High purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
View Patent ↗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 ).
1. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells, comprising the steps of:
collecting viable sperm cells from a male of a species of non-human mammal;
staining said viable sperm cells to produce viable, stained sperm cells;
differentiating between X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells based upon fluorescent light emitted from said viable, stained sperm cells
which is detected with a photomultiplier tube operating at below 400 volts;
separating said X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells into a X-chromosome bearing population and a Y-chromosome bearing population at a separation rate between 4,000 to 10,000 separations per second of each sex;
and producing separate X-chromosome bearing and Y-chromosome bearing populations of sperm cells each having a purity of greater than 90%.
2. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 1 , wherein said step of differentiating between X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells of said viable, stained sperm cells comprises measuring an amount of DNA within a nucleus of said sperm cells.
3. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 2 , wherein said step of measuring an amount of DNA within a nucleus of said sperm cells comprises the steps of:
a. generating a beam of electromagnetic radiation having initial wave form characteristics;
b. traversing said sperm cell containing said amount of DNA with said beam of electromagnetic radiation;
c. altering said initial wave form characteristics by traversing said sperm cell; and
d. analyzing altered wave form characteristics.
4. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 3 , wherein said step of altering said initial wave form characteristics by traversing said sperm cell comprises the step of shifting phase of said initial wave form of said beam of electromagnetic radiation.
5. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 4 , further comprising the step of superimposing said beam of electromagnetic radiation having shifted phase on said beam of electromagnetic radiation having said initial wave form characteristics.
6. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 5 , further comprising the step of comparing electromagnetic radiation intensity of superimposed beams of electromagnetic radiation with intensity of said beam of electromagnetic radiation having initial wave form characteristics.
7. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 6 , further comprising the step of determining said amount of DNA based upon a difference in said light intensity between said superimposed beams of electromagnetic radiation and said beam of electromagnetic radiation having initial waveform characteristics.
8. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 2 , further comprising the steps of:
a. introducing said sperm cells into a fluid stream;
b. analyzing said sperm cells entrained in said fluid stream;
c. forming a plurality of droplets having one of said sperm cells entrained;
d. charging each of said droplets differentially based upon said detected fluorescent light of said sperm cells entrained in said droplets;
e. deflecting each of said droplets; and
f. differentially collecting each population of said sperm cells entrained in said droplets.
9. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 1 , further comprising the step of irradiating said stained sperm cells.
10. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 9 , wherein said step of irradiating said stained sperm cells comprises generating an irradiation beam pattern having a height about equal to the length of said sperm cells along the longitudinal axis to about three times the length of said sperm cells along the longitudinal axis.
11. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 10 , wherein said sperm cell has a longitudinal axis of about nine micrometers in length, and said height of said irradiation beam pattern is about 20 micrometers.
12. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 8 , wherein said step of forming droplets comprises forming droplets having sufficient size to encapsulate said sperm cell.
13. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 12 , further comprising the step of ejecting said fluid stream from a nozzle having an orifice, wherein said orifice has a diameter of 70 micrometers.
14. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 13 , further comprising the steps of:
a. orienting said sperm cells with respect to a detector;
b. detecting light having characteristics differentially responsive to sperm cell orientation to said detector;
c. converting said light having characteristics deferentially responsive to said sperm cell orientation into at least one signal containing sperm cell orientation information; and
d. determining orientation of said sperm cell with respect to said detector.
15. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 14 , further comprising the step of differentially collecting said droplets based upon determined orientation of said sperm cell with respect to said detector.
16. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claims 1 , 3 , 12 , or 14 , wherein said purity of said X-chromosome bearing and said Y-chromosome bearing populations of sperm cells is 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%, and between about 99% to about 100%.
17. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claims 1 , 3 , 12 , or 14 , further comprising the step of establishing a separable event rate up to about 35,000 separable events per second.
18. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 12 or 14 wherein said step of forming droplets each having one of said sperm cells entrained comprises a droplet formation rate up to about 200,000 droplets per second.
19. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 1 , wherein said species of mammal comprises a bovine mammal.
20. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 1 , wherein said species of mammal comprises an equine mammal.
21. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 1 , wherein said species of mammal comprises an ovine mammal.
22. A method of isolating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells as described in claim 1 , further comprising the step of operating said photomultiplier tube at a voltage selected from a group consisting of about 280 volts, about 200 volts, just above 0 volts, and below 300 volts.