Chemically labile peptide-presenting surfaces for cellular self-assembly

Patent metadata

Field	Value
Publication number	US-10066204-B2
Application number	US-201414486600-A
Country	US
Kind code	B2
Filing date	Sep 15, 2014
Priority date	Mar 15, 2013
Publication date	Sep 4, 2018
Grant date	Sep 4, 2018

Abstract

Methods of cell culture using patterned SAM arrays are disclosed. Advantageously, the disclosed methods use SAM arrays presenting adhesion peptides to grow confluent monolayers that can invaginate to form an embryoid body.

First claim

What is claimed is:

1. A method of controlling the formation of a cell culture aggregate, the method comprising:
forming on a substrate, at least one alkanethiolate self-assembled monolayer spot wherein the spot is conjugated to a cellular adhesive peptide consisting of SEQ ID NO: 4 using a labile covalent bond, wherein the spot is part of a self-assembled monolayer array, and wherein the self-assembled monolayer array is prepared using a method selected from the group consisting of microcontact printing, microfluidics, stamping, photochemistry, locally removing a region in a fully formed self-assembled monolayer and reforming a new self-assembled monolayer in the region;
culturing at least one cell on the alkanethiolate self-assembled monolayer spot for a sufficient time to form a confluent monolayer of cells; and
detaching the confluent monolayer of cells from the array spot by latent nucleophilic cleavage of the labile covalent bond between the cellular adhesive peptide and the alkanethiolate, wherein the detached confluent monolayer of cells forms the cell culture aggregate; and
collecting the cell culture aggregate.

2. The method of claim 1 , wherein the confluent monolayer is cultured for a period of from about 6 hours to about 144 hours.

3. The method of claim 1 , further comprising culturing the confluent monolayer for a sufficient time to allow the confluent monolayer to invaginate.

4. The method of claim 3 , wherein the confluent monolayer is cultured for a period of from about 6 hours to about 144 hours.

5. The method of claim 1 , wherein the cell is selected from the group consisting of an induced pluripotent stem cell, a mesenchymal stem cell, an umbilical vein endothelial cell, a dermal fibroblast, a fibrosarcoma cell, an embryonic stem cell, an iPS IMR90-4 cell, an iPS-derived endothelial cell, and combinations thereof.

6. The method of claim 1 , wherein the specified diameter of the array spot is from about 600 μm to about 6 mm.

7. The method of claim 1 , wherein said aggregates comprises either a uniform size or a specified shape.

8. The method of claim 7 , wherein the specified shape is selected from the group consisting of a circle, an oval, and oval cross, a star, and a hand.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Xie Angela W
• Murphy William L

Classifications

• C12N5/0068

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C07K7/00

  Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof · CPC title

• C12N2531/00

  Microcarriers · CPC title

• C07K5/12

  Cyclic peptides {with only normal peptide bonds in the ring} · CPC title