System and method for sorting cells

• US 9,365,822 B2
• Filed: 02/11/2013
• Issued: 06/14/2016
• Est. Priority Date: 12/31/1997
• Status: Expired due to Fees

• Current Assignee: XY, LLC (Inguran LLC)
• Original Assignee: XY, LLC (Inguran LLC)
• Inventors: Seidel, George E., Herickhoff, Lisa A., Schenk, John L.
• Primary Examiner(s): Kim, Taeyoon
• Assistant Examiner(s): GOUGH, TIFFANY MAUREEN
• Application Number:
  US13/764,408
• Publication Number:
  US 20130150663A1
• Time in Patent Office: 1,219 Days
• US Class Current: 1/1
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: A61D 19/02 for artificial insemination
• CPC Class Codes: A61D 19/04 for embryo transplantation
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

Improved flow cytometer system particularly adapted to use for sex-selected sperm sorting include enhanced sheath fluid and other strategies which minimize stress on the sperm cells, including a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems and techniques for the process are described so that commercial applications of sperms samples as well as the resulting animals may be achieved.

Claims

1. A method of producing at least one sexed embryo comprising:

producing a stream containing sperm cells, wherein the stream comprises sperm cells from a cell source surrounded by sheath fluid, wherein the sheath fluid surrounding the sperm cells includes an amount of citrate;

identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream;

collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid; and

fertilizing at least one egg with the collected sperm to form at least one sexed embryo.

2. The method of claim 1, wherein the at least one egg is fertilized in vitro with the collected sperm.

3. The method of claim 1, wherein the citrate comprises a chemical in the citric acid cycle.

4. The method of claim 1, wherein the at least one egg is fertilized in vivo.

5. The method of claim 4, wherein the step of fertilizing in vivo further comprises the step of delivering sperm cells into both uterine horns of a uterus.

6. The method of claim 5, the step of delivering sperm cells into both uterine horns further comprises inserting said sperm cells approximately between 12 to 24 hours inclusive after detecting estrus.

7. The method of claim 1, wherein the steps of producing a stream containing sperm, identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream, and collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid further comprises:

supplying sperm cells to a nozzle from the cell source;

creating a sheath fluid environment in the nozzle in which sperm cells are surrounded by the sheath fluid, wherein the sheath fluid environment comprises a citrate;

producing a stream containing the sperm cells surrounded by the sheath fluid;

sensing a property of the sperm cells in the stream, wherein said property corresponds to the presence of an X-chromosome or a Y-chromosome;

identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream; and

collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid.

8. The method of claim 7, wherein the sperm cells are present in a pre-sort environment prior to being supplied to the nozzle and wherein the sperm cells are present in a post-sort environment at collection.

9. The method of claim 8, wherein the pre-sort and/or the post-sort environments are coordinated with the sheath fluid environment.

10. The method of claim 9, wherein the pre-sort and/or post-sort environments comprise the same concentration of citrate as the sheath fluid environment.

11. A method of producing at least one sexed embryo comprising:

producing a stream containing sperm cells, wherein the stream comprises sperm cells from a cell source surrounded by sheath fluid, wherein the sheath fluid surrounding the sperm cells includes a citric acid;

identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream;

collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid which includes a citric acid; and

fertilizing at least one egg with the collected sperm cells to form at least one sexed embryo.