US5364756A
Expired / Lapsed
US
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Method of cryopreserving a suspension of biological material
Assignee: LifeCell Corp
•
Filing: 1993-02-16
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Granted: 1994-11-15
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Expires: 2013-02-16
Abstract
Abstract This invention relates to methods, apparatus and solutions for cryopreserving microscopic biological materials for biologically extended periods of time. The method comprises treating a suspension of biological material, in an appropriate buffer, with a cryoprotectant or combination of cryoprotectants which raises the glass transition temperature range of the sample. One or more dry protectants may be added to the cryosolution. The cryosolution is then nebulized and rapidly cooled with novel apparatus, dried by molecular distillation, stored and then rehydrated in a buffer prior to its use. The solutions comprise novel mixtures of cryoprotectants.
Inventors
- 👤 Stephen A. Livesey
- 👤 Anthony A. del Campo
- 👤 Abhijit Nag
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 10:51
CPC Classifications
A; A01; A01N; A01N1/00; A01N1/10; A01N1/12; A01N1/122; A01N1/125; A01N1/14; A01N1/142; A01N1/144; A01N1/145; A61; A61K; A61K35/00; A61K35/12; A61K35/14; A61K35/18; A61K39/00; A61K39/12; A61K39/125; A61K39/13; C; C12; C12N; C12N1/00; C12N1/04; F; F25; F25C; F25C1/00; F25C1/12; F25C1/14; F25C1/142; F26; F26B; F26B5/00; F26B5/04; F26B5/06; F26B5/065; A61L; A61L2430/00; A61L2430/40; C12N2770/00; C12N2770/00011; C12N2770/32011; C12N2770/32611; C12N2770/32634
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
115 words
A method for preserving a suspension of biological material comprising: (a) preparing a cryosolution of a suspension of biological material, said cryosolution comprising a buffer, a cryoprotectant and a biological material; (b) nebulizing said cryosolution with a low frequency, ultrasonic nebulizer to form microdroplets; (c) cooling the microdroplets at a rate which results in the formation of amorphous phase ice and/or cubic phase ice with or without hexagonal phase ice in said cryosolution by spraying the microdroplets on a solid cryogenic surface; (d) continuously removing said cryosolution from said solid cryogenic surface after cooling to replenish said surface for further cooling of additional microdroplets; and (e) drying the cooled cryosolution to form a dried cryosolution.
Claim 2
Independent Claim
115 words
A method for preserving a suspension of biological material comprising: (a) preparing a cryosolution of a suspension of biological material, said cryosolution comprising a buffer, a cryoprotectant and a biological material; (b) nebulizing said cryosolution with a low frequency, ultrasonic nebulizer to form microdroplets; (c) cooling the microdroplets at a rate which results in the formation of amorphous phase ice and/or cubic phase ice with or without hexagonal phase ice in said cryosolution by spraying the microdroplets on a solid cryogenic surface; (d) continuously removing said cryosolution from said solid cryogenic surface after cooling to replenish said surface for further cooling of additional microdroplets; and (e) drying the cooled cryosolution to form a dried cryosolution.
Claim 3
Dependent Claim
20 words
The method of claim 1 wherein said dried cryosolution has a final residual moisture content of less than about 5%.
Claim 4
Dependent Claim
13 words
The method of claim 1 wherein said cryosolution further comprises a dry protectant.
Claim 5
Dependent Claim
30 words
The method of claim 2 further comprising the step of sealing said dried cryosolution in a container without increasing the residual water content of said dried cryosolution above about 5%.
Claim 6
Dependent Claim
21 words
The method of claim 4 wherein said sealing is under low pressure or in the presence of a dry inert gas.
Claim 7
Dependent Claim
27 words
The method of claim 1 wherein said buffer is selected from the group consisting of 2-amino-2-hydroxymethyl-1,3-propanediol; 2-ethanesulfonic acid; 2-amino-2-methyl-1,3-propanediol; 3-propanesulfonic acid; piperazine-N-N'-bis; 1,4-piperazinediethanesulfonic acid and combinations thereof.
Claim 8
Dependent Claim
146 words
The method of claim 1 wherein said buffer is a low salt buffer mixture selected from the group consisting of (1) 8 gms glycine, 20 mls of 0.15M phosphate buffer pH 6.7-8.4 and 1.8 gm sodium chloride made up to 1000 ml., pH 6.7; (2) 16 gms glycine, 20 mls of 0.15M phosphate buffer pH 6.7-8.4, 1.8 gm sodium chloride, 5.0 gms glucose, 0.06 gm calcium chloride and 0.1 gms adenine made up t 1000 ml., pH 6.7; (3) a solution containing 0.9% NaCl, 0.75% mannitol, 2.2% dextrose and 0.027% adenine; (4) a solution containing 0.875% dextrose, 0.9% NaCl, 0.214% mannitol, 0.008% adenine, 0.2555% sodium citrate, 0.029% citric acid and 0.022% monobasic sodium phosphate; (5) 16 mM phosphate buffer, pH 7.4, 5 mM KCl, 4 g/dl BSA and 6.9 g/lit. lactose; and (6) 16 mM phosphate buffer, pH 7.4, 5 mM KCl and 15 g/lit. lactose.
Claim 9
Dependent Claim
41 words
The method of claim 1 wherein said cryoprotectant is selected from the group consisting of dimethyl sulfoxide, 2-3 butane diol, polyvinylpyrrolidone, propylene glycol, 1,2 propanediol, glycerol, fructose, trehalose, raffinose, hydroxyethyl starch, dextran, sucrose, sorbitol, proline, human serum albumin and combinations thereof.
