Hydrogel microwell with pneumatic soft actuator for compression formation of three-dimensional cellular tissue

Abstract

Three-dimensional cellular tissue reproduces the structure and function similar to the tissues in the body. So far, as a means for the formation and maturation of three-dimensional cellular tissues, various mechanical stimuli have been applied to cells. Compression by mechanical actuators is a popular method for applying mechanical stimulation to cells. However, the use of linear or rotary actuators limits the direction of compressive stimulation to one axis and requires the cell to be adhered to a scaffold for fixation. Here, we propose a method to form three-dimensional cellular tissue cultured by a hydrogel microwell in a pneumatic soft actuator as a hybrid soft actuator. The hydrogel microwell deformed by the pneumatic soft actuator envelopes and three-dimensionally compresses the cells in the hydrogel microwell even shortly after cell seeding due to no need to fix the cells on the scaffold. In this study, ATDC5 cells, which are a mouse chondrogenic progenitor cell line having high activity to compressive stimuli, were cultured to apply enveloping compressive stimulation to the cells. By applying compressive stimulation to the cells in the hydrogel microwell using the pneumatic soft actuator, the formation of cellular tissue was promoted at an early stage before the cells began to adhere to each other. Moreover, the cellular tissue compressed by the pneumatic soft actuator expressed Sox9 and collagen II as well as the control without compressive stimuli. This proposed culture with the pneumatic soft actuator is a promising three-dimensional culture method capable of compressing cells, which can be applied for regenerative medicine and drug screening.

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1. Introduction

Three-dimensional culture is superior to two-dimensional culture for maturing cells due to its characteristic of mimicking the environment in vivo. The formation of three-dimensional cellular tissue, such as spheroid,¹ sheet,^2,3 and fiber,^4–6 reproduces cell–cell communication, biological interactions between cells and extracellular matrix (ECM), and high differentiation potential. This formation of three-dimensional cellular tissue is crucial for the current regenerative medicine process, cancer research, and drug screening.^7,8 Hence, a variety of methods to form cellular tissue have been studied, including the surface tension of droplets,⁹ a template made of non-adherent material,¹⁰ hydrogel wells,¹¹ a standing wave,¹² and an acoustic streaming¹³ for the formation of spheroids. Therefore, numerous attempts have been made to form three-dimensional cellular tissues, although improving the function of the tissues remains a challenge.

In tissue engineering, mechanical stimulation is one of the solutions for cell maturation to reproduce cellular tissue more similar to in vivo. This is because the cellular tissue composing our body is constantly exposed to mechanical stimuli from gravity, physical activity, and exercise.^14,15 Mechanical stimuli promote differentiation by increasing cell activity and triggering the expression of specific genes.¹⁶ Thus, providing mechanical stimuli to cells and cellular tissues during culture is a key factor for mimicking the cellular tissues in vivo. One way to apply mechanical stimulation to cells is compression by actuators. For instance, a microfluidic device inflated by gas pressure applying compression on a cell monolayer¹⁷ and a stretchable actuator compressing cells¹⁸ have been reported. However, compressive stimulation to cellular tissue is limited to one axis due to the use of linear or rotary actuators.^19,20 Additionally, cellular tissue typically requires fixation to a scaffold for the application of compressive stimulation. Thus, the application of compressive stimulation until the cells adhere to each other to form tissue was difficult. That is, single cells immediately after seeding do not allow compression due to their lack of adhesion to the scaffold.

Here, we propose a method to form three-dimensional cellular tissue cultured by multidirectional pneumatic hydrogel-microwell guided retention system (MPHGS, Fig. 1). In this system, a pneumatic soft actuator compresses the cells seeded in the hydrogel microwell through the inflating and deflating polydimethylsiloxane (PDMS) chamber when applying air pressure to the pneumatic soft actuator. In the pneumatic soft actuator, the cells are compressed and aggregated in the hydrogel microwell to form three-dimensional cellular tissue. For evaluating the pneumatic soft actuator, the actuation performance, the mechanical stimulation on cellular tissue, and the compressive strength of the pneumatic soft actuator were measured. Finally, ATDC5 cells were cultured in the pneumatic soft actuator. After culture, the formation of cell tissues was evaluated based on the density and size of the cellular tissues. Additionally, the cell nucleus, cytoskeleton, SRY-box9 (Sox9), and collagen II were immunostained to observe the maturity of the ATDC5 cells. Consequently, this proposed culture by hydrogel microwells and pneumatic stimulation indicated the possibility of facilitating the growth of cellular tissues at the early stage of culturing.

Fig. 1 Concept of cell culture by a pneumatic soft actuator with a hydrogel microwell. Inflating the pneumatic soft actuator repetitively by air pressure applied compressive stimulation to the cells in the hydrogel microwell for promoting the formation of three-dimensional cellular tissue.

2. Materials and methods

2.1 Fabrication of pneumatic soft actuator

To culture three-dimensional cellular tissue while applying compressive stimulation to cells (Fig. 2), the pneumatic soft actuator consisted of an air inlet (VFI116, Nordson MEDICAL), a PDMS chamber, a cover glass (A219, Sunlead Glass Corp.), and an acrylic enclosure (Fig. 3a and b). The acrylic enclosure held the PDMS chamber when applying air pressure to the chamber. The PDMS chamber had space inside, which was inflated by air pressure through the air inlet. Also, the cover glass set at the bottom of the pneumatic soft actuator enabled observing the cells in the hydrogel microwell formed in the pneumatic soft actuator by an inverted microscope.

