Non-contact culturing of vascular endothelial cells on wall surface following retention using acoustic radiation force and lipid bubbles

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Utilizing the propulsive force acting on cells under ultrasound exposure when the cells were surrounded by lipid bubbles, the conditions of the acoustic field were investigated. First, cells were cultured in the presence of lipids without ultrasound to derive the optimal concentration of lipids. Next, cells were retained on the inner surface of the flow path using various acoustic fields, which include single-focal, multifocal, and bar-shaped fields. After culturing the cells in the path without flow for 24 h, the cultured area of cells were measured to evaluate the series of performance. In the experiment of cell culturing without ultrasound, the cultured area decreased inversely proportional to the lipid concentration, thus deriving the optimal concentration of bubbles. When the bar-shaped fields were used for the retention process, significant cell engraftment was observed compared to other fields, even though the acoustic intensity of SATA (Spatial average temporal average) and the retained area of the cells were similar. Those results suggest that conditions of acoustic field, including the distribution and magnitude of sound pressure according to the flow direction, are dominant for non-contact culturing of cells following retention. We succeeded in culturing cells at desired position on inner wall of the path, regardless of the direction of gravity. Acoustic field Bubble-surrounded cell Lipid bubbles 2D array transducer Time-reversal Vascular endothelial cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction T he development of artificial organs has emerged as a pivotal technology for regenerative medicine, since artificial organs can replicate in vivo disease conditions and transform therapeutic methods by constructing complex structures, such as spheroids or organoids (Lee, et al., 2017 ; Mi, et al., 2019 ; De Bartolo, et al., 2022; Wang, et al., 2019). Because the vascular network is essential to any artificial organ, the three-dimensional fabrication of artificial blood vessels is critical in advancing these technologies. Some studies utilized 3D printing technology to fabricate artificial blood vessels using vascular cells (Hou, et al., 2023 ; Choi, et al., 2023 ; Hu, et al., 2021 ). Typically, cell aggregates were deposited as droplets through inkjet or laser-assisted bioprinting to build cell blocks. However, one important limitation of this approach is its capacity to accurately replicate key features of blood vessels, such as their multiple bifurcations. In case that laminating the cells is left to gravity, which takes time, especially to form tubular structures. Furthermore, fabricating tubular structures with arbitrary shape with diameters less than several mm presents a challenge, primarily due to the requirements involved in layering blocks (Su, et al., 2019 ). To address these challenges, photopolymerization was used to facilitate the construction of smaller artificial blood vessels (Wang, et al., 2020 ; Hoch, et al., 2014 ; Liu, et al., 2021 ). Using this method, the medium containing vascular cells is hardened through exposure to laser light outside the vascular template. While this technique has been successful in creating blood vessels with diameters with several mm, replicating the multi-layered structure of actual blood vessels, including the integumentum, mesothelium, and endothelium layers, remains challenging. Therefore, there is a need for a new technology to create tubular structures that can arbitrarily design the distribution of cells, including situations that defy gravity, and that can be used for multi-layer culturing. In our previous experiments, we manipulated cell dynamics of not only T-cells (Oitate, et al., 2017 ; Oitate, et al., 2018 ; Masuda, et al., 2018 ; Seki, et al., 2019 ; Chikaarashi, et al., 2022 ; Kajita, et al., 2023 ), Colon-26 cells (Shigehara, et al., 2013 ; Demachi, et al., 2015 ) but also vascular endothelial cells (Ito, et al., 2022 ; Ogawa, et al., 2024 ; Noguchi, et al., 2024 ), using bubbles, where the bubble-surrounded cells (BSCs) were produced. Figure 1 explains the senario of this study; difference in propulsive force between a cell and a BSC under ultrasound exposure in Fig. 1 (a), and the trajectory of a BSC passing through the flow under ultrasound exposure in Fig. 1 (b). In the absence of bubbles shown in the upper part of Fig. 1 (a), the propulsive force hardly produced since ultrasound penetrate through the cell since the acoustic impedances of the elastic tissues are between 1.38 MRayls (fat) and 1.70 MRayls (muscle), which is similar to that of the medium (water, 1.5 MRayls). On the other hand, if there are bubbles (gas, less than 0.001 MRayls) attached on the cell surface shown in the lower part of Fig. 1 (a), a propulsive force acting on the cell is enhanced by the traveling wave reflected off the cell surface, attributable to the boundary in acoustic impedance. Furthermore, the oscillation of the bubbles enables the Bjerknes force (Louisnard, et al., 2012; Garbin, et al., 2011 ) to propel the cell in the direction of the traveling wave. As shown in Fig. 1 (b), when traveling wave of ultrasound was exposed to a BSC passing through the flow from a direction different from the flow direction, the trajectory of the BSC was distract in the direction of ultrasound propagation. In case that the multiple BSCs were contained in the flow, there might be some BSCs propelled against the upper wall depending on the relationship between the flow velocity, sound pressure, and the concentration of BSCs. Thus, using ultrasound and bubbles, it would be possible to apply a non-contact external force to the cells in the flow, control their behavior, and fabricate cellular structures in any shape and position in the flow path. Meanwhile, it is necessary to take into account that cell damage occurs due to cavitation caused by the destruction of bubbles under ultrasound exposure. To fabricate a structure made of cells by retaining BSCs, we must confirm that the cells retained on the wall surface of a flow path via an acoustic force, regardless of the direction of gravity, can be engrafted and cultured in situ after retention. Furthermore, considering that the bubbles are attached to the cell surface, it is crucial to determine how the presence of the bubbles affects the cell culture process. This study performed a preliminary cell culture trial following retention using acoustic radiation force and examined the conditions of the acoustic field and cell proliferation. 2. Materials and Methods 2.1 Cells, lipid, and lipid bubbles In this study, we used bovine-derived carotid endothelial cells (Ito, et al., 2022 ; Ogawa, et al., 2024 ; Noguchi, et al., 2024 ) (referred to as “cells”, hereinafter), which were cultured at 37°C with a CO 2 concentration of 5%, using Dulbecco's modified Eagle medium (DMEM, Thermo Fisher Scientific) with 10% fetal calf serum. We also prepared a phospholipid, which consisted of 1,2-distearoyl-sn-glycero-3-phosphatidyl-choline (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-polyethylene glycol 2000 (DSPE-PEG2k) (Suzuki, et al., 2008 ; Wada, et al., 2016 ; Negishi, et al., 2013 ). To compose lipid bubbles (referred to as “LBs”, hereinafter) containing perfluoropropane (PFP, C 3 F 8 ) gas, DSPC:DSPE-PEG2k was dissolved (molar ratio of 94:6) in mixed organic solvents (each containing 4 ml of chloroform). Then, phosphate-buffered saline (PBS) was added to the lipid solution and sonicated before the organic solvent evaporated. The obtained LBs had the size distribution (Ogawa, et al., 2024 ), which occurs naturally through the manufacturing process, with an average diameter of 1 µm and were encapsulated in a liposome using the phosphate buffer solution. Finally, we modified the LBs by conjugating a ligand of cyclic-RGD (cRGD) peptides, which covalently adhere to vascular endothelial cells via the DSPE-PEG on the LB surfaces. The conjugation of DSPE-PEG3.4k-Mal and cRGD ligand was performed during a 1-hour incubation using a rotary mixer. The molar ratio of DSPC:DSPE-PEG2k:DSPE-PEG3.4k-cRGD was 94:5:1. To prepare the LB suspension, 2 ml of liposome suspension (lipid concentration: 1 mg/ml) was diluted to yield the desired PBS concentration. The concentration [mg/ml] was derived from the weight of the lipid divided by the volume of the suspension. In our previous studies (Chikaarashi, et al., 2022 ; Ito, et al., 2022 ; Noguchi, et al., 2024 ), the controllability of the cells, which was evaluated as the amount of cell retention on the wall surface of a flow path, increased in proportion to the LB concentration. However, the presence of surrounding LBs may affect not only the cells damage via cavitation but also the subsequent cell engraftment and culture process. In the following experiments, the cell concentration in the suspension was set to 1.0 × 10 5 /ml, and the LBs were diluted at a concentration within the range of 0–0.5 mg/ml. Here, the cell concentration was expressed with the cell number per ml, whereas the LBs concentration was with lipid weight per ml (Kajita, et al., 2023 ; Ogawa, et al., 2024 ). 