Growth Kinetics of Human Mesenchymal Stem Cells in a 3-L Single-Use, Stirred-Tank Bioreactor - The authors describe the growth characteristics of human mesenchymal stem cells cultured in a stirred


Growth Kinetics of Human Mesenchymal Stem Cells in a 3-L Single-Use, Stirred-Tank Bioreactor
The authors describe the growth characteristics of human mesenchymal stem cells cultured in a stirred-tank bioreactor.

BioPharm International
Volume 26, Issue 4, pp. 28-38

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Samples were withdrawn from bioreactors daily except on weekends.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 2.1104 cells/cm2 for inducing adipogenesis and at 4.2103 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. 1g 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 95C 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 5105 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.) for analysis.

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.

Statistical analysis

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.

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