Fig. 2 The comparison of compressive stimulation applied to cells with and without hydrogel microwells. (a) Applying compressive stimulation to cells by deforming rubber under pressure without hydrogel microwells. With only rubber, the cells are pushed up by compression along with the cell suspension, making it impossible to keep the suspension of cells in the culture space. (b) Applying compression through a hydrogel microwell to cells by deforming rubber under pressure. By combining the rubber and the hydrogel microwell, the cells can be enclosed in the hydrogel microwell. When compression is applied, the culture medium is pushed out into the hydrogel. At this time, the cells are trapped in the hydrogel mesh, allowing compressive stimulation to be applied to the cells (MPHGS).

Fig. 3 Culture system with pneumatic soft actuator and control system. (a) Design of the pneumatic soft actuator. The pneumatic soft actuator consisted of an air inlet, a PDMS chamber, a cover glass, and an acrylic enclosure. (b) Fabricated pneumatic soft actuator. The pneumatic soft actuator had a width of 44 mm, a length of 44 mm, and a height of 10 mm. (c) Fabrication process of the PDMS chamber. Firstly, PDMS was poured into the molds and evacuated. Then, the PDMS in the molds was heated until the PDMS was cured. Afterward, the PDMS cover and bottom were retrieved. The PDMS chamber was fabricated by bonding the PDMS cover and bottom. (d) Control system. Modulated air pressure by a microcomputer and electro-pneumatic regulators was applied to the pneumatic soft actuators. The air pressure was cyclically applied to the pneumatic soft actuators at 0–0.06 MPa, accuracy ±8% FS, and 0.13 Hz during culturing.

The PDMS chamber was fabricated by bonding two PDMS parts, a PDMS cover and a PDMS bottom, to provide an enclosed space inside the PDMS chamber. Before the bonding, the PDMS solution was prepared by stirring PDMS base and PDMS curing agent (70417543, Dow TORAY) in a ratio of 10 : 1.

Afterward, the mixed solution was stirred and degassed by a planetary centrifugal mixer (AR-100, Thinky Corporation) for 2 min, respectively. Then, the solution was additionally degassed with a vacuum pump (VR-16N, HITACHI) for 20 min. After degassing the solution, the solution was poured into the molds of the PDMS cover and bottom (Fig. 3c). When pouring the solution into the molds of the PDMS cover, a cylindrical pin (MSHSM6-15, MISUMI) was placed at the center of the molds to form the compressed section of the pneumatic soft actuator. In addition, a cross-shaped mold was used for curing the PDMS cover to prevent the air bubbles from forming, which improves the durability of the pneumatic soft actuator. Then, the solution in the molds was degassed again with the vacuum pump for 1 h. Finally, the solution in the molds was heated with a dry heat sterilizer (NDO-520, TOKYO RIKAKIKAI CO., LTD.) at 90 °C for 2 h and cured.

After curing the PDMS solution, the PDMS cover and bottom were detached from the molds. The retrieved PDMS cover was made with a small hole and connected to the air inlet through the hole before bonding the PDMS cover and bottom. After connecting the air inlet, the PDMS bottom and the cover glass were set in the acrylic enclosure. For bonding the PDMS cover and bottom, the PDMS solution prepared by the same process above was poured at the bottom of the acrylic enclosure and on top of the PDMS bottom. Note that the amounts of pouring on the bottom of the acrylic enclosure and the top of the PDMS bottom were 500 μL and 1000 μL. Next, the acrylic enclosure containing the PDMS bottom, the cover glass, and the PDMS solution was degassed for 1 h. Afterward, the solution was poured at the bottom of the acrylic enclosure and the top of the PDMS bottom to refill the solution reduced by degassing. Then, the PDMS cover was placed on the PDMS bottom with the cylindrical pin at the center of the acrylic enclosure and heated with the dry heat sterilizer at 90 °C for 2 h. Lastly, after bonding the PDMS cover and bottom, the pin was retrieved.

2.2 Control system

The pneumatic soft actuators were pressured with a control system (Fig. 3d). The control system consisted of a pump (MD-0304PA, PAOCK), a computer, a microcomputer (A000066, Arduino.cc), and electro-pneumatic regulators (ITV0030-2L, SMC Corporation). The pneumatic soft actuators and control system were connected through polymer tubes (SCFJ00003, Saint-Gobain Life Sciences). Through the polymer tubes, the electro-pneumatic regulators output air pressure commanded from a microcomputer with open-loop control. The microcomputer sent command values to the regulators. The command value was an analog voltage transmitted via an ADC chip. In cell culture, the maximum command value was 0.59 V. Moreover, the command value was updated every 300 ms. While applying air pressure to the pneumatic soft actuators containing cells, the pneumatic soft actuators were placed inside an incubator (MCO-5ACUV-PJ, Panasonic). On the other hand, the control system was installed outside the incubator.

2.3 Simulation of an inflated pneumatic soft actuator with the finite element method

The behavior of the inflated pneumatic soft actuator by the finite element method was calculated by the 3D CAD software (COMSOL Multiphysics, COMSOL ver. 6.2, Inc.). When simulating, a hydrogel microwell made from agarose gel was placed at the center of the pneumatic soft actuator. In a previous study, Young's modulus of agarose gel ranged from several to hundreds of kPa, depending on the concentration of the agarose solution.²¹ In the simulation, the Young's modulus of agarose gel was set to 43.1 kPa according to the physical properties of the agarose gel (A2576, Sigma-Aldrich) in a previous study.²² Lastly, the surfaces inside the PDMS chamber were pressurized perpendicularly to the surfaces at 0.1 MPa. The detailed simulation parameters were described in SI 1.

