Microcarriers for stem cell culture

• US 9,458,431 B2
• Filed: 10/29/2013
• Issued: 10/04/2016
• 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, Li, Jian, Birch, William Richard Nicholas
• Primary Examiner(s): Lankford, Blaine
• Assistant Examiner(s): Visone, Thomas J
• Application Number:
  US13/968,214
• Publication Number:
  US 20140315300A1
• Time in Patent Office: 1,071 Days
• Field of Search: None
• US Class Current: 1/1
• 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 2533/10 Mineral substrates
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• 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 2533/70 Polysaccharides
• CPC Class Codes: C12N 2533/78 Cellulose
• CPC Class Codes: C12N 2533/80 Hyaluronan
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

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 propagating stem cells in suspension culture in vitro, the method comprising:

(i) attaching stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes, wherein the surface of the microcarriers is coupled to poly-L-lysine and is coated in laminin or vitronectin;

(ii) prior to agitation, culturing the microcarrier-stem cell complexes under static culture conditions, such that stem cell-microcarrier aggregates are formed; and

iii) culturing the microcarrier-stem cell complexes in suspension culture, wherein the suspension culture is subject to at least 24 hours continuous agitation, wherein the number of stem cells in the culture is thereby expanded,

wherein stem cells in the culture after step (iii) are pluripotent or multipotent.

2. The method of claim 1, wherein prior to agitation of the suspension culture the method comprises forming stem cell-microcarrier aggregates having a concentration of at least 30 aggregates per ml and/or a mean average size of greater than 200 μ

m.

3. The method of claim 2, wherein the concentration of aggregates is selected from the group consisting of:

at least 35 aggregates per ml, at least 40 aggregates per ml, at least 45 aggregates per ml, at least 50 aggregates per ml, at least 55 aggregates per ml, at least 60 aggregates per ml, at least 65 aggregates per ml, at least 70 aggregates per ml, at least 75 aggregates per ml, at least 80 aggregates per ml, at least 85 aggregates per ml, at least 90 aggregates per ml, at least 95 aggregates per ml, or at least 100 aggregates per ml.

4. The method of claim 2, wherein the mean average size of aggregate is selected from the group consisting of:

greater than 210 μ

m, greater than 220 μ

m, greater than 230 μ

m, greater than 240 μ

m, greater than 250 μ

m, greater than 260 μ

m, greater than 270 μ

m, greater than 280 μ

m, greater than 290 μ

m, greater than 300 μ

m, greater than 310 μ

m, greater than 320 μ

m, greater than 330 μ

m, greater than 340 μ

m, greater than 350 μ

m, greater than 360 μ

m, greater than 370 μ

m, greater than 380 μ

m, greater than 390 μ

m, or greater than 400 μ

m.

5. The method of claim 1 wherein prior to agitation of the suspension culture the method comprises culturing the microcarrier-stem cell complexes under static culture conditions such that stem cell-microcarrier aggregates are formed, wherein the time period of static culture is selected from the group consisting of:

at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, or at least 168 hours.

6. The method of claim 1, wherein the microcarrier is a polystyrene microcarrier.

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

8. The method of claim 1, wherein the stem cells are human.