Cryopreservation of articular cartilage

Abstract

The invention relates generally to methods and compositions for the cryopreservation and/or vitrification of tissue including articular cartilage and the preparation of said tissue for clinical or research use, including but not limited to joint replacement and the treatment and prevention of osteoarthritis.

1 . A kit comprising cryopreserving agents (CPAs), at least two of which CPAs being different CPAs from each other and including a first CPA and a second CPA, the kit having instructions for carrying out a method for cryopreserving articular cartilage using more than one CPA, the method described in the instructions comprising:
 permeating a sample of articular cartilage with a sequence of at least two different CPAs comprising the first CPA and the second CPA, the second CPA being permeated into the sample after permeating the sample with the first CPA, to form combined CPAs having a concentration distribution within the sample;   the concentration distribution of the combined CPAs being selected so that upon cooling of the sample, the combined CPAs vitrify and cryopreserve the sample and   vitrifying the sample by cooling the sample below the glass transition temperature of the permeated sample of articular cartilage in a sequence of cooling steps corresponding to the sequential addition of CPAs.   
     
     
         2 . The kit of  claim 1  further comprising a third CPA and in which in the method described in the instructions the sequence of at least two different CPAs comprises the third CPA permeated into the sample after permeating the sample with the second CPA. 
     
     
         3 . The kit of  claim 2  further comprising a fourth CPA and in which in the method described in the instructions the sequence of at least two different CPAs comprises the fourth CPA permeated into the sample after permeating the sample with the third CPA. 
     
     
         4 . The kit of  claim 1  in which the first CPA is selected from the group comprising dimethyl sulfoxide (D), ethylene glycol (EG), propylene glycol (PG), glycerol (G) and formamide (F). 
     
     
         5 . The kit of  claim 1  in which the second CPA is selected from the group comprising dimethyl sulfoxide (D), ethylene glycol (EG), propylene glycol (PG), glycerol (G) and formamide (F). 
     
     
         6 . The kit of  claim 1  in which the third CPA is selected from the group comprising dimethyl sulfoxide (D), ethylene glycol (EG), propylene glycol (PG), glycerol (G) and formamide (F). 
     
     
         7 . The kit of any  claim 1  in which the CPAs are selected from the group comprising dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, formamide, methanol and ethanol. 
     
     
         8 . The kit of  claim 1  in which the fourth CPA is selected from the group comprising dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol. 
     
     
         9 . The kit of  claim 1  in which the kit and sequence of at least two different CPAs comprises five, six, or seven different CPAs. 
     
     
         10 .- 11 . (canceled) 
     
     
         12 . The kit of  claim 1  in which the CPAs are chosen from the group comprising dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, formamide, methanol and ethanol. 
     
     
         13 . The kit of  claim 1  in which either the first CPA or the second CPA is glycerol. 
     
     
         14 . The kit of  claim 3  in which the sequence of CPAs is D-G-PG-EG, G-EG-D-F, EG-G-D-PG, EG-G-F-D or D-G-EG-F. 
     
     
         15 - 18 . (canceled) 
     
     
         19 . The kit of  claim 1  in which in the method described in the instructions either a) one or more of the CPAs in the sequence of CPAs are combined with chondroitin sulphate or b) one or more of the CPAs in the sequence of CPAs is combined with hyaluronic acid, or a) and b). 
     
     
         20 . The kit of  claim 1  in which in the method described in the instructions each succeeding CPA is permeated into the sample along with each preceding CPA and the preceding CPA added with each succeeding CPA has a concentration substantially equal to the concentration of the preceding CPA in the sample cartilage. 
     
     
         21 .- 22 . (canceled) 
     
     
         23 . The kit of  claim 1  in which in the method described in the instructions the sequence of CPAs comprises one of the given examples in Tables 2a-2ll and the kit further comprises the CPAs in the given example. 
     
     
         24 .- 25 . (canceled) 
     
     
         26 . A method for cryopreserving articular cartilage using more than one cryopreserving agent, comprising:
 (i) obtaining an articular cartilage sample;   (ii) adding one CPA first at a temperature above the freezing point of the tissue and CPA at the outset for a sufficient period of time to obtain a desired degree of CPA tissue permeation;   (iii) moving the tissue to another solution that contains at least one or more CPAs at a temperature equal to or lower than the temperature in step “(ii)”, but higher than the freezing point of the solution and tissue at the end of step (ii) above, for a sufficient period of time to obtain a desired degree of CPA tissue permeation;   (iv) repeating step (iii) with CPAs at a temperature equal to or lower than previously used but higher than the solution and tissue freezing point, until a high enough CPA concentration in the tissue is achieved of all the different CPAs to vitrify the solution and effectively cryopreserve the tissue.   
     
     
         27 - 28 . (canceled) 
     
     
         29 . Articular cartilage (AC) tissue cryopreserved using a method for cryopreserving AC using more than one cryopreserving agent (CPA), the method comprising:
 permeating a sample of articular cartilage with a sequence of at least two different CPAs comprising a first CPA and a second CPA, the second CPA being permeated into the sample after permeating the sample with the first CPA, to form combined CPAs having a concentration distribution within the sample;   the concentration distribution of the combined CPAs being selected so that upon cooling of the sample, the combined CPAs vitrify and cryopreserve the sample; and   vitrifying the sample by cooling the sample below the glass transition temperature of the permeated sample of articular cartilage in a sequence of cooling steps corresponding to the sequential addition of CPAs.