2.4 Evaluation of pneumatic soft actuator

The contraction of the compressed section of the pneumatic soft actuator containing the hydrogel microwell made from agarose gel was measured when applying air pressure to the pneumatic soft actuator. The hydrogel microwell was stained with fluorescent particles (30-01-251, COREFRONT) to clarify the boundary between the pneumatic soft actuator and the hydrogel. The images of the hydrogel at 0–0.1 MPa were acquired with a confocal microscope (ECLIPSE Ti2, AX, Nikon Instruments Inc.). Afterward, to calculate the contraction of the compressed section, the diameter of the boundary between the pneumatic soft actuator and the hydrogel was measured and analyzed with an image analysis software (ImageJ, National Institutes of Health). In this contraction measurement, contractions of various pneumatic soft actuators were measured.

Moreover, to measure the compressive strength of the pneumatic soft actuator, the pneumatic soft actuators were pressurized without the hydrogel microwell until the breaking point of the pneumatic soft actuator. Before pressurizing, the pneumatic soft actuator was connected to a 100 mL disposable syringe (S75-2188-05, Narika Corporation) and a pressure sensor (SEU11-4UA, Nihon Pisco Co., Ltd.) through the polymer tubes. Also, while applying air pressure to the pneumatic soft actuator by pushing the syringe, the video of the pressurized device was acquired with a stereo microscope (SMZ25, Nikon Instruments Inc.). Afterward, the contraction and breaking point of the pneumatic soft actuator were analyzed with ImageJ. The contraction of the compressed section was measured from the size change of the compressed section of the pneumatic soft actuator.

2.5 Fabrication of hydrogel microwell

Firstly, autoclaved agarose was mixed with sterilized water (2 w/v%). Then, the agarose solution was heated for 6 cycles of 30 s with a microwave oven (NE-FL100, Panasonic) and poured into the center of the pneumatic soft actuator sterilized by disinfectant solution (42293, Medical SARAYA). When pouring the agarose solution, a stainless-steel wire, which had a diameter of 500 μm, was installed at the center of the pneumatic soft actuator to form the hydrogel microwell using a microwell jig. The tip of the stainless-steel wire was processed roundly. In addition, the stainless-steel wire was sterilized beforehand with an autoclave (LBS-245, TOMY DIGITAL BIOLOGY CO., LTD.) and placed 0.5 mm away from the cover glass at the bottom of the pneumatic soft actuator. After pouring the agarose solution and setting the stainless-steel wire, the solution was cooled for 20 min. Finally, the stainless-steel wire was withdrawn, and then the hydrogel microwell was fabricated.

2.6 Measurement of the hydrogel microwell compressive strain

Before the measurement of the compressive strain of the hydrogel microwell, the agarose gel was stained with fluorescent particles, the same as the contraction measurement of the compressed section of the pneumatic soft actuator. In this measurement, two types of hydrogel microwells made from low- and high-gelling temperature agarose (A2576 and A9539, Sigma-Aldrich) were prepared and compressed with the pneumatic soft actuator at 0–0.06 MPa. At each pressure, the images of the hydrogel microwells were acquired with the confocal microscope. After analyzing the images, the diameter of the hydrogel microwell was measured with ImageJ, and then the compressive strain of the hydrogel microwell was calculated. The hydrogel microwells made from the low-gelling temperature agarose were formed in various pneumatic soft actuators. The compressive strain in the hydrogel microwells made from the high-gelling temperature agarose was also measured, the same as the low-gelling temperature agarose.

2.7 Cell culture

ATDC5 cells (a mouse chondrogenic progenitor cell line) were cultured to prepare a cell suspension. The cell suspension was prepared at a cell density of 1 × 10⁶ cells per mL with culture medium (048-29785, FUJIFILM Wako Pure Chemical Corporation). Then, 50 μL of the cell suspension was dropped on each hydrogel microwell in the pneumatic soft actuator. After dropping, the pneumatic soft actuators containing the cells and the hydrogel microwell were placed in the incubator at 37 °C and 5% CO₂ for 1 h. Next, 150 μL of the culture medium was added to the hydrogel microwell. Moreover, a piece of flexible film (PM996, Amcor PLC) was set on the culture medium in the pneumatic soft actuator. Afterward, the pneumatic soft actuators, which compress the cells, were connected with the control system through the polymer tubes and placed in the incubator. On the other hand, the non-pressurized pneumatic soft actuators containing the cells were simply placed in the incubator at 37 °C and 5% CO₂ without connecting the polymer tubes as a control. The cells in the pneumatic soft actuators were cultured in the incubator for 6 h, 12 h, and 24 h. During culturing, the cultural conditions of the controls were the same as the compressed cells except for compressing the cells. While incubating, air pressure was applied to the pneumatic soft actuators at 0–0.06 MPa, accuracy ±8% FS and 0.13 Hz (Fig. S1), repeatedly.

2.8 Cellular tissue observation

The cellular tissues were immunostained after incubation. Firstly, the cells in the hydrogel microwell were washed with phosphate-buffered saline (PBS). Then, 4% paraformaldehyde phosphate buffer solution (163-20145, FUJIFILM Wako Pure Chemical Corporation) was poured into the hydrogel microwell. Afterward, 0.1% non-ionic surfactant (X100, Sigma-Aldrich) diluted with 1% bovine serum albumin (BSA) (A2153, Sigma-Aldrich) was poured into the hydrogel microwell, and then the pneumatic soft actuators were placed in the incubator for 1 h. The 1% BSA was prepared with PBS. Next, anti-Sox9 antibody (AB5535, Sigma-Aldrich) was diluted with 1% BSA at a ratio of 1 : 100 and poured into the hydrogel microwell. Then, the pneumatic soft actuators stayed overnight at 4 °C. After staying overnight, the hydrogel microwell was washed with PBS. Furthermore, the cells were stained with Alexa Fluor 680 (A-21076, Thermo Fisher Scientific K.K.) diluted with PBS at a ratio of 1 : 50 for 12 h at room temperature. After staining, the hydrogel microwell was washed with 1% BSA again. Next, collagen type II antibody (bs-0709R, Bioss Inc.) diluted with 1% BSA at a ratio of 1 : 100 was poured into the hydrogel microwell. Then, the cells in the pneumatic soft actuators stayed overnight at 4 °C and were washed with PBS. After washing, the cells were stained with Alexa Fluor 594 (A-11012, Thermo Fisher Scientific K.K.) diluted with PBS at a ratio of 1 : 100 at room temperature for 12 h. Afterward, the hydrogel microwell was washed with PBS. In addition, DAPI (D523, DOJINDO LABORATORIES) diluted with PBS at a ratio of 1 : 100 and phalloidin (PHDG1-A, Cytoskeleton, Inc.) diluted with PBS at a ratio of 21 : 1000 were poured into the hydrogel microwell. Moreover, the pneumatic soft actuators stayed in the incubator for 12 h. Then, the cells were washed with PBS. Finally, the stained cellular tissues in the hydrogel microwells were observed with the confocal microscope and analyzed with ImageJ.

