A Novel Method for the Expansion of Mesenchymal Stem Cells using an Eppendorf New Brunswick™ S41i CO₂ Incubator Shaker
Khandaker Siddiquee and Ma Sha, Eppendorf Inc., Enfeld, CT, U.S.A

Abstract
The expansion of stem cells, including mesenchymal stem cells (MSC), has been successfully demonstrated using microcarrier-based small bioreactors such as spinner flasks. In this study, we explored a simple alternative for microcarrier-based MSC expansion using conventional shake flasks. This method relies on a new type of CO2 incubator with built-in shaking capability, i.e. New Brunswick S41i CO 2 Incubator Shaker. The expansion of adipose-derived mesenchymal stem cells (AdMSCs) was compared between shake flasks and spinner flasks using microcarriers. The AdMSCs were seeded at a density of 3x103 cells/cm2 in both setups, each containing 0.5 g plastic microcarriers and 40 ml of stem cell growth medium.
The cell culture comparisons were conducted for 12 days and samples were collected daily for analysis of cell growth, biochemistry and metabolites. Cell density comparisons revealed that AdMSCs cultured under shake flask conditions achieved between 1.6 to 1.8-fold higher cell count over spinner flask culture during early log phase (day 4) and stationary phase (day 9) of growth. Daily metabolite analyses revealed that the spinner culture had elevated ammonium levels early in the culture not seen in the shake flask culture, indicating possible stem cell damage due to the shear force generated by the spinner rod.
Lastly, the AdMSCs expanded using the shake flask method remained high quality stem cells, which was evident by CD44 and CD90 stem cell marker assays and their ability to differentiate into either adipocytes or osteocytes.

Introduction
Stem cells are undifferentiated cells which have the capability of self-renewal and the potential to divide for a long period of time. They have the ability to differentiate into various specialized cells when appropriate growth factors and conditions are provided. Stem cells can be broadly classifed as: embryonic, adult, and induced pluripotent stem cells (iPS). Adult stem cells can be further characterized by their tissue of origin, such as: hematopoietic, mammary, intestinal, mesenchymal, endothelial, neural, and hair follicle stem cells. Most of the studies performed on adult stem cells utilize either hematopoietic or adipose-derived mesenchymal stem cells1. Like other adult stem cells, adipose-derived mesenchymal stem cells (AdMSCs) express all of the common stem cell markers and can be differentiated into various types of specialized cells under appropriate growth conditions. AdMSCs have advantages over other mesenchymal stem cells (MSCs), since they can be isolated in large quantities from fat tissue and are resistant to apoptosis2.
Although MSCs have enormous advantages for regenerative medicine, drug screening and drug discovery, their applications are limited by the quantities required for industrial or clinical applications3. In this study, we developed a simple shake flask culture technique to expand MSCs on microcarrier beads which can be used to scale-up into large-scale bioreactors. The microcarrier shake flask culture, which requires both agitation and CO2 gas control, was conducted in the Eppendorf New Brunswick S41i CO2 incubator shaker.

The New Brunswick S41i CO 2 Incubator Shaker, designed for both non-adherent and adherent cell culture applications, combines the precise temperature and CO2 control of an incubator with the reliable New Brunswick laboratory shaker drive mechanism. Key features include sealed inner/outer doors, high-temperature disinfection, and reduced CO 2 consumption compared to competitor models4.

Materials and Methods
Initial cell culture in T-Flasks
AdMSCs were obtained from ATCC^® (PCS-500-011) at passage 2 and cells were seeded at a density of 5,000 cells/cm2 into a T-75 cm2 fl ask (Eppendorf) using 15 ml of mesenchymal stem cell basal medium (ATCC) supplemented with 2% fetal bovine serum, 5ng/ml rh FGF basic, 5ng/ml rh acidic, 5 ng/ml rh EGF and 2.4 mM L-Alanyl-L- Glutamine (ATCC). Cultivation of cells on microcarriers Prior to start of the experiment, 0.5 g of 125-212 micron polystyrene microcarriers (SoloHill^®) (180 cm2 for a 50 mL culture) was transferred into a siliconized (Sigmacoat^®; Sigma) 250 ml spinner fl ask (Corning^®) and shake fl asks (Schott®, Duran^®) along with 25-30 ml of PBS. The fl asks were then autoclaved at 121 °C for at least 30 minutes. Microcarriers were allowed to settle to the bottom of the shake/spinner fl asks and the autoclaved PBS buff ers were carefully aspirated with the electronic pipetting aid easypet^® (Eppendorf) equipped with a 25 or 50 mL pipette. The AdMSCs were initially seeded at a density of 3x103 cells/cm2 into both fl asks, each containing 40 ml of basal mesenchymal stem cell medium. For the initial attachment of cells, the agitation speed of the New Brunswick S41i CO₂ incubator shaker and rotation speed of the spinner (housed inside of an Eppendorf Galaxy® 170 R CO₂ incubator) were both kept at 50 rpm and incubated for 2 hrs at 37 °C with 5% CO ₂. After incubation, the cell culture volume was adjusted to 50 ml total with 10 ml of medium containing serum to reach a f nal FBS concentration of 4% and targeted f nal concentration of growth supplements (10 ng/ml f nal concentration of rh FGF basic, rh FGF acidic & rh EGF and 4.8 mM f nal concentration of L-Alanyl-LGlutamine). Following the addition of FBS and growth supplements, the rotation speed of the spinner and the agitation speed of New Brunswick S41i CO₂ incubator shaker were both raised to 70 rpm. After 18 to 24 hrs of incubation, 1 ml of homogeneous samples containing both media and microcarriers were collected for microscopic observations, cell counting as well as biochemistry analysis. Cell counting
Cells on microcarrier beads were counted by hemocytometer. To accomplish this, microcarrier beads were suspended in citric acid solution containing crystal violet (0.1% crystal violet in 0.1 M citric acid solution) equal to the volume of supernatant removed from the tube. The contents of the tube were incubated for 1 hr or overnight at 37 °C and vortexed for a few seconds to release the stained nuclei. The nuclei were counted with hemocytometer.
Biochemistry and metabolites analysis The supernatants collected during cell counting were used for biochemistry and metabolite measurements using an automated YSI^® 2950 Bio-analyzer.
Stem cell surface marker assay
To assess the quality of AdMSCs after expansion and to conf rm that the stem cell markers were retained during the microcarrier-based culture, CD44 and CD90-specif c fl uorescent immunoassays were performed using the following procedure. 5 ml samples were collected from both the spinner and shake fl asks near the end of microcarrier culture. After the microcarriers settled to the bottom, the supernatants were removed and the microcarrier beads containing cells were gently washed 3 times with PBS at room temperature. Cells on the microcarrier beads were then f xed with 4% paraformaldehyde for 30 minutes followed again by PBS washing 3 times. Cellcontaining microcarrier beads were blocked with 5% FBS at room temperature for 1hr and immunostained with FITC-conjugated antihuman CD44 (BioLegend^®) and APC-conjugated antihuman CD90 (BioLegend^®) antibody solutions, also for 1hr at room temperature. The beads were washed 5 times with room temperature PBS for 5 min and visualized using an EVOS^® FL fl uorescence microscope.