Stem cell differentiation assays
AdMSCs were harvested from both shake and spinner flasks into 50 ml tubes. Once the microcarrier beads settled to the bottom of the tube, the supernatants were removed and cells were washed with DPBS. Afterwards, the microcarrier beads were treated with 5 ml of prewarmed trypsin-EDTA solution at 37 °C for 10 min. During incubation, the tubes were occasionally vortexed for 2 sec and then neutralized by adding an equal volume of trypsin neutralizing solution. Microcarrier beads were a llowed to settle to the bottom of the tube and the supernatants were collected as soon as possible. Microcarrier beads were washed 2-3 times with DPBS and as much supernatant as possible was collected into a 50 ml tube. Following washing, AdMSCs were collected to bottom of the tube by centrifugation at 120 xg for 5 min and resuspended in 5 ml of mesenchymal stem cell medium. Cells were seed at a density of 18,000 cells/cm2 into a 24-well plate. Adipocytes and osteocyte differentiations were performed on those cells using differentiation assay kits from ATCC. Adipocyte and osteocyte differentiated cells were identifed by cell-type specifc staining with either Oil red O or Alizarin red S kits (ScienCell^®) according to manufacturer instructions and visualized using an OLYMPUS^® CK40 microscope.
Results and Discussion
To compare between shake flask and spinner flask cultures, AdMSCs were seeded at a density of 3x103 cells/cm2 in both systems. Cell culture comparisons were conducted for 12 days and samples were collected for cell growth, biochemistry and metabolite analysis daily. Cell growth comparisons revealed that AdMSCs cultured under shake flask conditions achieved between 1.6 and 1.8-fold higher cell count over spinner flask cultures during early log phase (day 4) and stationary phase (day 9) of growth (Figure 1A). Biochemistry and metabolite analysis revealed that glucose concentrations decreased from 1.09 g/l to 0.548 g/l (for shake flask culture) and 0.798 g/l (for spinner culture), whereas lactate concentrations increased from 0.042 g/l to 0.396 g/l (for shake flask culture) and 0.259g/l (for spinner culture) after 12 days of culture (Figure 1B &C). The higher glucose consumption and lactate production rate seen in the shake flask culture supports the fnding that the stem cells grew at a faster rate under the shake flask conditions. Furthermore, during early growth phase (day 4); the amount of ammonium accumulated in spinner flask culture (2.4 mM) was 1.8-fold higher than shake flask culture (1.3 mM) (Figure 1D). It has been shown that even low level of ammonium (1.9 mM) inhibits MSC growth5. The spinner culture has shown ammonium level exceeding 2 mM early and throughout the culture process, which indicates the slower growth by the spinner method could be a result of ammonium toxicity-induced growth inhibition. The fact that spinner culture had elevated ammonium levels early in the culture not seen in the shake flask also indicates possible stem cell damage due to shear force by the spinner rod. The spinner rod was observed to display a “stop & go” motion at low speeds; precise speed control is not possible, especially at low rotation speeds.
To determine whether or not AdMSCs retained their stem cell properties during their growth under shake flask conditions, immunostaining of stem cell surface markers and differentiation assays were performed. Microcarrier beads that contained AdMSCs were immunostained with stem cell surface marker antibodies such as: FITC conjugated antihuman CD44 and APC-conjugated antihuman CD90 and revealed that AdMSCs retained stem cell surface markers during growth under shake flask culture condition (Figure 2A&B). For the adipocyte and osteocyte differentiation assays, AdMSCs were collected from the microcarrier beads and seeded into 24 well plates that contained either adipocyte or osteocyte differentiation medium. After 17 days of culture, the plates were stained with Oil Red O or Alizarin Red S staining solutions, respectively. Microscopic observation indicated that most of the AdMSCs from shake flask culture differentiated into either adipocytes or osteocytes successfully (Figure 3A&B).

Figure 1. Comparison of AdMSCs between shake fl ask and spinner fl ask cultures: A) growth; B) glucose utilization; C) lactate production and D) ammonium production.() shake fl ask and () spinner fl ask.
 

Figure 2. Stem cell marker identif cation assay for AdMSCs expanded on microcarriers in shake fl ask. A) AdMSCs on microcarrier beads are positive for CD90 stem cell marker, as indicated in red by fl uorescence imaging. B) AdMSCs on microcarrier beads are positive for CD 44 stem cell marker, as indicated in green by Fluorescence Imaging. Blue color indicates stem cell nuclear staining by DAPI.
 

Figure 3. Diff erentiation assays for AdMSCs expanded on microcarriers in shake fl ask. A) Adipogenic diff erentiation formed lipid droplets as indicated by Oil red O positive staining. B) Osteogenic diff erentiation caused calcium mineralization of extracellular matrix as indicated by Alizarin Red S positive staining.

Conclusions

Stem cell expansion using shake flask conditions appears to be a viable and simple alternative to the spinner flask system. This novel method relies on a new type of CO2 incubator with built-in shaking capability, such as the New Brunswick S41i CO 2 Incubator Shaker. The New Brunswick S41i reduces shearing, eliminates potential cell damage by the spinner rod, decrease the risk of contamination associated with inserting a magnetic stirrer base into the CO 2 incubator and reduces experimental complexity. This method also greatly increases the cell culture capacity whereby a large number of shake flasks can be placed in the New Brunswick S41i simultaneously. In the case of spinner flask culture, a typical incubator without active cooling can only handle the heat emitted from a very limited number of magnetic stirrer bases before causing temperature setpoint overshoot, a signifcant limitation to the scale-up potential of the spinner method. This reinforces the superiority of New Brunswick S41i CO 2 Incubator Shaker as an alternative to incubator/spinner based stem cell culture. This method eliminates a scale-up bottleneck while providing the highest quality stem cell culture for inoculation of large scale industrial bioreactors for the production of clinical material.

References
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