Method for cryopreserving blood vessels

• US 5,158,867 A
• Filed: 01/23/1990
• Issued: 10/27/1992
• Est. Priority Date: 08/21/1987
• Status: Expired due to Term

• Current Assignee: Cryolife Incorporated
• Original Assignee: Cryolife Incorporated, Medical University of South Carolina (State of South Carolina)
• Inventors: Brockbank, Kelvin G. M., Heacox, Albert E., McNally, Robert T., Bank, Harvey L., McCaa, Cameron
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/436,365
• Time in Patent Office: 1,008 Days
• Field of Search: 435/1, 435/2
• US Class Current: 435/1.3
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time

Abstract

A device for use in cryopreservation of blood vessels comprising a pair of stylets insertable into the ends of a dissected blood vessel segment. The stylets are mountable on a support track whereby the blood vessel can be distended and supported during cryopreservation procedures. Also disclosed is a freezing and thawing profile capable of maximizing endothelial cell survival. The use of chondroitin sulfate or similar compound is discussed as a novel cryoprotectant.

Claims

1. A method of maintaining intact a layer of endothelial cells present in a blood vessel during cryopreservation comprising:

i) contacting said blood vessel with an effective amount of a cryoprotectant composition comprising a medium for freezing said blood vessel, a cryoprotectively effective concentration of a cell penetrating cryoprotectant and a cryoprotectively effective concentration of a glycosaminoglycan; and

ii) maintaining said blood vessel in contact with said composition at a temperature below -100°

C.

2. The method according to claim 1 wherein said composition further comprises an antibiotic.

3. The method according to claim 1 wherein the glycosaminoglycan is chondroitin sulfate.

4. The method according to claim 1 wherein said cell penetrating cryoprotectant is dimethylsulfoxide.

5. The method of claim 1 wherein the freezing schedule comprises:

(a) placing the blood vessel into a freezing chamber;

(b) cooling the sample at a suitable rate until the sample reaches about -2°

C.;

(c) cooling the sample after substantial completion of phase change at a suitable rate;

(d) removing the blood vessel from the chamber;

(e) placing the blood vessel in a liquid nitrogen refrigerator at a temperature of about -196°

C.

6. The method of claim 5 wherein the cooling rate is in a range of from about 0.1°

-100°

C./min.

7. The method of claim 6 wherein the cooling rate is in a range of from about 0.1°

-30°

C./min.

8. The method of claim 7 wherein the cooling rate is in a range of from about 0.3°

-1°

C./min.

9. The method of claim 8 wherein the cooling rate is in a range of from about 0.5°

-1°

C./min.

10. The method of claim 9 wherein the cooling rate is in a range of from about 0.5°

C./min.

11. The method of claim 5 wherein the freezing schedule comprises:

(a) placing the blood vessel into a freezing chamber, then

(b) cooling the chamber from an initial temperature of about -10°

C. at a rate of about 0.01°

C. per min. until the sample reaches about +4°

C.;

then

(c) cooling the chamber at a rate of about 0.3°

C. per min. until the sample reaches about -2°

C.;

then

(d) cooling the chamber at a rate of about 30°

C. per min. until the chamber reaches a temperature of about -70°

C.;

then

(e) warming the chamber at a rate of about 20°

C. per min. until the chamber reaches a temperature of about -60°

C.;

then

(f) holding the chamber at a temperature of about -60°

C. for 17 minutes;

then

(g) warming the chamber at a rate of about 10°

C. per min. until the chamber reaches a temperature of about -30°

C.;

then

(h) holding the chamber at a temperature of about -30°

C. for one min.;

then

(i) cooling the chamber at a rate of about 0.01°

C. per min. until the sample reaches a temperature of about -20°

C.;

then

(j) cooling the chamber at a rate of about 0.5°

C. per min. until the sample reaches a temperature of about -65°

C.;

then

(k) removing the blood vessel from the chamber; and

then

(l) placing the blood vessel in a liquid nitrogen refrigerator at a temperature of about -196°

C.