Method for producing high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa

• US 10,208,345 B2
• Filed: 01/28/2013
• Issued: 02/19/2019
• Est. Priority Date: 05/09/2000
• Status: Expired due to Fees

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY, LLC (Inguran LLC)
• Inventors: Evans, Kenneth M., van Munster, Erik B.
• Primary Examiner(s): Lankford, Blaine
• Assistant Examiner(s): Van Buren, Lauren K
• Application Number:
  US13/752,057
• Publication Number:
  US 20130203108A1
• Time in Patent Office: 2,213 Days
• Field of Search: None
• 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 method differentiating sperm cells comprising:

establishing a fluid stream containing sperm cells with a flow cytometer;

orienting the sperm cells with respect to a detector, wherein the detector comprises a photomultiplier tube that has a lower limit of linear operation;

operating the photomultiplier tube at a voltage below the lower limit of linear operation though an altered photomultiplier controller, said altered photomultiplier controller being altered to operate the photomultiplier tube throughout a range of nearly 0 volts to below 400 volts;

detecting fluorescence having characteristics differentially responsive to sperm cell orientation to the photomultiplier tube;

converting the fluorescence having characteristics differentially responsive to sperm cell orientation into at least one signal containing sperm cell orientation information;

analyzing the sperm cell orientation information;

using an analyzer that is configured to analyze at least one signal from said photomultiplier tube operated at a voltage range of nearly 0 volts to below 400 volts to differentiate between X chromosome bearing sperm and Y chromosome bearing sperm; and

generating a population of viable X chromosome and/or a population of viable Y chromosome bearing sperm cells.

2. The method as claimed in claim 1, wherein said fluid stream comprises a sheath fluid.

3. The method as claimed in claim 1, wherein said fluorescence emits from a light emission material bound to said sperm cells.

4. The method as claimed in claim 3, wherein at least one signal containing sperm cell orientation information comprises emission signal plots and further comprising the steps of:

integrating the areas of said emission signal plots; and

comparing the integrations of said emission signal plots to integrations of emission signal plots of oriented sperm cells.

5. The method as claimed in claim 4, further comprising excluding sperm cells based upon determined orientation of said sperm cells with respect to said detector from the generated populations of viable X chromosome bearing or a Y chromosome bearing sperm cells.

6. The method as claimed in claim 1, further comprising the step of ejecting said fluid stream from a nozzle having an orifice, wherein said orifice has a diameter of 100 micrometers.

7. The method as claimed in claim 6, wherein said X chromosome bearing and/or said Y chromosome bearing populations of sperm cells have a purity selected from the group consisting of between 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%, between about 99% to about 100%.

8. The method as claimed in claim 7, wherein said step of separating said sperm cells comprises a separation rate selected from the group consisting of at least 500 separations per second, at least 1,000 separations per second, at least 2,000 separations per second, at least 3,000 separations per second, at least 4,000 separations per second, at least 5,000 separations per second, at least 6,000 separations per second, at least 7,000 separations per second, at least 8,000 separations per second, at least 9,000 separations per second, at least 10,000 separations per second.

9. The method as claimed in claim 1, wherein said sperm cells comprise bovine sperm cells.

10. The method as claimed in claim 1, wherein said sperm cells comprise equine sperm cells.

11. The method as claimed in claim 1, wherein said sperm cells comprise ovine sperm cells.