2.9 Image processing and statistical analysis

After culture, the volume of the cellular tissues was analyzed by ImageJ. By accumulating the cytoskeleton of the cellular tissue z-stack images, the volume of the cellular tissue was acquired. Moreover, the cell density of the cellular tissues was measured from the cell nucleus density of the cellular tissue images by ImageJ. In these measurements of the volume and the density, the volume and density of the cellular tissues cultured in various pneumatic soft actuators were measured after one culture. The cell density of the cellular tissues was analyzed by Student's t-test. The value of *p < 0.05 was considered significant.

3. Results

3.1 Design and fabrication of pneumatic soft actuator

In order to form three-dimensional cellular tissues by compressing cells in the hydrogel microwell during incubation (MPHGS), the pneumatic soft actuator, mainly made from PDMS, was constructed. In comparison to only rubber as a traditional soft actuator with air pressure, a combination of rubber and hydrogel made it achievable to apply compressive stimulation while maintaining cells in the culture space (Fig. 2a and b). This compressive mechanism, by a rubber and a hydrogel microwell, softly enveloped and compressed the cells three-dimensionally while squeezing out the culture medium. The pneumatic soft actuator was composed of an air inlet, a PDMS chamber, a cover glass, and an acrylic enclosure (Fig. 3a and b). This PDMS chamber was fabricated by bonding the PDMS cover and the PDMS bottom, which enabled the PDMS chamber to have space inside. The pneumatic soft actuator compresses the hydrogel microwell during culturing in this study. Therefore, this enclosed space was required inside the pneumatic soft actuator to inflate when applying air pressure to the pneumatic soft actuator. In addition, to convey compressive stimulation to the cells three-dimensionally through the hydrogel microwell, the enclosed space needed to be placed surrounding the hydrogel microwell.

The PDMS cover was cured with the cross-shaped mold to reduce the air bubbles in the PDMS (Fig. 3c). The cover glass was adhered on the pneumatic soft actuator to observe the cells cultured in the pneumatic soft actuator (Fig. 3a). Also, the acrylic enclosure surrounding the PDMS chamber restrained the inflation of the chamber when applying air pressure, which enabled the PDMS chamber to inflate to the center direction of the pneumatic soft actuator and envelop the hydrogel microwell containing the cells (Fig. 1). During incubating, the hydrogel microwell was placed in the center of the pneumatic soft actuator to culture the cells in the pneumatic soft actuator. In addition, to prevent the hydrogel from drying, culture medium was filled into the hydrogel microwell. The pneumatic soft actuator and the hydrogel microwell were designed and arranged to have enough space for the culture medium on the hydrogel microwell. The culture system consisted of a pump, a computer, a microcomputer, electro-pneumatic regulators, and pneumatic soft actuators, which applied compressive stimulation in parallel to the cells in the pneumatic soft actuators (Fig. 3d).

3.2 Performance of pneumatic soft actuator

While culturing cells in the pneumatic soft actuators, the performance of the pneumatic soft actuator affected the formation of the three-dimensional cellular tissue. The PDMS chamber of the pneumatic soft actuator inflated and deflated when air pressure was applied to the pneumatic soft actuator, and then the center strain of the PDMS chamber conveyed the compressive force to the hydrogel microwell. To evaluate the pneumatic compression behavior of the pneumatic soft actuator containing the agarose gel, the finite element analysis (FEA) of the pneumatic soft actuator was conducted using COMSOL. In the simulation, the PDMS chamber was inflated at 0.1 MPa. To reproduce the environment of the inside of the pneumatic soft actuator pressurized by air pressure, the equalized pressure was applied on the inner surfaces of the pneumatic soft actuator. As a result, the top of the PDMS chamber largely deformed in the direction perpendicular to the cover glass. In addition, the compressed section of the PDMS chamber inflated in the center direction of the pneumatic soft actuator by up to ∼6% (Fig. 4a). In addition to the FEA of the pneumatic soft actuator, the actual contraction percentage of the pneumatic soft actuator was measured when applying air pressure (Fig. 4b).

Fig. 4 Performance of the pneumatic soft actuator. (a) Cross-sectional image of the finite element analysis (FEA) of the inflated device by air pressure. In the simulation, the hydrogel microwell was placed in the center of the pneumatic soft actuator. The applied air pressure to the pneumatic soft actuator was set at 0.1 MPa in the simulation. (b) Compressed section contraction ratio of the pneumatic soft actuator when applying air pressure to the pneumatic soft actuator containing the hydrogel microwell (mean ± S.D., n = 3). The diameter of the compressed section of the pneumatic soft actuator was 6 mm without applying air pressure. (c) The compressive strength of the pneumatic soft actuator without the hydrogel microwell when applying air pressure. The vertical axis represented the compressed section contraction ratio of the pneumatic soft actuator.

