CELL STRETCHING MICRODEVICE FOR EVALUATING CELLULAR BIOMECHANICS

BASED ON IN-SITU CELLULAR RESPONSE OBSERVATION Yuta Nakashima1, 2*, Ryo Monji2, Katsuya Sato3 and Kazuyuki Minami2

1Kumamoto University, JAPAN 2Yamaguchi University, JAPAN

3The University of Tokushima, JAPAN ABSTRACT

We developed a cell stretching microdevice to evaluate the cellular biomechanics based on in-situ cellular responses to mechanical stimuli. The device consists of an elastic culture chamber and leaf springs by which uniaxial stretch is applied to the culture chamber. When the slider is pushed by a microneedle, stretching arms moved toward the outside and stretched the culture chamber. We demonstrated the fundamental characteristics of a microdevice, and observed cellular response behavior. As a result, we succeeded in the in-situ observation of transition of the cellular calcium signaling response to stretch stimulation by optical microscopy using the microdevice. KEYWORDS: Cell stretching, Mechanical stimulation, Cellular response evaluation, Cell biomechanics

INTRODUCTION

The clarification of the mechanoreception mechanism and signal transduction pathways in cells by analyzing the cellular reactions and responses to mechanical stimuli in vitro is an important field of study in cellular biology and cell biomechanics [1, 2]. Several studies that evaluate the cellular response behavior to mechanical stress using self-made experimental devices have been carried out for clarifying cell biomechanics [3, 4]. In particular, evaluation of cellular responses to receive the stretch stimuli is commonly used for clarifying cell biomechanics because the physiological interpretation is easily. Also, detailed understanding of cellular responses to receive stretch stimuli is very important for not only understanding cellular functions in fundamental biology but also development of stem cell differentiation technique in regenerative medicine. However, in a conventional cell-stretching device, it was difficult to observe the initial cellular response to stretching with high spatial and temporal resolution because observed cells go out of the microscope field during stretch stimuli. We present a highly precise cell stretching microdevice to evaluate the cellular responses to a mechanical stimulus. The device permits the in-situ observation of single cell reaction behavior stimulated by stretching with high spatial and temporal resolution during the stretching operation.

Figure 1: Schematic of a cell stretching microdevice.

MATERIALS AND METHODS

A cell stretching microdevice consists of one elastic culture chamber made of PDMS, one pair of stretching arms by which the chamber is uniaxially stretched, parallel leaf springs that support the stretching arms, and the slider that drives the stretching arms (Fig.1). This device permits the in-situ observation of single cell reaction behavior during the stretching operation, because it is designed to have less rigid body displacement during stretching, and a culture chamber is almost the same size as the observation field of optical microscopes. Thus, we are able to clarify the cell biomechanics by stimulation control using the presented microdevice based on real-time cellular reaction. Figure 2 shows the principle of creating cell stretch stimuli. When the slider is pushed by microneedle connected to the micromanipulator, the pair of stretching arms is moved toward the outside by the slider and the culture chamber is stretched from both ends (Fig.2 (a)). In other words, cells cultured on the culture chamber receive the stretch stimuli (Fig.2 (b)). Mechanical stimulation to a cell can be controlled by regulating of the chamber contraction with the slider operation.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 374 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

Figure 2: Principle of creating cell stretch stimuli.

EXPERIMENTAL RESULT Figure 3 shows the photographs of each element of the fabricated cell stretching microdevice. The device was

fabricated by photolithography and PDMS molding. Each element, which is important for device driving, was successfully fabricated by three-dimensional micro fabrication technique using multiple exposures to SU-8 [5]. The width and length of bridge structures were 70 μm and 700 μm, respectively (Fig.3 (a, b)). We succeeded in the bridge structures in midair for preventing the detachment of the slider. Each leaf spring was 50 μm in width and 100 μm in height (Fig.3 (c)). The device permits the observation of cellular responses to stretching in real time because it is made of transparent materials when observed by optical microscope.

