Abstract
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 × 106 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 × 109 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.
| Original language | English |
|---|---|
| Pages (from-to) | 920-933 |
| Number of pages | 14 |
| Journal | Biotechnology and Bioengineering |
| Volume | 92 |
| Issue number | 7 |
| DOIs | |
| State | Published - Dec 30 2005 |
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Keywords
- Bioreactor agitation
- Cardiomyocytes
- Embryoid bodies
- Embryonic stem cells
- Process control
- Scale-up
ASJC Scopus subject areas
- Biotechnology
- Microbiology
Cite this
Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control. / Schroeder, Magnus; Niebruegge, Sylvia; Werner, Andreas; Willbold, Elmar; Burg, Monika; Ruediger, Manfred; Field, Loren; Lehmann, Juergen; Zweigerdt, Robert.
In: Biotechnology and Bioengineering, Vol. 92, No. 7, 30.12.2005, p. 920-933.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control
AU - Schroeder, Magnus
AU - Niebruegge, Sylvia
AU - Werner, Andreas
AU - Willbold, Elmar
AU - Burg, Monika
AU - Ruediger, Manfred
AU - Field, Loren
AU - Lehmann, Juergen
AU - Zweigerdt, Robert
PY - 2005/12/30
Y1 - 2005/12/30
N2 - 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 × 106 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 × 109 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.
AB - 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 × 106 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 × 109 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.
KW - Bioreactor agitation
KW - Cardiomyocytes
KW - Embryoid bodies
KW - Embryonic stem cells
KW - Process control
KW - Scale-up
UR - http://www.scopus.com/inward/record.url?scp=33644821605&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33644821605&partnerID=8YFLogxK
U2 - 10.1002/bit.20668
DO - 10.1002/bit.20668
M3 - Article
VL - 92
SP - 920
EP - 933
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 7
ER -