Method for improved sperm cell populations
The invention encompasses methods for reducing the proportion of sperm cells with abnormal morphology, and unviable sperm cells, in a sperm cell population.
1. A method of processing sperm cells comprising
a) selecting a population of sperm cells wherein each sperm cell has a cell long axis and wherein greater than 25% of sperm cells in the population have abnormal morphology;
b) staining the sperm cells in the population;
c) placing the sperm cells in the population in a channel configured to impart orienting forces on the sperm cells that defines a flow axis and through which the sperm cells flow; wherein the cells, when the cell long axis is parallel with the flow axis, have at least a portion that has a flow orthogonal, cell cross-section that is non-circular, wherein the flow orthogonal, cell cross-section has a flow orthogonal, cell cross-section long axis and a flow orthogonal, cell cross-section short axis that is orthogonal to the flow orthogonal, cell cross-section long axis, wherein the channel configured to impart orienting forces on the sperm cells defines an intended, flow orthogonal, cell cross section long axis alignment line and an intended, flow orthogonal, cell cross section short axis alignment line that is orthogonal to the intended, flow orthogonal, cell cross section long axis alignment line;
d) orienting cells with the channel such that a cell presented at full orientation during cell irradiation has the cell long axis parallel with the flow axis, the flow orthogonal, cell cross-section long axis aligned with the intended flow orthogonal, cell cross section long axis alignment line; and the flow orthogonal, cell cross-section short axis aligned with the intended, flow orthogonal, cell cross section short axis alignment line;
e) irradiating the sperm cells in the population with a source of electromagnetic radiation;
f) detecting fluorescence emitted by the sperm cells in the population using a detector, wherein the detector has a flow orthogonal collection angle that defines a flow orthogonal, detector axis and wherein the flow orthogonal, detector axis is substantially coaxial with the intended, flow orthogonal, cell cross section long axis alignment line; and
g) creating a gate that excludes a portion of sperm cells in the population, wherein the angle between an excluded cell's flow orthogonal, cell cross-section long axis and the intended, flow orthogonal, cell cross section long axis alignment line is greater than 5°.
2. The method of claim 1 , wherein the angle in step g) is greater than 10°.
3. The method of claim 1 , wherein the angle in step g) is greater than 15°.
4. A method of processing sperm cells comprising
a) selecting a population of sperm cells wherein each sperm cell has a cell long axis and wherein greater than 25% of sperm cells in the population have abnormal morphology;
b) staining the sperm cells in the population;
c) placing the sperm cells in the population in a channel configured to impart orienting forces on the sperm cells that defines a flow axis and through which the sperm cells flow; wherein the cells, when the cell long axis is parallel with the flow axis, have at least a portion that has a flow orthogonal, cell cross-section that is non-circular, wherein the flow orthogonal, cell cross-section has a flow orthogonal, cell cross-section long axis and a flow orthogonal, cell cross-section short axis that is orthogonal to the flow orthogonal, cell cross-section long axis;
d) irradiating the sperm cells in the population with a beam of electromagnetic radiation, the beam having a flow orthogonal optical axis;
e) detecting fluorescence emitted by the sperm cells in the population using a detector, wherein the detector has a flow orthogonal collection angle that defines a flow orthogonal, detector axis and wherein the flow orthogonal, detector axis is orthogonal to the flow orthogonal optical axis; and
f) creating a gate that excludes a portion of sperm cells in the population, wherein the angle between an excluded cell's flow orthogonal, cell cross-section long axis and the flow orthogonal, detector axis is greater than 5°.
5. The method of claim 4 , wherein the angle in step f) is greater than 10°.
6. The method of claim 4 , wherein the angle in step f) is greater than 15°.