2.2 Experimental setup Figure 2 (a) shows the experimental setup for observing the behavior of the cells in BSCs passing through the flow path during ultrasound exposure (Chikaarashi, et al., 2022 ; Kajita, et al., 2023 ; Ito, et al., 2022 ; Ogawa, et al., 2024 ). This setup comprised a fluorescence microscope (Olympus, BXFM with DP74) and the 2D array transducer, which has a central frequency of 3 MHz and 128 elements (Takano, et al., 2020 ; Ushimizu, et al., 2018 ) (12 × 12 elements forming a square, and the four elements at each corner were lacked). The driving equipment (ES1144-1, Microsonic Co., Ltd.) (Chikaarashi, et al., 2022 ; Suzuki, et al., 2017 ; Hosaka, et al., 2013 ; Koda, et al., 2014 ) generated a burst wave with a minimum pulse repetition time and delay pitch of 10 ms and 5 ns, respectively, to electrically oscillate the elements individually. The maximum sound pressure of 400 kPa-pp with a duty ratio of 60% is the output limitation of this equipment. The flow path was placed at the surface of the water. The transducer targeted the observation area in the flow path from a distance of l = 60 mm at the elevation angle of θ = 60°. Figure 2 (b) shows the schematic of the flow path made of polydimethylsiloxane (PDMS), which has the acoustic impedance of 1.03 MRayl at 3 MHz and 37 ˚C (Guillermic, et al., 2019 ). PDMS easily deforms in the behavior of elastic tissues. The inner cavity of the flow path has a length of 30 mm and a rectangular cross-section with a width w p = 2.1 mm and a height h p = 1.5 mm, respectively, as shown in Fig. 2 (c). From the viewpoint of fabricating a tubular structure, the cross-sectional shape of the flow path should be circular. However, in order to accurately measure the distribution of cells on the inner wall using microscopic images, a rectangular cross-section was produced so that the effect of depth of field could be ignored. The thickness of the upper and lower wall of the path was d up = 2.6 mm and d up = 1.4 mm, respectively. To coat the inner surface of the path with a basement membrane, the cavity of the flow path was filled with a mixture of Cellmatrix Type I-C (Nitta Gelatin Co., Ltd.) (Kojima, et al., 2020 ) and pure water, which was allowed to stand for 24 h to enable the collagen fibers to precipitate. The thickness of the membrane was estimated to be less than 10 µm. A BSC suspension was injected during the artificial flow of the medium through the flow path maintained at a velocity of 10 mm/s. The BSCs in the suspension passing through the flow path were exposed to an acoustic field for up to 60 s. The behavior of the BSCs, which were propelled to adhere to the upper surface of the cavity, was optically observed using a fluorescence microscope. To evaluate the cell retention performance in the observation area, the cells were stained using Calcein-AM solution (Dojindo) (Ogawa, et al., 2024 ; Uggeri, et al., 2004 ; Tawakoli, et al., 2013 ) in advance. The cells emitted fluorescence with a wavelength of 515 nm for 490 nm excitation light to distinguish them. The fluorescent images were binarized by applying a threshold based on the average background brightness in 256-scale (8-bit) to obtain a retained area of the cells as S ret . The images were obtained with a 1.25× objective lens (NA 0.04, Olympus). Following the retention process, the flow path was extracted from the water tank to be maintained in a CO 2 incubator for culturing 24 h without turning the flow path upside down. To evaluate the cultured area of cells, the cells were not stained in the above-mentioned retention process. After the suspension in the path was drained to remove floating cells, the flow path was turned upside down to stain the living cells engrafted on the basement membrane. The living cells in the fluorescent images were distinguished from the background and dead cells to obtain a cultured area of the cells as S cul , and the images were obtained with a 5× objective lens (NA 0.15, Olympus). 2.3 Formation of acoustic fields We designed several acoustic fields using the 2D array transducer to form an arbitrary shape of the acoustic field (Xu, et al., 2020 ; Riis, et al., 2024 ). First, a single focal (SF) acoustic field formed a single focal point in the center of the flow path. To extend the irradiation area along the path, a multifocal (MF) acoustic field was produced using tempo-spatial division emission (Takano, et al., 2020 ; Ushimizu, et al., 2018 ; Suzuki, et al., 2017 ), as shown in Fig. 3 (a), where the applied acoustic energy was distributed temporally and spatially. A focal point translates linearly as F i (2 ≤ i ≤ n ) along the y -direction with the spatial interval, r . Figure 3 (b) shows the time chart of the position transition of the focal point, where the repetition transition of the focal point is from F 1 to F n , each of which has emission duration, τ [s]. Number n indicates the number of preservable focal points, where one period of the tempo-spatial division emission is nτ [s]. In the following experiments, the MF acoustic field is denoted by MF n (e.g., MF3 includes three focal points). For comparison with the MF acoustic field, a bar-shaped (BS) acoustic field was produced using the time-reversal method (Fink, 2008 ; Jing, et al., 2012 ), which refocuses an incident acoustic field back to the position of the source, irrespective of the complexity of the propagation medium, using the k-Wave mathematical platform (Treeby, et al., 2012 ). A BS virtual sound source was established in the space to generate the acoustic field as shown in Fig. 4 . Thereafter, the ultrasound propagation was calculated to record the amplitudes and phases at the position of the 2D array. Finally, the recorded amplitudes and phases in all the elements were reproduced. In the following experiments, the BS acoustic field is denoted by BS n , where the bar length in the y -direction corresponds to ( n − 1) r , to be compared with the length of MF n acoustic field. Here, we defined the applied acoustic intensity of the aforementioned acoustic fields by measuring the distributions of sound pressure (Chikaarashi, et al., 2022 ; Noguchi, et al., 2024 ). The measured distribution ( x-y plane) was divided into small grid areas with a width of w . Assuming a mean sound pressure of P ij [Pa-pp] in a small area ( S ij ), an applied acoustic power E [W] can be estimated as follows: In the above equation, Z denotes the acoustic impedance of the medium. Coefficient D indicates the duty ratio in a small area. The applied acoustic intensity of spatial average temporal average (SATA) (Izadifar, et al., 2017 ; Acevedo, et al., 2002) was defined as I SATA = E / S [mW/cm 2 ]. S is the area of the acoustic field defined by the area, where the relative amplitude exceeds − 20 dB of the maximum point. In the following experiments, an identical coordinate of w = 0.5 mm was used. Additionally, the duty ratio was set to D = 0.6, which was fixed due to the device limitation as mentioned above. 2.4 Measurement of acoustic fields Figure 5 shows the normalized distributions of (a) SF, (b) MF3, (c) BS2, (d) BS3, and (e) BS4 acoustic fields. They were measured using an acoustic intensity measurement system (AIMS III, Onda Co., Ltd.) by translating the hydrophone (HNR-1000, Onda Co., Ltd.) (Seki, et al., 2019 ; Chikaarashi, et al., 2022 ; Kajita, et al., 2023 ) in degassed water without the flow path installed. The coordinate in Fig. 5 corresponds to that of the x-y plane ( z = 0), where the flow path was installed in Fig. 2 . In the MF acoustic fields, the emission duration, τ , was set to 0.01 ms, which is faster enough than cell motion. In the MF and BS acoustic fields, the spatial interval, r , was fixed at 3 mm. The beam width, defined as the half-width of the distribution in SF and MF3, was measured at 2.5 mm. In Fig. 5 (b), the three focal points appeared to occur simultaneously since the scanning speed of the hydrophone was approximately 10 mm/s. The BS2 acoustic field has one peak in the distribution shown in Fig. 5 (c), whereas there were two peaks appeared in the BS3 and BS4 acoustic fields as shown in Figs. 5 (d) and (e). Figure 6 shows the measured profiles of acoustic fields of SF, BS2, BS3, and BS4 along the (a) y -axis ( x = z = 0) and (b) x -axis ( y = z = 0). When the maximum sound pressure of SF was 400 kPa-pp, the acoustic intensity of I SATA was 143 mW/cm 2 . The maximum sound pressures in BS acoustic fields were adjusted to establish similar acoustic intensities, which of BS2, BS3 and BS4 were 138, 139, and 141 mW/cm 2 , respectively. Figure 7 shows the measured profiles of the acoustic fields of MF2, MF3, and MF4, where the SF acoustic field translated linearly in the y -direction, preserving the distribution of the SF field. 3. Results 3.1 Cell culture in the presence of lipids without ultrasound Considering that there is a potential for fragments of LBs disrupted by the ultrasound to remain, we examined the cell culture in the presence of LBs and lipids prior to their composition into LBs. The cells were cultured on the lower wall of the flow path shown in Fig. 2 (b) without ultrasound exposure and flow. In this experiment, the “lipids” being referred to are the same components as the LBs without PFP gas. We prepared two types of cell suspensions (lipid and LB suspensions), with lipid concentrations varying between 0 (only cells) and 0.4 mg/ml, where the cRGD ligand was incorporated into the lipid solution. After injection of suspension (0.16 ml) with the cell concentration of 1.0 × 10 5 /ml onto the bottom of the flow path, the cells were cultured in a CO 2 incubator to measure the S cul . Figure 8 shows the fluorescent images after 24 h of incubation. Based on the observed increase in lipid concentration, although the cultured area of the cells, S cul , decreased under both conditions, the presence of LBs negatively affected the culturing. The cells cultured with LBs were hardly visible at the concentration of 0.4 mg/ml. Figure 9 compares the S cul between the suspension of lipids and LBs. When the lipid concentration was below 0.3 mg/ml, no significant difference was observed between them until the LB concentration reached 0.4 mg/ml. In our previous study on retaining BSCs in the flow (Ito, et al., 2022 ), we observed that the retained area of the BSCs increased proportionally to the LB concentration and was saturated at 0.4 mg/ml. To prioritize retaining as many cells as possible, we set the LB concentrations to 0.3 mg/ml for the subsequent experiments. 3.2 Cell retention with various acoustic fields Next, we compared the cell retention performance using the prepared acoustic fields. A BSC suspension with the cell and LB concentrations of 1.0 × 10 5 /ml and 0.3 mg/ml, respectively, was flowed through the flow path at a flow velocity of 10 mm/s and an irradiation duration of 60 s. Figure 10 shows the transition of the distribution of the retained cells with an acoustic field of BS2 and the maximum sound pressure of 260 kPa-pp, which was shown in Fig. 6 (a). It can be confirmed that the retained area of the cells were gradually increased. Also, the cells that were not retained were propelled aside and accumulated in the corners of the path. Figure 11 shows fluorescent images 60 s after starting the retention with the acoustic fields of (a) SF, (b) MF3, (c) BS3, and (d) BS4, where the maximum sound pressures were adjusted to establish similar acoustic intensities (135–155 mW/cm 2 ) as appeared in Figs. 6 and 7 . The area of cell retention varied with the acoustic field and became longer along the flow direction in the MF3 and BS acoustic fields compared with that in the SF acoustic field. Figure 12 compares the retained area S ret versus the acoustic intensity I SATA for 30 different acoustic fields with various sound pressures. In the SF acoustic field, the increase in the retained area was limited as the acoustic intensity. In the MF acoustic fields, the retained area of the cells increased in proportional to I SATA , whereas the retained areas were inferior to that in the SF acoustic fields, except for MF3. Comparing between the shapes of acoustic fields, the BS acoustic fields were more effective to retain more cells than others. 