US5578485A
Expired / Lapsed
US
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Three-dimensional blood-brain barrier cell and tissue culture system
Assignee: Smith and Nephew Wound Management La Jolla
•
Filing: 1995-04-06
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Granted: 1996-11-26
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Expires: 2015-04-06
Abstract
Abstract The present invention relates to a three-dimensional cell culture system which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. In accordance with the invention, cells derived from a desired tissue are inoculated and grown on a pre-established stromal support matrix. The stromal support matrix comprises stromal cells, such as fibroblasts actively growing on a three-dimensional matrix. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.
Inventors
- 👤 Gail K. Naughton
- 👤 Brian A. Naughton
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 13:35
CPC Classifications
A; A61; A61L; A61L27/00; A61L27/36; A61L27/3604; A61B; A61B90/00; A61K; A61K35/00; A61K35/12; A61K35/28; A61L27/38; A61L27/3886; A61L27/50; A61L27/60; C; C12; C12N; C12N5/00; C12N5/06; C12N5/0697; C12N5/0698; A61B10/00; A61B10/02; A61B10/0233; A61B10/025; A61B2010/0258; A61K2035/124; C12N2500/00; C12N2500/30; C12N2500/34; C12N2501/00; C12N2501/10; C12N2501/11; C12N2501/30; C12N2501/33; C12N2501/335; C12N2501/38; C12N2501/39; C12N2502/00; C12N2502/09; C12N2502/091; C12N2502/094; C12N2502/13; C12N2502/1323; C12N2503/00; C12N2503/04; C12N2510/00; C12N2533/00; C12N2533/30; C12N2533/40; C12N2533/70; Y; Y10; Y10S; Y10S623/00; Y10S623/915
Claims Analysis
Total Claims: 47
Claim 1
Independent Claim
58 words
A three-dimensional blood-brain barrier cell culture system, comprising neuron cells and astrocytes cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent small blood vessel endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells.
Claim 2
Dependent Claim
58 words
A three-dimensional blood-brain barrier cell culture system, comprising neuron cells and astrocytes cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent small blood vessel endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells.
Claim 3
Dependent Claim
17 words
The three-dimensional cell culture of claim 1 in which the framework is composed of a biodegradable material.
Claim 4
Dependent Claim
23 words
The three-dimensional cell culture of claim 2 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
Claim 5
Dependent Claim
17 words
The three-dimensional cell culture of claim 1 in which the framework is composed of a non-biodegradable material.
Claim 6
Dependent Claim
33 words
The three-dimensional cell culture of claim 4 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
Claim 7
Dependent Claim
19 words
The three-dimensional cell culture of claim 2, 3, 4 or 5 in which the framework is pre-coated with collagen.
Claim 8
Dependent Claim
19 words
The three-dimensional cell culture of claim 1, 2, 3, 4 or 5 in which the framework is a mesh.
Claim 9
Dependent Claim
14 words
The three-dimensional cell culture of claim 6 in which the framework is a mesh.
Claim 10
Independent Claim
78 words
A method for culturing blood-brain barrier cell cultures in vitro, comprising: (a) culturing astrocytes inoculated onto a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent endothelial cells inoculated on a three-dimensional framework so that the confluent endothelial cells attach to and substantially envelope the framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; and further (b) inoculating further onto said astrocytes, neuron cells.
Claim 11
Independent Claim
78 words
A method for culturing blood-brain barrier cell cultures in vitro, comprising: (a) culturing astrocytes inoculated onto a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent endothelial cells inoculated on a three-dimensional framework so that the confluent endothelial cells attach to and substantially envelope the framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; and further (b) inoculating further onto said astrocytes, neuron cells.
Claim 12
Dependent Claim
16 words
The method according to claim 9 in which the framework is composed of a biodegradable material.
Claim 13
Dependent Claim
22 words
The method according to claim 10 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
Claim 14
Dependent Claim
16 words
The method according to claim 9 in which the framework is composed of a non-biodegradable material.
Claim 15
Dependent Claim
32 words
The method according to claim 12 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
Claim 16
Dependent Claim
18 words
The method according to claim 10, 11, 12 or 13 in which the framework is pre-coated with collagen.
Claim 17
Dependent Claim
18 words
The method according to claim 9, 10, 11, 12 or 13 in which the framework is a mesh.
Claim 18
Dependent Claim
13 words
The method according to claim 14 in which the framework is a mesh.
Claim 19
Independent Claim
74 words
A method for transplantation or implantation of a three-dimensional blood-brain barrier cell culture comprising implanting in vivo neuron cells and astrocytes cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent small blood vessel endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells so that a tissue equivalent is formed.
Claim 20
Independent Claim
74 words
A method for transplantation or implantation of a three-dimensional blood-brain barrier cell culture comprising implanting in vivo neuron cells and astrocytes cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent small blood vessel endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells so that a tissue equivalent is formed.
Claim 21
Dependent Claim
16 words
The method according to claim 17 in which the framework is composed of a biodegradable material.
Claim 22
Dependent Claim
22 words
The method according to claim 18 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
Claim 23
Dependent Claim
16 words
The method according to claim 17 in which the framework is composed of a non-biodegradable material.
