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Introduction ex vivo expansion processes depends on a variety of fac- Natural killer (NK) cells are innate immune cells that tors, and culture system is a key factor to overcome the lyse tumor cells in a non-major histocompatibility com- technical obstacle. The large-scale production of cells plex (MHC) restricted manner, which makes it a promis- is mainly achieved in bioreactors. Bioreactor systems ing candidate for adoptive immunotherapy (Wang et al. have the advantages of providing a homogenous culture 2017; Ljunggren and Kärre 1990; Wang et al. 2017). How- environment, real-time monitoring and control of cell ever, there are technical challenges in obtaining sufficient culture process, which enables transparent and control- numbers of functionally active NK cells from a patient’s lable. Bioreactors create a consistent culture condition blood since they represent only 10% of the lymphocytes and guarantee homogeneous supply of cultured cells with and are often dysfunctional (Klingemann et al. 2016; nutrients and gasses for efficient expansion strategy. Zhang et al. 2019). NK-92 is a highly activated NK cell For immune cell expansion of bioreactor systems such line which was originally established from a non-Hodg- as the shake flasks, rotating wall vessels, WAVE bioreac - kin’s lymphoma. And NK-92 is homogenous cell popula- tor, and stirring bioreactor have been investigated (Meng − + tions, which has a typical NK profile mainly CD3 CD56 et al. 2018; Zhang et al. 2018; Ou et al. 2019; Kaiser et al. population, compared to NK cells (Gong et al. 1994; 2015). In stirring bioreactors, mixing of culture medium Luetke-Eversloh et al. 2013; Verheyden and Demanet that exposed the cells with impeller cause to exert the 2008). They can be expanded substantially in the pres - fluid shear stress. However, immune cells do not have ence of interleukin-2 (IL-2) without the need for alloge- cell wall and are sensitive to shear force. Then different neic feeder cells, which makes them suitable for cancer types of impellers produce different shear forces during immunotherapy (Wang et al. 2017; Suck et al. 2016). operation, and spherical agitation produces less shear Since NK-92 is readily available from a current (c)-GMP- force than impeller type agitation (Collignon et al. 2010; compliant master cell bank, predictable and reproducible Hosseinizand et al. 2016; Mckee and Chaudhry 2017). expansion of an extensively characterized potent NK cell Rotating bioreactors generate a low-shear stress culture agent holds great promise for clinical application (Suck environment, allowing to partially overcome the limi- et al. 2016). tations of stirred tank devices. However, the complex- NK cells need to be expanded before clinical infu- ity of the technological solutions adopted for rotation sion to meet the dosage demand. The optimization of make these devices not easily scalable and unsuitable for Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 3 of 11 Fig. 1 Device for generating different types of magnetic fields. A Static magnetic field device. B Intermittent magnetic field device continuous medium replacement and real-time moni- 120 mT maximum uniform pulsed magnetic fields expo - toring (Rodrigues et al. 2011). Sutlu et al. expand large sure increased NK cytotoxic activity (De Seze et al. 1993). numbers of clinical-grade NK cells in a Wave bioreac- However, 50 Hz, 2 mT magnetic field suppressed NK cell tor without feeder cells. Expanded cells consisted 38% activity in guinea pigs (Canseven et al. 2006). The viabil - − + CD3 CD56 NK cells and have a higher cytotoxic capac- ity and cytotoxicity of human NK cell line were enhanced ity than cells expanded in flask (Sutlu et al. 2010). Mean - when cultured under 0.4-T static magnetic field. Lin et al. der type bioreactors which is the directed laminar flow (2019) observed that strong static magnetic field (10 T) of medium and minimize cell stress, achieved extensive decreased naive peripheral blood T cells, while no dif- expansion of highly pure (> 85%) and potent anticancer ference in number of NK cell subpopulation was found active NK cells (Bröker et al. 2019). Therefore, an optimal (Onodera et al. 2003). It is indicated that magnetic fields bioreactor system is needed for the expansion of effector of different types, field intensity or frequencies can lead cells. to diverse results. Magnetic fields within reasonable At present