3.3 Cell culturing following retention The retained cells were continuously cultured in situ in the flow path without ultrasound exposure. The flow path was left undisturbed for 30 min before being placed in a CO 2 incubator for 23.5 h, allowing the cells to adhere to the upper surface of the flow path. Figure 13 shows the fluorescent images of cells cultured for 24 h after retention with the same acoustic fields as in Figs. 10 and 11 . Cultured cells were hardly found using the acoustic fields of SF and MF3, indicating that the cells were either almost extinct or had detached from the surface of the flow path, as well as other MF acoustic fields. In contrast, cultured cells were widely spread along the flow path using the BS acoustic fields. Figure 14 compares the S ret and S cul with various acoustic fields, which include all BS acoustic fields appeared in Fig. 12 , and SF and MF n acoustic fields with I SATA ranged between 135 and 155 mW/cm 2 . Furthermore, BS acoustic fields were classified by I SATA into three ranges: 135 to 155, 100 to 110, and 70 to 80 mW/cm 2 . It is obvious that the BS acoustic fields were superior in both cell retention and cell culturing with I SATA ranged between 135 and 155 mW/cm 2 , although a similar retained area of the cells was observed with the MF3 field. Comparing between the BS acoustic fields, in BS3 and BS4, both the occupied areas of the cells of S ret and S cul were almost proportional to the acoustic intensity I SATA . In BS2, in contrast, there was little correlation between S ret and S cul , while S ret tended to be high. Figure 15 indicates the statistical analysis of the cultured area of the cells shown in Fig. 14 , comparing between the acoustic fields and the value ranges of I SATA . In each of the acoustic fields of BS2, BS3, and BS4, the retained area of the cells significantly increased as I SATA increased. Furthermore, comparing the acoustic fields with I SATA of 135–155 mW/cm 2 , although there was no significant difference between BS2 and BS3 and between BS3 and BS4, there was a significant difference between BS2 and BS4. These results suggest that there are optimal conditions for the acoustic field, such as the distribution of acoustic intensity and/or sound pressure, for non-contact culturing of cells following retention. 4. Discussion This study investigated the possibility of the non-contact culturing of vascular endothelial cells on the wall surface following retention using acoustic force and LBs. First, for the results shown in Figs. 8 and 9 , the effects of LBs and lipids on the cell culture without ultrasound were evaluated. As the concentration of LBs increased, the cell culture performance significantly decreased with an increase in the LB and lipid concentrations, where the surrounding LBs inhibited cell adhesion to the wall. In our previous study (Ito, et al., 2022 ), which focused on the retention of BSCs on an elastic wall using acoustic radiation force, the area of cell retention reached saturation at an LB concentration of 0.4 mg/ml. This suggests that the LBs adhering to the cell surface nearly covered the entire surface at this concentration. Therefore, an LB concentration of 0.3 mg/ml was the less saturated condition on the cell surface, where a cell was not isolated from its surrounding medium. Contrarily, when the LBs were replaced with lipids (without gas), although lipid adhesion to the cell surface occurred similarly to the LBs, the physical volume of the lipids, compared with that of LBs, probably limited their effect on the cell culture. In the subsequent experiments, because of ultrasound exposure during the retention process, the LB concentration was expected to be less than 0.3 mg/ml due to the destruction of bubbles. However, it might be necessary to remove the lipids around the cells in future studies on culturing. Here, it should be noted that the lipid concentration used in this study ranged 0.1–0.4 mg/ml, which was comparable to suspensions used in clinical applications (Sridharan, et al., 2021 ). As shown in Figs. 10 and 11 , the cell retention performance was executed with various acoustic fields. Using the MF and BS fields, we succeeded to extend the retained area of the cells along the flow direction compared to the SF field. The reason why the sound pressure distribution shown in Fig. 5 differs from the distribution of the retained cells shown in Figs. 10 and 11 is because the cells were propelled away from higher sound pressure and retained to form the contour of the sound pressure distribution. In the MF fields, the retained area of the cells tended to increase in proportion to the acoustic intensity as shown in Fig. 12 . In contrast, in the BS fields, it was possible to retain more cells at low acoustic intensities than other fields. Here, it should be mentioned that the distributions of cultured cells shown in Fig. 13 were not preserved the distributions of the retained cells. Especially in the BS fields, the shape of the cell clusters had a strong tendency to be stacked in the corners of the cross-section of the path, where the flow was stagnant. This may be one of the reason of larger retained areas with the BS fields than others, as shown in Fig. 12 . The important result in this research was the success of non-contact culturing of cells following retention using the BS fields, as shown in Fig. 13 , which indicates that the cells were not only sufficiently propelled and attached to the wall, but also survived or recovered from damage (Ito, et al., 2022 ; Ogawa, et al., 2024 ) under the effects of cavitation caused by the destruction of LBs. In our previous study (Wada, et al., 2016 ) using the same LBs as in this study, higher sound pressure than 300 kPa-pp accelerates the damage to the cells due to the destruction of LBs. Considering that the cells accumulated in the corners were detached during the culture process, the cultured area of the cells with BS fields shown in Fig. 14 indicates that the cells have engrafted and proliferated. Due to the constraints of the driving equipment, acoustic intensities more than 150 mW/cm 2 were not applied in the BS fields. It is believed that more cells can be cultured in proportion to the acoustic intensity. On the other hand, higher sound pressures may increase damage to cells due to cavitation effects. We are going to continue investigating the relationship between acoustic field conditions and cell culture efficiency. The above results suggest that the 3D shape of the acoustic field, particularly the distribution and magnitude of the sound pressure, influenced the boundary of culturing of cells following retention in the flow. In the SF field shown in Fig. 6 (a), the sound pressure decreases with distance from the focal point. If the magnitude of the sound pressure is dominant for the non-contact culturing, the distribution of cells should form a ring shape. However, cultured cells were barely observed with the SF field or the MF fields as shown in Fig. 13 . Therefore, in the evaluation of the non-contact culturing of cells following retention, it is necessary to consider not only the magnitude of sound pressure but also the spatial variation, which is the gradient of sound pressure in the direction of the suspension flow. Finally, we discuss the limitations of this method and future developments. Since the cells are propelled by acoustic radiation force to travel a distance through the flow, there might be the minimum size of blood vessels to be fabricated with this method, which is supposed to be a diameter of around 100 µm. In further developments, we are going to reveal quantitative investigation of these limitations. Since the cells flowed in one way through the flow path, there was a limitation to the retention efficiency relative to the number of injected cells. To enhance the retained cells on the wall, the experimental setup should be improved, for example, by the circulation of a suspension. Furthermore, since the cells were suppressed on a flat plane, we are going to consider to establish uneven surface of the inner wall according to the shape of the acoustic field. In addition, a series of procedures should be extended using a flow path with a circular cross-section and bifurcations. Further research will focus on the development of artificial blood vessels by introducing a variation of acoustic fields corresponding to cell dynamics to realize the non-contact culturing of cells in a wider area. The results presented in this article can therefore serve as a basis for future developments. 5. Conclusions This study achieved the non-contact culturing of vascular endothelial cells on the inner surface of the flow path, following the cell retention process enabled by acoustic force and LBs. First, the effect of lipids and LBs upon culturing the cells was investigated to derive an optimal LB concentration to facilitate retention and culturing. Then, the cells were retained using various acoustic fields, which were a single-focal (SF), multifocal (MF), and bar-shaped (BS), to compare cultured area of the cells. As the results, significant cell engraftment was confirmed using the BS acoustic fields compared to other fields, even when the acoustic intensity ( I SATA ) and the retained area of the cells were similar. This study suggested that the non-contact culturing of cells following the retention process required the distribution of the sound pressure dependent on the shape of the acoustic field, and the gradient in the flow direction. Declarations Competing Interests: The authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Author Contribution Author Contributions S.W., Y.I. and K.M. designed the experiments. Y.M., D.O. and R.S. contributed the preparation of the experiments. S.W., A.N. and K.K. performed the experiments. S.W. and K.M. wrote the main manuscript text. All authors reviewed the manuscript. Y.M., R.S. and K.M. secured the funding. Acknowledgment This work was supported in part by a grant from the Japan Society for the Promotion of Science (JSPS) through KAKENHI Grant Number 20H04547, and the Terumo Life Science Foundation. The authors express a great thankfulness to Dr. Hiroya Takada, in Nippon Medical School, Japan, for technical assistance. References S. Lee, S.P. Jin, Y.K. Kim, G.Y. Sung, J.H. Chung, J.H. Sung, Construction of 3D multicellular microfluidic chip for an in vitro skin model, Biomed. Microdevices, 19 , 22 (2017). S. Mi, S. Yang, T. Liu, Z. Du, Y. Xu, B. Li, W. Sun, A Novel Controllable Cell Array Printing Technique on Microfluidic Chips, IEEE Trans. Biomed. Eng., 66 , 2512-2520 (2019). L. De Bartolo, D. Mantovani, Bioartificial Organs: Ongoing Research and Future Trends, Cells Tissues Organs, 211 , 365-367 (2022). X. 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Das-Gupta, The measurement of the spatial average temporal average intensity I sata and ultrasonic power W in composite ultrasonic transducers for medical application, Ultrasonics, 40 , 819-821 (2002). A. Sridharan, J.R. Eisenbrey, F. Forsberg, N. Lorenz, L. Steffgen, A. Ntoulia, Ultrasound contrast agents: microbubbles made simple for the pediatric radiologist, Pediatr Radiol., 51 (12), 2117-2127 (2021). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Biomedical Microdevices → Version 1 posted Editorial decision: Revision requested 19 Jun, 2025 Reviews received at journal 19 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviews received at journal 01 Jun, 2025 Reviewers agreed at journal 22 May, 2025 Reviewers invited by journal 20 May, 2025 Editor assigned by journal 19 May, 2025 Submission checks completed at journal 13 May, 2025 First submitted to journal 10 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6635839","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459277213,"identity":"bcb500eb-7864-4714-aff0-6feed0316629","order_by":0,"name":"Shunya Watanabe","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shunya","middleName":"","lastName":"Watanabe","suffix":""},{"id":459277214,"identity":"7f46e61c-d5f7-43c9-84c8-227211f16e97","order_by":1,"name":"Yuki Ichikawa","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Ichikawa","suffix":""},{"id":459277215,"identity":"38b68721-52dc-4abb-be8f-5b0c37bab12e","order_by":2,"name":"Kota Konishi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Kota","middleName":"","lastName":"Konishi","suffix":""},{"id":459277216,"identity":"098213ad-c56a-45f4-863d-dd40216406c8","order_by":3,"name":"Ayako Noguchi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ayako","middleName":"","lastName":"Noguchi","suffix":""},{"id":459277217,"identity":"bfbced92-78e0-4944-b394-468bf35e1e4c","order_by":4,"name":"Yoshitaka Miyamoto","email":"","orcid":"","institution":"National Center for Child Health and Development","correspondingAuthor":false,"prefix":"","firstName":"Yoshitaka","middleName":"","lastName":"Miyamoto","suffix":""},{"id":459277218,"identity":"4c02268f-55cb-43ea-b477-4fd133fb53b7","order_by":5,"name":"Daiki Omata","email":"","orcid":"","institution":"Teikyo University","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Omata","suffix":""},{"id":459277219,"identity":"6f02a041-b045-45a2-8312-2851091b9a60","order_by":6,"name":"Ryo Suzuki","email":"","orcid":"","institution":"Teikyo University","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Suzuki","suffix":""},{"id":459277222,"identity":"bd210794-a560-400c-978a-a5002d9735bd","order_by":7,"name":"Kohji Masuda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYLACHgYbFH4CMVrSgCQzXDFRWg6jaMEPdNuPP5N4U3Perp+B/5jEzx8M8vwNDM8e4NNidibHTHLOsdvJMxuY2SR7EhgMZxxgSDfAq+VADps0D9vtZIMDzGwSPAkMjBsYGNIk8Go5//yZNM+/c2Atkn8SGOwJa7mRYCbN23bADqRFGmhLIhFa3hhbzu1LTpBsZja2lkmTSJ5xmJBfzqc/vPHmm509P3vjw5tvbGxs+9t70h7g0wIDiQ3MYBroJGaeNGJ0MNgjsdmPEaVlFIyCUTAKRgwAAMLNQo/YMUDUAAAAAElFTkSuQmCC","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":true,"prefix":"","firstName":"Kohji","middleName":"","lastName":"Masuda","suffix":""}],"badges":[],"createdAt":"2025-05-10 16:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6635839/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6635839/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10544-025-00771-0","type":"published","date":"2025-10-31T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83297499,"identity":"37a8739d-8277-4c91-ab07-99fc685befb9","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":244808,"visible":true,"origin":"","legend":"\u003cp\u003eSenario of this study: (a) Production of propulsive force with a cell without microbubbles (above) and a bubble-surrounded cell (BSC, below), and (b) trajectory of a BSC passing through the flow under ultrasound exposure.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/327c4bb0030ea3df8fb13ab2.png"},{"id":83298336,"identity":"32e76b2c-c47b-4478-916d-e03220344c74","added_by":"auto","created_at":"2025-05-22 14:29:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111809,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup for observing the behavior of BSCs in the flow during ultrasound exposure. (a) Entire view, (b) flow path design, and (c) cross section of the path.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/26f0a86c8ce9859a6097dd4a.png"},{"id":83299902,"identity":"af1a934f-5b03-47fa-b687-b002ef7287ab","added_by":"auto","created_at":"2025-05-22 14:45:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67790,"visible":true,"origin":"","legend":"\u003cp\u003eDefinition of multifocal (MF) acoustic field. (a) Spatial distribution of focal points along the flow path. (b) Time chart of repetition transition of the focal point.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/521c4415888574d05ceb0002.png"},{"id":83298971,"identity":"b4ef98c9-cf68-46e2-ac54-5219cf7ed788","added_by":"auto","created_at":"2025-05-22 14:37:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22977,"visible":true,"origin":"","legend":"\u003cp\u003eDefinition of bar-shaped (BS) acoustic field explaining the length along the flow path.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/c208e22487cf805d8e97372c.png"},{"id":83297503,"identity":"8c9477e0-1683-457d-b821-9ee6240d5b71","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":711549,"visible":true,"origin":"","legend":"\u003cp\u003eMeasured distributions of the acoustic fields of (a) SF, (b) MF3, (c) BS2, (d) BS3, and (e) BS4, applied on the flow path.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/336724643c0dd9373387ecef.png"},{"id":83297507,"identity":"ede88ba1-735c-454c-9f15-cccdc60d9161","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128553,"visible":true,"origin":"","legend":"\u003cp\u003eMeasured profiles of the acoustic fields of SF, BS2, BS3, and BS4 along the (a) \u003cem\u003ey\u003c/em\u003e-axis (\u003cem\u003ex\u003c/em\u003e = \u003cem\u003ez\u003c/em\u003e = 0) and (b) \u003cem\u003ex\u003c/em\u003e-axis (\u003cem\u003ey\u003c/em\u003e = \u003cem\u003ez\u003c/em\u003e = 0).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/f0ffeeb082938162136971d4.png"},{"id":83298339,"identity":"2e709ce2-b26e-4c2d-b6bb-c676ef427f1b","added_by":"auto","created_at":"2025-05-22 14:29:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":68218,"visible":true,"origin":"","legend":"\u003cp\u003eMeasured profiles of the acoustic fields of MF2, MF3, and MF4 along the \u003cem\u003ey\u003c/em\u003e-axis (\u003cem\u003ex\u003c/em\u003e= \u003cem\u003ez\u003c/em\u003e = 0).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/e05689a26e68ef0fa08f0eb6.png"},{"id":83298977,"identity":"71e19165-906a-46ff-b2c9-174500209a87","added_by":"auto","created_at":"2025-05-22 14:37:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":919724,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent images after culturing the cells with the concentration of 1.0 × 10\u003csup\u003e5\u003c/sup\u003e /ml for 24 h in the presence of lipids (left) and lipid bubbles (right) without ultrasound exposure.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/dc149551f7b2a433ac6f56b1.png"},{"id":83297517,"identity":"938cfc3a-dbaa-42bd-b48b-fde36ccbb0f4","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":78747,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the occupied areas of the cultured cells in the presence of lipids and lipid bubbles without ultrasound exposure.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/25250c6b616526a92232bf10.png"},{"id":83297510,"identity":"6aabd8a4-5ade-44d3-a263-62ecac07874f","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":241416,"visible":true,"origin":"","legend":"\u003cp\u003eTransition of the distribution of retained cells with an BS2 acoustic field of the maximum sound pressure of 260 kPa-pp, where the distribution of sound pressure was shown in Fig. 6.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/69d8c1289e3e5853ad478fb0.png"},{"id":83297512,"identity":"ceab3293-1f0f-455b-87bd-f2997b00ae8e","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":283413,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent images of retained cells 60 s after starting the retention with the acoustic fields of (a) SF, (b) MF3, (c) BS3, and (d) BS4, where the distributions of sound pressure were shown in Figs. 6 and 7.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/19581e7d52255531351e0d7d.png"},{"id":83298344,"identity":"8be7ef16-7ef8-4fc9-b2cd-5d3e68d4c6f3","added_by":"auto","created_at":"2025-05-22 14:29:37","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":48412,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between\u003cem\u003e I\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA.\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e \u003c/sub\u003eand \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e with various conditions of SF, MF\u003cem\u003en\u003c/em\u003e, and BS\u003cem\u003en\u003c/em\u003e acoustic fields.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/e3e60114156264a78b54e8f1.png"},{"id":83297522,"identity":"eef02a4c-e63c-4ba0-bbc2-844eb801efad","added_by":"auto","created_at":"2025-05-22 14:21:37","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":460255,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent images of cells cultured for 24 h after the process of retention with the SF, MF3, BS2, BS3, and BS4 acoustic fields, where the acoustic intensity \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e ranged between 135 and 155 mW/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/5c6c5715707f1e21864d9ea0.png"},{"id":83298354,"identity":"85b9d02d-db53-41d2-aebe-a0372d8ba4ac","added_by":"auto","created_at":"2025-05-22 14:29:37","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":167034,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the retained area of the cells (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e) and the cultured area of the cells (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e) using SF, MF and BS acoustic fields. In BS acoustic fields, they were classified by \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e into three ranges: 135 to 155, 100 to 110, and 70 to 80 mW/cm\u003csup\u003e2\u003c/sup\u003e. In SF and MF acoustic fields, \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e ranged between 135 and 155 mW/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/a2211d1e52e59d1d031fa0e4.png"},{"id":83298981,"identity":"bfe120ed-c7ec-4ebf-a3fa-8125cf2fec53","added_by":"auto","created_at":"2025-05-22 14:37:37","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":74195,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis of the cultured area of the cells shown in Fig. 14, comparing between the shapes of BS acoustic fields and the value ranges of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/7ca179ae3027b4fbfac2ebd2.png"},{"id":95040994,"identity":"006d0cc1-c104-42ca-ad93-7ad45ad01502","added_by":"auto","created_at":"2025-11-03 