Microcarriers for stem cell culture

• US 8,716,018 B2
• Filed: 03/17/2009
• Issued: 05/06/2014
• 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, Lecina, Marti, Choo, Andre, Reuveny, Shaul, Zweigert, Robert, Chen, Allen
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US12/921,599
• Publication Number:
  US 20110014693A1
• Time in Patent Office: 1,876 Days
• Field of Search: 435/363, 435/403, 435/377
• US Class Current: 435/363
• 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 embryonic stem cells, or human induced pluripotent stem cells, in suspension in vitro, the method comprising:

(i) attaching human embryonic stem cells or human induced pluripotent 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 pluripotent, thus achieving stable, long-term culture and expansion of human embryonic stem cells or human induced pluripotent stem cells 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) having the ability to differentiate into endoderm, ectoderm, and mesoderm and retaining 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).

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

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

4. 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.

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

6. 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.

7. The method of claim 1 wherein the microcarriers have a substantially spherical shape.

8. 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.

9. 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.

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

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

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

13. 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.

14. 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.

15. 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 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.

16. The method of claim 15 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.

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

and wherein the culture is free of feeder cells.

20. The method of claim 1 wherein the microcarrier is a macroporous or microporous carboseed microcarrier.

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

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

23. The method of claim 1 wherein the microcarrier has a positive surface charge.

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

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

18. A method of culturing and differentiating human embryonic stem cells, or human induced pluripotent stem cells, in vitro, the method comprising:

(i) attaching human embryonic stem cells or human induced pluripotent 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 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 pluripotent, 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 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.

26. The method of claim 18 wherein the method comprises culturing the stem cells in serum-free media.