Claim 10
Dependent Claim
149 words
The method of claim 8 wherein said cryoprotectant is selected from the group consisting of (1) a vitrification solution comprising a mixture of 0.5M DMSO, 0.5M propylene glycol, 0.25M 2-3 butanediol, 1.0M proline, 2.5% raffinose, 15% PVP with an average molecular weight of about 40,000 and 15% dextran with an average molecular weight from about 40,000 to about 70,000; (2) a vitrification solution comprising a mixture of 0.5M DMSO, 0.5M propylene glycol, 0.25M 2-3 butanediol, 10% raffinose, 6% trehalose, 6% sucrose, 12% PVP with an average molecular weight of about 40,000 and 12% dextran with an average molecular weight from about 40,000 to about 70,000; and (3) a vitrification solution comprising a mixture 0.5M DMSO, 0.5M propylene glycol, 0.25M 2-3 butanediol. 2.5% raffinose, 12% sucrose, 15% PVP with an average molecular weight of about 40,000 and 15% dextran with an average molecular weight from about 40,000 to about 70,000.
Claim 11
Dependent Claim
27 words
The method of claim 3 wherein said dry protectant is selected from the group consisting of: sucrose, raffinose, trehalose, zinc, proline, myristic acid, spermine and combinations thereof.
Claim 12
Dependent Claim
11 words
The method of claim 1 wherein said drying comprises freeze drying.
Claim 13
Dependent Claim
12 words
The method of claim 1 wherein said drying comprises molecular distillation drying.
Claim 14
Dependent Claim
13 words
The method of claim 1 wherein said biological material comprises red blood cells.
Claim 15
Dependent Claim
13 words
The method of claim 1 wherein said biological material comprises mammalian cultured cells.
Claim 16
Dependent Claim
11 words
The method of claim 1 wherein said biological material comprises platelets.
Claim 17
Dependent Claim
11 words
The method of claim 1 wherein said biological material comprises leukocytes.
Claim 18
Dependent Claim
12 words
The method of claim 1 wherein said biological material comprises Factor VIII.
Claim 19
Dependent Claim
11 words
The method of claim 1 wherein said biological material comprises sperm.
Claim 20
Dependent Claim
12 words
The method of claim 1 wherein said biological material comprises pancreatic islets.
Claim 21
Dependent Claim
13 words
The method of claim 1 wherein said biological material comprises bone marrow cells.
Claim 22
Dependent Claim
12 words
The method of claim 1 wherein said biological material comprises a virus.
Claim 23
Dependent Claim
12 words
The method of claim 1 wherein said biological material comprises a vaccine.
Claim 24
Dependent Claim
11 words
The method of claim 1 further comprising rehydrating the dried cryosolution.
Claim 25
Independent Claim
151 words
A method for preserving a suspension of a microscopic biological material comprising: (a) preparing a cryosolution of a microscopic biological material, said cryosolution comprising a buffer, a cryoprotectant, and a biological material; (b) nebulizing said cryosolution With a low frequency, ultrasonic nebulizer to form discrete droplets, said droplets being less than about 200 μm in diameter; (c) rapidly cooling said droplets resulting in the formation of amorphous phase ice, cubic phase ice and hexagonal phase ice by spraying the droplets on a solid cryogenic surface; (d) continuously removing the rapidly cooled cryosolution from said solid cryogenic surface after said ice has been formed to replenish said surface for cooling of additional droplets; and (e) drying said rapidly cooled cryosolution to remove said amorphous phase ice, cubic phase ice and hexagonal ice to form a dried cryosolution, said drying being completed to a final residual moisture content of less than about 5%.
Claim 26
Independent Claim
151 words
A method for preserving a suspension of a microscopic biological material comprising: (a) preparing a cryosolution of a microscopic biological material, said cryosolution comprising a buffer, a cryoprotectant, and a biological material; (b) nebulizing said cryosolution With a low frequency, ultrasonic nebulizer to form discrete droplets, said droplets being less than about 200 μm in diameter; (c) rapidly cooling said droplets resulting in the formation of amorphous phase ice, cubic phase ice and hexagonal phase ice by spraying the droplets on a solid cryogenic surface; (d) continuously removing the rapidly cooled cryosolution from said solid cryogenic surface after said ice has been formed to replenish said surface for cooling of additional droplets; and (e) drying said rapidly cooled cryosolution to remove said amorphous phase ice, cubic phase ice and hexagonal ice to form a dried cryosolution, said drying being completed to a final residual moisture content of less than about 5%.
Claim 27
Dependent Claim
30 words
The method of claim 24 further comprising the step of sealing said dried cryosolution in a container without increasing the residual water content of said dried cryosolution above about 5%.
Claim 28
Dependent Claim
30 words
The method of claim 25 wherein said sealing is performed in a dry inert gas atmosphere at a low pressure of from about 10 -6 mbar to 10 -1 mbar.
Claim 29
Dependent Claim
11 words
The method of claim 24 further comprising rehydrating the dried cryosolution.
References Cited (Backward Citations)
GB351132A
Cited
US3228838A
Cited
FR1522286A
Cited
FR2104349A5
Cited
GB1482785A
Cited
CH614532A5
Cited
US4205132A
Cited
US4229544A
Cited
US4329787A
Cited
US4559298A
Cited
US4567847A
Cited
US4865871A
Cited
US5024830A
Cited
US4688387A
Cited
EP0267026A2
Cited
US4707998A
Cited
US4807442A
Cited
US4980277A
Cited
US5171661A
Cited
US5178884A
Cited
US4874690A
Cited
US5045446A
Cited
US5153004A
Cited
US4964280A
Cited
WO1991018504A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-11-14 | 1998-05-16 | 1998-11-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-11-14 | 2002-05-16 | 2002-11-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-11-14 | 2006-05-16 | 2006-11-15 | $7700.00 | Window Closed / Lapsed |