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

• US 8,703,418 B2
• Filed: 01/28/2013
• Issued: 04/22/2014
• 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 Michael, van Munster, Erik B.
• Primary Examiner(s): HURST, JONATHAN M
• Application Number:
  US13/752,068
• Publication Number:
  US 20130206648A1
• Time in Patent Office: 449 Days
• Field of Search: 435/2, 435/6.1, 435/288.7
• US Class Current: 435/6.1
• 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 of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells wherein the X-chromosome bearing sperm cells and the Y-chromosome bearing sperm cells have differing sperm head volumes, the method comprising:

a) generating a beam of electromagnetic radiation having initial waveform characteristics;

b) traversing the volume of each sperm head with the beam of electromagnetic radiation having initial waveform characteristics, wherein the initial waveform characteristics are altered by traversing the volume of each sperm head;

d) analyzing the altered wave form characteristics of the beam of electromagnetic radiation;

e) assessing the volume of each sperm head by determining a phase shift in the electromagnetic radiation;

f) assessing an orientation of each sperm head by determining a phase shift in the electromagnetic radiation; and

g) differentiating between X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells based upon the orientation and the volume of each sperm head.

2. The method as claimed in claim 1, wherein the step of generating a beam of electromagnetic radiation having initial waveform characteristics further comprises the step of adjusting polarization of the beam.

3. The method as claimed in claim 2, wherein the step of adjusting the polarization of the beam further comprises polarizing portions of the beam with different polarization angles.

4. The method as claimed in claim 1, wherein the step of altering the initial wave form characteristics by traversing the volume of the sperm head comprises shifting phase of the initial waveform characteristics of the beam of electromagnetic radiation.

5. The method as claimed in claim 4, further comprising the step of superimposing the initial waveform characteristics and phase shifted waveform characteristics.

6. The method as claimed in claim 5, further comprising the step of comparing an intensity of the initial waveform characteristics with superimposed waveform characteristics.

7. The method as claimed in claim 6, further comprising the step of determining the volume of a sperm head containing DNA based upon the difference in the intensity between the superimposed waveform characteristics and the initial waveform characteristics.

8. The method of isolating as claimed in claim 1, further comprising the steps of:

a) introducing sperm cells into a fluid stream;

b) forming droplets a plurality having one of the sperm cells entrained;

c) charging each of the droplets differentially based upon the assessed volume of the sperm head;

d) deflecting each of the droplets;

e) differentially collecting each of the droplets based upon the volume of the sperm head; and

f) generating X-chromosome bearing and Y-chromosome bearing populations of the sperm cells.

9. The method as claimed in claim 8, further comprising the step of maintaining the an amount of DNA within the nucleus of the sperm cells unstained.

10. The method as claimed in claim 8, further comprises the steps of:

a) detecting a signal having sperm cell orientation characteristics;

b) comparing the signal having sperm cell orientation characteristics to a signal having oriented sperm cell characteristics;

c) determining whether the sperm cell has unoriented sperm cell characteristics; and

d) differentially collecting said sperm cell having unoriented sperm cell characteristics.