SCALABLE FABRICATION OF A SELF-ASSEMBLED CELL SHEET AND USES THEREOF
This disclosure relates to methods of fabricating self-assembled 3D cell sheets. In one embodiment, the cell sheets are formed by seeding cells to a monolayer density of at least 65% with a culture medium on to a substantially flat substrate having low surface energy and incubating cells on the substrate under time and temperature conditions to form a cell sheet.
1 . A method of forming a cell sheet, comprising:
seeding cells with a culture medium on to a substantially flat substrate, the substrate having low surface energy and/or ultralow adhesion; and
incubating cells on the substrate under time and temperature conditions to form a cell sheet;
wherein the cells are seeded on the substrate to a monolayer density of at least 65%.
2 . The method of claim 1 , wherein the cells are seeded on the substrate to a monolayer density of 90 to 100%.
3 . The method of claim 1 , wherein the incubation time ranges from 20 minutes to 10 hours.
4 . The method of claim 1 , wherein the incubation temperature ranges from 25° C. to 39° C.
5 . The method of claim 1 , wherein the cell sheet is a 3-dimensional (3D) cell sheet.
6 . The method of claim 1 , wherein seeding the cells comprises providing the cells to a mold, the bottom of the mold comprising the substrate, optionally wherein the mold defines the shape of the cell sheet.
7 . The method of claim 1 , wherein the substantially flat substrate comprises at least one topographical feature.
8 . The method of claim 1 , wherein the substrate is selected from the group consisting of a silicone polymer, a fluoropolymer, a passivated or native polyolefin, a denser than water liquid, or a combination thereof.
9 . The method of claim 8 , wherein the silicone polymer comprises polydimethylsiloxane (PDMS), optionally pristine PDMS.
10 . The method of claim 1 , wherein the cells are selected from human cells or animal cells.
11 . The method of claim 1 , wherein two or more types of cells are seeded and incubated at different areas of the substrate.
12 . The method of claim 11 , wherein the two or more types of cells are seeded and incubated at different areas divided by an insert on the substrate.
13 . The method of claim 12 , wherein the insert has a thickness that ranges from 0.1 mm to 10 mm, optionally less than 4 mm.
14 . The method of claim 12 , wherein the insert is used to segment the substrate area for selective seeding of cells and is removed within 10 minutes to 1 hour of cell seeding.
15 . The method of claim 1 , wherein the culture medium is selected from the group consisting of a biological media, optionally plasma, serum, or diluted tissue extract, a synthetic media, optionally DMEM, EBM, EBM-2, F10, or F12, or a combination thereof.
16 . A method of constructing a multi-layer cell sheet, comprising: stacking two or more of the cell sheets produced by the method of claim 1 in a culture medium, wherein the cell sheets mutually fuse under time and temperature conditions.
17 . The method of claim 16 , wherein the time ranges from 10 minutes to 10 hours and/or wherein the temperature ranges from 25° C. to 39° C.
18 . A method of constructing a two-dimensional (2D) or a three-dimensional (3D) cell construct comprising: overlapping the edges of two or more of the cell sheets produced by the method of claim 1 in a culture medium, wherein the edges mutually fuse under time and temperature conditions.
19 . The method of claim 18 , wherein the time ranges from 10 minutes to 10 hours and/or wherein the temperature ranges from 25° C. to 39° C.
20 . A cell sheet made according to the method of claim 1 , wherein the cell sheet is an artificial meat product or is for wound healing, regenerative medicine or drug discovery applications.