Method for culturing stem cells

• US 8,956,867 B2
• Filed: 11/09/2009
• Issued: 02/17/2015
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Kamp, Timothy J., Zhang, Jianhua, Mohr, Jeffrey C., Depablo, Juan J., Palecek, Sean P.
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/615,044
• Publication Number:
  US 20100197013A1
• Time in Patent Office: 1,926 Days
• Field of Search: 435/377, 435/395, 435/366, 435/372
• US Class Current: 435/377
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

A three-dimensional microwell system that supports long term pluripotent cell culture and formation of homogeneous embryoid bodies (EBs) is described. Microwell-cultured pluripotent cells remain viable and undifferentiated for several weeks in culture and maintain undifferentiated replication when passaged to Matrigel®-coated, tissue culture-treated polystyrene dishes. Microwell-cultured pluripotent cells maintain pluripotency, differentiating to each of the three embryonic germ layers. Pluripotent cell aggregates released from microwells can be passaged for undifferentiated replication or differentiated to monodisperse EBs. The ability to constrain pluripotent cell growth in three dimensions advantageously provides for more efficient, reproducible culture of undifferentiated cells, high-throughput screening, and the ability to direct pluripotent cell differentiation by generating monodisperse EBs of a desired size and shape. Cardiomyocyte-rich EBs are obtained from pluripotent cells cultured in microwells of defined size and shape.

Claims

1. A method for culturing human pluripotent stem cells to obtain a contracting embryoid body in which at least 50% of cells of the contracting embryoid body express MF20 or MLC2a, the method comprising the steps of:

harvesting aggregates or colonies of substantially undifferentiated human pluripotent stem cells from a microwell that constrains the growth of the human pluripotent stem cells in three dimensions and having a lateral dimension of about 100 microns;

differentiating the harvested aggregates or colonies of pluripotent stem cells under unconstrained culture conditions for a period sufficient to form a population of embryoid bodies comprising a plurality of contracting embryoid bodies, wherein at least 50% of the cells of each contracting embryoid body of the plurality express MF20 or MLC2a.

2. The method of claim 1, wherein the population of embryoid bodies have a substantially uniform size and shape.

3. The method of claim 1, wherein each embryoid body comprises a substantially uniform number of cells.

4. The method of claim 1, wherein the microwell is rectangular.

5. The method of claim 1, wherein the microwell has a depth between about 10 microns and about 1000 microns.

6. The method of claim 1, wherein the harvesting step comprises shearing the pluripotent stem cells from the microwell.

7. The method of claim 1, wherein the dimension-constrained microwell has cell-attracting and cell-repulsing portions.