US5071741A
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US
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Cryoprotective agent and its use in cryopreservation of cellular matter
Assignee: Artivion Inc
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Filing: 1988-04-18
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Granted: 1991-12-10
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Expires: 2008-04-18
Abstract
Abstract A novel class of nonpermeating cryoprotectants which, when mixed with certain known penetrating cryoprotectants, provide a useful medium for protection of living cells during a cryopreservation process. Algae-derived polysaccharides such as agarose and alginate are useful as nonpermeating cryoprotectants as they form a gel matrix when cooled, protect against ice crystal formation, and yield improved viability of cells when thawed.
Inventors
- 👤 Kelvin G. M. Brockbank
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 13:38
CPC Classifications
A; A01; A01N; A01N1/00; A01N1/10; A01N1/12; A01N1/122; A01N1/125; Y; Y10; Y10S; Y10S128/00; Y10S128/27; Y10T; Y10T436/00; Y10T436/10; Y10T436/108331
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
21 words
A cryoprotective agent comprised of a mixture of a cell-permeating cryoprotectant and a non-cell permeating, macromolecular polysaccharide cryoprotectant derived from algae.
Claim 2
Dependent Claim
21 words
A cryoprotective agent comprised of a mixture of a cell-permeating cryoprotectant and a non-cell permeating, macromolecular polysaccharide cryoprotectant derived from algae.
Claim 3
Dependent Claim
16 words
The cryopreservative agent of claim 1, wherein said nonpermeating cryoprotectant comprises a mixture of macromolecular polysaccharides.
Claim 4
Dependent Claim
20 words
The cryoprotective agent of claim 1 wherein said macromolecular polysaccharide is selected from the group consisting of agarose and alginate.
Claim 5
Dependent Claim
20 words
The cryoprotective agent of claim 1 wherein said cell-permeating cryoprotectant is selected from the group consisting of glycerol and dimethylsulfoxide.
Claim 6
Dependent Claim
25 words
The cryopreservative agent of claim 4, wherein said cell-permeating cryoprotectant is dimethylsulfoxide and said dimethylsulfoxide is present in a concentration from about 0.5M to 3.0M.
Claim 7
Dependent Claim
17 words
The cryopreservative agent of claim 5, wherein said dimethylsulfoxide is present in a concentration of about 1M.
Claim 8
Independent Claim
109 words
A method for cryopreserving viable cellular matter comprising: (a) treating viable cellular matter with a cryoprotective agent comprising a mixture of a non-cell permeating cryoprotectant and a cell permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide including from about 3 to about 3000 saccharide units such that said mixture gels at temperatures below ambient or when contacted by divalent ions, said non-cell permeating and cell permeating cryoprotectants being present in the mixture in concentrations effective for enhancing cellular viability upon transplant, and said non-cell permeating cryoprotectant being effective for encapsulating said cellular matter; (b) freezing said encapsulated cellular matter and (c) storing said frozen cellular matter.
Claim 9
Independent Claim
109 words
A method for cryopreserving viable cellular matter comprising: (a) treating viable cellular matter with a cryoprotective agent comprising a mixture of a non-cell permeating cryoprotectant and a cell permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide including from about 3 to about 3000 saccharide units such that said mixture gels at temperatures below ambient or when contacted by divalent ions, said non-cell permeating and cell permeating cryoprotectants being present in the mixture in concentrations effective for enhancing cellular viability upon transplant, and said non-cell permeating cryoprotectant being effective for encapsulating said cellular matter; (b) freezing said encapsulated cellular matter and (c) storing said frozen cellular matter.
Claim 10
Dependent Claim
15 words
The method of claim 7, wherein said nonpermeating cryoprotectant comprises a mixture of macromolecular polysaccharides.
Claim 11
Independent Claim
49 words
A method of cryopreserving viable cellular matter comprising: treating viable cellular matter with a cryoprotective agent comprised of a cell-permeating cryoprotectant and a non-cell permeating, algae-derived macromolecular polysaccharide cryoprotectant, and freezing the treated viable cellular matter to a temperature at least as low as the temperature of nitrogen vapor.
Claim 12
Independent Claim
49 words
A method of cryopreserving viable cellular matter comprising: treating viable cellular matter with a cryoprotective agent comprised of a cell-permeating cryoprotectant and a non-cell permeating, algae-derived macromolecular polysaccharide cryoprotectant, and freezing the treated viable cellular matter to a temperature at least as low as the temperature of nitrogen vapor.
Claim 13
Dependent Claim
23 words
The method of claim 9, wherein said macromolecular polysaccharide is composed of a material selected from the group consisting of agarose and alginate.
Claim 14
Dependent Claim
20 words
The method of claim 9, wherein said cell-permeating cryoprotectant agent is selected from the group consisting of glycerol and dimethylsulfoxide.
Claim 15
Dependent Claim
18 words
The method of claim 11, wherein said dimethylsulfoxide is present in a concentration from about 0.5M to 3M.