At this time, the hydrogel microwell was formed in the pneumatic soft actuator. In the contraction evaluation, the deformation of the compressed section in the pneumatic soft actuator containing the hydrogel microwell was measured. Consequently, the contraction percentage increased in proportion to the air pressure. Moreover, the maximum contraction percentage was ∼6% at 0.1 MPa. Apart from the contraction percentage due to pneumatic drive, the compressive strength of the pneumatic soft actuator was also measured considering a long-term culture. In the evaluation of the compressive strength of the pneumatic soft actuator, the PDMS chamber of the pneumatic soft actuator was broken from 0.16 to 0.25 MPa (Fig. 4c). From these results, considering the safety factor of the pneumatic soft actuator when pressurizing repeatedly into the pneumatic soft actuator, the air pressure ranging from 0–0.06 MPa, accuracy ±8% FS and 0.13 Hz was cyclically applied to the pneumatic soft actuators while culturing.

3.3 Performance of hydrogel microwell compressed by pneumatic soft actuator

In order to culture cells in a three-dimensional shape, hydrogel microwells with a rounded hole were fabricated. Specifically, the hydrogel microwell was formed in the center of the PDMS chamber with the stainless-steel wire and the microwell jig (Fig. 5a and b). The hydrogel microwell in the pneumatic soft actuator was molded to the shape of the stainless-steel wire. The hydrogel microwells were stained with fluorescent particles to clarify the contour of the hydrogel microwell (Fig. 5c). In order to promote cell-to-cell adhesion, the tip of the stainless-steel wire for forming the hydrogel microwell was processed into a round shape. After forming the hydrogel microwell, the hydrogel was compressed with the pneumatic soft actuator. This compression shrank the hydrogel microwell, then the compressive strain conveyed the compressive force to the cells in the culturing part of the hydrogel microwell. Referring to previous studies,^23–25 mechanical stimulation of ∼10% displacement was targeted. Therefore, the compressive strain of the culturing part was adjusted up to ∼10%. To choose a suitable agarose gel in this pneumatic soft actuator, agarose gels with various gelation temperatures used for cell culture^26,27 were evaluated. Then, the compressive strain of the two different hydrogel microwells, low- and high-gelling temperature agarose gels (AG_low and AG_high), were measured (Fig. 5d). Although the structural stability of AG_low was low, AG_low had a low-gelling temperature and melting point, which allowed live cells to be embedded in the gel and retrieved from the gel without damaging the cells afterward. On the other hand, AG_high had high structural stability under the condition above 37 °C, which enabled the gel to maintain its structure for a long time. The maximum compressive strain of the culturing parts made from AG_low and AG_high showed ∼11% and ∼10% at 0.06 MPa, respectively (Fig. 5d). Other than the compressive strain evaluation, the durability of the two agarose gels against repeated strain was also evaluated. After compressing the two agarose gels for 24 h, the AG_high was not broken, while the AG_low cracked (Fig. 5e). The agarose gel in MPHGS needed to be durable to the deformation of the PDMS chamber. Therefore, the AG_high was selected for culturing the cells due to its higher durability against repeated strain than the AG_low.

Fig. 5 Performance of hydrogel microwell. (a) The stainless-steel wire for forming a hydrogel microwell. The diameter of the stainless-steel wire was 0.5 mm. (b) Microwell jig for holding stainless-steel wires. (c) Fabricated hydrogel microwell stained by fluorescent particles from the side and top views. In the culturing part of the hydrogel microwell, cells were cultured. (d) Comparison of the compressive strain in the hydrogel microwells made from agarose gels with low- and high-gelling temperatures (AG_low and AG_high) when applying air pressure to the pneumatic soft actuator (mean ± S.D., n = 3). The diameter of the culturing part of the hydrogel microwell was 0.5 mm without applying air pressure. (e) The top images of the hydrogel microwells (AG_low and AG_high) after applying compressive stimulation to the hydrogel microwells for 24 h. Air pressure was cyclically applied to the pneumatic soft actuators at 0–0.06 MPa, accuracy ±8% FS, and 0.13 Hz for 24 h. All scale bars = 1 mm.

3.4 Cell culture with compressive stimulation

To evaluate the effects of compressive stimulation by the pneumatic soft actuator, ATDC5 cells were cultured in the culturing part of the hydrogel microwell formed in the pneumatic soft actuator. The cellular tissue was formed with compressive stimulation by air pressure during incubation (Fig. 6). Before culturing the cells, the ATDC5 cells were seeded at a density of 1 × 10⁶ cells per mL into the hydrogel microwells. The incubation times were 6, 12, and 24 h. In the cell culture, the compressed cells and the controls were cultured separately. Also, the air pressure ranging from 0 to 0.06 MPa, accuracy ±8% FS and 0.13 Hz was applied into the pneumatic soft actuators during incubation. The controls were the cells cultured for 6, 12, and 24 h in the hydrogel microwells formed in the pneumatic soft actuators without compressing the cells during incubation. For the measurement of the volume of the cellular tissues, the cytoskeleton was observed by the confocal microscope (Fig. 6a). Then, the volume of the cellular tissues stimulated by compression formed more largely than the controls. Moreover, the cell density of the cellular tissues was evaluated from the porosity per area of the cellular tissues from the cell nucleus images observed by the confocal microscope. In addition to the increase in volume, the cell density of the cellular tissues stimulated by compression was higher than that of the controls at 6 h (Fig. 6a and b). Consequently, the cellular tissues compressed for 6 h were as dense and large as the controls cultured for 12 h. Also, the cells adhered to each other and shaped a three-dimensional cellular tissue after the compressive stimulation for 6 h. The increase in the cell density and cellular volume indicated that compressive stimulation of MPHGS facilitated the cellular aggregation. From these results, the cellular tissues compressed by the pneumatic soft actuator showed an early cellular tissue formation compared with the controls. Moreover, the proposed method formed the cellular tissues of a size comparable to cellular tissues cultured for one and two days in a shorter formation time than previous studies (Fig. 7).^28–33 Lastly, after incubation, the cell nucleus, cytoskeleton, Sox9, and collagen II of the cellular tissues were immunostained respectively (Fig. 6c). Sox9 is a transcription factor essential for chondrocyte differentiation.³⁴ Also, collagen II exists abundantly in cartilage.³⁵ In consequence, from qualitative evaluation based on the observation of the images of the immunostained cellular tissues, the cellular tissues stimulated by compression expressed Sox9 and collagen II, the same as the controls.

