Fig 1.
The automated agitation device to maintain colloidal suspension during syringe pump perfusion.
Front (A) and top (B) views without a syringe pump. The device alongside a syringe pump from the front (C) with an arrow indicating perfusion direction and a closeup, transverse view of the syringe (D). Labeled components include the hooked attachments with mounted neodymium magnets (a), the lead screw assembly (b), the lead screw (c), guide rods (d), the controller housing (e) containing the DC motor, joint coupler, microcontroller, motor controller, potentiometers, push buttons, and liquid crystal display, the end-stop limit switches (f), a syringe (g), a collection Eppendorf tube (h), a syringe pump (i), and the magnetic stir bar (j).
Table 1.
Primers used for gene expression.
Fig 2.
Stir bar velocity in a syringe can be externally controlled.
The velocity of the stir bar in HBSS (left) or 1% w/v alginate (right) solution was measured with varied external magnet velocity. Best fit lines were plotted using linear regression with y-intercept = 0 (R2>0.96). All data are from n = 3 independent experiments. Error bars indicate standard deviation.
Table 2.
Translational velocities of both the external and stir bar magnets during agitation.
Fig 3.
Automated stirring maintains cell concentration over time and cell viability.
(A) Cell concentrations as a function of time for each agitation condition. The data were fitted to a constant followed by one-phase exponential decay curve for no stirring (t0=12.4 min, t1/2=38.7 min, plateau after decay = 1.92 million; R2>0.95), and a linear curve with slope = 0 for other groups. P<0.05 via two-way ANOVA. (B) Standard deviation of cell concentrations for each agitation condition. ***p<0.001, **p<0.01 via one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Percentage of viable cells at 120 minutes of agitation at agitation conditions. n = 3 independent experiments, each performed in two replicates. Error bars represent standard deviation.
Table 3.
Mean and standard deviation of cell concentrations at each time point and agitation condition.
Simple main effects were analyzed for each time point and agitation condition. Statistical analysis was done by two-way ANOVA.
Fig 4.
Agitation does not alter the expression of mechanosensitive genes at a shorter timescale.
Gene expression analysis of (A) Piezo1, (B) Acta2, (C) Ctgf in MSCs from different tested conditions after 120 min. n = 3 independent experiments, each performed in three replicates. Error bars represent standard deviation.