Claim 16
Dependent Claim
16 words
The method of claim 12, wherein said dimethylsulfoxide is present in a concentration of about 1M.
Claim 17
Dependent Claim
17 words
The method of claim 9, wherein said cryopreservative agent comprises at least one algae-derived polysaccharide and dimethylsulfoxide.
Claim 18
Dependent Claim
39 words
The method of claim 9, wherein said cellular matter is selected from the group consisting of heart valves, corneas, pancreas, blood vessels, bone, bone marrow, endothelial cells, saphenous veins, liver cells, nerves, connective tissue endocrine glands and exocrine glands.
Claim 19
Dependent Claim
20 words
The method of claim 9 wherein said cellular matter is selected from the group consisting of ligaments, tendons and cartilage.
Claim 20
Dependent Claim
11 words
The method of claim 9 wherein said cellular matter is skin.
Claim 21
Dependent Claim
13 words
The method of claim 9 wherein said cellular matter is islets of Langerhans.
Claim 22
Dependent Claim
52 words
In a process for cryopreserving transplantable cellular matter wherein transplantable cellular matter is harvested from a donor and then cryopreserved at a temperature at least as low as the temperature of nitrogen vapor, the improvement comprising the step of encapsulating the transplantable cellular matter in an algae-derived polysaccharide gel prior to cryopreservation.
Claim 23
Dependent Claim
52 words
In a process for cryopreserving transplantable cellular matter wherein transplantable cellular matter is harvested from a donor and then cryopreserved at a temperature at least as low as the temperature of nitrogen vapor, the improvement comprising the step of encapsulating the transplantable cellular matter in an algae-derived polysaccharide gel prior to cryopreservation.
Claim 24
Dependent Claim
83 words
In a process for cryopreserving transplantable cellular matter wherein transplantable cellular matter is harvested from a donor and then cryopreserved at a temperature at least as low as the temperature of nitrogen vapor, the improvement comprising the steps of: (a) treating the cellular matter with a mixture of at least one algae-derived polysaccharide and dimethylsulfoxide in amounts which are effective for reducing ice crystal formation; and (b) cooling said treated cellular matter such that a cell is formed around said treated cellular matter.
Claim 25
Dependent Claim
83 words
In a process for cryopreserving transplantable cellular matter wherein transplantable cellular matter is harvested from a donor and then cryopreserved at a temperature at least as low as the temperature of nitrogen vapor, the improvement comprising the steps of: (a) treating the cellular matter with a mixture of at least one algae-derived polysaccharide and dimethylsulfoxide in amounts which are effective for reducing ice crystal formation; and (b) cooling said treated cellular matter such that a cell is formed around said treated cellular matter.
Claim 26
Dependent Claim
87 words
A cryopreservative agent comprising a mixture of a non-cell permeating cryoprotectant and a cell-permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide having about 3 to about 3000 saccharide units, said non-cell permeating and cell permeating cryoprotectants being present in the mixture at concentrations which are effective (a) for forming a gel at a temperature below ambient temperature or upon contact with divalent ions, and (b) improving the biological function of the transplantable, cryopreserved tissue upon transplant into a patient in need of such treatment.
Claim 27
Dependent Claim
87 words
A cryopreservative agent comprising a mixture of a non-cell permeating cryoprotectant and a cell-permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide having about 3 to about 3000 saccharide units, said non-cell permeating and cell permeating cryoprotectants being present in the mixture at concentrations which are effective (a) for forming a gel at a temperature below ambient temperature or upon contact with divalent ions, and (b) improving the biological function of the transplantable, cryopreserved tissue upon transplant into a patient in need of such treatment.
Claim 28
Dependent Claim
12 words
A cryoprotective agent of claim 21 wherein the cell-permeating cryoprotectant is dimethylsulfoxide.
Claim 29
Independent Claim
106 words
A method of improving the post-transplantable biological function of a transplantable tissue comprising: treating the transplantable tissue prior to cryopreservation with a mixture of a non-cell permeating cryoprotectant and a cell-permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide containing about 3 to about 3000 saccharide units per molecule, said non-cell permeating and cell-permeating cryoprotectants being present in the mixture in concentrations which are effective (a) for forming a gel at a temperature below ambient temperature or upon contact with divalent ions, and (b) for improving the biological function of the transplantable, cryopreserved tissue upon transplant into a patient in need of such treatment.
Claim 30
Independent Claim
106 words
A method of improving the post-transplantable biological function of a transplantable tissue comprising: treating the transplantable tissue prior to cryopreservation with a mixture of a non-cell permeating cryoprotectant and a cell-permeating cryoprotectant, said non-cell permeating cryoprotectant comprising an algae-derived macromolecular polysaccharide containing about 3 to about 3000 saccharide units per molecule, said non-cell permeating and cell-permeating cryoprotectants being present in the mixture in concentrations which are effective (a) for forming a gel at a temperature below ambient temperature or upon contact with divalent ions, and (b) for improving the biological function of the transplantable, cryopreserved tissue upon transplant into a patient in need of such treatment.
