Microcarriers for stem cell culture

• US 8,691,569 B2
• Filed: 11/01/2010
• Issued: 04/08/2014
• 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:
  US12/917,210
• Publication Number:
  US 20110129919A1
• Time in Patent Office: 1,254 Days
• Field of Search: 435/363, 435/403, 435/377
• US Class Current: 435/363
• 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 for achieving stable, long-term culture, expansion, and differentiation of 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 an extracellular matrix component;

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

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

(iv) repeating steps (i)-(iii) through 7 passages,

wherein stem cells are expanding between each passage, wherein stem cells in the culture after step (iv) are pluripotent or multipotent, whereby stable, long-term culture and expansion of stem cells are achieved in suspension in vitro, wherein the achievement of stable, long-term culture is characterized by 50% or more of the stem cells in the culture after step (iv) have the ability to differentiate, if pluripotent, into endoderm, ectoderm, and mesoderm, if multipotent, into cells of a limited number of types within a particular lineage, and retain at least one stem cell characteristic selected from the group consisting of expression of a pluripotency marker, cell viability, and normal karyotype, and wherein the achievement of expansion is characterized by the number of cultured, expanded cells at the end of step (ii) being at least 0.2 order of magnitude greater than the number of cells attached to the microcarriers in step (i);

(v) transferring pluripotent or multipotent stem cells obtained from (iv) to a plurality of second microcarriers to form microcarrier-stem cell complexes, wherein the surface of the second microcarriers 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 stem cells.

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 extracellular matrix component of (i) and the matrix of (v) are the same.

6. The method of claim 1 wherein the extracellular matrix component of (i) and the matrix of (v) are different.

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

(vii) transferring differentiated stem cells obtained from step (vi) to a plurality of third microcarriers to form microcarrier-stem cell complexes, where the surface of the third microcarriers is coated in a second 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.

8. The methods of claim 1 wherein in step (iv), steps (i)-(iii) are repeated through one of:

at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, at least 20 passages, at least 21 passages, at least 22 passages, at least 23 passages, at least 24 passages, at least 25 passages, at least 30 passages, at least 40 passages, at least 50 passages, at least 60 passages, at least 70 passages, at least 80 passages, at least 90 passages, or at least 100 passages.

9. The method of claim 1 wherein the matrix comprises one or more of hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulfate, dextran, dextran sulphate, and chondroitin sulphate.

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

11. The method of claim 1 wherein the microcarrier comprises or consists of one or more of cellulose, dextran, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, and silicone.

12. The method of claim 1 wherein the stem cells are pluripotent, and the conditions in step (vi) induce the differentiation of the stem cells into endoderm, ectoderm, or mesoderm.

13. The method of claim 1 wherein the stem cells are pluripotent, and the conditions in step (vi) induce the differentiation of the stem cells into an embyroid body.

14. The method of claim 1 wherein the conditions in step (vi) induce the differentiation of the stem cells into one of cardiomyocytes, cardiac mesoderm, adipocytes, osteocytes, hepatocytes, hepatic endoderm, pancreatic islet cells, pancreatic endoderm, insulin producing cells, neural tissue, neuroectoderm, epidermal tissue, surface ectoderm, bone, cartilage, muscle, ligament, tendon, and other connective tissue.

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