TY - JOUR
T1 - Umbilical cord derived stem cell culture
T2 - Multiple-harvest explant method
AU - Adiwinata, Jeanne
AU - Liem, Isabella Kurnia
AU - Budiyanti, Ecie
AU - Fasha, Iqbal
AU - Feroniasanti, Lauda
AU - Jamaan, Taufik
AU - Sumapradja, Kanadi
PY - 2014
Y1 - 2014
N2 - Published methods of umbilical cord explant culture require removing the explant after the MSCs are confluent. However, we observed that the explants were still attached after the MSCs were detached using TrypLE Select. Therefore, the objective of this study was to do explant cultures in xeno-free media, and reculture the explants after the first harvest to test whether the explant could grow further to yield more stem cells. In this study, umbilical cord explant culture were done in 10% platelet rich plasma (PRP) containing alpha minimal essential medium (αMEM) or Dulbecco's modified Eagle's medium (DMEM), or 10% human AB or cord blood serum (CBS) containing αMEM. Three explants were cultured in each well of a twelve well plate in triplicate. The cells were harvested using TrypLE Select, and cell yield was counted after cell growth achieved 80-90% confluence. The remaining attached explants were re-cultured in the respective media for several times after harvesting, until the explants were detached or until there was no more cell growth from the explants. Cell counts for every harvest were noted. Cumulative cell yield from each well for each medium was computed and tabulated. Our results showed that the time needed for cell growth was variable between explants. Therefore, in one well, the three explants might grow with different speed, and in many instances, harvests were done before optimal growth in all explants occurred. The maximal number of harvest was four times, which occurred in 10% human AB serum supplemented αMEM (two times) followed by two harvest in 10% autologous CBS supplemented αMEM, and yielded a cumulative amount of 144,000 viable cells in 37 days. In conclusion, we have developed a multi-harvest explant method to culture umbilical cord stem cells in various xeno-free media that yield far more numerous viable cells compared to standard explant method.
AB - Published methods of umbilical cord explant culture require removing the explant after the MSCs are confluent. However, we observed that the explants were still attached after the MSCs were detached using TrypLE Select. Therefore, the objective of this study was to do explant cultures in xeno-free media, and reculture the explants after the first harvest to test whether the explant could grow further to yield more stem cells. In this study, umbilical cord explant culture were done in 10% platelet rich plasma (PRP) containing alpha minimal essential medium (αMEM) or Dulbecco's modified Eagle's medium (DMEM), or 10% human AB or cord blood serum (CBS) containing αMEM. Three explants were cultured in each well of a twelve well plate in triplicate. The cells were harvested using TrypLE Select, and cell yield was counted after cell growth achieved 80-90% confluence. The remaining attached explants were re-cultured in the respective media for several times after harvesting, until the explants were detached or until there was no more cell growth from the explants. Cell counts for every harvest were noted. Cumulative cell yield from each well for each medium was computed and tabulated. Our results showed that the time needed for cell growth was variable between explants. Therefore, in one well, the three explants might grow with different speed, and in many instances, harvests were done before optimal growth in all explants occurred. The maximal number of harvest was four times, which occurred in 10% human AB serum supplemented αMEM (two times) followed by two harvest in 10% autologous CBS supplemented αMEM, and yielded a cumulative amount of 144,000 viable cells in 37 days. In conclusion, we have developed a multi-harvest explant method to culture umbilical cord stem cells in various xeno-free media that yield far more numerous viable cells compared to standard explant method.
KW - Explant culture
KW - Multiple harvests
KW - Stem cell
KW - Thrombocyte concentrate
KW - Umbilical cord
UR - http://www.scopus.com/inward/record.url?scp=84904973052&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:84904973052
SN - 0974-4304
VL - 6
SP - 1202
EP - 1208
JO - International Journal of PharmTech Research
JF - International Journal of PharmTech Research
IS - 4
ER -