Stem cell culture method

• US 9,005,607 B2
• Filed: 03/13/2013
• Issued: 04/14/2015
• Est. Priority Date: 08/17/2012
• Status: Active Grant

• Current Assignee: Keele University
• Original Assignee: Keele University
• Inventors: Kumar, Deepak, Yang, Ying, Forsyth, Nicholas
• Primary Examiner(s): Long, Scott
• Application Number:
  US13/800,325
• Publication Number:
  US 20140050704A1
• Time in Patent Office: 762 Days
• US Class Current: 424/93.7
• CPC Class Codes: A61L 2400/12 Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
• CPC Class Codes: A61L 27/18 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: A61L 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: C08L 67/04 Polyesters derived from hydroxycarboxylic acids, e.g. lactones C08L67/06 takes precedence
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Methods for culturing pluripotent stem cells on fiber scaffolds are provided which result in the expansion of the number of stem cells without loss of pluripotency. Cells obtained by such methods, implants containing such cells and medical methods using such cells are also disclosed.

Claims

1. A method of culturing human embryonic stem cells (hESC) the method comprising seeding hESC onto an uncoated fiber scaffold in which the fibers are not coated or treated with, and do not incorporate, an extracellular matrix (ECM) component, and wherein the fibers have a diameter of less than about 500 nm, and incubating the hESC loaded fiber scaffold in a gas atmosphere in which the percentage of oxygen is in the range 0.25% to 15%, wherein the cultured hESC maintain stemness.

2. The method of claim 1 wherein the fiber scaffold comprises nanofibers.

3. The method of claim 1 wherein the fiber scaffold comprises electrospun fibers.

4. The method of claim 1 wherein the percentage of oxygen is in the range 0.5% to 10%.

5. The method of claim 1 wherein the percentage of oxygen is about 2%.

6. The method of claim 1 wherein the fibers have a diameter of between 100 nm and 300 nm.

7. The method of claim 1 wherein the fiber scaffold comprises aligned fibers.

8. The method of claim 1 wherein the fibers comprise a synthetic polymer.

9. The method of claim 1 wherein the fibers comprise poly-ε

-caprolactone (PCL).

10. The method of claim 1 wherein the cultured human embryonic stem cells (hESC) are incubated for 10 days without loss of pluripotency.

11. The method of claim 1 further comprising the step of inducing the cultured human embryonic stem cells (hESC) to differentiate.

12. The method of claim 11 wherein inducing the cultured human embryonic stem cells (hESC) to differentiate comprises adding differentiation media to the fibers seeded with pluripotent stem cells.

13. The method of claim 12 wherein inducing the cultured human embryonic stem cells (hESC) to differentiate comprises removing the pluripotent stem cells from the fibers before adding differentiation medium.

14. A method of preparing a population of human embryonic stem cells (hESC) that are suitable for clinical use comprising expanding a population of hESC by the method of claim 1.

15. The method according to claim 1, wherein the fiber scaffold is, or is connected to an implant.

16. The method of claim 1, further comprising introducing the incubated hESC loaded fiber scaffold to a patient in need of treatment.

17. The method according to claim 1 wherein the percentage of oxygen is in the range 1% to 5%.