High density cultures of embryonic stem cells.
Embryonic stem cells (ESC) have the unique ability to differentiate into a variety of tissue types. However, the realization of regenerative medicine will require the production of large quantities of ESC which subsequently have to be differentiated into the final phenotype. Thus, we sought to develop a simple and scaleable bioprocess to increase densities of ESC to achieve this goal. Using mouse embryonic stem cells (mESC) as a model, by combining automated feeding and culture of mESC on petriperm dishes, cell densities were enhanced up to 6.4 x 10(6) cells/cm2 compared to conventional petri dish culture which only reached 0.2 to 1.4 x 10(6) cells/cm2. It was found that mESC from all experiments maintained excellent viability, pluripotency, and genetic stability after growing for 6 days in petriperm cultures with automated feeding. The expression of Oct-4 transcription factor was observed in all cultures, mESC formed embryoid bodies in differentiated cultures and teratomas in SCID mice, confirming their pluripotency, and karyotype of the cultures was normal. This culture method was stable for routine passaging and a second mESC cell line was shown to perform in a similar manner on petriperm with automated feeding. This work represents an important step towards achieving high density cultures of ESC.
Oh
Steve K W
SK
Bioprocessing Technology Institute, 20 Biopolis Way, #06-01, Centros, Singapore. steve_oh@bti.a-star.edu.sg
Fong
Wey Jia
WJ
Teo
Yawen
Y
Tan
Heng Liang
HL
Padmanabhan
Jayanthi
J
Chin
Angela C P
AC
Choo
Andre B H
AB
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D007211:Indoles; D007621:Karyotyping; D016256:Lewis X Antigen / Q000378:metabolism; D008297:Male; D051379:Mice; D016513:Mice, SCID; D050814:Octamer Transcription Factor-3; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013724:Teratoma / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism
D015415:Biomarkers; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007211:Indoles; D016256:Lewis X Antigen; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors; C007293:DAPI; D000469:Alkaline Phosphatase