Granted Patent B2

US 12,331,312 · App. 17/225,708 · Granted Jun 17, 2025

Inventors: Cagri A. Savran (West Lafayette, IN); Rohil Jain (West Lafayette, IN); Chun-Li Chang (West Lafayette, IN)

Assignee: Purdue Research Foundation

C12N5/0062C12M21/08C12M23/16C12M23/34C12M41/48C12N5/0693C12N5/0697G01N33/5011G01N33/5088C12N2513/00

Abstract

Claims (26)

1. A method for deterministically culturing one or more single cells into respective multi-cell tissues, the method comprising:

(a) depositing one or more separate droplets of about 0.1 μL to about 100 μL per droplet of a liquid matrix material onto a surface of a culture vessel to form one or more three-dimensional culturing islands attached to the surface of the culture vessel, wherein each culturing island is separated and not in contact with any other culturing island, and wherein each separate culturing island can comprise one or more droplets of a liquid matrix;

(b) allowing the matrix material in each culturing island to solidify, or solidifying each culturing island, wherein the one or more culturing islands become solid or semi-solid;

(c) adding a culture medium to the culture vessel in an amount sufficient to contact, but not completely submerge, each of the culturing islands, wherein the top of each culturing island is not submerged in the culture medium;

(d) selecting one or more parameters of each of the matrix and the culture medium based on one or more micro-environment selection criteria comprising one or more of chemical composition, stiffness, pH, porosity, or presence of one or more specific molecules that facilitate cell attachment, probe cell proliferation, or cause cells to differentiate into organ-specific cells;

(e) selecting one or more single cells from a population of tumor cells, stem cells, or fetal cells using one or more cell selection criteria comprising one or more of an expression level of one or more cell-surface molecules, a morphology of the cell, a shape of the cell, a circularity and aspect ratio of the cell, a stemness of the cell, a proliferation potential of the cell, a type of the cell, a morphology of the nucleus, a presence of nuclear foci, a genetic composition, epigenetic modifications, and/or a secretion of one or more cytokines or chemokines by the cells;

(f) seeding one or more selected single cells onto or into each of the culturing islands, with one single cell seeded per island, and providing a time sufficient to enable the one or more single cells to adhere to a surface of a respective culturing island or adhere to the matrix within the respective culturing island, wherein each of the one or more culturing islands then includes a single cell;

(g) adding additional culture medium in an amount sufficient to submerge the one or more seeded single cells and the one or more culturing islands in the culture medium; and

(h) culturing the one or more single cells on or within the one or more culturing islands for a time and under conditions sufficient to grow one multi-cell tissue from each seeded single cell.

2. The method of claim 1 , wherein the matrix comprises one or more components of an extracellular matrix, selected from the group consisting of collagen, a gelatinous protein mixture that resembles extracellular matrix, elastin, and laminin.

3. The method of claim 2 , wherein the matrix is solidified by cross-linking.

4. The method of claim 1 , wherein the diameter of each of the culturing islands is about 1 mm to about 10 mm.

5. The method of claim 1 , comprising a plurality of culturing islands.

6. The method of claim 5 , wherein a distance between the culturing islands is at least about 1 mm.

7. The method of claim 5 , wherein the culturing islands are separated in individual compartments.

8. The method of claim 1 , wherein the volume of the culturing medium is about 1.0 μL to about 10 μL.

9. The method of claim 1 , wherein the microenvironment mimics a cancer microenvironment.

10. The method of claim 1 , wherein each of the single cells is selected and seeded using a microfluidic device.

11. The method of claim 1 , wherein the culture medium comprises a biological fluid or a liquid extracted from a tissue or gland.

12. The method of claim 11 , wherein the biological fluid is blood, plasma, or lymph fluid.

13. The method of claim 1 , wherein the single cell on each respective culturing island is cultured for about 8 to about 11 days.

14. The method of claim 1 , wherein each single cell is a single tumor cell.

15. The method of claim 14 , wherein the tumor is a colorectal tumor or a breast tumor.

16. The method of claim 14 , wherein the single tumor cell is isolated from a patient.

17. The method of claim 1 , further comprising separating individual cells from the multi-cell tissue(s) and culturing the separated individual cells.

18. The method of claim 1 , further comprising administering a therapeutic agent to the multi-cell tissue(s), thereby testing an effect of the therapeutic agent on the multi-cell tissue(s).

Assignments (2)

Continuity (2)

References Cited (67)