Inverse patterning process for three-dimensional multi-compartmental micro-organization of multiple cell types

Patent metadata

Field	Value
Publication number	US-10072257-B2
Application number	US-201314381866-A
Country	US
Kind code	B2
Filing date	Feb 28, 2013
Priority date	Feb 29, 2012
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

The invention features an “inverse patterning” or “Intaglio-Void/Embed-Relief Topographic (In VERT) molding” manufacturing process for generating high-resolution three-dimensional (3D) multi-cellular microstructures in distinct cellular compartments of a single hydrogel. The platform has general utility in the development of engineered tissues for human therapies, drug testing, and disease models. Additionally, the platform can serve as a model system for studying 3D cell-cell interactions in fields as diverse as stem cell biology to the development of cancer therapeutics.

First claim

We claim:

1. A method of making a three-dimensional, multiple cell type tissue construct, comprising
introducing a first population of cells into recessed features of a patterned cell capture substrate;
encapsulating said first cell population in a first polymerizable biomaterial;
polymerizing said first polymerizable biomaterial;
removing and inverting said encapsulated first cell population thereby exposing an inverse pattern of the recessed features containing the first cell population in the first polymerizable biomaterial;
contacting the inverse pattern of the recessed features comprising the, first cell population with a second population of cells in a second polymerizable biomaterial;
encapsulating said second population in said second polymerizable biomaterial; and
polymerizing said second polymerizable biomaterial,
such that the three-dimensional, multiple cell type tissue construct is made.

2. The method of claim 1 , wherein said first population of cells is incubated under conditions sufficient for formation of cell-cell junctions between cells in said features of said patterned cell capture substrate prior to encapsulating said first cell population in said first polymerizable biomaterial.

3. The method of claim 1 , wherein the patterned cell capture substrate consists of polydimethyl siloxane (PDMS) comprising micro-scale features.

4. The method of claim 1 , wherein the first population of cells is introduced into the features of the patterned cell capture substrate in a media or pre-polymer solution.

5. The method of claim 1 , wherein said first population of cells is incubated for a period of about 6 to about 24 hours, to permit formation of cell-cell junctions between said cells.

6. The method of claim 1 , wherein said first and/or second polymerizable biomaterial is a hydrogel material.

7. The method of claim 6 , wherein the hydrogel material is agarose, fibrin, or polyethylene hydrogel.

8. The method of claim 7 , wherein the hydrogel material is photopolymerized polyethylene glycol (PEG) hydrogel.

9. The method of claim 1 , wherein the first or second cell population, or both the first and second cell populations comprise parenchymal cells.

10. The method of claim 1 , wherein the first or second cell population, or both the first and second cell populations comprise non-parenchymal cells.

11. The method of claim 1 , wherein the first or second cell population, or both the first and second cell populations comprise a combination of parenchymal and non-parenchymal cells.

12. The method of claim 9 , wherein the parenchymal cells are human parenchymal cells.

13. The method of claim 5 , wherein the hydrogel is derivatized with one or more cell-adhesive peptides, or comprises one or more soluble factors supporting cell growth and/or differentiation.

14. The method of claim 1 , wherein the first or second cell populations, or both the first and second cell populations are encapsulated at a concentration of from about 8×10 6 cells/ml to about 24×10 6 cells/ml.

15. The method of claim 1 , wherein the polymerizable biomaterial is biodegradable.

16. The method of claim 1 , wherein one or more of the populations of cells is engineered to express a reporter protein.

17. The method of claim 1 , wherein said first population of cells is incubated for a period of about 8 to about 16 hours to permit formation of cell-cell junctions between said cells.

18. The method of claim 1 , wherein said first population of cells is incubated for a period of about 12 hours to permit formation of cell-cell junctions between said cells.

Assignees

• Massachusetts Inst Technology

Inventors

• Bhatia Sangeeta N
• Stevens Kelly R

Classifications

• C12N5/0012

  Cell encapsulation · CPC title

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• C12N11/08

  the carrier being a synthetic polymer · CPC title

• C12N11/04 Primary

  entrapped within the carrier, e.g. gel or hollow fibres · CPC title

• C12N5/0671

  Three-dimensional culture, tissue culture or organ culture; Encapsulated cells · CPC title