Figure 6.

  Timeline showing stages required to isolate tLDA‐mMSCs from heterogeneous bone marrow cultures. (a) Bone marrow adherent cell cultures were initiated in a plastic dish. Non‐adherent cell population was removed on day 3 and remaining adherent cells were cultured to near confluence with medium changes twice weekly. (b) By day 8, mMSCs clone developed, with some HCs adhering to the plastic surface of the dish, while others engaged on mMSCs. (c) After trypsin digestion, some engaged HCs were lifted with mMSCs (most HCs were segregated from mMSCs, whereas some of them still adhered to mMSCs). Plastic‐adherent trypsin‐insensitive HCs were depleted. (d) To isolate mMSCs using the tLDA method, non‐dispersed cells were first removed using a 30 μm mesh after trypsin digestion. (e) mMSCs could be segregated from HCs using lower‐density culture (1.25 × 10⁴ cells/cm²) on plastic within 2.5 h. After being lifted by trypsin digestion, mMSCs were retrieved from the cultures, whereas trypsin‐insensitive HCs were depleted from recovered cells as they remained adherent to plastic. (f) tLDA‐mMSCs could be obtained with good proliferative and therapeutic potentials when cells were replated at a density of 5 × 10⁴ cells/cm².