16:10:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4149478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6635839/v1/b789fe34-68bb-45d2-8215-73c1af76b4fb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Non-contact culturing of vascular endothelial cells on wall surface following retention using acoustic radiation force and lipid bubbles","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eT\u003c/span\u003ehe development of artificial organs has emerged as a pivotal technology for regenerative medicine, since artificial organs can replicate in vivo disease conditions and transform therapeutic methods by constructing complex structures, such as spheroids or organoids (Lee, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mi, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; De Bartolo, et al., 2022; Wang, et al., 2019). Because the vascular network is essential to any artificial organ, the three-dimensional fabrication of artificial blood vessels is critical in advancing these technologies. Some studies utilized 3D printing technology to fabricate artificial blood vessels using vascular cells (Hou, et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Choi, et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hu, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Typically, cell aggregates were deposited as droplets through inkjet or laser-assisted bioprinting to build cell blocks. However, one important limitation of this approach is its capacity to accurately replicate key features of blood vessels, such as their multiple bifurcations. In case that laminating the cells is left to gravity, which takes time, especially to form tubular structures. Furthermore, fabricating tubular structures with arbitrary shape with diameters less than several mm presents a challenge, primarily due to the requirements involved in layering blocks (Su, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To address these challenges, photopolymerization was used to facilitate the construction of smaller artificial blood vessels (Wang, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hoch, et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Using this method, the medium containing vascular cells is hardened through exposure to laser light outside the vascular template. While this technique has been successful in creating blood vessels with diameters with several mm, replicating the multi-layered structure of actual blood vessels, including the integumentum, mesothelium, and endothelium layers, remains challenging. Therefore, there is a need for a new technology to create tubular structures that can arbitrarily design the distribution of cells, including situations that defy gravity, and that can be used for multi-layer culturing.\u003c/p\u003e \u003cp\u003eIn our previous experiments, we manipulated cell dynamics of not only T-cells (Oitate, et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oitate, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Masuda, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Seki, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chikaarashi, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kajita, et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Colon-26 cells (Shigehara, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Demachi, et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) but also vascular endothelial cells (Ito, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ogawa, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Noguchi, et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), using bubbles, where the bubble-surrounded cells (BSCs) were produced. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e explains the senario of this study; difference in propulsive force between a cell and a BSC under ultrasound exposure in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a), and the trajectory of a BSC passing through the flow under ultrasound exposure in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b). In the absence of bubbles shown in the upper part of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a), the propulsive force hardly produced since ultrasound penetrate through the cell since the acoustic impedances of the elastic tissues are between 1.38 MRayls (fat) and 1.70 MRayls (muscle), which is similar to that of the medium (water, 1.5 MRayls). On the other hand, if there are bubbles (gas, less than 0.001 MRayls) attached on the cell surface shown in the lower part of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a), a propulsive force acting on the cell is enhanced by the traveling wave reflected off the cell surface, attributable to the boundary in acoustic impedance. Furthermore, the oscillation of the bubbles enables the Bjerknes force (Louisnard, et al., 2012; Garbin, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) to propel the cell in the direction of the traveling wave. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b), when traveling wave of ultrasound was exposed to a BSC passing through the flow from a direction different from the flow direction, the trajectory of the BSC was distract in the direction of ultrasound propagation. In case that the multiple BSCs were contained in the flow, there might be some BSCs propelled against the upper wall depending on the relationship between the flow velocity, sound pressure, and the concentration of BSCs. Thus, using ultrasound and bubbles, it would be possible to apply a non-contact external force to the cells in the flow, control their behavior, and fabricate cellular structures in any shape and position in the flow path. Meanwhile, it is necessary to take into account that cell damage occurs due to cavitation caused by the destruction of bubbles under ultrasound exposure. To fabricate a structure made of cells by retaining BSCs, we must confirm that the cells retained on the wall surface of a flow path via an acoustic force, regardless of the direction of gravity, can be engrafted and cultured in situ after retention. Furthermore, considering that the bubbles are attached to the cell surface, it is crucial to determine how the presence of the bubbles affects the cell culture process. This study performed a preliminary cell culture trial following retention using acoustic radiation force and examined the conditions of the acoustic field and cell proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Cells, lipid, and lipid bubbles\u003c/h2\u003e\n \u003cp\u003eIn this study, we used bovine-derived carotid endothelial cells (Ito, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ogawa, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Noguchi, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) (referred to as \u0026ldquo;cells\u0026rdquo;, hereinafter), which were cultured at 37\u0026deg;C with a CO\u003csub\u003e2\u003c/sub\u003e concentration of 5%, using Dulbecco\u0026apos;s modified Eagle medium (DMEM, Thermo Fisher Scientific) with 10% fetal calf serum. We also prepared a phospholipid, which consisted of 1,2-distearoyl-sn-glycero-3-phosphatidyl-choline (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-polyethylene glycol 2000 (DSPE-PEG2k) (Suzuki, et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wada, et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Negishi, et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). To compose lipid bubbles (referred to as \u0026ldquo;LBs\u0026rdquo;, hereinafter) containing perfluoropropane (PFP, C\u003csub\u003e3\u003c/sub\u003eF\u003csub\u003e8\u003c/sub\u003e) gas, DSPC:DSPE-PEG2k was dissolved (molar ratio of 94:6) in mixed organic solvents (each containing 4 ml of chloroform). Then, phosphate-buffered saline (PBS) was added to the lipid solution and sonicated before the organic solvent evaporated. The obtained LBs had the size distribution (Ogawa, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e), which occurs naturally through the manufacturing process, with an average diameter of 1 \u0026micro;m and were encapsulated in a liposome using the phosphate buffer solution. Finally, we modified the LBs by conjugating a ligand of cyclic-RGD (cRGD) peptides, which covalently adhere to vascular endothelial cells via the DSPE-PEG on the LB surfaces. The conjugation of DSPE-PEG3.4k-Mal and cRGD ligand was performed during a 1-hour incubation using a rotary mixer. The molar ratio of DSPC:DSPE-PEG2k:DSPE-PEG3.4k-cRGD was 94:5:1. To prepare the LB suspension, 2 ml of liposome suspension (lipid concentration: 1 mg/ml) was diluted to yield the desired PBS concentration. The concentration [mg/ml] was derived from the weight of the lipid divided by the volume of the suspension.\u003c/p\u003e\n \u003cp\u003eIn our previous studies (Chikaarashi, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ito, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Noguchi, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e), the controllability of the cells, which was evaluated as the amount of cell retention on the wall surface of a flow path, increased in proportion to the LB concentration. However, the presence of surrounding LBs may affect not only the cells damage via cavitation but also the subsequent cell engraftment and culture process. In the following experiments, the cell concentration in the suspension was set to 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e /ml, and the LBs were diluted at a concentration within the range of 0\u0026ndash;0.5 mg/ml. Here, the cell concentration was expressed with the cell number per ml, whereas the LBs concentration was with lipid weight per ml (Kajita, et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ogawa, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Experimental setup\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (a) shows the experimental setup for observing the behavior of the cells in BSCs passing through the flow path during ultrasound exposure (Chikaarashi, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kajita, et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ito, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ogawa, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). This setup comprised a fluorescence microscope (Olympus, BXFM with DP74) and the 2D array transducer, which has a central frequency of 3 MHz and 128 elements (Takano, et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ushimizu, et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) (12 \u0026times; 12 elements forming a square, and the four elements at each corner were lacked). The driving equipment (ES1144-1, Microsonic Co., Ltd.) (Chikaarashi, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Suzuki, et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hosaka, et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Koda, et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) generated a burst wave with a minimum pulse repetition time and delay pitch of 10 ms and 5 ns, respectively, to electrically oscillate the elements individually. The maximum sound pressure of 400 kPa-pp with a duty ratio of 60% is the output limitation of this equipment.