Fig. 6 Cellular tissues cultured by pneumatic compressive stimulation. ATDC5 cells were compressed and cultured by MPHGS. Air pressure was cyclically applied to the pneumatic soft actuators at 0–0.06 MPa, accuracy ±8% FS, and 0.13 Hz during culturing. (a) Volume of the cellular tissues after culturing for 6 h (mean ± S.D., n = 3). (b) Porosity per area in the cellular tissue after culturing for 6 h (mean ± S.D., n = 3, *p < 0.05). (c) The confocal images showing z-axis projections and cross-sectional views of immunostained cellular tissues after culturing for 6, 12, and 24 h. The cell nucleus, cytoskeleton, Sox9, and collagen II were immunostained with blue, green, violet, and red, respectively. All scale bars = 100 μm.

Fig. 7 Comparison of culture time and size of chondrocyte spheroids in non-adherent wells in previous studies.^28–33

4. Discussion

In this study, ATDC5 cells were stimulated by compression during incubation with pneumatic soft actuators. After culturing, the cellular tissues compressed for 6 h showed an increase in volume and cell density in comparison with the controls for 6 h despite seeding the same amount of cells into each hydrogel microwell. These differences suggested that the pneumatic soft actuator promoted cell aggregation at the early stage of cellular tissue formation. This early mechanical stimulation of cells was achieved by constructing a device integrating the pneumatic soft actuator and the hydrogel microwell. The pneumatic soft actuator is suited to gently enveloping the target cells as a soft actuator. Also, combining a pneumatic soft actuator and a hydrogel microwell enabled cells to be stimulated shortly after seeding. In the soft actuator with only rubber (Fig. 2a), pressing spreads the cells. Meanwhile, the cells were trapped by the hydrogel microwell to prevent the cells from spreading in this study (Fig. 2b). The PDMS chamber is constrained from the sides and bottom due to the acrylic enclosure. Conversely, the top and center directions of the PDMS chamber are free boundaries. Thus, the upper part of the compressed section of the PDMS chamber inflates more in comparison with the lower part of the compressed section when applying air pressure to the pneumatic soft actuator. Consequently, the PDMS chamber compresses the hydrogel microwell diagonally downward. In addition, at this time, this diagonal downward compression to the hydrogel microwell causes a downward force to the cover glass at the bottom of the pneumatic soft actuator, which creates a reaction force to the bottom of the hydrogel microwell. Therefore, the hydrogel microwell deformed by the pneumatic soft actuator compresses the culture medium containing the cells at the bottom of the microwell from above at a diagonal angle and the bottom of the microwell, despite being open to the atmosphere in this proposed method.

To confirm the effectiveness of hydrogel, the cells were cultured with and without hydrogel in the pneumatic soft actuator. To be specific, the cells were compressed during culturing in the hydrogel microwell (W/HG_M), in the microwell replaced with PDMS (W/PM_M), and in the pneumatic soft actuator without a hydrogel microwell (W/O HG_M), respectively (Fig. 8a–c). As a result (Fig. S2), only the cells in the W/HG_M aggregated and formed a cellular tissue, whereas the cells in the W/PM_M and W/O HG_M were scattered. Especially in the W/PM_M, the cells were hardly seen compared to the other two conditions. This difference is because the air in the W/PM_M hindered the culture medium containing the cells from entering the microwell. The cell suspension falls into the W/HG_M due to the mesh of the hydrogel (Fig. 8d), allowing the air in the microwell to escape (Fig. 8e). However, the W/PM_M had no routes for the air to escape (Fig. 8f), which prevented the cells from entering the microwell. Therefore, hydrogel enables cells to aggregate in the microwell, in addition to promoting effective compression of the cells. Forming spheroids by wells made of hydrogel has been researched.^36,37 However, the method of compressing microwells formed with hydrogel by air-driven soft actuators to apply three-dimensional compression stimulation to cells in the wells is unique to this study.

Fig. 8 Comparison of a pneumatic soft actuator with a hydrogel microwell, with a PDMS microwell, and without a hydrogel microwell. (a) The microwell with hydrogel formed in the pneumatic soft actuator (W/HG_M). (b) The microwell with PDMS formed in the pneumatic soft actuator (W/PM_M). (c) Without a hydrogel microwell in the pneumatic soft actuator (W/O HG_M). (d) The cells in the culture medium falling on the mesh of the hydrogel. (e) The cell suspension entering the hydrogel microwell. The air in the hydrogel microwell escapes through the mesh of the hydrogel. (f) The cell suspension staying on the PDMS microwell. The air in the PDMS microwell has no routes to escape.

The pneumatic soft actuator in this study mainly consisted of PDMS and agarose gel. In this study, the pneumatic soft actuator deforms by air pressure; therefore, high elasticity and durability were necessary for the deformation part of the pneumatic soft actuator. Additionally, the deformation part of the pneumatic soft actuator touches the culture medium and the cells. Thus, the material of the deformation part needed to have biocompatibility. PDMS was used as the material of the pneumatic soft actuator because of the elasticity, durability, and biocompatibility of PDMS. However, PDMS can be replaced with other rubbers as long as the material of the deformation part has elasticity, durability, and biocompatibility, such as silicone and polyurethane.

