Ex vivo culture, proliferation and expansion of intestinal epithelium

Patent metadata

Field	Value
Publication number	US-9464275-B2
Application number	US-54575509-A
Country	US
Kind code	B2
Filing date	Aug 21, 2009
Priority date	Aug 21, 2008
Publication date	Oct 11, 2016
Grant date	Oct 11, 2016

Abstract

Methods are provided for long term culture of mammalian intestinal cells. Cultures are initiated with fragments of mammalian intestinal tissue, which are then maintained embedded in a gel substrate that provides an air-liquid interface. Intestinal epithelium in cultures of the invention can be continuously grown for extended periods of time. Mammalian intestinal cells cultured by the methods of the invention recapitulate features of intestinal growth in vivo.

First claim

What is claimed is:

1. A long term culture system of mammalian intestinal cells, wherein said culture provides for multilineage differentiation that maintains (a) the three-dimensional structure of intestinal epithelium and (b) differentiation markers characteristic of intestinal absorptive enterocytes, goblet cells and/or enteroendocrine cells, in which intestinal cells grow, differentiate and are viable for more than 3 months and up to 350 days in culture, comprising intestinal tissue cultured in a gel with an air-liquid interface,
wherein the culture system is initiated by:
mixing fragments of intestinal tissue with a gel solution,
pouring the gel solution containing the fragments of intestinal tissue over a layer of gel substrate formed in a transwell container with a lower semi-permeable support;
placing the transwell in an outer dish comprising culture medium, wherein the level of the culture medium is maintained such that the gel containing the tissue fragments is not submerged in the culture medium;
wherein the long term culture system recapitulates features of intestinal growth in culture comprising cellular ultrastructure, presence of enterocytes, goblet and enteroendocrine cells, and Wnt-dependent proliferation.

2. The method of claim 1 , wherein the fragments of intestinal tissue are neonatal, juvenile or adult.

3. The long term culture of claim 1 , wherein said culture comprises exogenous R-spondin, and wherein the growth of intestinal cells is increased relative to cells in a culture in which R-spondin is absent.

4. The long term culture of claim 1 , wherein the mammalian intestinal cells are experimentally modified prior to or during culture.

5. The in vitro culture of claim 1 , wherein the cells are modified by introduction of a pathogen.

6. The in vitro culture of claim 1 , wherein the cells are modified by introduction of a genetic agent.

Assignees

• Kuo Calvin Jay
• Ootani Akifumi
• Univ Leland Stanford Junior

Inventors

• Kuo Calvin Jay
• Ootani Akifumi

Classifications

• G01N33/5005

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

• G01N33/5073

  Stem cells · CPC title

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N5/0012

  Cell encapsulation · CPC title

• C12N5/0679 Primary

  Cells of the gastro-intestinal tract · CPC title