\u003c/p\u003e\n \u003cp\u003eThe flow path was placed at the surface of the water. The transducer targeted the observation area in the flow path from a distance of \u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;60 mm at the elevation angle of \u003cem\u003e\u0026theta;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;60\u0026deg;. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (b) shows the schematic of the flow path made of polydimethylsiloxane (PDMS), which has the acoustic impedance of 1.03 MRayl at 3 MHz and 37 ˚C (Guillermic, et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). PDMS easily deforms in the behavior of elastic tissues. The inner cavity of the flow path has a length of 30 mm and a rectangular cross-section with a width \u003cem\u003ew\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e = 2.1 mm and a height \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ep\u003c/em\u003e\u003c/sub\u003e = 1.5 mm, respectively, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (c). From the viewpoint of fabricating a tubular structure, the cross-sectional shape of the flow path should be circular. However, in order to accurately measure the distribution of cells on the inner wall using microscopic images, a rectangular cross-section was produced so that the effect of depth of field could be ignored. The thickness of the upper and lower wall of the path was \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003eup\u003c/em\u003e\u003c/sub\u003e = 2.6 mm and \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003eup\u003c/em\u003e\u003c/sub\u003e = 1.4 mm, respectively. To coat the inner surface of the path with a basement membrane, the cavity of the flow path was filled with a mixture of Cellmatrix Type I-C (Nitta Gelatin Co., Ltd.) (Kojima, et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) and pure water, which was allowed to stand for 24 h to enable the collagen fibers to precipitate. The thickness of the membrane was estimated to be less than 10 \u0026micro;m.\u003c/p\u003e\n \u003cp\u003eA BSC suspension was injected during the artificial flow of the medium through the flow path maintained at a velocity of 10 mm/s. The BSCs in the suspension passing through the flow path were exposed to an acoustic field for up to 60 s. The behavior of the BSCs, which were propelled to adhere to the upper surface of the cavity, was optically observed using a fluorescence microscope. To evaluate the cell retention performance in the observation area, the cells were stained using Calcein-AM solution (Dojindo) (Ogawa, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Uggeri, et al., \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Tawakoli, et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) in advance. The cells emitted fluorescence with a wavelength of 515 nm for 490 nm excitation light to distinguish them. The fluorescent images were binarized by applying a threshold based on the average background brightness in 256-scale (8-bit) to obtain a retained area of the cells as \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e. The images were obtained with a 1.25\u0026times; objective lens (NA 0.04, Olympus).\u003c/p\u003e\n \u003cp\u003eFollowing the retention process, the flow path was extracted from the water tank to be maintained in a CO\u003csub\u003e2\u003c/sub\u003e incubator for culturing 24 h without turning the flow path upside down. To evaluate the cultured area of cells, the cells were not stained in the above-mentioned retention process. After the suspension in the path was drained to remove floating cells, the flow path was turned upside down to stain the living cells engrafted on the basement membrane. The living cells in the fluorescent images were distinguished from the background and dead cells to obtain a cultured area of the cells as \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e,\u003c/sub\u003e and the images were obtained with a 5\u0026times; objective lens (NA 0.15, Olympus).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Formation of acoustic fields\u003c/h2\u003e\n \u003cp\u003eWe designed several acoustic fields using the 2D array transducer to form an arbitrary shape of the acoustic field (Xu, et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Riis, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). First, a single focal (SF) acoustic field formed a single focal point in the center of the flow path. To extend the irradiation area along the path, a multifocal (MF) acoustic field was produced using tempo-spatial division emission (Takano, et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ushimizu, et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Suzuki, et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e), as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (a), where the applied acoustic energy was distributed temporally and spatially. A focal point translates linearly as \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (2\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003ei\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003en\u003c/em\u003e) along the \u003cem\u003ey\u003c/em\u003e-direction with the spatial interval, \u003cem\u003er\u003c/em\u003e. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (b) shows the time chart of the position transition of the focal point, where the repetition transition of the focal point is from \u003cem\u003eF\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e to \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e, each of which has emission duration, \u003cem\u003e\u0026tau;\u003c/em\u003e [s]. Number \u003cem\u003en\u003c/em\u003e indicates the number of preservable focal points, where one period of the tempo-spatial division emission is \u003cem\u003en\u0026tau;\u003c/em\u003e [s]. In the following experiments, the MF acoustic field is denoted by MF\u003cem\u003en\u003c/em\u003e (e.g., MF3 includes three focal points).\u003c/p\u003e\n \u003cp\u003eFor comparison with the MF acoustic field, a bar-shaped (BS) acoustic field was produced using the time-reversal method (Fink, \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Jing, et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e), which refocuses an incident acoustic field back to the position of the source, irrespective of the complexity of the propagation medium, using the k-Wave mathematical platform (Treeby, et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). A BS virtual sound source was established in the space to generate the acoustic field as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Thereafter, the ultrasound propagation was calculated to record the amplitudes and phases at the position of the 2D array. Finally, the recorded amplitudes and phases in all the elements were reproduced. In the following experiments, the BS acoustic field is denoted by BS\u003cem\u003en\u003c/em\u003e, where the bar length in the \u003cem\u003ey\u003c/em\u003e-direction corresponds to (\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1)\u003cem\u003er\u003c/em\u003e, to be compared with the length of MF\u003cem\u003en\u003c/em\u003e acoustic field.\u003c/p\u003e\n \u003cp\u003eHere, we defined the applied acoustic intensity of the aforementioned acoustic fields by measuring the distributions of sound pressure (Chikaarashi, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Noguchi, et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The measured distribution (\u003cem\u003ex-y\u003c/em\u003e plane) was divided into small grid areas with a width of \u003cem\u003ew\u003c/em\u003e. Assuming a mean sound pressure of \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eij\u003c/em\u003e\u003c/sub\u003e [Pa-pp] in a small area (\u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eij\u003c/em\u003e\u003c/sub\u003e), an applied acoustic power \u003cem\u003eE\u003c/em\u003e [W] can be estimated as follows:\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"312\" height=\"71\"\u003e\u003c/p\u003e\n \u003cp\u003eIn the above equation, \u003cem\u003eZ\u003c/em\u003e denotes the acoustic impedance of the medium. Coefficient \u003cem\u003eD\u003c/em\u003e indicates the duty ratio in a small area. The applied acoustic intensity of spatial average temporal average (SATA) (Izadifar, et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Acevedo, et al., 2002) was defined as \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e = \u003cem\u003eE\u003c/em\u003e/\u003cem\u003eS\u003c/em\u003e [mW/cm\u003csup\u003e2\u003c/sup\u003e]. \u003cem\u003eS\u003c/em\u003e is the area of the acoustic field defined by the area, where the relative amplitude exceeds \u0026minus;\u0026thinsp;20 dB of the maximum point. In the following experiments, an identical coordinate of \u003cem\u003ew\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5 mm was used. Additionally, the duty ratio was set to \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.6, which was fixed due to the device limitation as mentioned above.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Measurement of acoustic fields\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the normalized distributions of (a) SF, (b) MF3, (c) BS2, (d) BS3, and (e) BS4 acoustic fields. They were measured using an acoustic intensity measurement system (AIMS III, Onda Co., Ltd.) by translating the hydrophone (HNR-1000, Onda Co., Ltd.) (Seki, et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chikaarashi, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kajita, et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) in degassed water without the flow path installed. The coordinate in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e corresponds to that of the \u003cem\u003ex-y\u003c/em\u003e plane (\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0), where the flow path was installed in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eIn the MF acoustic fields, the emission duration, \u003cem\u003e\u0026tau;\u003c/em\u003e, was set to 0.01 ms, which is faster enough than cell motion. In the MF and BS acoustic fields, the spatial interval, \u003cem\u003er\u003c/em\u003e, was fixed at 3 mm. The beam width, defined as the half-width of the distribution in SF and MF3, was measured at 2.5 mm. In Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (b), the three focal points appeared to occur simultaneously since the scanning speed of the hydrophone was approximately 10 mm/s. The BS2 acoustic field has one peak in the distribution shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (c), whereas there were two peaks appeared in the BS3 and BS4 acoustic fields as shown in Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (d) and (e).