In vitro expansion of postpartum-derived cells in roller bottles

• US 8,741,638 B2
• Filed: 12/19/2006
• Issued: 06/03/2014
• Est. Priority Date: 12/19/2005
• Status: Active Grant

• Current Assignee: Depuy Synthes Products Incorporated (Johnson & Johnson)
• Original Assignee: DePuy Synthes Products LLC (Johnson & Johnson)
• Inventors: Harmon, Alexander M.
• Primary Examiner(s): HIBBERT, CATHERINE S
• Application Number:
  US11/612,872
• Publication Number:
  US 20070141700A1
• Time in Patent Office: 2,723 Days
• Field of Search: None
• US Class Current: 435/366
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly

Abstract

Methods for the maximizing parameter of the in vitro growth and expansion of mammalian cells, specifically postpartum-derived cells in containers such as roller bottles is described. Methods of optimizing growth rate and cell yield in such culture systems are provided. The methods are particularly adapted for human postpartum-derived cells, such as umbilicus-derived cells.

Claims

1. A method of simultaneously maximizing doubling rate, harvest density, and the total number of population doublings for postpartum cells in a roller bottle culture system comprising:

using a rotational speed from 0.65-0.9 rpm;

using at least 300 ml of growth medium in an 850 square centimeter culture bottle;

using a seeding density of less than 2,500 cells per square centimeter; and

incubating for between 5.5 to 6.5 days,

wherein the postpartum cells that are cultured are characterized as having an identical marker profile as starting cells with respect to each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-ABC, CD31, CD34, CD45, CD117, CD141, and HLA-DRDPDQ, and

wherein the postpartum cells are positive for expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-ABC, and negative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DRDPDQ.

2. The method of claim 1 wherein the rotational speed is about 0.7, and the incubation time is about 6 to 6.3 days.

3. The method of claim 1 wherein the rotational speed is about 0.85, and the incubation time is about 6 to 6.3 days.

4. The method of claim 1 wherein a population doubling time is less than about 39 hours, the population achieves at least 3 doublings, and the density of cells at harvest is at least 1.8 E+4 cells per square centimeter.

5. The method of claim 1 wherein the postpartum cells are umbilicus-derived cells.

6. The method of claim 5 wherein the postpartum cells are ATCC Nos PTA-6067 and PTA-6068.