Also, agarose gel can be replaced with other gels. In this study, the cells were prevented from adhering to a scaffold in order to promote adhesion to each other; therefore, the gel needed to have non-cellular adhesion. Although collagen and Matrigel are used as materials for the ECM, these two gels are not suitable for the material of the hydrogel microwell because of the cell adhesion. One alternative material to agarose gel is alginate due to the non-cellular adhesion. However, alginate has some problems. First, in general, alginate forms a gel by dipping alginate into a CaCl₂ solution, resulting in non-uniformity of gel shape. Secondly, alginate gelates immediately after touching a CaCl₂ solution, leading to an imperfect microwell shape. In contrast, agarose gelates more slowly than alginate at room temperature or below after heating the agarose solution and is easy to form desired shapes. Therefore, agarose was used as the material for the hydrogel microwell.

A feature of this system is that it combines a hydrogel well with a rubber air actuator, eliminating the need to fix cells to a scaffold. Multiple cell cultures by mechanical stimulation require the cells to be fixed to a scaffold while the stimulation is applied, which restricts cell harvesting and the direction of mechanical stimulation.^38–40 Compression is one of the mechanical stimuli to cells. Generally, to stimulate cells and cellular tissues, one-dimensional compression devices are used.^41–44 Mechanical stimuli from one-dimensional compression devices promote cell differentiation, such as osteoblasts^41,42 and chondrocytes.⁴⁵ Meanwhile, these one-dimensional compressions require cell seeding onto or into scaffolds before compressing the cells. Additionally, cell culture by one-dimensional compression needs time for cell adhesion to the scaffolds beforehand. In contrast, hydraulic pressure can unidirectionally compress cells and cellular tissues without cell adhesion to scaffolds.^43,44 However, unidirectional compressions by hydraulic pressure basically cannot compress from cells to cellular tissues through one process while culturing. Also, in regard to the deformation of cells caused by compression, while one-dimensional compression tends to cause the deformation of cells in a specific direction,⁴⁶ the shapes of the cells compressed by three-dimensional compression do not show significant anisotropy in cell morphology.⁴⁷ The proposed method in this study three-dimensionally compresses the cells through the pneumatic soft actuator and hydrogel microwell without fixing the cells to a scaffold. This compression of the pneumatic soft actuator with the hydrogel microwell enabled cell culture from cells to cellular tissues continuously. Moreover, the proposed method formed the cellular tissues of a size comparable to chondrocyte spheroids cultured in non-adherent wells for one and two days in a shorter time than previous studies (Fig. 7). In addition to these advantages of the proposed method, cell culture by the pneumatic soft actuator and hydrogel microwell compresses from multiple directions as opposed to one-dimensional compression methods, which prevents significant cell deformation in a specific direction. This is because the hydrogel microwell deformed by the pneumatic soft actuator encapsulates the bottom of the hydrogel microwell and presses the cells directly (Fig. 2b). The results of the cell culture suggested that the effects of the compression in this study stimulated the cells, facilitating cell aggregation. Therefore, this proposed method has the potential for applications to cells highly active to compressive stimuli: mesenchymal stem cells (MSCs), osteoblasts, and fibroblasts, besides chondrocytes.^48–50 The compressive stimulation of this method can be used for regenerative medicine and drug discovery research: joint regeneration, bone metabolism disorders, and drug research for cancer treatment.

MPHGS facilitated the early cellular formation of chondrocytes in the cell culture. Regarding this early cell formation, two factors are mentioned: one common to all cells and another specific to chondrocytes. One factor is that the expression of integrin and cadherin promoted by compression enhanced intercellular adhesion and accelerated cell aggregation. Integrin β1 and integrin α5 are the major mediators of mechanical signals in chondrocytes. In a previous study,⁵¹ hydrostatic compressive forces increased the expression of integrin β1, activating integrin-focal adhesion kinase (FAK)–ERK (extracellular signal-regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway. FAK, ERK, and PI3K regulate cell proliferation, growth, and survival. This activation increased the viability of chondrocytes. In addition, the increase in integrin β1 promotes the expression of N-cadherin, which enhances intercellular adhesion and cell aggregation.⁵² These suggest that the compression by the pneumatic soft actuator caused the increase of integrin β1, which promoted the expression of N-cadherin in ATDC5 cells, suggesting cell aggregation at the early stage.

Another factor is the increase in aggrecan and collagen II of the chondrocytes. Compressive stimulation upregulates the expression of aggrecan and collagen II of chondrocytes.⁵³ Aggrecan and collagen II are major components of the ECM, which constitutes cartilage.^54,55 Collagen II forms the majority of the structural framework of cartilage.^56,57 Also, aggrecans contain multiple glycosaminoglycan (GAG) chains⁵⁸ and fill the spaces between the fibrils of the network of collagen. Due to the high hydrophilic and polyanionic properties of the GAG chains, the chains attract water molecules, leading to high osmotic and swelling pressure within the network of collagen.⁵⁹ Additionally, these high osmotic and swelling pressure play a role in the lubricity of cartilage tissue, which leads to the resistance of mechanical forces.^57,60 Thus, these aggrecan and collagen II stabilize the structure of chondrocyte tissue⁵⁹ and promote the cellular formation.⁶¹ As in the previous studies,^61,62 the increase of aggrecan and collagen II in this study was likely promoted by the compression of the pneumatic soft actuator with the hydrogel microwell, which probably affected the cellular formation. Therefore, early tissue formation in this study would be attributed to the increase in integrin, cadherin, aggrecan, and collagen II of the chondrocytes due to the compression of the pneumatic soft actuator with the hydrogel microwell, thereby contributing to tissue organization and differentiation.

