Sperm processing method, apparatus and related media compositions

Patent metadata

Field	Value
Publication number	US-2022186182-A1
Application number	US-202217666325-A
Country	US
Kind code	A1
Filing date	Feb 7, 2022
Priority date	Jan 20, 2017
Publication date	Jun 16, 2022
Grant date	—

Abstract

Embodiments of the present invention relate generally to processes, systems, and compositions useful in manipulating a ratio of viable X chromosome bearing sperm to viable Y chromosome bearing sperm in at least one sperm population and useful for preserving the resulting manipulated sperm population. In some embodiments a cryoprotectant may be incorporated into various medias used in manipulating the sperm sample, such as in a staining media, a sheath fluid, and a collection media.

First claim

We claim:

1 . A method of freezing a manipulated sperm sample comprising:
contacting a stained sperm sample with a sheath fluid in a flow path; manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population; collecting the manipulated sperm sample in the presence of a polyalcohol; concentrating the collected sperm sample; resuspending the concentrated sperm sample in a freezing extender; and freezing the resuspended sperm sample.

2 . The method of claim 1 , further comprising the step of cooling sperm for a period of 120 minutes or a period of less than 120 minutes.

3 . The method of claim 1 , wherein the step of collecting a mixture further comprises: collecting the mixture in a container.

4 . The method of claim 3 , wherein the step of collecting a mixture further comprises: collecting the mixture in a container having between about 5 ml and about 50 ml of collection media.

5 . The method of claim 3 , wherein the step of collecting a mixture further comprises collecting a sorted sperm sample from a flow cytometer until the container is filled to between about 60% to about 90% of the container's capacity.

6 . The method of claim 5 , wherein the step of concentrating the collected mixture is performed in the container.

7 . The method of claim 1 , wherein the step of concentrating the collected sperm sample follows the step of collecting the manipulated sperm sample in the presence of a polyalcohol without any further dilution occurring between the steps.

8 . The method of claim 1 , wherein the freezing extender comprises less than 6% polyalcohol vol./vol. or wt./vol.

9 . The method of claim 1 , wherein the freezing extender comprises between 3.5% and 5.5% polyalcohol vol./vol. or wt./vol.

10 . The method of claim 1 , wherein the freezing extender comprises between 4% and 5% polyalcohol vol./vol. or wt./vol.

11 . The method of claim 1 , wherein the resuspended mixture is held for between 2 hours and 18 hours before freezing.

12 . The method of claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises adding the polyalcohol to the sperm sample during a step of staining.

13 . The method of claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises adding the polyalcohol to the sheath fluid which is collected together with the sperm sample in the step of collecting.

14 . The method of claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises collecting the manipulated sperm sample in a container having a collection media, the collection media comprising the polyalcohol.

15 . The method of claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises incorporating the polyalcohol into one or more of: a staining media used to stain the sperm sample, the sheath fluid, and the collection media.

16 . The method of claim 1 , wherein the step of manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population further comprises classifying cells in the flow path and selecting sperm based on their classification.

17 . The method of claim 1 , wherein the step of manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population further comprises either forming droplets expected to contain selected sperm and deflecting those droplets for collection or photo-damaging selected sperm in the flow path.

Assignees

• Inguran Llc

Inventors

• Gilligan Thomas B
• Gonzalez-Marin Clara
• Vishwanath Ramakrishnan

Classifications

• G01N1/30

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title

• A01N1/126

  Physiologically active agents, e.g. antioxidants or nutrients · CPC title

• A01N1/125

  Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators · CPC title

• A01N1/124

  Disinfecting agents, e.g. antimicrobials · CPC title

• A01N1/12

  Chemical aspects of preservation · CPC title