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 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 (alphaMHC) 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.
Schroeder
Magnus
M
Institute of Cell Culture Technology, University of Bielefeld, 33501 Bielefeld, Germany.
Niebruegge
Sylvia
S
Werner
Andreas
A
Willbold
Elmar
E
Burg
Monika
M
Ruediger
Manfred
M
Field
Loren J
LJ
Lehmann
Juergen
J
Zweigerdt
Robert
R
D000818:Animals; D019149:Bioreactors; D002454:Cell Differentiation / Q000502:physiology*; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*