Three-dimensional co-culture process

A method for culturing at least two distinct originating types of mammalian cells to produce in vitro multi-cellular, three-dimensional, differentiated, organized, living tissues, said method including the steps of:

• 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 upgoing 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 to 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.