Mesencult acf plus culture kit

ADSCs cultured in FBS containing media, DMEM (HyClone) supplemented with 10% FBS (HyClone), SFM (CellCor, Xcell Therapeutics). StemPro MSC SFM XenoFree (Thermo Fisher Scientific), or MesenCult-ACF Plus Culture Kit (STEMCELL Technologies) were compared. ADSCs were seeded at 4 × 10³ cells/cm² in T25 flasks (Corning) and passaged every 4 days, except every 3 days for SFM. Flask of FBS containing media and SFM group did not need to be pre-coated. When using StemPro MSC SFM XenoFree and the MesenCult-ACF Plus Culture Kit, the flasks were pre-coated with CELLstart Substrate (Thermo Fisher Scientific) and Animal Component-Free Cell Attachment Substrate (STEMCELL Technologies), respectively. At each passage, ADSCs were detached and counted using a NucleoCounter NC-250 (ChemoMetec, Allerod, Denmark) and seeded in new T25 flasks. The population doubling time (PDT) was determined at each passage by the formula: PDT = culture period in hours / {log (number of cells T₀) – log (number of cells T₁)} / log(2), where T₀ is the seeding cell number and T₁ is the cell number at harvest. The accumulation cell number (ACN) was determined at each passage by using formula: ACN = (ACN of previous passage / T₀) x T₁.

Using the plastic adherence method, MSCs were purified and expanded from bone marrow cells harvested from femurs of C57BL/6 mice in murine MesenCult medium with supplements (STEMCELL Technologies, Canada).^{4 (link),16 (link)} Non-adherent cells were removed every 48 h by changing the medium. Phenotypic and functional characterizations of MSCs, as set forth by “The International Society for Cellular Therapy,” were completed prior to use.^{17 (link)} Human bone marrow-derived MSCs (STEMCELL Technologies) were purchased and expanded using MesenCult™-ACF Plus Culture Kit (STEMCELL Technologies). MSCs from the second or third passage were used in the following experiments.