Three-Dimensional Microporous Scaffold Device for Cell Culture

The method of any one of the preceding claims, wherein step (a) comprises seeding a volume of a solution comprising the pancreatic progenitor cells onto a three-dimensional, porous scaffold at, wherein the volume is not more than about 50 μL, optionally, not more than about 30 to about 35 μL.

The method of any one of the preceding claims, wherein the pancreatic progenitor cells are Stage 4 pancreatic progenitor cells, optionally, wherein the Stage 4 pancreatic progenitor cells are derived from pluripotent stem cells.

The method of any one of the preceding claims, further comprising (i) culturing Stage 0 hPSCs to obtain Stage 1 cells, (ii) culturing Stage 1 cells to obtain Stage 2 cells, (iii) culturing Stage 2 cells to obtain Stage 3 cells, (iv) culturing Stage 3 cells to obtain Stage 4 pancreatic progenitor cells, or (v) a combination thereof, wherein, when the method comprises all of (i) to (iv), the method comprises culturing for about 8 to about 12 days, optionally, about 10 days.

The method of any one of the preceding claims, wherein the pancreatic progenitor cells are seeded on both faces of the three-dimensional, porous scaffold.

The method of any one of the preceding claims, wherein, prior to step (a), the method comprises (i) treating the pancreatic progenitor cells with a cell dissociation agent, (ii) drying the scaffold, or (iii) a combination thereof.

The method of any one of the preceding claims, wherein step (b) comprises culturing the cells on the scaffold in vitro for at least or about 4 to about 10 days to obtain insulin-producing cell clusters, and optionally culturing the cells on the scaffold in vitro for more than or about 14 days.

The method of any one of the preceding claims, wherein step (b) comprises culturing the cells on the scaffold in vitro in an air-liquid interface cell culture system comprising a culture medium and a transwell membrane aligned with the air-liquid interface of the system, wherein the scaffold is positioned on top of the transwell membrane.

The method of any one of the preceding claims, wherein the insulin-producing cell clusters have an average diameter which is about the same as the average diameter of the pores of the scaffold after step (b).

The method of any one of the preceding claims, wherein the insulin-producing cell clusters comprise mature islet β-cells.

The method of any one of the preceding claims, further comprising preparing the cell clusters and the scaffold for transplantation into a subject.

The method of any one of the preceding claims, further comprising assaying the cell clusters for expression of β cell maturation markers.

The method of any one of the preceding claims, further comprising assaying the cell cluster for insulin production rate in response to exposure to glucose.

The method of any one of the preceding claims, wherein the cells seeded onto the scaffold express and secrete extracellular matrix (ECM) proteins within the scaffold.

The method of any one of the preceding claims, wherein step (a) comprises seeding the pancreatic progenitor cells onto an ECM protein-free, three-dimensional, porous scaffold, optionally, wherein the scaffold is not coated with one or more ECM proteins prior to the seeding step or optionally wherein the scaffold is protein-free.

The method of any one of the preceding claims, wherein the only source of ECM proteins present in the culture comprising the cells seeded onto the scaffold are ECM proteins expressed and secreted by the seeded cells.

The method of any one of the preceding claims, wherein the cells seeded onto the scaffold express at least one β-cell maturation marker during and/or after step (b).

The method of any one of the preceding claims, wherein cells of the cell cluster produce and secrete insulin at a rate greater than about 0.5 μlU per 103 cells in response to a one-hour exposure to 28 mM glucose.

The method of any one of the preceding claims, wherein greater than about 85% of the seeded cells are viable for at least one week after step (a), optionally, for at least two weeks after step (a).

The method of any one of the preceding claims, wherein greater than about 90% of the seeded cells are viable for at least two weeks after the seeding step.

The method of any one of the preceding claims, wherein the average pore diameter is about 300 μm to about 535 μm.

The method of any one of the preceding claims, wherein the scaffold comprises poly(ethylene glycol) (PEG) or poly(lactide-co-glycolide) (PLG), or a combination thereof.

A composition comprising a scaffold comprising insulin-producing cell clusters, wherein the insulin-producing cell clusters are prepared in accordance with the method of any one of the preceding claims.