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows the measured profiles of acoustic fields of SF, BS2, BS3, and BS4 along the (a) \u003cem\u003ey\u003c/em\u003e-axis (\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0) and (b) \u003cem\u003ex\u003c/em\u003e-axis (\u003cem\u003ey\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0). When the maximum sound pressure of SF was 400 kPa-pp, the acoustic intensity of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e was 143 mW/cm\u003csup\u003e2\u003c/sup\u003e. The maximum sound pressures in BS acoustic fields were adjusted to establish similar acoustic intensities, which of BS2, BS3 and BS4 were 138, 139, and 141 mW/cm\u003csup\u003e2\u003c/sup\u003e, respectively. Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e shows the measured profiles of the acoustic fields of MF2, MF3, and MF4, where the SF acoustic field translated linearly in the \u003cem\u003ey\u003c/em\u003e-direction, preserving the distribution of the SF field.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cell culture in the presence of lipids without ultrasound\u003c/h2\u003e \u003cp\u003eConsidering that there is a potential for fragments of LBs disrupted by the ultrasound to remain, we examined the cell culture in the presence of LBs and lipids prior to their composition into LBs. The cells were cultured on the lower wall of the flow path shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b) without ultrasound exposure and flow. In this experiment, the \u0026ldquo;lipids\u0026rdquo; being referred to are the same components as the LBs without PFP gas. We prepared two types of cell suspensions (lipid and LB suspensions), with lipid concentrations varying between 0 (only cells) and 0.4 mg/ml, where the cRGD ligand was incorporated into the lipid solution. After injection of suspension (0.16 ml) with the cell concentration of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e /ml onto the bottom of the flow path, the cells were cultured in a CO\u003csub\u003e2\u003c/sub\u003e incubator to measure the \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the fluorescent images after 24 h of incubation. Based on the observed increase in lipid concentration, although the cultured area of the cells, \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e, decreased under both conditions, the presence of LBs negatively affected the culturing. The cells cultured with LBs were hardly visible at the concentration of 0.4 mg/ml.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e compares the \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e between the suspension of lipids and LBs. When the lipid concentration was below 0.3 mg/ml, no significant difference was observed between them until the LB concentration reached 0.4 mg/ml. In our previous study on retaining BSCs in the flow (Ito, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we observed that the retained area of the BSCs increased proportionally to the LB concentration and was saturated at 0.4 mg/ml. To prioritize retaining as many cells as possible, we set the LB concentrations to 0.3 mg/ml for the subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cell retention with various acoustic fields\u003c/h2\u003e \u003cp\u003eNext, we compared the cell retention performance using the prepared acoustic fields. A BSC suspension with the cell and LB concentrations of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e /ml and 0.3 mg/ml, respectively, was flowed through the flow path at a flow velocity of 10 mm/s and an irradiation duration of 60 s. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e shows the transition of the distribution of the retained cells with an acoustic field of BS2 and the maximum sound pressure of 260 kPa-pp, which was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a). It can be confirmed that the retained area of the cells were gradually increased. Also, the cells that were not retained were propelled aside and accumulated in the corners of the path.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e shows fluorescent images 60 s after starting the retention with the acoustic fields of (a) SF, (b) MF3, (c) BS3, and (d) BS4, where the maximum sound pressures were adjusted to establish similar acoustic intensities (135\u0026ndash;155 mW/cm\u003csup\u003e2\u003c/sup\u003e) as appeared in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The area of cell retention varied with the acoustic field and became longer along the flow direction in the MF3 and BS acoustic fields compared with that in the SF acoustic field.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e compares the retained area \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e versus the acoustic intensity \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e for 30 different acoustic fields with various sound pressures. In the SF acoustic field, the increase in the retained area was limited as the acoustic intensity. In the MF acoustic fields, the retained area of the cells increased in proportional to \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e, whereas the retained areas were inferior to that in the SF acoustic fields, except for MF3. Comparing between the shapes of acoustic fields, the BS acoustic fields were more effective to retain more cells than others.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Cell culturing following retention\u003c/h2\u003e \u003cp\u003eThe retained cells were continuously cultured in situ in the flow path without ultrasound exposure. The flow path was left undisturbed for 30 min before being placed in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 23.5 h, allowing the cells to adhere to the upper surface of the flow path. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e shows the fluorescent images of cells cultured for 24 h after retention with the same acoustic fields as in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. Cultured cells were hardly found using the acoustic fields of SF and MF3, indicating that the cells were either almost extinct or had detached from the surface of the flow path, as well as other MF acoustic fields. In contrast, cultured cells were widely spread along the flow path using the BS acoustic fields.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e compares the \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e with various acoustic fields, which include all BS acoustic fields appeared in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e, and SF and MF\u003cem\u003en\u003c/em\u003e acoustic fields with \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e ranged between 135 and 155 mW/cm\u003csup\u003e2\u003c/sup\u003e. Furthermore, BS acoustic fields were classified by \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e into three ranges: 135 to 155, 100 to 110, and 70 to 80 mW/cm\u003csup\u003e2\u003c/sup\u003e. It is obvious that the BS acoustic fields were superior in both cell retention and cell culturing with \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e ranged between 135 and 155 mW/cm\u003csup\u003e2\u003c/sup\u003e, although a similar retained area of the cells was observed with the MF3 field. Comparing between the BS acoustic fields, in BS3 and BS4, both the occupied areas of the cells of \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e were almost proportional to the acoustic intensity \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e. In BS2, in contrast, there was little correlation between \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003ecul\u003c/em\u003e\u003c/sub\u003e, while \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eret\u003c/em\u003e\u003c/sub\u003e tended to be high.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e indicates the statistical analysis of the cultured area of the cells shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e, comparing between the acoustic fields and the value ranges of \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e. In each of the acoustic fields of BS2, BS3, and BS4, the retained area of the cells significantly increased as \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e increased. Furthermore, comparing the acoustic fields with \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e of 135\u0026ndash;155 mW/cm\u003csup\u003e2\u003c/sup\u003e, although there was no significant difference between BS2 and BS3 and between BS3 and BS4, there was a significant difference between BS2 and BS4. These results suggest that there are optimal conditions for the acoustic field, such as the distribution of acoustic intensity and/or sound pressure, for non-contact culturing of cells following retention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study investigated the possibility of the non-contact culturing of vascular endothelial cells on the wall surface following retention using acoustic force and LBs. First, for the results shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the effects of LBs and lipids on the cell culture without ultrasound were evaluated. As the concentration of LBs increased, the cell culture performance significantly decreased with an increase in the LB and lipid concentrations, where the surrounding LBs inhibited cell adhesion to the wall. In our previous study (Ito, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which focused on the retention of BSCs on an elastic wall using acoustic radiation force, the area of cell retention reached saturation at an LB concentration of 0.4 mg/ml. This suggests that the LBs adhering to the cell surface nearly covered the entire surface at this concentration. Therefore, an LB concentration of 0.3 mg/ml was the less saturated condition on the cell surface, where a cell was not isolated from its surrounding medium. Contrarily, when the LBs were replaced with lipids (without gas), although lipid adhesion to the cell surface occurred similarly to the LBs, the physical volume of the lipids, compared with that of LBs, probably limited their effect on the cell culture. In the subsequent experiments, because of ultrasound exposure during the retention process, the LB concentration was expected to be less than 0.3 mg/ml due to the destruction of bubbles. However, it might be necessary to remove the lipids around the cells in future studies on culturing. Here, it should be noted that the lipid concentration used in this study ranged 0.1\u0026ndash;0.4 mg/ml, which was comparable to suspensions used in clinical applications (Sridharan, et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, the cell retention performance was executed with various acoustic fields. Using