Composition comprising three-dimensional astrocyte bundles of bi-polar and aligned astrocyte processes and a method of making thereof
AssigneeThe Trustees of the University of Pennsylvania
InventorD. Kacy Cullen
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Abstract
The invention includes a composition comprising an astrocyte comprising aligned and elongated astrocyte processes resulting from ex vivo machine-driven, physical stretching of the astrocyte maintained in culture, as well as a method of making it. The invention also includes a composition comprising an astrocyte comprising aligned bi-polar processes resulting from growth within hydrogel micro-columns in culture, as well as a method of making it. The invention also provides methods of treatment of nervous system injury or degeneration by implanting the compositions of the invention.
Claims25
A composition comprising three-dimensional astrocyte bundles of bi-polar and aligned astrocyte processes, wherein the astrocyte bundles are enclosed in a micro-column comprising a biocompatible matrix on the inner surface of the micro-column.
A composition comprising at least one three-dimensional bundle of bi-polar and aligned astrocyte processes made by a method comprising seeding a micro-column comprising a biocompatible matrix and an inner diameter in a range of about 0.1 mm to about 0.9 mm, or about 0.3 mm to about 0.35 mm with a plurality of astrocytes at a density in a range of 9-12×105 cells/ml;
- and culturing the astrocytes in the micro-column.
A method of producing three-dimensional astrocyte bundles of bi-polar aligned astrocyte processes, comprising (i) seeding a micro-column comprising a biocompatible matrix with a plurality of astrocytes at a density in a range of 9-12×105 cells/ml.;
- and (ii) culturing the micro-column so seeded of (i) in a culture medium, wherein the culture medium comprises Neurobasal® (A neuronal culture medium for cultivating neuronal cells from hippocampus, cortex and other regions of the brain and containing no L-glutamine, L-glutamic acid, or aspartic acid), B-27® supplement (a neuronal culture supplement comprising insulin, transferrin, progesterone, putrescine, selenium, thyroid hormone T3, fatty acids, and antioxidants including vitamin E and glutathione), G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 formulation for growth and expression of glial cells and astrocytes) and L-glutamine.
A method of producing aligned and elongated astrocyte processes from an astrocyte comprising:
- a) culturing an astrocyte in a culture medium, wherein the culture medium comprises Neurobasal® (a neuronal culture medium for cultivating neuronal cells from hippocampus, cortex and other regions of the brain and containing no L-glutamine, L-glutamic acid or aspartic acid), B-27® supplement (a neuronal culture supplement comprising insulin, transferrin, progesterone, putrescine, selenium, thyroid hormone T3, fatty acids, and antioxidants including vitamin E and glutathione), G-5 supplement (a chemically defined serum free supplement based on Bottenstein's G-5 formulation for growth and expression of glial cells and astrocytes) and L-glutamine;
- b) plating the cultured astrocyte onto an overlying membrane and an underlying membrane so that the cultured astrocyte adheres to both membranes, wherein the overlying membrane or the underlying membrane is selected from polystyrene, ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film), collagen coated ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film) or MATRIGEL® (a gelatinous protein mixture) coated ACLAR® film (a clear, poly-chloro-trifluoroethylene (PCTFE) film), and wherein the overlying membrane is a towing membrane and the underlying membrane is a stationary membrane, or wherein the underlying membrane is the towing membrane and the overlying membrane is the stationary membrane;
- and
- c) moving one of the two membranes—the overlying membrane and the underlying membrane-across the other under an ex vivo force at a stretching rate of 0.1-0.5 millimeter/day so that astrocyte processes from the astrocyte are stretched and aligned along the ex vivo force.
Dependencies are inferred from the claim text.