Templated islet cells and small islet cell clusters for diabetes treatment

• US 8,735,154 B2
• Filed: 04/06/2010
• Issued: 05/27/2014
• Est. Priority Date: 10/30/2006
• Status: Active Grant

• Current Assignee: University of Kansas
• Original Assignee: University of Kansas
• Inventors: Berkland, Cory, Stehno-Bittel, Lisa A., Siahaan, Teruna, Ramachandran, Karthik
• Primary Examiner(s): Stanfield, Cherie M
• Assistant Examiner(s): Pyla, Paul D
• Application Number:
  US12/798,529
• Publication Number:
  US 20100233239A1
• Time in Patent Office: 1,512 Days
• Field of Search: None
• US Class Current: 435/395
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61L 27/18 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: A61L 27/3882 Hollow organs, e.g. bladder, esophagus, urether, uterus
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C08L 67/04 Polyesters derived from hydroxycarboxylic acids, e.g. lactones C08L67/06 takes precedence
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

A scaffold having islet cells or small islet cell clusters attached thereto in a multilayer, and a micro-mold having divots for culturing islets, wherein islet formation is influenced by the shape and dimensions of the divots are disclosed.

Claims

1. A non-implantable substrate for culturing cells comprising:

a non-adherent, divoted surface comprising a substantially planar top surface, and a plurality of divots etched into the substantially planar top surface of the substrate using a metal mask, and being free of coatings that increase cell adhesion;

wherein each divot comprises an interior bottom surface and an interior sidewall surface, wherein the bottom surface is rounded and wherein each divot is between 100-200 μ

m in diameter and 50-150 μ

m in depth;

wherein cells can be distributed into individual divots in an approximately 20-150 cells per 1 divot ratio, wherein the cells do not adhere to the divoted surface, such that the cells are removable from said divots.

2. A device for culturing cells comprising the substrate of claim 1 additionally comprising a system for holding said substrate, wherein the system forms a bottom surface and an interior side wall surface and an interior volume, the bottom and side walls being substantially tight to liquids, wherein cells and culture medium are distributed into the interior volume.

3. The substrate of claim 1, wherein the cells are islet cells.

4. The substrate of claim 1, wherein the cells are non-islet cells.

5. The substrate of claim 1, wherein the divots are 100 μ

m in diameter and 60 μ

m in depth.

6. The substrate of claim 1, wherein the substrate is made of glass.

7. The substrate of claim 1, wherein the substrate is sterilizable.

13. A method for testing a plurality of cell samples for response to an introduced chemical comprising:

(a) culturing cells in a device according to claim 2,

(b) introducing a test chemical to each divot, and

(c) analyzing the response of the cells to the test chemical.

15. The substrate of claim 1, wherein the average distance between divots is less than 30 μ

m.

16. The substrate of claim 1, wherein said substrate surface is free of cell adhesion molecules to facilitate individual cell attachment to the surface.

8. A method for culturing cells comprising introducing cells to a non-implantable substrate comprising a divoted surface comprising a substantially planar top surface and a plurality of divots etched into the substantially planar top surface of the substrate using a metal mask, said substrate being free of coatings that increase cell adhesion, said cells settling into the divots in an approximately 20-150 cells per 1 divot ratio, wherein each divot is between 100 and 200 μ

m in diameter and 50 and 150 μ

m in depth, and exposing the surface to cell culture conditions, wherein the cells do not adhere to the divoted surface, and are removable therefrom.

9. The method of claim 8, wherein the cells are islet cells and wherein the cells reaggregate into small islets after exposure to the substrate and supportive culture conditions.

10. The method of claim 8, wherein the cells are dispersed from an islet culture and chemicals or biological molecules are incorporated into the resulting small islets.

11. The method of claim 8, wherein the cells are non-islet cells.

12. The method of claim 8, wherein the divots are 100 μ

m in diameter and 60 μ

m in depth.

14. The method of claim 8, wherein the cells aggregate into 3-dimensional cell aggregates after exposure to the divoted surface and supportive culture conditions.