Bioreactor inoculation, culture, and sampling
hMSCs just thawed or harvested from tissue-culture flasks were seeded directly into the Mobius CellReady 3-L Bioreactor (EMD
Millipore) with microcarriers. To help cells attach to microcarriers, the agitation was set low, at around 25–35 rpm. The
temperature of the bioreactor was set at 37 °C and supplemented with 5% CO2 from the bioreactor overlay.
Samples were withdrawn from bioreactors daily except on weekends. Cells and microcarriers were spun down at 200 rpm for 5
min and cell numbers were measured using a NucleoCounter NC100 (New Brunswick Scientific). Supernatants were collected and
read by BioProfile FLEX (Nova Biomedical, Waltham, Mass.) for metabolic profile analysis.
hMSC differentiation to adipocytes and osteocytes
Mesenchymal stem cells cultured in bioreactors were trypsinized off the microcarriers and plated on 24-well tissue culture
plates at seeding densities of 2.1×104 cells/cm2 for inducing adipogenesis and at 4.2×103 cells/cm2 for inducing osteogenesis. The induction of differentiation was carried out as per the instructions provided with the human
mesenchymal stem cell functional identification kit (R&D Systems, Minneapolis, Minn.). After 21 d in culture, the cells were
fixed and stained with Oil red O (EMD Millipore) to confirm the differentiation to adipocytes or with Alizarin red (EMD Millipore)
to confirm the differentiation to osteocytes. Also, RNA was isolated from these differentiated cells to look at expression
levels of differentiation markers using real-time polymerase chain reaction (RT-PCR).
RNA isolation, cDNA synthesis and RT–PCR
RNA was isolated from mesenchymal stem cells using RNeasy Mini Kit (Qiagen, Valencia, Calif.) as per the instructions in the
protocol. 1µg total RNA was used for cDNA synthesis using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, Md.). Real-time–polymerase
chain reaction (RT–PCR) to quantify relative gene expression was carried out using custom PCR Array plates (SABiosciences,
Valencia, Calif.) using the CFX96 Touch Real-time PCR system (Bio-Rad, Hercules, CA). PCR reaction was done with PerfeCTa
SYBR Green SuperMix (Quanta Biosciences) as per the following cycling conditions: Initial denaturation and activation of AccuStart
Taq DNA polymerase at 95°C for 10 min; PCR cycling for 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The specificity of
the amplification product was assessed by the melt curve method. The ΔΔCT method was used to quantify the relative gene expression
by normalizing to the house-keeping gene.
Fluorescence-activated cell sorting
Cell pellets were resuspended in DMEM (Life Technologies) plus 2% BSA (Sigma-Aldrich) to bring the cell concentration to 5×105 cells/mL. Cells were aliquoted into a round bottomed 96-well plate and antibody diluted in PBS (Cellgro, Manassas, Va.)
was added. The plate was incubated at room temperature protected from light for 20 min. PBS was added after incubation and
the plate was centrifuged to pellet the cells. The supernatant was discarded and cells resuspended in PBS. The plate was read
on a guava easyCyte 8HT Flow Cytometry System (EMD Millipore). Raw data were imported into FlowJo (Treestar, Ashland, Ore.)
The antibody and isotype control were listed as following: CD 105-FITC, CD 73-PE (AbCam, Cambridge, Mass.), CD 90-FITC, CD
11b-PE, CD 79α-PE, CD 14-FITC, CD45-FITC, CD 34-FITC, CD 19-FITC, HLA-DR-FITC (EMD Millipore), CD 146-FITC, CD 44-FITC, CD
45-FITC, CD 106-FITC, CD 274-FITC (AbD Serotec, Raleigh, N.C.), IgG1k FITC, IgG2ak FITC (BD Bioscience, Sparks, Md.), IgG1
PE, IgG2a PE (R & D Systems,), and IgG2b FITC (AbD Serotec).
Karyotyping and StemArray assays
hMSCs harvested from bioreactors or CellStack were harvested and seeded at 3000 cells/cm2 in T25 flasks. Flasks were filled with fresh medium and shipped to Cell Line Genetics Inc. for characterization. Karyotyping,
FISH, and StemArray assays were done according to their protocols.
Data are expressed as mean± standard deviation unless stated otherwise. Statistical analysis including ANOVA was performed
using Minitab (Minitab Inc, State College, PA). P values less than 0.05 were considered as significant.