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US 5,308,764 AGranted
Multi-cellular, three-dimensional living mammalian tissue
AssigneeThe United States of America as represented by the Administrator of the National Aeronautics and Space Administration
InventorsThomas J. Goodwin, David A. Wolf
Life of this patent
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Abstract
The present invention relates to a multicellular, three-dimensional, living mammalian tissue. The tissue is produced by a co-culture process wherein two distinct types of mammalian cells are co-cultured in a rotating bioreactor which is completely filled with culture media and cell attachment substrates. As the size of the tissue assemblies formed on the attachment substrates changes, the rotation of the bioreactor is adjusted accordingly.
Claims22
A multi-cellular, three-dimensional, differentiated, living mammalian tissue of at least two distinct originating cell types produced in vitro by:
- providing a bioreactor having a culture chamber rotatable about an approximately horizontal longitudinal axis, means to controllably rotate said culture chamber, means to oxygenate said culture chamber and means to remove metabolic waste products therefrom;
- filling said culture chamber completely with a fluid nutrient media, cell attachment substrates, and individually dispersed mammalian cells of at least two distinct types to establish a culturing environment;
- rotating said culture chamber about its longitudinal axis, oxygenating the culturing environment within said culture chamber and removing metabolic waste products therefrom;
- controlling the rotating of said culture chamber such that the culturing environment except during transient periods has the following simultaneous properties:
- a. collocation of cells and attachment substrates with similar or differing sedimentation properties in a similar spatial region,
- b. freedom for three-dimensional spatial orientation of tissues formed by the culturing of the cells, and
- c. low shear and essentially no relative motion of said culturing environment with respect to the walls of the culture chamber;
- and
- determining the trajectory of the tissues, in response to said determining step:
- increasing the speed of rotation of the culture chamber if the tissues fall excessively inward and downward on the downward side of the rotational cycle and excessively outward and insufficiently upward on the ongoing side of the rotational cycle to prevent wall impact;
- or decreasing the speed of rotation of the culture chamber in response to excessive accumulation of the tissues near the outer wall of the culture chamber so as not restrict three-dimensional assembly, and as the size of the tissues increase beyond the capability to fully satisfy the above three properties by further increase of the speed of rotation, selecting a rotational rate to visually obtain minimal collision frequency and intensity.
8 more elements
A multi-cellular, three-dimensional, living mammalian tissue of at least two distinct originating cell types having complex polypoid structures and specific areas of cellular differentiation produced in vitro by:
- providing a bioreactor having a culture chamber rotatable about an approximately horizontal longitudinal axis, means to controllably rotate said culture chamber, means to oxygenate said culture chamber and means to remove metabolic waste products therefrom;
- filling said culture chamber completely with a fluid nutrient media, cell attachment substrates, and individually dispersed mammalian cells of at least two distinct types to establish a culturing environment;
- rotating said culture chamber about its longitudinal axis, oxygenating the culturing environment within said culture chamber and removing metabolic waste products therefrom;
- controlling the rotation of said culture chamber such that the culturing environment except during transient periods has the following simultaneous properties:
- a. collocation of cells and attachment substrates with similar or differing sedimentation properties in a similar spatial region,
- b. freedom for three-dimensional spatial orientation of tissues formed by the culturing of the cells, and
- c. low shear and essentially no relative motion of said culturing environment with respect to the walls of the culture chamber;
- and
- determining the trajectory of the tissues, in response to said determining step:
- increasing the speed of rotation of the culture chamber if the tissues fall excessively inward and downward on the downward side of the rotational cycle and excessively outward and insufficiently upward on the ongoing side of the rotational cycle to prevent wall impact;
- or decreasing the speed of rotation of the culture chamber in response to excessive accumulation of the tissues near the outer wall of the culture chamber so as not restrict three-dimensional assembly, and as the size of the tissues increase beyond the capability to fully satisfy the above three properties by further increase of the speed of rotation, selecting a rotational rate to visually obtain minimal collision frequency and intensity.
8 more elements
A multi-cellular, three-dimensional, living mammalian tissue of at least two distinct originating cell types residing on a matrix formed of molecularly permeable microcarrier beads produced in vitro by:
- providing a bioreactor having a culture chamber rotatable about an approximately horizontal longitudinal axis, means to controllably rotate said culture chamber, means to oxygenate said culture chamber and means to remove metabolic waste products therefrom;
- filling said culture chamber completely with a fluid nutrient media, cell attachment substrates, and individually dispersed mammalian cells of at least two distinct types to establish a culturing environment;
- rotating said culture chamber about its longitudinal axis, oxygenating the culturing environment within said culture chamber and removing metabolic waste products therefrom;
- controlling the rotation of said culture chamber such that the culturing environment except during transient periods has the following simultaneous properties:
- a. collocation of cells and attachment substrates with similar or differing sedimentation properties in a similar spatial region,
- b. freedom for three-dimensional spatial orientation of tissues formed by the culturing of the cells, and
- c. low shear and essentially no relative motion of said culturing environment with respect to the walls of the culture chamber;
- and
- determining the trajectory of the tissues, in response to said determining step:
- increasing the speed of rotation of the culture chamber if the tissues fall excessively inward and downward on the downward side of the rotational cycle and excessively outward and insufficiently upward on the ongoing side of the rotational cycle to prevent wall impact;
- or decreasing the speed of rotation of the culture chamber in response to excessive accumulation of the tissues near the outer wall of the culture chamber so as not restrict three-dimensional assembly, and as the size of the tissues increase beyond the capability to fully satisfy the above three properties by further increase of the speed of rotation, selecting a rotational rate to visually obtain minimal collision frequency and intensity.
8 more elements
Dependencies are inferred from the claim text.