novel equipment is continuously developed range could be applied to support ex vivo expansion of to keep shear forces in large-scale bioreactors low, and NK cells in bioreactor and maintain function integrity. magnetically controlled bioreactor should represent a In this study, the magnetically controlled bioreactor potential alternative. Magnetically controlled bioreactor was developed for ex vivo expansion of NK-92 cells. The is mainly composed of magnetic material and magnetic effects of magnetic field on the growth and activity of fields. The spherical mixer is controlled by non-contact NK-92 cells were investigated. Further, the optimal cul- mode to blend the culture condition. Through the move - ture system parameters were determined based on cell ment of the spherical magnetic material, the concen- growth. The expansion folds, immunophenotype, and tration gradient of the substance is eliminated and the killing activity of expanded NK-92 cells were evaluated culture condition is evenly homogeneous. At the same to validate effect of the culture system. The magnetically time, the shear force is lower than the shear force gen- controlled bioreactor supported ~ 70-fold NK-92 cell erated by the traditional stirring impeller, avoiding the expansion within 8 days of culture without feeder cells. mechanical damage to cells. Magnetic field is divided into This study provides a promising platform for ex vivo weak magnetic fields (< 1mT), medium magnetic fields expansion of immune cells. (1mT–1 T), strong magnetic fields (1–20 T) and ultras - trong magnetic fields (> 20 T), depending on the mag- Materials and methods netic field intensity. Furthermore, according to the type Magnetic field exposure device of magnetic field, it can be divided into static magnetic Static magnetic field and intermittent magnetic field field, alternating magnetic field, pulsed magnetic field were set for cell exposure. A static magnetic field was and rotating magnetic field. The effect of magnetic field produced by placing a permanent neodymium magnet on cell growth and function has been investigated, and on the lower layer of the bracket, where different static the type and intensity of magnetic field act as the main magnetic field intensity was generated at different posi - parameters. Although lots of ex vivo and in vivo experi- tions on the upper layer of the bracket (Fig. 1A). Inten- ments have been studied, the effects of magnetic field on sity of the obtained static magnetic field were determined biological systems, experimental coherence among differ - by Gauss meter at about 10 mT, 50 mT and 100 mT, ent studies is still lacking. Early study showed that 0.8 Hz, respectively. Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 4 of 11 Flow cytometry The intermittent magnetic field was produced by fixing Cells were harvested from the magnetically controlled the magnet on the crossbar, and the well plate was placed bioreactor and T25 flasks on day 8. A total of 1 × 10 on the bracket above the magnet (Fig. 1B). The rotation of cells were washed with phosphate buffered saline (PBS) the crossbar drives the magnet to make a circular motion, and resuspended. Then, cells were incubated with FITC- and the magnet passes under the well plate at intervals conjugated anti-human CD3 antibody and PE-conjugated to form different intermittent magnetic field around the anti-human CD56 antibody (BD, USA) for 30 min at orifice plate. Intensity of the obtained intermittent mag - 4 °C in dark. Samples were analyzed by flow cytometer netic field was determined at about 10 mT, 50 mT and (FACS Aria I, BD, USA) to determine the proportions of 100 mT, respectively, and the intermittent frequency of − + CD3 CD56 cells in total cell population. the intermittent magnetic field was 0.5 Hz. During the experiment, the magnetic field device was introduced to Physiological function assays of expanded NK‑92 cells the CO incubator. Physiological function of expanded NK-92 cells was determined by their cytotoxic capacity on tumor cells. Preparation of magnetic beads Expanded NK-92 cells were collected as effector cells The magnetic beads were prepared by a physical cross- (E) and K562 cells, from Shanghai cell bank of Chinese linking method. Simply put, 2.5 g of sodium alginate Academy of Sciences, as target cells (T). K562 cells was fully dissolved in 100 ml 1.0% (v/v) acetic acid. 3 g (5 × 10 cells/ml) were cultured in DMEM (Gibco, USA) of Fe O nanoparticles were mixed to the solution. The 3 4 with 10% serum (Hyclone, USA). 