Figure 1 illustrates our proposed and the conventional cell detachment method involved in the subculture process (Thermo Fisher Scientific, 2024; Vanderbilt University, 2024).

In the proposed method, as illustrated in Figure 1A, trypsin is initially added to the cell culture vessel. The cells are then incubated briefly to partially degrade the proteins that mediate cell adhesion. Prior to complete cell detachment, trypsin is removed and fresh medium is introduced. This process results in the cells being suspended in the culture medium with reduced adhesion. Subsequently, the cells are detached through the application of a physical stimulus, specifically forced vibration of the culture vessel.

In the conventional method, as shown in Figure 1B, trypsin is added to the cell culture vessel. The cells are then incubated for several minutes to facilitate the degradation of proteins that mediate cell adhesion to the culture substrate. Complete detachment of the cells is achieved through tapping the culture vessel or pipetting. Following detachment, the enzymatic activity of trypsin is halted by the addition of culture medium. To remove trypsin, the cell suspension is transferred to a centrifuge tube and subjected to centrifugation. The supernatant is then replaced with fresh medium, and the resulting cell suspension is prepared for reseeding into a new culture vessel.

In this study, a 60-mm dish was utilized as a culture vessel, and cell detachment was achieved through forced vibration generated by a shaker (BT300 Vibration Speaker 36W, Adin) (Figure 2). Vibrations of 500 Hz, 1000 Hz, and 2000 Hz were employed during the experiments. Amplitudes at the center of the dish were measured using a laser Doppler vibrometer (Laser Vibrometer LV-1800, Ono Sokki). Given that the maximum amplitude achievable by the shaker at 2000 Hz was 2.08 µm, the vibration conditions for different frequencies were adjusted as outlined in Table 1 to ensure that the maximum acceleration at the center of the dish was approximately equal across all frequencies.

We utilized the Chinese hamster ovary-K1 (CHO-K1) cell line in this study. The CHO-K1 (RCB0285; Riken Bio Resource Center) were cultured in Ham’s F-12 Nutrient Mix (11,765,054; Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (2917354H; MP Biomedicals) and 2% Antibiotic-Antimycotic (100) (15,240,062; Gibco, Thermo Fisher Scientific) at 37 ° C and 5% CO 2 . Cells were passaged twice a week using 0.05% trypsin-EDTA (25,300,054; Gibco, Thermo Fisher Scientific). For the experiments, cells were prepared at a density of 5.0 × 10 ⁵ live cells per 60-mm dish (Iwaki, AGC Techno Glass) and incubated for 48 h. To determine cell counts, dead cells were stained with 0.4% Trypan blue solution (15,250,061; Gibco, Thermo Fisher Scientific), and the numbers of live and dead cells were quantified using a hemocytometer (A116; Asone).

To determine the optimal trypsinization time required to weaken cell adhesion in the proposed method, we conducted experiments to identify the time at which cells begin to detach. Initially, the prepared cells were washed twice with 1 mL of phosphate-buffered saline (PBS). Subsequently, 1 mL of 0.05% trypsin-EDTA was added to the dish, and the cells were incubated at 37 ° C with 5% CO 2 . After incubation, the trypsin-EDTA solution was collected. Trypsinization times were varied from 0.5 to 2 min in 0.5-min increments. Detached cells were then collected using a pipette. The cells remaining on the dish surface were subjected to an additional 3-min trypsinization, after which they were pipetted and collected. It should be noted that the specified trypsinization times refer to the incubation period, with an additional 1 minute required for handling before and after incubation, thus ensuring a constant total operation time. The number of cells collected during the initial trypsinization (denoted as N ₁ ) and the number collected during the second trypsinization (denoted as N ₂ ), representing the number of cells remaining after the initial trypsinization, were counted. The ratio of N ₁ to the total number of cells (N ₁ + N ₂ ), denoted as R ₁ , was calculated as Equation 1. R 1 = N 1 N 1 + N 2 (1) This ratio was used to evaluate the cell detachment capability at each trypsinization time.

Figure 3 illustrates the values of R ₁ at various trypsinization times. Statistical analysis (cf. 2.7) revealed a significant difference only at a trypsinization time of 2 min. This result suggests that a substantial number of cells began to detach after 2 min of trypsinization. Given that trypsinization primarily affects cell adhesion and that trypsin-EDTA must be thoroughly removed before cell detachment in the proposed method, we selected a trypsinization time of 1.5 min. This time did not exhibit significant differences in detachment ratio against shorter trypsinization time, indicating its suitability for optimal cell detachment.

To evaluate the effectiveness of the proposed method in the centrifugation-free subculture procedure, CHO-K1 cells were detached using the proposed technique. Initially, cells prepared in a 60-mm dish were washed twice with 1 mL of phosphate-buffered saline (PBS). Subsequently, 1 mL of trypsin-EDTA was added, and the cells were incubated at 37 ° C for 1.5 min, as decided in Section 2.4. After removing the trypsin-EDTA solution, 5 mL of fresh medium was added to the dish. The dish was then subjected to forced vibration under the conditions outlined in Table 1. Following the forced vibration, the medium containing suspended cells was collected.