Claim 24
Dependent Claim
32 words
The method according to claim 20 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
Claim 25
Dependent Claim
18 words
The method according to claim 18, 19, 20 or 21 in which the framework is pre-coated with collagen.
Claim 26
Dependent Claim
18 words
The method according to claim 17, 18, 19, 20 or 21 in which the framework is a mesh.
Claim 27
Dependent Claim
13 words
The method according to claim 22 in which the framework is a mesh.
Claim 28
Independent Claim
103 words
A method for determining the effect of a drug on neuronal cells or astrocytes cells in culture comprising: (a) exposing a three-dimensional blood-brain barrier cell culture to the drug, in which the three-dimensional cell culture comprises neuron cells and astrocytes grown on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent small blood vessel endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; and (b) determining the effect of the drug on the neuronal cells or astrocyte cells in culture.
Claim 29
Independent Claim
103 words
A method for determining the effect of a drug on neuronal cells or astrocytes cells in culture comprising: (a) exposing a three-dimensional blood-brain barrier cell culture to the drug, in which the three-dimensional cell culture comprises neuron cells and astrocytes grown on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent small blood vessel endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; and (b) determining the effect of the drug on the neuronal cells or astrocyte cells in culture.
Claim 30
Dependent Claim
23 words
The method for determining the effect of a drug according to claim 25 in which the framework is composed of a biodegradable material.
Claim 31
Dependent Claim
29 words
The method for determining the effect of a drug according to claim 26 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
Claim 32
Dependent Claim
23 words
The method for determining the effect of a drug according to claim 25 in which the framework is composed of a non-biodegradable material.
Claim 33
Dependent Claim
39 words
The method for determining the effect of a drug according to claim 28 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
Claim 34
Dependent Claim
25 words
The method for determining the effect of a drug according to claim 26, 27, 28 or 29 in which the framework is pre-coated with collagen.
Claim 35
Dependent Claim
18 words
The method according to claim 25, 26, 27, 28 or 29 in which the framework is a mesh.
Claim 36
Dependent Claim
13 words
The method according to claim 30 in which the framework is a mesh.
Claim 37
Independent Claim
129 words
A method for studying the mechanism of a disease or condition in a patient, which disease or condition has a detectable effect on neuronal cells or astrocytes comprising: (a) inoculating astrocytes from a sample obtained from the patient onto a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; (b) inoculating further onto said astrocytes neuron cells obtained from the patient; (c) culturing the inoculated living stromal tissue in a nutrient medium so that the inoculated astrocytes and neuron cells proliferate in culture; and (d) analyzing the proliferated cells in culture for markers of the disease or condition.
Claim 38
Independent Claim
129 words
A method for studying the mechanism of a disease or condition in a patient, which disease or condition has a detectable effect on neuronal cells or astrocytes comprising: (a) inoculating astrocytes from a sample obtained from the patient onto a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; (b) inoculating further onto said astrocytes neuron cells obtained from the patient; (c) culturing the inoculated living stromal tissue in a nutrient medium so that the inoculated astrocytes and neuron cells proliferate in culture; and (d) analyzing the proliferated cells in culture for markers of the disease or condition.
Claim 39
Dependent Claim
16 words
The method according to claim 33 in which the framework is composed of a biodegradable material.
Claim 40
Dependent Claim
22 words
The method according to claim 34 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
Claim 41
Dependent Claim
16 words
The method according to claim 33 in which the framework is composed of a non-biodegradable material.
Claim 42
Dependent Claim
32 words
The method according to claim 36 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluorethylene, or a nitrocellulose compound.
Claim 43
Dependent Claim
18 words
The method according to claim 34, 35, 36 or 37 in which the framework is pre-coated with collagen.
Claim 44
Dependent Claim
18 words
The method according to claim 33, 34, 35, 36 or 37 in which the framework is a mesh.
Claim 45
Dependent Claim
13 words
The method according to claim 38 in which the framework is a mesh.
Claim 46
Independent Claim
110 words
A method for testing the ability of a substance to cross a blood-brain barrier comprising: (a) exposing an amount of the substance which has a detectable effect on the astrocytes or neuron cells to the endothelial surface of a three-dimensional culture system comprising astrocytes and neuron cells grown on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; and (b) detecting the effect of the substance on the astrocytes or neuron cells of the inoculated three-dimensional stromal matrix.
Claim 47
Independent Claim
110 words
A method for testing the ability of a substance to cross a blood-brain barrier comprising: (a) exposing an amount of the substance which has a detectable effect on the astrocytes or neuron cells to the endothelial surface of a three-dimensional culture system comprising astrocytes and neuron cells grown on a living stromal tissue prepared in vitro, said living stromal tissue comprising confluent endothelial cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the endothelial cells; and (b) detecting the effect of the substance on the astrocytes or neuron cells of the inoculated three-dimensional stromal matrix.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-11-26 | 2000-05-27 | 2000-11-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-11-26 | 2004-05-27 | 2004-11-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-11-26 | 2008-05-27 | 2008-11-26 | $7700.00 | Window Closed / Lapsed |