Microcarriers for stem cell culture

• US 8,828,720 B2
• Filed: 11/18/2010
• Issued: 09/09/2014
• 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, Reuveny, Shaul, Chen, Allen
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US12/949,172
• Publication Number:
  US 20110111498A1
• Time in Patent Office: 1,391 Days
• Field of Search: 435/363, 435/403, 435/377
• US Class Current: 435/363
• CPC Class Codes: C12M 23/20 Material Coatings immunocoatings C12M25/00
• CPC Class Codes: C12M 25/16 Particles; Beads; Granular material; Encapsulation chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• 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 expanding pluripotent stem cells in suspension culture in vitro, the method comprising:

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

(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 7 passages,

wherein the cells in the culture after step (iv) comprise pluripotent stem cells and said pluripotent stem cells are expanded, thereby producing expanded pluripotent stem cells in culture, and wherein the number of said expanded pluripotent stem cells at the end of step (ii) is at least 0.2 order of magnitude greater than the number of cells attached to the microcarriers in step (i).

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

3. The method of claim 2 wherein the pluripotent stem cells are human or primate.

4. The method of claim 1 wherein steps (i)-(iii) are repeated through at least 10 passages.

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

6. The method of claim 1 wherein in each repeat cycle the stem cells of step (i) are obtained from the passaged cells of step (iii) of the preceding repeat cycle.

7. The method of claim 1 further comprising the step of inducing differentiation of the stem cells obtained after step (iv).

8. The method of claim 1 further comprising the step of inducing differentiation of the stem cells obtained after step (iv), wherein the method comprises placing the microcarrier-stem cell complexes under conditions which induce the differentiation of the stem cells.

9. The method of claim 1 wherein after step (iv) the method comprises the step of separating stem cells from the microcarriers and culturing the separated stem cells in non-microcarrier culture under conditions which induce differentiation of the stem cells.

10. The method of claim 1 further comprising the differentiation of pluripotent stem cells, comprising:

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

11. The method of claim 1 wherein the method comprises continuous or intermittent agitation of the cell culture.

12. The method of claim 1 wherein the method does not comprise continuous or intermittent agitation of the cell culture.

16. The method of claim 1 wherein the method comprises culturing the stem cells in serum-free media.

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

(i) attaching pluripotent stem cells to 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 7 passages,

wherein the cells in the culture after step (iv) comprise pluripotent stem cells and said pluripotent stem cells are expanded, thereby producing expanded pluripotent stem cells in culture, and wherein the number of said expanded pluripotent stem cells at the end of step (ii) is at least 0.2 order of magnitude greater than the number of cells attached to the microcarriers in step (i);

the method further comprising;

(v) attaching pluripotent 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 uncoated; and

(vi) culturing the microcarrier-stem cell complexes of (v) in suspension culture under conditions that induce the differentiation of the pluripotent stem cells,

wherein at least the first matrix is a matrix comprising an extracellular matrix component, and at least one of the first and second matrix is laminin.

14. The method of claim 13 wherein the stem cells are embryonic stem cells, or induced pluripotent stem cells.

15. The method of claim 14 wherein the stem cells are human or primate.

17. The method of claim 13 wherein the method comprises culturing the stem cells in serum-free media.