The cells were enumerated at each stage, and the following parameters were defined: the number of cells detached by trypsinization before the forced vibration (N _t ), the number of cells detached by forced vibration (N _v ), and the number of cells remaining on the dish surface after the forced vibration (N _r ). The ratios of these quantities to the total number of cells (N _t + N _v + N _r ) were calculated as Equations 1–4, R t = N t N t + N v + N r (2) R v = N v N t + N v + N r (3) R r = N r N t + N v + N r (4) where, R _t , R _v , and R _r represent the proportions of cells detached by trypsinization, by forced vibration, and those remaining on the dish surface, respectively.

To assess the viability of cells detached using the proposed method, we performed a subculture procedure. Cells were detached under the following conditions: a trypsinization duration of 1.5 min, a forced vibration frequency of 500 Hz, and a vibration duration of 5 min. Following detachment, 2.5 × 1 0 5 cells were plated into 60-mm dishes and incubated for 24, 48, and 72 h. For comparison, cells detached using conventional trypsinization (as illustrated in Figure 1B) were incubated under the same conditions, with a trypsinization duration of 3 min. After incubation, the cells are collected with a trypsinization and enumerated.

Values are expressed as means ± SDs. Statistical significance was evaluated using Student’s t-test for comparisons between two means, and ANOVA and Tukey’s multiple comparisons for comparisons between three or more means. Results with p values of less than 0.05 were considered significant.

Figure 4 illustrates the proportions of cells detached via pre-trypsinization (R _t ), by forced vibration (R _v ), and those remaining adherent to the dish surface (R _r ), at frequencies of 0 Hz, 500 Hz, 1000 Hz, and 2000 Hz, respectively. At 0 Hz, indicating no applied mechanical stimulation, approximately 30% of cells were detached. This detachment is attributed to minor medium flows associated with procedural operations, such as the addition or removal of liquids, or the handling of the dish when placing it in or removing it from the incubator. At frequencies of 500 Hz and 1000 Hz, a higher proportion of cells were detached compared to 0 Hz, suggesting that forced vibration contributed to cell detachment. Conversely, at 2000 Hz, the detachment levels were comparable to those observed at 0 Hz, indicating that the forced vibration at this frequency did not significantly influence cell detachment. Comparing the results at 500 Hz and 1000 Hz, where cell detachment was observed, it is evident that more cells were detached at 500 Hz. These findings imply that the maximum acceleration of forced vibration is not the primary factor influencing cell detachment; rather, the frequency, amplitude of the vibration, and the resultant medium flow are crucial factors.It has been shown that cell detachment is predominantly influenced by the shear flow of the surrounding medium (Kurashina et al., 2019). Additionally, Goldasteh et al. investigated particle removal from a substrate under fluid flow, demonstrating that the drag force magnitude depends on the shear velocity, which is, in turn, proportional to frequency and amplitude (Goldasteh et al., 2013). Based on this understanding, the shear velocity is likely maximized at 500 Hz, decreasing to half and one-quarter of this value at 1000 Hz and 2000 Hz, respectively. From this perspective, the cell detachment results presented in Figure 4 appear to be reasonable.

Focusing on the effect of vibration time, it was observed at 500 Hz that the proportion of cells detached by forced vibration (R _v ) increased with longer vibration times. However, the rate of increase in R _v diminished as vibration time continued to increase. At 1000 Hz, R _v also increased with vibration time from 0.2 min to 5 min, similar to the trend observed at 500 Hz. However, R _v decreased at 25 min. This decrease can be attributed to an anomalously low experimental value at 25 min, which caused a reduction in the mean value and an increase in the standard deviation. When considering this outlier, the mean R _v at 25 min is 74.1%, with a standard deviation of 12.4%. The trend at 1000 Hz shows that the increase in R _v with longer vibration times becomes less pronounced over time. These findings suggest that while increasing vibration time generally leads to greater cell detachment, there is an upper limit to the detachment ratio that depends on the vibration frequency.

The optimal results were achieved with a pre-trypsinization time of 1.5 min, a vibration frequency of 500 Hz, and a vibration duration of 5 min. Under these conditions, cells were exposed to 0.05% trypsin-EDTA for only 1.5 minutes—half the duration typically required for conventional trypsinization—while the cell detachment ratio was effectively controlled by the forced vibration. This approach may offer an additional advantage by potentially eliminating the need for centrifugation in the subculture process. Consequently, it could mitigate the adverse effects of trypsin on cell viability and function (Danika et al., 2006).

Figure 5 presents the cell counts following incubation in the subsequent subculture for cells detached using both methods. A reader may doubt the negative effect of forced vibration on cell viability; however, no statistically significant difference in cell numbers was observed between cells detached by the proposed method and those detached by the conventional method. These results suggest that the proposed method does not have a detrimental effect on cell proliferation, suggesting that the proposed method is compatible for cell passage.