C2C12 cell myoblasts were purchased from ATCC (VA, USA) and were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS, Gibco). At 80–90% confluence of cells, media was changed to DMEM containing 1% normal horse serum (NHS, Gibco) to differentiate C2C12 cell myoblasts to myotubes.
Isolation, culture and characterization of hUCB-MSCs were performed as described previously40 (link),41 (link). hUCB samples were obtained from the Seoul City Boramae medical center cord blood bank (Allcord) and obtained samples were harvested with the mother’s informed consent. The samples were mixed with Hetasep (Stem cell Technologies, Canada) at a ratio of 5:1 and incubated 1 hr at RT. Then, supernatant was collected with Ficoll and centrifuged 2,500 rpm, 20 min for separating mononuclear cells. The isolated cells were seeded at density 2 × 105 cells per cm2 on plates in growth media containing D-media (Formula 78–5470EF, Gibco, USA), EGM-2 singlequot and 10% fetal bovine serum (FBS, Gibco, USA). After 3 days, the non-adherent cells were washed out and the adherent cell colonies were cultured and grew to sharp, spindle shaped hUCB-MSCs. This process was performed under regulations and guidelines approved by the Institutional Review board (IRB) of Boramae medical center and Seoul national university (IRB.no 1608/001-021).
Isolation, culture and characterization of hUCB-MSCs were performed as described previously40 (link),41 (link). hUCB samples were obtained from the Seoul City Boramae medical center cord blood bank (Allcord) and obtained samples were harvested with the mother’s informed consent. The samples were mixed with Hetasep (Stem cell Technologies, Canada) at a ratio of 5:1 and incubated 1 hr at RT. Then, supernatant was collected with Ficoll and centrifuged 2,500 rpm, 20 min for separating mononuclear cells. The isolated cells were seeded at density 2 × 105 cells per cm2 on plates in growth media containing D-media (Formula 78–5470EF, Gibco, USA), EGM-2 singlequot and 10% fetal bovine serum (FBS, Gibco, USA). After 3 days, the non-adherent cells were washed out and the adherent cell colonies were cultured and grew to sharp, spindle shaped hUCB-MSCs. This process was performed under regulations and guidelines approved by the Institutional Review board (IRB) of Boramae medical center and Seoul national university (IRB.no 1608/001-021).
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