Figure 4 shows the operation test of the fabricated device. When the slider of the device was pushed from the initial state by microneedle connected to the micromanipulator (Fig.4 (a)), the culture chamber was stretched from both ends in the directions to the right and left in the figure (Fig.4 (b)). The amount of the culture chamber deformation was controlled by the amount of the stretching arm displacement. It was shown that the device was actually able to work under observation using the inverted optical microscope.

A cell stretch stimuli experiment was carried out by the fabricated microdevice. Osteoblastic cell line MC3T3-E1 cells were stained by fluorescent dye (Fluo-3 AM, Dojindo Molecular Technologies, Inc.) and used as samples in the experiments. We observed the changes of intracellular calcium concentration before and after stretch stimulation on the fabricated device (Fig.5). The cultured cells in the chamber received the stretch stimuli via the stretched culture chamber by stretching arms. The transition of the intracellular calcium signaling response to stretching was observed by optical microscopy using the fabricated microdevice. When cells received the stretch stimuli on the device, the cells were quick to respond to stretch stimulation and intracellular calcium concentration around nucleus began to increase immediately. Then the calcium concentration of cell body was gradually increased as time passed. We succeeded in the in-situ observation of first reaction of cells that received stretch stimulation. The fabricated device can be used in the in-situ observation experiments of cellular reaction such as the alteration of cellular morphology and cell differentiation induction to mechanical stimuli.

Figure 3: Photograph of each elements of the fabricated cell stretching microdevice.

Figure 4: Operating image of the cell stretching microdevice. (a) The initial state. (b) The stretched state.

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Figure 5: Observation of the intracellular calcium signaling response to the stretch stimuli using the fabricated device. (a-d) are transmitted phase contrast images and (e-h) are fluorescence images of the first reaction of cells that received stimulation.

CONCLUSION The cell stretching microdevice for evaluating cellular biomechanics based on in-situ cellular responses was

fabricated by three-dimensional micro fabrication technique using multiple exposures to SU-8. The fabricated microdevice was actually able to work under observation using the inverted optical microscope during the stretching operation. Also, the fabricated device succeeded in observing the first reaction and transition of the intracellular calcium concentration of cells that received stretch stimuli. In the near future, we will evaluate the threshold level of amount of strain which a cell detects. ACKNOWLEDGEMENTS

This work was partly supported by the Grant-in-Aid for Young Scientists (B) 23760232 and 24760078 of the fund from the Japan Society for the Promotion of Science (JSPS) KAKENHI. Further, the PDMS (KE-106) and the fluorine polymer (DURASURF DS-3304Z) were provided by Shin-Etsu Chemical Co., Ltd. and HARVES Co., Ltd., respectively. REFERENCES [1] M. Chiquet, A. S. Renedo, F. Huber and M. Fluck, "How do fibroblasts translate mechanical signals into changes

in extracellular matrix production?," Matrix Biology, vol. 22, pp. 73-80, 2003. [2] T. Adachi, K. Sato and Y. Tomita, "Directional dependence of osteoblastic calcium response to mechanical

stimuli," Biomechanics and Modeling in Mechanobiology, vol. 2, pp. 73-82, 2003. [3] S. Yano, M. Komine, M. Fujimoto, H. Okochi and K. Tamaki, "Mechanical stretching in vivo regulates signal

transduction pathways and cellular proliferation in human epidermal kertinocytes," Journal of Investigative Dermatology, vol. 122, pp. 783-790, 2004.

[4] K. Sato, S. Kamada and K. Minami, "Development of microstretching device to evaluate cell membrane strain field around sensing point of mechanical stimuli," International Journal of Mechanical Sciences, vol. 52, Issue 2 pp. 251-256, 2010.

[5] I. G. Foulds and M. Parameswaran, "A planar self-sacrificial multilayer SU-8-based MEMS process utilizing a UV-blocking layer for the creation of freely moving parts," Journal of Micromechanics and Microengineering, vol. 16, pp. 2109-2115, 2006

CONTACT *Yuta Nakashima, tel: +81-96-342-3754; yuta-n@mech.kumamoto-u.ac.jp

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