CONTROL OF CELL GROWTH AND AGGREGATE SIZE IN BIOREACTORS
Methods of repeated aggregate dissociation and reformation of pluripotent stem cells (PSCs) within the same bioreactor until a desired final cell number is achieved. A preferred step-wise process for controlled growth of PSCs and aggregate size using periodic dissociation with a dissociation medium which contains either proteolytic enzymes or chemical reagents, mechanical agitation, or a combination of these methods.
1 . A method of controlling the growth of cells and aggregates thereof, comprising:
a. seeding a bioreactor with anchorage-dependent cells;
b. operating the bioreactor for an initial period of time such that the cells form cell aggregates that will continue to grow in size and thus also increase the total number of cells in the bioreactor;
c. dissociating the cell aggregates within the same bioreactor; and
d. repeating the steps of operating and dissociating within the same bioreactor until a desired number of cells has been reached or the capacity of the bioreactor is fully utilized.
2 . The method of claim 1 , wherein the cells are selected from the group consisting of pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), human primary cells, or any other anchorage-dependent cells that require aggregate formation for growth.
3 . The method of claim 1 where dissociating the cell aggregates can be accomplished using either a dissociation medium containing proteolytic enzymes or chemical reagents, mechanical agitation, or a combination of these methods.
4 . The method of claim 1 where the timing of dissociation is dictated by cell aggregates reaching a predetermined threshold size, or range of sizes.
5 . A method of controlling the growth of cells and aggregates thereof, comprising:
a. seeding a bioreactor with anchorage-dependent cells as suspended single cells, along with microcarriers;
b. operating the bioreactor for an initial period of time such that the cells first attach to the surface of microcarriers before cell-to-cell attachment leads to formation of aggregates comprised of both cells and microcarriers;
c. dissociating the aggregates comprised of cells and microcarriers within the same bioreactor; and
d. repeating the steps of operating and dissociating within the same bioreactor until a desired number of cells has been reached or the capacity of the bioreactor is fully utilized.
6 . The method of claim 5 , wherein the cells are selected from the group consisting of pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), human primary cells, or any other anchorage-dependent cells.
7 . The method of claim 1 where dissociating the cell-and-microcarrier aggregates can be accomplished using either a dissociation medium containing proteolytic enzymes or chemical reagents, mechanical agitation, or a combination of these methods;
8 . The method of claim 1 c, where the timing of dissociation is dictated by the cell-and-microcarrier aggregates reaching a predetermined threshold size, or range of sizes.