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Wang et al. Microstructures 2023;3:2023042 https://dx.doi.org/10.20517/microstructures.2023.46 Page 5 of 16

Triplicate samples were used for each measurement.

Anticancer effect of Fe O NPs in different matrices
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The anticancer effect of free Fe O NPs, agarose/Fe O hydrogels, and gelatin/Fe O porous scaffolds was
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investigated by incubating breast cancer cells in the different matrices under AMF irradiation [55-61] . Three
culture modes were used to simulate the cells directly adhered to, near, or far away from the matrices
[Figure 1]. The cells were seeded in wells of cell culture plates, and then free Fe O NPs, agarose/Fe O
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hydrogels, or gelatin/Fe O porous scaffolds were added to the adhered cells. The hydrogels and porous
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scaffolds were sitting on the adhered cells (sitting mode), which simulated the cells near the matrices. The
second mode was a transwell mode by seeding cells in the bottom wells of the transwell plates and placing
the hydrogels and porous scaffolds in the inserts, which simulated the cells far away from the matrices. The
third mode was an adhesion mode by seeding cells on the hydrogels or in the porous scaffolds to allow the
cells to adhere to the hydrogels or in the pores of the porous scaffolds, which simulated the cells directly
adhering to the matrices.
Anticancer effect of free Fe O NPs
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The free Fe O NPs could only be added in the culture medium. Therefore, only the sitting mode was used
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for investigating the anticancer effect of free Fe O NPs. The sub-cultured MDA-MB-231-Luc cells were
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harvested and resuspended in a culture medium at a concentration of 2.5 × 10 cells mL . A 200 μL cell
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suspension solution was seeded in the wells of a 48-well plate (5 × 10 cells well ). After culture in a
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humidified incubator (5% CO , 37 °C) for 24 h, the culture medium was removed, and another 200 μL fresh
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culture medium was added. Then, 300 μL medium, without or with free Fe O -5, free Fe O -10 and free
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Fe O -20, was added to the wells, respectively. After co-incubation for 2 h, the wells were exposed to AMF
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(frequency: 373.6 kHz of; field intensity: 130 Gauss) for 10 min. Cell viability before and after AMF
irradiation was visualized by live/dead staining and quantitatively analyzed by WST-1 assay. Triplicate
samples were used for each measurement.
Anticancer effect of agarose/Fe O hydrogels
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The three culture modes were used for the investigation of the anticancer effect of the agarose/Fe O
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hydrogel. For the sitting mode, the MDA-MB-231-Luc cells were seeded and cultured in the wells of a 48-
well plate, as mentioned above. After the culture medium was changed with another 200 μL fresh medium,
the agarose and agarose/Fe O hydrogel discs (Φ10 mm × H4 mm) were placed on the cells. After co-
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incubation for 2 h, the wells were exposed to AMF (frequency: 373.6 kHz of; field intensity: 130 Gauss) for
10 min. Cell viability was investigated by live/dead staining and WST-1 assay before and after AMF
irradiation. Triplicate samples were used for each measurement.
For the transwell mode, the MDA-MB-231-Luc cells were seeded in the centers of the wells of 24-well plate
by using donut-shaped silicone rings (inner diameter 10 mm, outer diameter: 16 mm, height: 2 mm). The
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seeded cell number was the same (5 × 10 cells well ). The agarose/Fe O hydrogel discs were placed on the
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transwell inserts and co-cultured with the cells on the bottom wells. The transwell plates containing cells
and discs were irradiated by AMF (frequency: 373.6 kHz; magnetic field: 130 Gauss) for 10 min. Before and
after AMF irradiation, cell viability was investigated, as mentioned above. Triplicate samples were used for
each measurement.
For the adhesion mode, the agarose/Fe O hydrogel discs (Φ10 mm × H4 mm) were put in the wells of a 48-
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well plate. Subsequently, 200 μL of cell suspension solution was seeded on the agarose/Fe O hydrogel discs.
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After 2 h incubation, the plates were exposed to AMF (frequency: 373.6 kHz; magnetic field: 130 Gauss) for