Method of lyophilization of mammalian sperm cells

• US 5,849,473 A
• Filed: 02/15/1991
• Issued: 12/15/1998
• Est. Priority Date: 02/15/1991
• Status: Expired due to Fees

• Current Assignee: Cobe Cardiovascular Incorporated (LivaNova Plc)
• Original Assignee: Cobe Cardiovascular Incorporated (LivaNova Plc)
• Inventors: Cabrera, Gonzalo M., Goodrich, Raymond P. Jr.
• Primary Examiner(s): Saucier, Sandra E.
• Application Number:
  US07/656,194
• Time in Patent Office: 2,860 Days
• Field of Search: 435/2, 424/561
• US Class Current: 435/2
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Abstract

A method is provided for the lyophilization of mammalian sperm cells which can be stored and reconstituted to provide morphologically intact cells. The method is advantageous in that the sperm cells may be stored at ambient temperatures for extended periods of time and recovered morphologically intact with DNA-containing heads and intact flagella. The lyophilization medium comprises a carbohydrate and a polymer selected from PVP, HES, dextran and poloxamers or mixtures thereof.

Claims

1. A method for lyophilizing mammalian sperm cells comprising the steps of mixing a composition containing said sperm cells with a buffered solution comprising;

a carbohydrate and a polymer or mixture of polymers selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, dextran and poloxamers, said polymers having a number average molecular weight in the range of about 1K to 600K; and

drying said cells by sublimation of water.

2. A method according to claim 1 wherein said composition comprises egg yolk.

3. A method according to claim 1 wherein the sperm cell concentration in said composition is in a range of 10% to 50% by volume.

4. A method according to claim 1 wherein said sperm cells are selected from the group consisting of porcine, bovine and human sperm cells.

5. A method according to claim 1 wherein said average molecular weight is in the range of 1K to 350K.

6. A method according to claim 1 wherein the concentration of said polymers in said solution is in the concentration range from 0.5 to 30% weight/volume.

7. A method according to claim 1 wherein said carbohydrate comprises a monosaccharide.

8. A method according to claim 7 wherein said monosaccharide comprises glucose.

9. A method according to claim 1 wherein the concentration of said carbohydrate and said solution is in a range of 0.1 to 2.6 molar.

10. A method for preparing reconstituted sperm cells comprising the step of lyophilizing said sperm cells with a buffered solution comprising a carbohydrate and a polymer or mixture of polymers selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch, dextran and poloxamers having a number average molecular weight in the range of about 1K to 600K;

reconstituting the lyophilized composition containing said sperm cells with a sufficient volume of reconstitution buffer to rehydrate said sperm cells in a morphologically intact form with intact DNA containing heads and intact tails.

11. A method according to claim 10 wherein said polymers have a molecular weight in the range of about 1K to 30K.

12. A method according to claim 11 wherein said polymers have an average molecular weight of about 15K.

13. A method according to claim 10 wherein said reconstitution buffer comprises phosphate-buffered saline with glucose and adenine.

14. A method according to claim 13 wherein said reconstitution buffer further comprises cell metabolites.