Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable l...

Full description

Saved in:
Bibliographic Details
Published in:Biotechnology and bioengineering 2005-12, Vol.92 (7), p.920-933
Main Authors: Schroeder, M, Niebruegge, S, Werner, A, Willbold, E, Burg, M, Ruediger, M, Field, L.J, Lehmann, J, Zweigerdt, R
Format: Article
Language:English
Subjects:
Animals
automation
Biological and medical sciences
bioreactor agitation
Bioreactors
Biotechnology
cardiomyocytes
Cell Differentiation - physiology
Embryo, Mammalian - cytology
Embryo, Mammalian - physiology
embryoid bodies
embryonic stem cells
Fundamental and applied biological sciences. Psychology
Mice
Myocytes, Cardiac - cytology
Myocytes, Cardiac - physiology
process control
scale-up
Stem Cells - cytology
Stem Cells - physiology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (αMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.20668