendered chemically immotile during the sorting process in a suspension comprising potassium and sodium, wherein the molar ratio of potassium to sodium is greater than 1:

1, respectively.

17. The method of claim 16, wherein the molar ratio of potassium to sodium is greater than 1.25:

1, respectively.

18. The method of claim 16, wherein the molar ratio of potassium to sodium is greater than 1.5:

1, respectively.

19. The method of claim 16, wherein the molar ratio of potassium to sodium is greater than 1.75:

1, respectively.

20. The method of claim 16, wherein the molar ratio of potassium to sodium is greater than 2:

1, respectively.