Three-dimensional cell and tissue culture system
First Claim
1. A method for culturing stromal cells transfected with an exogenous gene under the control of an expression element, comprising culturing in vitro the transfected stromal cells inoculated on a three-dimensional framework so that the transfected stromal cells and connective tissue proteins naturally secreted by the stromal 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 stromal 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.
117 Citations
50 Claims
- 1. A method for culturing stromal cells transfected with an exogenous gene under the control of an expression element, comprising culturing in vitro the transfected stromal cells inoculated on a three-dimensional framework so that the transfected stromal cells and connective tissue proteins naturally secreted by the stromal 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 stromal cells.
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11. A method for culturing a genetically engineered dermis comprising culturing in vitro stromal cells transfected with an exogenous gene under the control of an expression element comprising culturing the transfected stromal cells inoculated on a three-dimensional framework so that the transfected stromal cells and connective tissue proteins naturally secreted by the stromal 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 stromal cells.
- 12. A method for culturing a three-dimensional cell culture comprising culturing in vitro parenchymal cells inoculated onto the transfected living stromal tissue comprising stromal cells transfected with an exogenous gene under the control of an expression element, so that the transfected stromal cells and connective tissue proteins naturally secreted by the stromal cells are attached to and substantially envelope a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
- 21. A method for culturing transfected parenchymal cells which contain an exogenous gene under the control of an expression element, comprising culturing the transfected parenchymal cells inoculated onto a living stromal tissue prepared in vitro, in which stromal cells and connective tissue proteins naturally secreted by the stromal cells are attached to and substantially envelope a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
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37. A method for culturing transfected astrocytes containing an exogenous gene under the control of an expression element, comprising culturing transfected astrocytes inoculated into a living stromal tissue prepared in vitro, 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 stromal cells.
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38. A method for culturing transfected neuron cells containing an exogenous gene under the control of an expression element, comprising culturing the transfected neuron cells inoculated onto a living stromal tissue prepared in vitro, and 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 stromal cells.
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39. A method for producing a biological product in a genetically engineered living stromal tissue comprising:
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(a) culturing stromal cells transfected with an exogenous gene under the control of an expression element so that the exogenous gene product is expressed in culture, and in which the transfected stromal cells and connective tissue proteins naturally secreted by the transfected stromal cells are attached to and substantially envelope a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and (b) isolating the exogenous gene product from the culture.
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40. A method for producing a biological product in a three-dimensional cell culture comprising:
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(a) culturing parenchymal cells inoculated onto a genetically engineered living stromal tissue, comprising stromal cells transfected with an exogenous gene under the control of an expression element, so that the exogenous gene product is expressed in culture, and in which the transfected stromal cells and connective tissue proteins naturally secreted by the transfected stromal 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 transfected stromal cells; and (b) isolating the exogenous gene product from the culture. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48)
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49. A method for producing a biological product in a genetically engineered dermis comprising:
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(a) culturing stromal cells transfected with an exogenous gene under the control of an expression element so that the exogenous gene product is expressed in culture, and in which the transfected stromal cells and connective tissue proteins naturally secreted by the transfected stromal cells are attached to and substantially envelope a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and (b) isolating the exogenous gene product from the culture.
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50. A method for producing a biological product in a genetically engineered three-dimensional cell culture, comprising:
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(a) culturing transfected parenchymal cells inoculated onto a living stromal tissue prepared in vitro comprising stromal cells and connective tissue proteins naturally secreted by the stromal 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 stromal cells, so that the inoculated cells proliferate, in which the transfected parenchymal cells contain an exogenous gene under the control of an expression element so that the exogenous gene product is expressed in the culture; and (b) isolating the exogenous gene product from the culture.
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Specification