IN VITRO EXPANSION OF POSTPARTUM-DERIVED CELLS IN ROLLER BOTTLES

• US 20070141700A1
• Filed: 12/19/2006
• Published: 06/21/2007
• Est. Priority Date: 12/19/2005
• Status: Active Grant

• Current Assignee: Depuy Synthes Products Incorporated (Johnson & Johnson)
• Original Assignee: Ethicon Incorporated (Johnson & Johnson)
• Inventors: Harmon, Alexander
• Granted Patent: US 8,741,638 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• 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 maximizing the number doublings of a culture of postpartum cells in a roller bottle culture system comprising using a rotational speed of at least about 0.85 rpm, using a media volume of at least about 100 ml in an 850 square centimeter culture bottle;

using a seeding density of less than about 2500 cells per square centimeter; and

incubating for at least about 5.5 days.

2. The method of claim 1 wherein the rotational speed is about 1 rpm;

the media volume is about 112 ml in an 850 square centimeter culture bottle; and

the incubation time is about 6.2.

3. The method of claim 1 which results in about 3 population doublings.

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

5. The method of claim 4 wherein the postpartum cells are ATCC Nos PTA-6067 and PTA-6068.

6. A method of maximizing the rate of doubling of a culture of postpartum cells in a roller bottle culture system comprising using a rotational speed of at least about 0.85 rpm, using about 300 ml of growth medium in an 850 square centimeter culture bottle;

using a seeding density of less than about 2500 cells per square centimeter; and

incubating for less than about 6 days.

7. The method of claim 6 wherein the rotational speed is about 0.93 rpm and the incubation time is less than about 5.5 days.

8. The method of claim 6 wherein the time for a population to double is less than about 30 hours.

9. The method of claim 6 wherein the postpartum cells are umbilicus-derived cells.

10. The method of claim 9 wherein the postpartum cells are ATCC Nos PTA-6067 and PTA-6068.

11. A method of maximizing the density of cells at harvest for postpartum cells in a roller bottle culture system comprising using a rotational speed of about 0.5-1 rpm, using about 300 ml of growth medium in an 850 square centimeter culture bottle;

using a seeding density of about 10,000 cells per square centimeter; and

incubating for between about 5.5 to 6.7 days.

12. The method of claim 11 wherein the rotational speed is about 0.98 rpm and the incubation time is about 6.7 days.

13. The method of claim 11 wherein the density of the cells is about at least about 3×

E+4 cells/cm sq.

14. The method of claim 11 wherein the postpartum cells are umbilicus-derived cells.

15. The method of claim 14 wherein the postpartum cells are ATCC Nos PTA-6067 and PTA-6068.

16. 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 of about 0.65-0.9 rpm, using at least about 300 ml of growth medium in an 850 square centimeter culture bottle;

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

incubating for between about 5.5 to 6.5 days.

17. The method of claim 16 wherein the rotational speed is about 0.7, and the incubation time is about 6 to 6.3 days.

18. The method of claim 16 wherein the rotational speed is about 0.85, and the incubation time is about 6 to 6.3 days.

19. The method of claim 16 wherein a population doubling time is les than about 39 hours, the population achieves at least about 3 doublings, and the density of cells at harvest is at least about 1.8E+4 cells/cm sq.

20. The method of claim 16 wherein the postpartum cells are umbilicus-derived cells.

21. The method of claim 20 wherein the postpartum cells are ATCC Nos PTA-6067 and PTA-6068.