Microcarriers for stem cell culture

• US 9,340,770 B2
• Filed: 12/19/2013
• Issued: 05/17/2016
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Zweigerdt, Robert, Oh, Steve, Lecina, Marti, Choo, Andre, Reuveny, Shaul, Chen, Allen
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US14/134,275
• Publication Number:
  US 20140193903A1
• Time in Patent Office: 880 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• 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
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

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 and expansion of human pluripotent stem cells or human multipotent stem cells in suspension culture in vitro, the method comprising:

(i) attaching human pluripotent stem cells or human 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 comprising 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 at least 7 passages, wherein stem cells in the culture after step (iv) are respectively pluripotent or multipotent, thus achieving stable, long-term culture and expansion of human pluripotent stem cells or human multipotent stem cells in suspension culture in vitro.

2. The method of claim 1 wherein the matrix comprises one or more of hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate, or a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.

3. 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, silicone.

4. The method of claim 1 wherein the microcarrier is coupled with protamine or polylysine.

5. The method of claim 1 wherein the microcarrier is positively charged.

6. The method of claim 1 wherein the microcarrier is hydrophilic.

7. 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.

8. The method 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 10passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, at least 20passages, 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 50passages, at least 60passages, at least 70 passages, at least 80 passages, at least 90 passages, at least 100 passages.

9. The method of claim 1 wherein after step (iv) at least 60% of the stem cells in the culture are respectively pluripotent or multipotent.

10. The method of claim 1 wherein after step (iv) at least 60% of the pluripotent stem cells in the culture express one, two, three or all of Oct4, SSEA4, TRA-1-60 and Mab84.

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

12. 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.

13. 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.

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

15. The method of claim 14 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 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.

18. The method of claim 1, wherein the method further comprises differentiating the human pluripotent stem cells or human multipotent stem cells in culture after step (iv), 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 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.

19. The method of claim 18, 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 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.

16. A method of differentiating stem cells in vitro comprising attaching human pluripotent stem cells or human 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 comprising an extracellular matrix component, and culturing the microcarrier-stem cell complexes in suspension culture under conditions that induce the differentiation of the stem cells to one of ectoderm cells, endoderm cells, mesoderm cells, cardiomyocytes, cardiac mesoderm cells, hepatocytes, hepatic endoderm cells, pancreatic islet cells, pancreatic endoderm cells, insulin-producing cells, neural cells, neuroectoderm cells, epidermal cells, surface ectoderm cells, bone cells, cartilage cells, muscle cells, ligament cells, tendon cells, or other connective tissue cells, wherein at least about 15% of the cells in the culture differentiate.

17. The method of claim 16 wherein the matrix comprises one or more of laminin, fibronectin, vitronectin, hyaluronic acid, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate, or a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.