Three-dimensional blood-brain barrier cell and tissue culture system
First Claim
1. 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.
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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.
109 Citations
41 Claims
- 1. 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.
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9. A method for culturing blood-brain barrier cell cultures in vitro, comprising:
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(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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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- 17. 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.
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25. A method for determining the effect of a drug on neuronal cells or astrocytes cells in culture comprising:
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(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. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32)
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33. 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:
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(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. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40)
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41. A method for testing the ability of a substance to cross a blood-brain barrier comprising:
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(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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Specification