50 μL density adjusted Fe O nanoparticles were purchased from Aladdin. The 3 4 effector cells and target cells suspension were added into magnetic beads were obtained by pipetting the solution the same well of 96-well culture dish, used as the experi- to 300 mmol/l of calcium chloride and placing it on a mental group. At the same time, 50 μL density adjusted 130 r/min shaker for 30 min. Then, the magnetic beads effector cells and target cells suspension were added were transferred to a chitosan solution of 5 g per liter and into different wells, and then 50 μL medium was added, placed on a shaker at 130 revolutions per minute for 1 h respectively, used as the control group. Cells were incu- to obtain sodium alginate–chitosan magnetic beads. The bated for 24 h in 5% CO cell incubator at 37 °C. Finally, obtained magnetic beads were repeatedly washed three 10 μL CCK8 reagent was added to each well, and incu- times with ultrapure water to remove unreacted chitosan bated in 5% C O cell incubator at 37 °C for 1–4 h. The on the surface, and finally soaked in a medium for use. OD value at 450 nm was detected and expressed by OD , OD and OD , respectively. The mortality of K562 cells Cell culture T ET was calculated according to the following formula as the NK-92 cells are from American Type Culture Collection killing activity of effector cells: (ATCC). NK-92 cells were seeded at 2 × 10 cells/ml and cultured in serum-free medium T009 (Bioengine, Shang- OD − OD ET E hai) containing 1000 U/ml of IL-2 (PeproTech, USA). Killing activity = 1 − × 100% . OD The cells were passaged every 2 days. Fresh medium and 1000 U/ml IL-2 were added to maintain the cell density at 2 × 10 cells/ml for a total 8 days of culture. Similarly, Statistical analysis NK-92 cells were seeded into the bioreactor at 2 × 10 Data were presented as mean ± standard error. Student’s cells/ml while adding the appropriate amount of mag- t-test was applied to evaluate the significance of differ - netic beads according to the volume. ences. P < 0.05 was considered as statistically significant. Cell viability, proliferation rate, and cell counting Results and discussion Cell viability was determined by trypan blue staining. Magnetically controlled bioreactor development For cell counting, supernatants in the bioreactor and T25 The magnetically controlled bioreactor including reactor flasks were mixed sufficiently and collected every other holder, reaction vessel, drive machine and magnets was day. The kinetics of cell growth was calculated according designed (Fig. 2). The reactor bracket was divided into to the following equation: upper and lower layers, wherein a groove was designed in the middle of the lower layer to fix the driving machine, ln N 2 − ln N 1 Speciﬁc growth rate:μ= , (1) and a plurality of grooves were arranged on the upper t2 − t1 layer to fix the reaction container. In addition, the mag - where μ was the specific growth rate of cells, N1 was the netic beads were filled within the reaction vessel. The number of cells at the time point of t1, N2 was the num- magnets consisted of two parts, a part of which was a ber of cells at the time point of t2. ring magnet and placed on the top of the reaction vessel Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 5 of 11 Fig. 2 A, B Reality images of a magnetically controlled bioreactor. C, D Schematic diagram of a magnetically controlled bioreactor. 1, magnet; 2, magnetic hydrogel beads; 3, reaction vessel; 4, bracket; 5, drive machine to attract the magnetic beads to suspend on the surface growth rate and cell cycle distribution of most cells of the liquid. The other part was fixed on the crossbar of were not affected by SMFs (Miyakoshi 2005). We the link driver and performed circular motion with the assessed the effect of magnetic field on the cell growth, rotation of the crossbar. When the magnet moved to the phenotype and cytolytic function of NK-92 cells by bottom of the reaction vessel, the magnetic beads are culturing NK-92 cells without magnetic field (control) attracted to the bottom. The magnetic beads resuspend or 10 mT, 50 mT and 100 mT static magnetic field, when the magnet left. The frequency of the magnetic respectively. The results showed that the viability of beads floating is closely related to the rotational speed of NK-92 cells remained above 92% in both the control the crossbar. For example, under the condition of 10 r/ and magnetic field groups during the 8-day culture min, the magnetic ball moves every 3 s to complete a process (Fig. 3A). The expansion folds of NK-92 cells in round of floating up and down, and the magnetic beads static magnetic field were 38.26 ± 1.63, 44.96 ± 9.32 and can be instantly adsorbed to one end and hover there. 