Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.
Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.
Prowse
Andrew B J
AB
The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia.
Doran
Michael R
MR
Cooper-White
Justin J
JJ
Chong
Fenny
F
Munro
Trent P
TP
Fitzpatrick
Jane
J
Chung
Tung-Liang
TL
Haylock
David N
DN
Gray
Peter P
PP
Wolvetang
Ernst J
EJ
D000595:Amino Acid Sequence; D019149:Bioreactors; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013002:Somatomedins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D019096:Vitronectin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B