Microcarriers for stem cell culture

• US 8,637,309 B2
• Filed: 08/16/2011
• Issued: 01/28/2014
• 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: Oh, Steve, Lecina, Marti, Choo, Andre, Reuveny, Shaul, Zweigert, Robert, Chen, Allen
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/210,475
• Publication Number:
  US 20110294210A1
• Time in Patent Office: 896 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 multipotent stem cells in suspension culture in vitro, the method comprising:

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

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

3. 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.

4. 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.

5. The method of claim 1 further comprising the differentiation of multipotent stem cells, comprising:

(v) transferring 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.

6. The method of claim 1 wherein the stem cells are adult stem cells, or multipotent stem cells derived from pluripotent stem cells.

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

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

9. The method of claim 1 wherein the matrix comprises a mixture of laminin, collagen I, heparin sulphate proteoglycans, and entactin 1.

10. 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.

11. The method of claim 1 wherein 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.

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