Human clonal marrow stem cell lines were generated from human bone marrow aspirates using a new protocol, called the subfractionation culturing method.
Human bone marrow aspirates (1 ml), taken from the iliac crest of two donors after obtaining informed consent (approved by the Inha University Medical School Institutional Review Board), were mixed with 15 ml complete growth medium: DMEM containing high glucose or α-minimal essential medium (α-MEM), and with fetal bovine serum (FBS; 20%) and penicillin/streptomycin (1%), and then incubated in a 100-mm culture dish. After incubation for 2 h at 37°C with 5% CO2, only the cell culture supernatant was transferred to a new 100-mm dish. After the second 2-h incubation, the supernatant was again transferred to a new dish (D1) and incubated for a further 2 h. The supernatant was transferred to another new dish (D2) and incubated for 1 day, and then transferred to yet another new dish (D3) and incubated for 1 day. This process was repeated two more times with 1- and 2- day incubations (D4 and D5, respectively). The single-cell derived colonies that appeared in the D3, D4, and D5 dishes were first transferred to a six-well plate and then to larger culture flasks, where they continued to expand. After 10–14 days in the 100-mm dishes, the cells were harvested with trypsin (0.25%) and EDTA (1 mM), suspended at 1x10^6 cells/ml in DMSO (10%) and FBS (40%), and frozen in 1-ml aliquots in liquid nitrogen (passage 1). The single colonies were detached and isolated using a 1- to 2-min treatment with trypsin/EDTA in cloning cylinders. Once the cells reached 80–90% confluence, they were recovered with trypsin/EDTA and replated at a density of 50–100 cells/cm2.