Methods and Apparatus for High Purity X-Chromosome Bearing and Y-Chromosome Bearing Populations Of Spermatozoa

• US 20080233635A1
• Filed: 05/01/2008
• Published: 09/25/2008
• Est. Priority Date: 05/09/2000
• Status: Active Grant

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY Incorporated (Inguran LLC)
• Inventors: Evans, Kenneth M., van Munster, Erik B.
• Granted Patent: US 9,145,590 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: A61D 19/00 Instruments or methods for reproduction or fertilisation
• CPC Class Codes: B03B 9/00 General arrangement of separating plant, e.g. flow sheets
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: G01F 17/00 Methods or apparatus for determining the capacity of containers or cavities, or the volume of solid bodies measuring linear dimensions to determine volume G01B
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 21/17 Systems in which incident light is modified in accordance with the properties of the material investigated where the material investigated is optically excited causing a change in wavelength of the incident light G01N21/63

Abstract

Isolated non-naturally occurring populations of spermatozoa (15) having high purity and technologies to differentiate spermatozoa (28) based on characteristics such as mass, volume, orientation, or emitted light including methods of analysis and apparatus such as beam shaping optics (30) and detectors (32).

Claims

1. A particle differentiation apparatus, comprising:

a. particles having at least one particle differentiation characteristic;

b. a light emission source differentially responsive to said particle differentiation characteristics;

c. at least one photomultiplier tube, wherein said at least one photomultiplier tube converts light from said light emission source into at least one differentiation signal, and wherein said photomultiplier tube is operated at below about 400 volts; and

d. an analyzer responsive to said at least one differentiation signal which differentiates particles based upon said particle differentiation characteristics.

2. A particle differentiation apparatus as described in claim 1, wherein said photomultiplier tube is operated at a voltage selected from the group consisting of about 280 volts, about 200 volts, just above about 0 volts, and below about 300 volts.

3. A particle differentiation apparatus as described in claim 1, wherein said photomultiplier tube is operated in a range from just above about 0 volts to about 300 volts.

4. A particle differentiation apparatus as described in claim 1, further comprising an irradiation source generating an irradiation beam responsive to said particles.

5. A particle differentiation apparatus as described in claim 4, wherein said light emission source comprises a light emission material bound to said particles which emits said light in response to said irradiation beam.

6. A particle differentiation apparatus as described in claim 5, wherein said light emission material bound to said particles comprises a stain bound to the nuclear DNA of sperm cells.

7. A particle differentiation apparatus as described in claim 6, wherein said particle differentiation characteristic comprises a difference in amount of said stain bound the nuclear DNA of X-chromosome sperm cells and the nuclear DNA of Y-chromosome bearing sperm cells.

8. A particle differentiation apparatus as described in claim 5, wherein said particle comprises an asymmetric particle.

9. A particle differentiation apparatus as described in claim 8, further comprising optics to focus said irradiation beam responsive to said particle, wherein said optics focus a irradiation pattern having a height of about equal to the length of said asymmetrical particle along the longitudinal axis to about three times the length of said asymmetrical particle along the longitudinal axis.

10. A particle differentiation apparatus as described in claim 9, further comprising a fluid stream into which said particles are introduced.

11. A particle differentiation apparatus as described in claim 10, wherein said at least one particle differentiation characteristic comprises orientation of said asymmetric particle within said fluid stream, and wherein said photomultiplier tube is deferentially responsive to said light emitted from said light emission material based upon said particle orientation characteristics, and wherein said analyzer coupled to said photomultiplier tube differentiates between said asymmetric particles based upon orientation within said fluid stream.

12. A particle differentiation apparatus as described in claim 11, wherein said asymmetrical particles comprise sperm cells having sperm cell heads.

13. A particle differentiation apparatus as described in claim 12, wherein said sperm cell heads of said sperm cell have a length along the longitudinal axis of about nine micrometers, and wherein said irradiation pattern has a height of about 20 micrometers.

14. A particle differentiation apparatus as described in claim 1, wherein said particle differentiation characteristic comprises a difference between X-chromosome bearing and Y-chromosome bearing sperm cells.

15. A particle differentiation apparatus as described in claim 14, further comprising a plurality of droplets breaking off from said fluid stream having one of said sperm cells entrained.

16. A particle differentiation apparatus as described in claim 15, wherein said droplets breaking off from said fluid stream have sufficient size to encapsulate said one of said sperm cells.

17. A particle differentiation apparatus as described in claim 16, further comprising a nozzle having an orifice of about 100 micrometers in diameter.

18. A particle differentiation apparatus as described in claim 15, further comprising a droplet charger coupled to said analyzer, wherein said droplets receive a charge differentially based upon said difference in amount of said stain bound the nuclear DNA of X-chromosome sperm cells and the nuclear DNA of Y-chromosome bearing sperm cells.

19. A particle differentiation apparatus as described in claim 18, further comprising a droplet separator, wherein said droplet separator separates said droplet based upon charge of said droplet.

20. A particle differentiation apparatus as described in claim 19, further comprising collection containers responsive to said droplet separator, wherein X-chromosome bearing sperm cell and Y-chromosome bearing sperm cell populations are collected.

21. An apparatus for differentiating particles as described in claim 20, wherein said X-chromosome bearing sperm cell population and said Y-chromosome bearing population of said sperm cells are selected from the group consisting of between about 90% to about 100%, between about 91% to about 100%, between about 92% to about 100%, between about 93% to about 100%, between about 94% to about 100%, between about 95% to about 100%, between about 96% to about 100%, between about 97% to about 100%, between about 98% to about 100%, and between about 99% to about 100%.

22. A particle differentiation apparatus as described in claim 21, further comprising the step of establishing a separable event rate of up to about 35,000 separable events per second.

23. A particle differentiation apparatus as described in claim 16, wherein said droplets having one of said sperm cells entrained comprises a droplet formation rate of up to about 200,000 droplets per second.

24. A particle differentiation apparatus as described in claim 6, wherein said sperm cells comprise sperm cells from a bovine mammal.

25. A particle differentiation apparatus as described in claim 6, wherein said sperm cells comprise sperm cells from an equine mammal.

26. A particle differentiation apparatus as described in claim 6, wherein said sperm cells comprise sperm cells from an ovine mammal.

27. A particle differentiation apparatus as described in claim 1 and further comprising a particle differentiation system selected from the group consisting of flow cytometry, sorting flow cytometry, and flow separation.