MATERIALS AND METHODS
Isolation of hMSCs from unprocessed, fresh bone marrow
Unprocessed, fresh human bone marrows were purchased from Lonza (Walkersville, Md.). Upon receipt, unprocessed bone marrows
were divided between 50-mL conical tubes with addition of an equal volume of phosphate-buffered saline (PBS, EMD Millipore).
After spinning down at 1500 rpm for 5 min, supernatants were removed. Twenty-five mL of Red Blood Cell Lysing Buffer (Sigma-Aldrich,
St. Louis, Mo.) was added and tubes were incubated on ice for 3 min. Cells plus lysis buffer were spun down at 1500 rpm for
5 min and supernatant was removed. After three cycles of lysis and spinning, cells were resuspended in 10% fetal bovine serum/Dulbecco's
modified Eagle's medium (FBS/DMEM, Life Technology, Carlsbad, Calif.) and seeded at 10×106 cells/cm2 onto tissue-culture treated flasks. To calculate cumulative population doubling (cPD) by counting colonies, cells were placed
at 1-, 10-, and 100×103 cells/cm2 in treated tissue-culture flasks.
Human mesenchymal stem-cell culture
In-house isolated hMSCs were cultured on 0.1% gelatin-coated (EMD Millipore) tissue-culture dishes in 5% CO2/95% air at 37 °C as described previously (1). The culture medium consisted of DMEM (Life Technologies) supplemented with
10% MSC-screened FBS (Life Technologies), 2 mM L-glutamine (EMD Millipore), 1× EmbryoMax Penicillin-Streptomycin Solution
(EMD Millipore), and 8 ng/mL human recombinant basic fibroblast growth factor (bFGF, EMD Millipore). Cells were fed every
other day by changing 100% of the medium. To protect hMSCs in bioreactors, medium was supplemented with 0.02% Pluronic-F68
surfactant (Sigma-Aldrich) and 37.5 ppm anti-foam C emulsion (Sigma-Aldrich).
For passaging, the cells were incubated with Trypsin-EDTA (EMD Millipore) in PBS and cell clumps were dissociated by gentle
pipetting. The cell suspension was spun down at 200×g for 5 min, and after removing the supernatant, the cell pellet was resuspended
in fresh medium and plated on tissue culture flasks at 3000 cells/cm2.
Preparation of microcarriers
Cytodex 1 and 3 (GE Healthcare, Piscataway, N.J.), collagen-coated and Hillix microcarriers (Solohill, Ann Arbor, Mich.),
Cultispher G and S (Sigma-Aldrich) were prepared according to the manufacturer's instructions, including washing with PBS,
sterilization by autoclave, and washing again with culture medium.
hMSC expansion on micro-carriers under static conditions
To evaluate the most suitable microcarrier for hMSCs culture in bioreactor, six different microcarriers from three vendors
were tested. In particular, Cytodex 1 and 3 (GE Healthcare), collagen-coated and Hillix microcarriers (Solohill), Cultispher
G and S (Sigma-Aldrich) were used for screening.
An equivalent total surface area of 250 cm2 and the same amount of hMSCs were used for each microcarrier in 10-cm low adherent petri dishes. Cell attachment efficiency
was measured 24 hr after inoculation by calculating the ratio of the number of cells attached on microcarriers to the number
of seeded cells. After 6 d of culture, cells were trypsinized and harvested from the beads. The cell release rate was calculated
by the ratio of number of cells recovered from the beads to the total number of cells on the beads measured by NucleoCounter
NC100 (New Brunswick Scientific, Enfield, Ct.).