Methods for high throughput sperm sorting

Patent metadata

Field	Value
Publication number	US-10662408-B2
Application number	US-201313830365-A
Country	US
Kind code	B2
Filing date	Mar 14, 2013
Priority date	Mar 14, 2013
Publication date	May 26, 2020
Grant date	May 26, 2020

Abstract

This disclosure relates to methods for sorting sperm cells in a microfluidic chip. In particular, various steps are incorporated to align and orienting sperm in flow channels, as well as, to determining sperm orientation and measure relative DNA content for analysis and/or sorting.

First claim

The invention claimed is:

1. A method of processing sperm in a microfluidic chip comprising:
surrounding a core stream of sample fluid containing sperm with a sheath fluid at a sample injection location in a flow channel of a microfluidic chip;
orienting and focusing sperm within the core stream by:
focusing the core stream in the flow channel laterally;
diverting a first portion of the sheath fluid through a first looping channel;
diverting a second portion of the sheath fluid through a second looping channel;
reintroducing the first portion sheath fluid at a first location in the flow channel downstream of the sample injection location from a single side to influence the core stream in a first vertical direction;
reintroducing the second portion sheath fluid at a second location in the flow channel downstream of the first location from a single side of the flow channel to influence the core stream in a second vertical direction,
wherein the first and second portions of sheath fluids are reintroduced to the flow channel from opposite vertical sides of the flow channel;

flowing the sperm through an inspection region in the flow channel;
interrogating sperm at the inspection region to determine sperm characteristics based on the response of sperm or a stain associated with the sperm to the interrogation; and
differentiating sperm in the flow channel based on the sperm characteristics.

2. The method of claim 1 , further comprising the steps of:
providing an electromagnetic radiation source;
manipulating electromagnetic radiation produced from the electromagnetic radiation source for interrogating the inspection region.

3. The method of claim 2 , wherein the step of manipulating electromagnetic radiation further comprises: splitting the electromagnetic radiation produced by the electromagnetic radiation source to interrogate more than one inspection region in more than one flow channel.

4. The method of claim 1 , further comprising selecting a subpopulation of sperm based on the detected sperm characteristics and collecting the selected sperm.

5. The method of claim 1 , further comprising the step of differentiating oriented sperm from un-oriented sperm.

6. The method of claim 1 , further comprising the steps of:
generating a first signal with a forward fluorescence detector in response to emitted electromagnetic radiation of sperm at the inspection region, wherein the first signal comprises waveform pulses having detectable pulse characteristics.

7. The method of claim 6 , further comprising the step of generating a second signal with a side fluorescence detector.

8. The method of claim 7 , wherein the step of generating a second signal with a side fluorescence detector further comprises associating a reflective element with the flow channel for reflecting the side florescence outward and detecting the side fluorescence in parallel with a forward fluorescence detected by the forward fluorescence detector.

9. The method of claim 8 , further comprising the step of detecting the forward fluorescence through a first mask and the side fluorescence through a second mask.

10. The method of claim 1 , further comprising the step of:
generating a signal with a fluorescence detector in response to emitted electromagnetic radiation of sperm at the inspection region, wherein the signal embeds first waveform pulses having detectable pulse characteristics and second waveform pulses having detectable pulse characteristics; and
deconvolving the first waveform pulses and the second waveform pulses from the generated signal.

11. The method of claim 10 , wherein the deconvolved first waveform pulses and second waveform pulses provide information on the sperm orientation within the flow channel.

12. The method of claim 1 , further comprising the steps of generating a plurality of waveform pulses with a single detector in response to single sperm, wherein the plurality of waveform pulses provide orientation information about the sperm cell.

13. The method of claim 12 , further comprising the step of measuring laser extinction to determine sperm orientation.

14. The method of claim 7 , further comprising the steps of:
generating a second signal with a first side fluorescence detector, wherein the second signal comprises waveform pulses having detectable pulse characteristics; and
generating a third signal with a second side fluorescence detector, wherein the second signal comprises waveform pulses having detectable pulse characteristics.

15. The method of claim 14 , wherein the pulse characteristics are selected from the group consisting of: peak height, pulse width, pulse peak lag, pulse slope, pulse area, and combinations thereof.

16. The method of claim 14 , further comprising the step of comparing the pulse characteristics of the second signal to the pulse characteristics of the third signal to determine sperm orientation.

17. The method of claim 4 , further comprising the step of sorting the selected sperm.

18. The method of claim 1 , further comprising: orienting and focusing sperm within a plurality of core streams in a plurality of flow channels on the microfluidic chip.

Assignees

• Inguran Llc

Inventors

• Sharpe Johnathan Charles
• Sedoglavich Nemanya
• Morad Blair
• Perrault Jr Donald Francis

Classifications

• C12N5/0612 Primary

  sorting of gametes, e.g. according to sex or motility · CPC title

• G01N15/1459

  the analysis being performed on a sample stream · CPC title

• G01N15/1484

  microstructural devices · CPC title

• G01N15/149

  specially adapted for sorting particles, e.g. by their size or optical properties · CPC title