Scalable passaging of adherent human pluripotent stem cells.
Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive, result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs, we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate, formulated as a hypertonic solution, gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium, retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together, this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology, high levels (>80%) of pluripotency markers OCT4, SSEA-4, TRA-1-60 andTRA-1-81, a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers, both in vivo and in vitro.
Cell Therapy Development Services, Lonza-Walkersville, Inc., Walkersville, Maryland, United States of America.
Ying
Y
Nie
Cell Therapy Development Services, Lonza-Walkersville, Inc., Walkersville, Maryland, United States of America.
Patrick
P
Walsh
Cell Therapy Development Services, Lonza-Walkersville, Inc., Walkersville, Maryland, United States of America.
Diana L
DL
Clarke
Cell Therapy Development Services, Lonza-Walkersville, Inc., Walkersville, Maryland, United States of America.
Jon A
JA
Rowley
Cell Therapy Development Services, Lonza-Walkersville, Inc., Walkersville, Maryland, United States of America.
Thomas
T
Fellner
D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D002951:Citrates / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000502:physiology*; D007621:Karyotyping / Q000379:methods; D000077559:Sodium Citrate
D002951:Citrates; D000077559:Sodium Citrate; D002118:Calcium