Claim 31
Dependent Claim
36 words
A method in accordance with claim 23 further comprising: encapsulating the transplantable tissue in an algae derived polysaccharide, and cryopreserving the encapsulated transplantable tissue at a temperature effective for improving the biological function of the tissue.
Claim 32
Independent Claim
56 words
The method of cryopreserving viable cellular matter comprising: treating viable cellular matter with a mixture of at least one algae-derived macromolecular polysaccharide and dimethylsulfoxide in amounts which are effective for improving the biological function of said cellular matter upon transplant, said algae-derived polysaccharide encapsulating said cellular matter, and freezing said encapsulated cellular matter in said mixture.
Claim 33
Independent Claim
56 words
The method of cryopreserving viable cellular matter comprising: treating viable cellular matter with a mixture of at least one algae-derived macromolecular polysaccharide and dimethylsulfoxide in amounts which are effective for improving the biological function of said cellular matter upon transplant, said algae-derived polysaccharide encapsulating said cellular matter, and freezing said encapsulated cellular matter in said mixture.
Claim 34
Independent Claim
93 words
A method of transplanting cellular matter from a donor into a recipient, comprising: (a) removing cellular matter from a donor; (b) treating the cellular matter with a mixture of at least one algae-derived polysaccharide and dimethylsulfoxide in amounts which are effective for improving the biological function of the cellular matter upon subsequent thawing and implant, and for encapsulating said cellular matter with the algae-derived polysaccharide; (c) freezing said encapsulated cellular matter; (d) storing said frozen cellular matter; (e) thawing said frozen cellular matter; and (f) implanting the thawed cellular matter into the recipient.
Claim 35
Independent Claim
93 words
A method of transplanting cellular matter from a donor into a recipient, comprising: (a) removing cellular matter from a donor; (b) treating the cellular matter with a mixture of at least one algae-derived polysaccharide and dimethylsulfoxide in amounts which are effective for improving the biological function of the cellular matter upon subsequent thawing and implant, and for encapsulating said cellular matter with the algae-derived polysaccharide; (c) freezing said encapsulated cellular matter; (d) storing said frozen cellular matter; (e) thawing said frozen cellular matter; and (f) implanting the thawed cellular matter into the recipient.
Claim 36
Independent Claim
60 words
A method for cryopreserving cellular matter comprising: (a) removing the cellular matter from a donor; (b) forming an algae-derived polysaccharide gel around said cellular matter; (c) adding at least one cell-penetrating cryoprotectant to said gel-enclosed cellular matter in an amount effective for reducing ice crystal formation during cryopreservation, and (d) storing the cellular matter for a desired period of time.
Claim 37
Independent Claim
60 words
A method for cryopreserving cellular matter comprising: (a) removing the cellular matter from a donor; (b) forming an algae-derived polysaccharide gel around said cellular matter; (c) adding at least one cell-penetrating cryoprotectant to said gel-enclosed cellular matter in an amount effective for reducing ice crystal formation during cryopreservation, and (d) storing the cellular matter for a desired period of time.
Claim 38
Independent Claim
84 words
A method of transplanting cellular matter from a donor to a recipient, comprising: (a) removing cellular matter from a donor; (b) forming a algae-derived polysaccharide gel around said cellular matter; (c) adding a cell-penetrating cryoprotectant to the gel-enclosed cellular matter in an amount effective for reducing ice crystal formation; (d) freezing the gel-enclosed cellular matter and cell-penetrating cryoprotectant; (e) storing the frozen cellular matter for a desired period of time; (f) thawing said frozen cellular matter, and implanting the cellular matter in a recipient.
Claim 39
Independent Claim
84 words
A method of transplanting cellular matter from a donor to a recipient, comprising: (a) removing cellular matter from a donor; (b) forming a algae-derived polysaccharide gel around said cellular matter; (c) adding a cell-penetrating cryoprotectant to the gel-enclosed cellular matter in an amount effective for reducing ice crystal formation; (d) freezing the gel-enclosed cellular matter and cell-penetrating cryoprotectant; (e) storing the frozen cellular matter for a desired period of time; (f) thawing said frozen cellular matter, and implanting the cellular matter in a recipient.
References Cited (Backward Citations)
US3303662A
Cited
US3758382A
Cited
US4097338A
Cited
US4004975A
Cited
US4237218A
Cited
US4473552A
Cited
US4489162A
Cited
US4559298A
Cited
US4840891A
Cited
US4874690A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-12-09 | 1995-06-10 | 1995-12-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-12-09 | 1999-06-10 | 1999-12-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-12-09 | 2003-06-10 | 2003-12-10 | $7700.00 | Window Closed / Lapsed |