35.02 ± 5.58, which showed no significant difference Under the set experimental conditions, it can be directly from that of control (34.33 ± 4.11) (Fig . 3B). Similarly, − + observed that the magnetic ball has enough time to move there was no difference in CD3 CD56 cell popula- from one end to the other end. By floating the magnetic tion and the cytotoxicity toward the K562 cells between beads up and down, culture system was homogenized. static magnetic field-expanded and control NK-92 cells (Fig. 3C, D). These results indicated that static mag - netic field showed no apparent effect on the ex vivo Static magnetic field did not affect the expansion expansion and function of NK-92 cells. and function of NK‑92 cells Although the research on NK cells at the cellular level Studies had shown that regardless of the magnetic point out that exposure to a 400 mT constant magnetic induction intensity, SMFs alone had no lethal effect field increased the viability of NK92-MI cells and their on cell survival under normal culture conditions, and ability to kill K562 tumor cells was also improved (Lin had no significant effect on genetic toxicity. Also, the Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 6 of 11 − + Fig. 3 Eec ff t of static magnetic field on NK ‑92 cells. A Fold expansion of total cells. B Cell viability. C Percentage of CD3 CD56 cells. D Cytotoxicity of the expanded NK‑92 cells. n = 3 et al. 2019), the possible reason was that the ratio of E:T group were both above 92% at all time points (Fig. 4A). and the magnetic field strength both were somewhat dif - Notably, the expansion folds of NK-92 cells cultured ferent. Overall, the medium static magnetic field men - in the intermittent magnetic field were 61.55 ± 4.93, tioned here at least supported the maintenance of the 59.77 ± 9.07 and 64.46 ± 5.42, respectively, which is sig- viabilities, proliferation and cytotoxicity of NK cells with- nificantly higher than 34.33 ± 4.11 in control (Fig. 4B). − + out biological toxicity. No differences in the frequency of CD3 CD56 cells and cytotoxicity activity were detected in NK-92 cells Intermittent magnetic field promoted the expansion expanded under intermittent magnetic field compared to and function of NK‑92 cells control cells (Fig. 4C, D). These findings suggested that It was known that the biological effects of magnetic fields intermittent magnetic fields improved NK-92 cell expan - − + can be influenced by the magnetic field types, strength, sion while maintaining the frequency of CD3 CD56 frequency, treatment time and other parameters, all of cells and cytotoxicity. which contribute to the mixed results of biological effects This is the first study to assess the effects of intermit - of magnetic field in the literature (Zhang et al. 2017). tent magnetic fields on the survival, expansion and func - Previous studies on the effect of MFs on NK cells were tion of NK cells. The mechanism of the magnetic field focused on individual level (House and Mccormick 2000; affecting cells mainly includes the generation of induced Onodera et al. 2003; Gobba et al. 2009), and there were currents, causing ion distribution and movement, and few studies on the influence of direct exposure to mag - changing the membrane potential, thereby changing netic fields on NK cells. To investigate the effect of inter - the permeability of the cell membrane (Dini et al. 2005). mittent magnetic fields on the growth of NK-92 cells, the Although the specific mechanism had not been explored, intensities were set to 10 mT, 50 mT and 100 mT with these results indicated that the magnetic field strength 30 Hz intermittent frequency. Similarly, cell viabilities and magnetic field type in this study can be safely applied of the NK-92 cells from magnetic field and the control to the in vitro expansion of NK cells, which provides an alternative for large-scale expansion of NK cells. Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 7 of 11 Fig. 4 Eec ff t of intermittent magnetic field on NK ‑92 cells NK ‑92 cells under. A Cell viability. B Fold expansion of total cells. C Percentage of − + CD3 CD56 cells. D Cytotoxicity of expanded NK‑92 cells. n = 3, *P < 0.05 Optimization of culture conditions in magnetically to effectively suspend the cells in the culture system. This controlled bioreactor result suggested that rotation speeds of 30 r/min and The optimized culture conditions of the magnetically magnetic beads density of 5 per ml are recommended controlled bioreactor for the cells were studied by setting for the cultivation of cells in the magnetically controlled different rotation speeds and different magnetic bead bioreactor. densities. The rotation speeds and magnetic bead densi - The homogeneity of liquid mixing within the reaction ties were key parameters that influenced the property of vessel was evaluated by color fading test. Results showed magnetically controlled bioreactor. K562 cells were used that the entire culture system completely mixed while the as model and cultured with different rotation speeds and crossbar rotates five times (Fig. 5C). The results indicated magnetic bead densities. We confirmed that the expan - that the magnetic beads inside the magnetron bioreactor sion of total cells was enhanced while the rotational can effectively mix the culture system. speeds above 10 rpm (Fig. 5A). At 30 rpm, the expansion fold was 18.59 ± 0.74, similar to 40 rpm, but significantly Characterization of flask and bioreactor for NK‑92 cells higher than the expansion fold of rotation speed at 20 r/ expansion min. Given that cells were incapable of suspending in After demonstrating the viability of ex vivo culture of the culture system at low rotational speeds, the optimal suspension cells, we further evaluated the NK-92 expan- number of magnetic beads in the magnetically controlled sion process in the magnetically controlled bioreactor. bioreactor need further determined. The expansion After 8 days of culture, these cells in the magnetically folds of K562 cells were 18.59 ± 0.74 and 19.17 ± 1.63 at controlled bioreactor were dispersive and translucent, 5 beads/ml and 6 beads/ml of magnetic bead, which was with clear edges. However, the flask conditions resulted significantly higher than the expansion with magnetic in aggregation and the single cells were small and dim bead density of 4 beads/ml (Fig. 5B). This result may be (Fig. 6). The result may be that bioreactors provide a due to the fact that the magnetic bead density is too low unified environment for cells. In the T25 flask, however, Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 8 of 11 Fig. 5 Optimization of culture conditions in magnetically controlled bioreactor. A NK‑92 cell expansion fold at different rotation speeds. B NK ‑92 cell expansion fold at different magnetic beads densities. C Images of the effect of mixing medium in the rotational frequency of the crossbar Fig. 6 Morphology of NK‑92 cells after 8 days of culture. A magnetically controlled bioreactor. B culture flask these cells sank to the bottom of the culture flask due to to 6 but decreased on day 8. Along with the changes of a lack of mixing. The microenvironment around cultured viable cell density, NK-92 cells in the magnetically con- cells is heterogeneous (Curcio et al. 2012; Sadeghi et al. trolled bioreactor exhibited higher specific growth rate 2011). than that in T 25 culture flask (Fig. 7C). Meanwhile, sig- Cell viabilities of the magnetically controlled biore- nificantly increased expansion was observed after in the actor with intermittent magnetic fields and T25 cul - magnetically controlled bioreactor (67.71 ± 10.60 folds) ture flask without magnetic fields were both above 90% and flask (22.41 ± 1.19 folds) cultures (Fig. 7D). To inves- that during the 8-day culture period (Fig. 7A). And the tigate the physiological function of expanded NK-92 cells maximum viable cell densities in the magnetically con- in bioreactor, cell phenotype and cell killing activity were 5 − + trolled bioreactor reached 8.04 ± 0.77 × 10 cells/ml, sig-assessed. CD3 CD56 frequencies showed no signifi - nificantly higher than 5.17 ± 0.24 × 10 cells/ml in T25 cant difference between the bioreactor and flask cultures. flask (Fig. 7B). Moreover, the specific growth rate (μ) of Similarly, there is no significant difference in the killing NK-92 cells was determined based on Eq. (1). The μ of activity of the NK-92 cells against the K562 cells between NK-92 cells in the magnetically controlled bioreactor and the experimental group and the control group. Taken T25 culture flask were increasing gradually from day 0 