the MF and BS fields, we succeeded to extend the retained area of the cells along the flow direction compared to the SF field. The reason why the sound pressure distribution shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e differs from the distribution of the retained cells shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e is because the cells were propelled away from higher sound pressure and retained to form the contour of the sound pressure distribution. In the MF fields, the retained area of the cells tended to increase in proportion to the acoustic intensity as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. In contrast, in the BS fields, it was possible to retain more cells at low acoustic intensities than other fields. Here, it should be mentioned that the distributions of cultured cells shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e were not preserved the distributions of the retained cells. Especially in the BS fields, the shape of the cell clusters had a strong tendency to be stacked in the corners of the cross-section of the path, where the flow was stagnant. This may be one of the reason of larger retained areas with the BS fields than others, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe important result in this research was the success of non-contact culturing of cells following retention using the BS fields, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e, which indicates that the cells were not only sufficiently propelled and attached to the wall, but also survived or recovered from damage (Ito, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ogawa, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) under the effects of cavitation caused by the destruction of LBs. In our previous study (Wada, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) using the same LBs as in this study, higher sound pressure than 300 kPa-pp accelerates the damage to the cells due to the destruction of LBs. Considering that the cells accumulated in the corners were detached during the culture process, the cultured area of the cells with BS fields shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e indicates that the cells have engrafted and proliferated. Due to the constraints of the driving equipment, acoustic intensities more than 150 mW/cm\u003csup\u003e2\u003c/sup\u003e were not applied in the BS fields. It is believed that more cells can be cultured in proportion to the acoustic intensity. On the other hand, higher sound pressures may increase damage to cells due to cavitation effects. We are going to continue investigating the relationship between acoustic field conditions and cell culture efficiency.\u003c/p\u003e \u003cp\u003eThe above results suggest that the 3D shape of the acoustic field, particularly the distribution and magnitude of the sound pressure, influenced the boundary of culturing of cells following retention in the flow. In the SF field shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a), the sound pressure decreases with distance from the focal point. If the magnitude of the sound pressure is dominant for the non-contact culturing, the distribution of cells should form a ring shape. However, cultured cells were barely observed with the SF field or the MF fields as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e. Therefore, in the evaluation of the non-contact culturing of cells following retention, it is necessary to consider not only the magnitude of sound pressure but also the spatial variation, which is the gradient of sound pressure in the direction of the suspension flow.\u003c/p\u003e \u003cp\u003eFinally, we discuss the limitations of this method and future developments. Since the cells are propelled by acoustic radiation force to travel a distance through the flow, there might be the minimum size of blood vessels to be fabricated with this method, which is supposed to be a diameter of around 100 \u0026micro;m. In further developments, we are going to reveal quantitative investigation of these limitations. Since the cells flowed in one way through the flow path, there was a limitation to the retention efficiency relative to the number of injected cells. To enhance the retained cells on the wall, the experimental setup should be improved, for example, by the circulation of a suspension. Furthermore, since the cells were suppressed on a flat plane, we are going to consider to establish uneven surface of the inner wall according to the shape of the acoustic field. In addition, a series of procedures should be extended using a flow path with a circular cross-section and bifurcations. Further research will focus on the development of artificial blood vessels by introducing a variation of acoustic fields corresponding to cell dynamics to realize the non-contact culturing of cells in a wider area. The results presented in this article can therefore serve as a basis for future developments.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study achieved the non-contact culturing of vascular endothelial cells on the inner surface of the flow path, following the cell retention process enabled by acoustic force and LBs. First, the effect of lipids and LBs upon culturing the cells was investigated to derive an optimal LB concentration to facilitate retention and culturing. Then, the cells were retained using various acoustic fields, which were a single-focal (SF), multifocal (MF), and bar-shaped (BS), to compare cultured area of the cells. As the results, significant cell engraftment was confirmed using the BS acoustic fields compared to other fields, even when the acoustic intensity (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSATA\u003c/em\u003e\u003c/sub\u003e) and the retained area of the cells were similar. This study suggested that the non-contact culturing of cells following the retention process required the distribution of the sound pressure dependent on the shape of the acoustic field, and the gradient in the flow direction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting Interests:\u003c/strong\u003e \u003cp\u003eThe authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions S.W., Y.I. and K.M. designed the experiments. Y.M., D.O. and R.S. contributed the preparation of the experiments. S.W., A.N. and K.K. performed the experiments. S.W. and K.M. wrote the main manuscript text. All authors reviewed the manuscript. Y.M., R.S. and K.M. secured the funding.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eThis work was supported in part by a grant from the Japan Society for the Promotion of Science (JSPS) through KAKENHI Grant Number 20H04547, and the Terumo Life Science Foundation. The authors express a great thankfulness to Dr. Hiroya Takada, in Nippon Medical School, Japan, for technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eS. Lee, S.P. Jin, Y.K. Kim, G.Y. Sung, J.H. Chung, J.H. Sung, Construction of 3D multicellular microfluidic chip for an in vitro skin model, Biomed. Microdevices, \u003cstrong\u003e19\u003c/strong\u003e, 22 (2017).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eS. Mi, S. Yang, T. Liu, Z. Du, Y. Xu, B. Li, W. Sun, A Novel Controllable Cell Array Printing Technique on Microfluidic Chips, IEEE Trans. Biomed. Eng., \u003cstrong\u003e66\u003c/strong\u003e, 2512-2520 (2019).\u003c/li\u003e\n \u003cli\u003eL. 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Utilizing the propulsive force acting on cells under ultrasound exposure when the cells were surrounded by lipid bubbles, the conditions of the acoustic field were investigated. First, cells were cultured in the presence of lipids without ultrasound to derive the optimal concentration of lipids. Next, cells were retained on the inner surface of the flow path using various acoustic fields, which include single-focal, multifocal, and bar-shaped fields. After culturing the cells in the path without flow for 24 h, the cultured area of cells were measured to evaluate the series of performance. In the experiment of cell culturing without ultrasound, the cultured area decreased inversely proportional to the lipid concentration, thus deriving the optimal concentration of bubbles. When the bar-shaped fields were used for the retention process, significant cell engraftment was observed compared to other fields, even though the acoustic intensity of SATA (Spatial average temporal average) and the retained area of the cells were similar. Those results suggest that conditions of acoustic field, including the distribution and magnitude of sound pressure according to the flow direction, are dominant for non-contact culturing of cells following retention. We succeeded in culturing cells at desired position on inner wall of the path, regardless of the direction of gravity.\u003c/p\u003e","manuscriptTitle":"Non-contact culturing of vascular endothelial cells on wall surface following retention using acoustic radiation force and lipid bubbles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 14:21:32","doi":"10.21203/rs.3.rs-6635839/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-19T14:45:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-19T10:05:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33830191122022883534348462682966402988","date":"2025-06-02T11:12:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-01T19:58:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168509174730529074500191763657282818454","date":"2025-05-23T02:28:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-20T08:39:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-19T14:45:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-13T09:48:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biomedical Microdevices","date":"2025-05-10T16:00:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biomedical-microdevices","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmmd","sideBox":"Learn more about [Biomedical Microdevices]()","snPcode":"10544","submissionUrl":"https://submission.nature.com/new-submission/10544/3","title":"Biomedical Microdevices","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d49d7bee-8382-4786-9fa1-fd92eae0bb75","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:08:12+00:00","versionOfRecord":{"articleIdentity":"rs-6635839","link":"https://doi.org/10.1007/s10544-025-00771-0","journal":{"identity":"biomedical-microdevices","isVorOnly":false,"title":"Biomedical Microdevices"},"publishedOn":"2025-10-31 15:57:19","publishedOnDateReadable":"October 31st, 2025"},"versionCreatedAt":"2025-05-22 14:21:32","video":"","vorDoi":"10.1007/s10544-025-00771-0","vorDoiUrl":"https://doi.org/10.1007/s10544-025-00771-0","workflowStages":[]},"version":"v1","identity":"rs-6635839","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6635839","identity":"rs-6635839","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}