Magnetically controlled bioreactor development
The magnetically controlled bioreactor including reactor holder, reaction vessel, drive machine and magnets was designed (Fig. 2). The reactor bracket was divided into upper and lower layers, wherein a groove was designed in the middle of the lower layer to fix the driving machine, and a plurality of grooves were arranged on the upper layer to fix the reaction container. In addition, the magnetic beads were filled within the reaction vessel. The magnets consisted of two parts, a part of which was a ring magnet and placed on the top of the reaction vessel to attract the magnetic beads to suspend on the surface of the liquid. The other part was fixed on the crossbar of the link driver and performed circular motion with the rotation of the crossbar. When the magnet moved to the bottom of the reaction vessel, the magnetic beads are attracted to the bottom. The magnetic beads resuspend when the magnet left. The frequency of the magnetic beads floating is closely related to the rotational speed of the crossbar. For example, under the condition of 10 r/min, the magnetic ball moves every 3 s to complete a round of floating up and down, and the magnetic beads can be instantly adsorbed to one end and hover there. Under the set experimental conditions, it can be directly observed that the magnetic ball has enough time to move from one end to the other end. By floating the magnetic beads up and down, culture system was homogenized.
Static magnetic field did not affect the expansion and function of NK-92 cells
Studies had shown that regardless of the magnetic induction intensity, SMFs alone had no lethal effect on cell survival under normal culture conditions, and had no significant effect on genetic toxicity. Also, the growth rate and cell cycle distribution of most cells were not affected by SMFs (Miyakoshi 2005). We assessed the effect of magnetic field on the cell growth, phenotype and cytolytic function of NK-92 cells by culturing NK-92 cells without magnetic field (control) or 10 mT, 50 mT and 100 mT static magnetic field, respectively. The results showed that the viability of NK-92 cells remained above 92% in both the control and magnetic field groups during the 8-day culture process (Fig. 3A). The expansion folds of NK-92 cells in static magnetic field were 38.26 ± 1.63, 44.96 ± 9.32 and 35.02 ± 5.58, which showed no significant difference from that of control (34.33 ± 4.11) (Fig. 3B). Similarly, there was no difference in CD3−CD56+ cell population and the cytotoxicity toward the K562 cells between static magnetic field-expanded and control NK-92 cells (Fig. 3C, D). These results indicated that static magnetic field showed no apparent effect on the ex vivo expansion and function of NK-92 cells.
Although the research on NK cells at the cellular level point out that exposure to a 400 mT constant magnetic field increased the viability of NK92-MI cells and their ability to kill K562 tumor cells was also improved (Lin et al. 2019), the possible reason was that the ratio of E:T and the magnetic field strength both were somewhat different. Overall, the medium static magnetic field mentioned here at least supported the maintenance of the viabilities, proliferation and cytotoxicity of NK cells without biological toxicity.
Intermittent magnetic field promoted the expansion and function of NK-92 cells
It was known that the biological effects of magnetic fields can be influenced by the magnetic field types, strength, frequency, treatment time and other parameters, all of which contribute to the mixed results of biological effects of magnetic field in the literature (Zhang et al. 2017). Previous studies on the effect of MFs on NK cells were focused on individual level (House and Mccormick 2000; Onodera et al. 2003; Gobba et al. 2009), and there were few studies on the influence of direct exposure to magnetic fields on NK cells. To investigate the effect of intermittent magnetic fields on the growth of NK-92 cells, the intensities were set to 10 mT, 50 mT and 100 mT with 30 Hz intermittent frequency. Similarly, cell viabilities of the NK-92 cells from magnetic field and the control group were both above 92% at all time points (Fig. 4A). Notably, the expansion folds of NK-92 cells cultured in the intermittent magnetic field were 61.55 ± 4.93, 59.77 ± 9.07 and 64.46 ± 5.42, respectively, which is significantly higher than 34.33 ± 4.11 in control (Fig. 4B). No differences in the frequency of CD3−CD56+ cells and cytotoxicity activity were detected in NK-92 cells expanded under intermittent magnetic field compared to control cells (Fig. 4C, D). These findings suggested that intermittent magnetic fields improved NK-92 cell expansion while maintaining the frequency of CD3−CD56+ cells and cytotoxicity.
This is the first study to assess the effects of intermittent magnetic fields on the survival, expansion and function of NK cells. The mechanism of the magnetic field affecting cells mainly includes the generation of induced currents, causing ion distribution and movement, and changing the membrane potential, thereby changing the permeability of the cell membrane (Dini et al. 2005). Although the specific mechanism had not been explored, these results indicated that the magnetic field strength and magnetic field type in this study can be safely applied to the in vitro expansion of NK cells, which provides an alternative for large-scale expansion of NK cells.