together, these results indicated that the magnetically Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 9 of 11 Fig. 7 Time profiles of ex vivo expansion and physiological function of NK92 cells in culture flask and the magnetically controlled bioreactor within 8 days (*compared with static cultures, P < 0.05). A Cell viability. B Viable cell density. C Specific growth rate of total cells. D Expansion folds of total − + cells. E The percentage of CD3 CD56 cells of ex vivo‑ expanded CIK cells. F Cytotoxic capacity of ex vivo‑ expanded CIK cells controlled bioreactor could improve NK-92 cell expan- was a potent method to overcome this disadvantage, − + sion without loss of C D3 CD56 cells and impairment of however, the growth of immune cells may be impaired as cytotoxic capacity (Fig. 7E, F). the increasing shear force in the dynamic culture system Due to the limited number of immune cells, in vitro (Badenes et al. 2016; Liu et al. 2006). In the present study, expansion of cells is required for successful cancer immu- a magnetically controlled bioreactor using magnetic notherapy. Conventional culture regimens were mainly bead agitation was developed for NK-92 cells expan- performed in static culture with flasks and gas-perme - sion. The bioreactor realized homogeneous distribution able bags that lack of concern for process parameters, of the environment through the dynamic magnetic field resulting in unstable quality and quantity of cell-products and magnetic bead. Unlike conventional agitated bio- and poor reproducibility. Dynamic suspension culture reactors, the agitator of the bioreactor does not require Liu et al. Bioresources and Bioprocessing (2022) 9:50 Page 10 of 11 Competing interests direct contact with the outside environment, avoiding The authors declare that they have no competing interests. bacterial contamination (Rodling et al. 2018). And that, the agitator of the bioreactor is spherical, which reduces Received: 31 December 2021 Accepted: 8 April 2022 the shearing force of the fluid generated during opera - tion. In addition, intermittent magnetic fields promoted cell expansion while maintaining cell viability and cel- References lular function, though it was not fully deciphered. That Badenes SM, Fernandes TG, Rodrigues CAV (2016) Microcarrier‑based plat ‑ might also account for the enhanced amplification of forms for in vitro expansion and differentiation of human pluripotent NK cells by the magnetic bioreactor. And it is necessary stem cells in bioreactor culture systems. J Biotechnol 234:71–82 Bröker K, Sinelnikov E, Gustavus D (2019) Mass production of highly active NK to explore the mechanism by which the magnetic field cells for cancer immunotherapy in a GMP conform perfusion bioreactor. affects the cells in the following research. In conclusion, a Front Bioeng Biotechnol 7:194 magnetically controlled bioreactor for ex vivo expansion Canseven AG, Seyhan N, Mirshahidi S (2006) Suppression of natural killer cell activity on Candida stellatoidea by a 50 Hz magnetic field. Electromagn of NK-92 cells was designed, providing a novel model for Biol Med 25:79–85 expansion of immune cells in the future. Collignon M‑L, Delafosse A, Crine M (2010) Axial impeller selection for anchor ‑ age dependent animal cell culture in stirred bioreactors: Methodology based on the impeller comparison at just‑suspended speed of rotation. Conclusions Chem Eng Sci 65:5929–5941 In this work, a magnetically controlled bioreactor system Curcio E, Piscioneri A, Salerno S (2012) Human lymphocytes cultured in 3‑D with a floating magnetic mixer controlled by an inter - bioreactors: influence of configuration on metabolite transport and reac‑ tions. Biomaterials 33:8296–8303 mittent magnetic field was constructed and successfully De Seze R, Bouthet C, Tuffet S (1993) Eec ff ts of time ‑ varying uniform magnetic used to culture NK-92 cells. While maintaining the vital- fields on natural killer cell activity and antibody response in mice. Bioel‑ ity and function of NK-92 cells, the bioreactor achieved ectromagnetics 14:405–412 Dini L, Abbro L (2005) Bioeffects of moderate ‑intensity static magnetic fields efficient expansion that was superior to the traditional on cell cultures. 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Bioresources and Bioprocessing – Springer Journals
Published: May 3, 2022
Keywords: NK-92 cells; Bioreactor; Magnetic field; Ex vivo expansion
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