To evaluate proteins related to chondrogenesis expressed in cells, these proteins were observed by confocal microscope. The Sox9 and collagen II in the cellular tissues compressed by the pneumatic soft actuator were expressed as well as the controls. This means that MPHGS did not interfere with cell tissue formation and the resulting tissue differentiation. The magnitude of compression applied by MPHGS was based on some previous studies in which a compressive strain of 10% increased chondrogenic gene expression in MSCs or chondrocytes.^23–25 Moreover, in one of these previous studies, high mRNA expression of type II collagen and the increase in the production of sulfated glycosaminoglycan (sGAG) in chondrocytes were exhibited at a continuous dynamic compression of compressive strain of 10% at 0.1 Hz. From these parameters and results, the compressive strain and frequency of MPHGS were set to 10% and 0.13 Hz, respectively. The frequency was determined to be 0.13 Hz, which is slightly faster than 0.1 Hz, considering the delay in compressive force transmission between the PDMS chamber, the hydrogel microwell, the culture medium, and the cells until the compressive stimulation reaches the cells. Therefore, how the magnitude or the frequency of compression applied by the pneumatic soft actuator with the hydrogel microwell affects cellular formation and cell differentiation is yet unclear. To understand how the compressive stimulation of this study was applied to the cells in the hydrogel microwell, a spherical tissuelike model compressed in the pneumatic soft actuator with the hydrogel microwell during culturing was simulated. In the simulation, the spherical tissuelike model was subjected to compression induced by the inflation of the pneumatic soft actuator at 0.06 MPa. Consequently, the model was compressed from both the cylindrical wall and the bottom of the hydrogel microwell (Fig. S3a). Additionally, from the simulation, fluid flow was shown in the hydrogel microwell (Fig. S3b). That said, the tissuelike model was assumed to be the cellular tissue after cell aggregation. In addition, the hydrogel microwell was assumed to be a wall without a hydrogel mesh in the simulation. Hence, these effects remain only part of several factors. In actual cell culture, various factors, including contact conditions,⁶³ friction,⁶⁴ mechanical confinement,⁶⁵ and time-dependent compaction,⁶⁶ affect the cells in the hydrogel microwell compressed by the pneumatic soft actuator. For instance, some cells float in the culture medium. Even when the compressive stimulation is applied in the hydrogel microwell, the cells are affected by the frictions of cell-to-cell or between the cells and the hydrogel microwell. Among these factors, intercellular distance may be one of the main factors involved in the facilitated cellular formation. Cell adhesion is promoted more effectively when cells are in closer proximity.⁶⁷ Upon compression, the shrinkage of the hydrogel microwell narrows the bottom space of the hydrogel microwell, which leads to a closer proximity among the cells. This reduction in intercellular distance may have facilitated intercellular adhesion in the early-stage aggregation. To accurately evaluate the effects of the compressive stimulation by the proposed method, particularly from the perspective of cell differentiation, a large number of samples is required. Thus, to realize the quantitative evaluation of the compressed cellular tissue in the future, parallelizing pneumatic soft actuator and increasing the number of samples are necessary. For instance, miniaturizing and integrating the pneumatic soft actuator into a well plate enables mass culture.

The proposed method with MPHGS has the potential to be applied to various fields in addition to regenerative medicine and drug discovery research. One application is to combine the proposed culture system with cultured meat.⁶⁸ Cultured meat is one of the artificial meats made by proliferating and differentiating animal-derived cells. Generally, the muscle tissue of cultured meat is constructed by culturing cells in three-dimensional culture using scaffolds or suspension culture using a bioreactor.^69,70 Applying three-dimensional compressive stimulation to cells using the soft actuator promotes muscle cell growth,¹⁹ differentiation,⁷¹ and muscle fiber formation,^19,71 thereby improving the quality of cultured meat. Moreover, the pneumatic soft actuator is expected to clarify the feedback characteristics of muscles, bones, and ligaments for determining exercise load during rehabilitation. Moderate compression facilitates the recovery of damaged muscles and bones in the human body.^72,73 Repetitive compression in the pneumatic soft actuator reproducing the internal environment of the human body while exercising is applicable for elucidating the moderate compression of muscle and bone tissue during the rehabilitation process. Therefore, this MPHGS should contribute to the food industry and the rehabilitation field in the future.

5. Conclusion

In the cell culture of this study, the compressive stimulation by pneumatic soft actuators increased the cell density and the volume of cellular tissues at the early stage of cellular tissue formation. In addition, the proteins related to chondrogenesis in the compressed cellular tissues were expressed at the same level as the controls. This proposed method with MPHGS is applicable to the fields of regenerative medicine, drug discovery research, food, and rehabilitation.

Author contributions

Ryota Kawamae: conceptualization, investigation, methodology, writing – original draft, visualization. Atsushi Takata: methodology, writing – review & editing. Kenjiro Takemura: supervision, writing – review & editing. Yuta Kurashina: conceptualization, methodology, funding acquisition, supervision, writing – review & editing.

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI).

Supplementary information: the SI includes the simulation conditions by COMSOL, the waveform of the air pressure applied to the pneumatic soft actuator during culturing, and the figures used in the discussion. In addition, the detailed values used in the graphs (Fig. 4b and c, 5d and 6a and b) are described in the SI. See DOI: https://doi.org/10.1039/d5lc00731c.

Acknowledgements

This work was partly supported by Tateisi Science and Technology Foundation, the Casio Science Promotion Foundation, the Fluid Power Technology Promotion Foundation, and the Uehara Memorial Foundation.

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