Microcarriers for Stem Cell Culture

• US 20110129919A1
• Filed: 11/01/2010
• Published: 06/02/2011
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Oh, Steve, Chen, Allen, Reuveny, Shaul
• Granted Patent: US 8,691,569 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 5/0075 using microcarriers
• 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

We disclose a particle comprising a matrix coated thereon and having a positive charge, the particle being of a size to allow aggregation of primate or human stem cells attached thereto. The particle may comprise a substantially elongate, cylindrical or rod shaped particle having a longest dimension of between 50 μm and 400 μm, such as about 200 μm. It may have a cross sectional dimension of between 20 μm and 30 μm. The particle may comprise a substantially compact or spherical shaped particle having a size of between about 20 μm and about 120 μm, for example about 65 μm. We also disclose a method of propagating primate or human stem cells, the method comprising: providing first and second primate or human stem cells attached to first and second respective particles, allowing the first primate or human stem cell to contact the second primate or human stem cell to form an aggregate of cells and culturing the aggregate to propagate the primate or human stem cells for at least one passage. A method of propagating human embryonic stem cells (hESCs) in long term suspension culture using microcarriers coated in Matrigel or hyaluronic acid is also disclosed. We also disclose a method for differentiating stem cells.

Claims

1. A method of culturing and differentiating stem cells in vitro, the method comprising:

(i) attaching stem cells to a plurality of first microcarriers to form microcarrier-stem cell complexes, wherein the surface of the first microcarriers is coated in a first matrix;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

(iii) passaging the cultured cells from (ii); and

(iv) repeating steps (i)-(iii) through at least 2 passages,

wherein stem cells in the culture after step (iv) are pluripotent or multipotent, the method further comprising;

(v) attaching pluripotent or multipotent stem cells obtained after step (iv) to a plurality of second microcarriers to form microcarrier-stem cell complexes, wherein the surface of the second microcarriers is coated in a second matrix or is uncoated; and

(vi) culturing the microcarrier-stem cell complexes from (v) in suspension culture under conditions that induce the differentiation of the stem cells to the neural cell lineage.

2. The method of claim 1 wherein the stem cells are embryonic stem cells, or induced pluripotent stem cells.

3. The method of claim 1 wherein the stem cells are human or primate.

4. The method of claim 1 wherein the microcarriers are rod-shaped.

5. The method of claim 1 wherein the first and second matrix are the same.

6. The method of claim 1 wherein the first and second matrix are different.

7. The method of claim 1 wherein the stem cells are differentiated into neural precursors, neurons, or astrocytes.

8. The method of claim 1 wherein the method further comprises:

(vii) attaching differentiated stem cells obtained from step (vi) to a plurality of third microcarriers to form microcarrier-stem cell complexes, wherein the surface of the third microcarriers is coated in a third matrix or is uncoated; and

(viii) culturing the microcarrier-stem cell complexes from (vii) in suspension culture under conditions that induce the further differentiation of the differentiated stem cells.

9. A differentiated cell of the neural lineage obtained by the method of claim 1.

10. A neural precursor cell, neuron, or astrocyte obtained by the method of claim 1.

11. A method of differentiating stem cells in vitro, comprising attaching pluripotent or multipotent stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes, wherein the surface of the microcarriers is coated in a matrix or is uncoated, and culturing the microcarrier-stem cell complexes in suspension culture under conditions that induce the differentiation of the stem cells to cells of the neural cell lineage.

12. The method of claim 11 wherein the stem cells are embryonic stem cells, or induced pluripotent stem cells.

13. The method of claim 11 wherein the stem cells are human or primate.

14. The method of claim 11 wherein the microcarriers are rod-shaped.

15. The method claim 11 wherein the matrix comprises one or more of laminin, fibronectin, vitronectin, Matrigel™

(BD Biosciences), hyaluronic acid, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate.

16. The method of claim 11 wherein the matrix comprises a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.

17. The method of claim 11 wherein the microcarrier comprises or consists of one or more of cellulose, dextran, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone.

18. The method of claim 11 wherein the stem cells are differentiated into neural precursors, neurons, or astrocytes.

19. A differentiated cell of the neural lineage obtained by the method of claim 11.

20. A neural precursor cell, neuron, or astrocyte obtained by the method of claim 11.