Optimization of culture conditions in magnetically controlled bioreactor
The optimized culture conditions of the magnetically controlled bioreactor for the cells were studied by setting different rotation speeds and different magnetic bead densities. The rotation speeds and magnetic bead densities were key parameters that influenced the property of magnetically controlled bioreactor. K562 cells were used as model and cultured with different rotation speeds and magnetic bead densities. We confirmed that the expansion of total cells was enhanced while the rotational speeds above 10 rpm (Fig. 5A). At 30 rpm, the expansion fold was 18.59 ± 0.74, similar to 40 rpm, but significantly higher than the expansion fold of rotation speed at 20 r/min. Given that cells were incapable of suspending in the culture system at low rotational speeds, the optimal number of magnetic beads in the magnetically controlled bioreactor need further determined. The expansion folds of K562 cells were 18.59 ± 0.74 and 19.17 ± 1.63 at 5 beads/ml and 6 beads/ml of magnetic bead, which was significantly higher than the expansion with magnetic bead density of 4 beads/ml (Fig. 5B). This result may be due to the fact that the magnetic bead density is too low to effectively suspend the cells in the culture system. This result suggested that rotation speeds of 30 r/min and magnetic beads density of 5 per ml are recommended for the cultivation of cells in the magnetically controlled bioreactor.
The homogeneity of liquid mixing within the reaction vessel was evaluated by color fading test. Results showed that the entire culture system completely mixed while the crossbar rotates five times (Fig. 5C). The results indicated that the magnetic beads inside the magnetron bioreactor can effectively mix the culture system.
Characterization of flask and bioreactor for NK-92 cells expansion
After demonstrating the viability of ex vivo culture of suspension cells, we further evaluated the NK-92 expansion process in the magnetically controlled bioreactor. After 8 days of culture, these cells in the magnetically controlled bioreactor were dispersive and translucent, with clear edges. However, the flask conditions resulted in aggregation and the single cells were small and dim (Fig. 6). The result may be that bioreactors provide a unified environment for cells. In the T25 flask, however, these cells sank to the bottom of the culture flask due to a lack of mixing. The microenvironment around cultured cells is heterogeneous (Curcio et al. 2012; Sadeghi et al. 2011).
Cell viabilities of the magnetically controlled bioreactor with intermittent magnetic fields and T25 culture flask without magnetic fields were both above 90% that during the 8-day culture period (Fig. 7A). And the maximum viable cell densities in the magnetically controlled bioreactor reached 8.04 ± 0.77 × 105 cells/ml, significantly higher than 5.17 ± 0.24 × 105 cells/ml in T25 flask (Fig. 7B). Moreover, the specific growth rate (μ) of NK-92 cells was determined based on Eq. (1). The μ of NK-92 cells in the magnetically controlled bioreactor and T25 culture flask were increasing gradually from day 0 to 6 but decreased on day 8. Along with the changes of viable cell density, NK-92 cells in the magnetically controlled bioreactor exhibited higher specific growth rate than that in T 25 culture flask (Fig. 7C). Meanwhile, significantly increased expansion was observed after in the magnetically controlled bioreactor (67.71 ± 10.60 folds) and flask (22.41 ± 1.19 folds) cultures (Fig. 7D). To investigate the physiological function of expanded NK-92 cells in bioreactor, cell phenotype and cell killing activity were assessed. CD3−CD56+ frequencies showed no significant difference between the bioreactor and flask cultures. Similarly, there is no significant difference in the killing activity of the NK-92 cells against the K562 cells between the experimental group and the control group. Taken together, these results indicated that the magnetically controlled bioreactor could improve NK-92 cell expansion without loss of CD3−CD56+ cells and impairment of cytotoxic capacity (Fig. 7E, F).
Due to the limited number of immune cells, in vitro expansion of cells is required for successful cancer immunotherapy. Conventional culture regimens were mainly performed in static culture with flasks and gas-permeable bags that lack of concern for process parameters, resulting in unstable quality and quantity of cell-products and poor reproducibility. Dynamic suspension culture was a potent method to overcome this disadvantage, however, the growth of immune cells may be impaired as the increasing shear force in the dynamic culture system (Badenes et al. 2016; Liu et al. 2006). In the present study, a magnetically controlled bioreactor using magnetic bead agitation was developed for NK-92 cells expansion. The bioreactor realized homogeneous distribution of the environment through the dynamic magnetic field and magnetic bead. Unlike conventional agitated bioreactors, the agitator of the bioreactor does not require direct contact with the outside environment, avoiding bacterial contamination (Rodling et al. 2018). And that, the agitator of the bioreactor is spherical, which reduces the shearing force of the fluid generated during operation. In addition, intermittent magnetic fields promoted cell expansion while maintaining cell viability and cellular function, though it was not fully deciphered. That might also account for the enhanced amplification of NK cells by the magnetic bioreactor. And it is necessary to explore the mechanism by which the magnetic field affects the cells in the following research. In conclusion, a magnetically controlled bioreactor for ex vivo expansion of NK-92 cells was designed, providing a novel model for expansion of immune cells in the future.