A cell culture module capable of cooling stimulus to collect cells efficiently on a metal culture substrate was developed. We evaluated the cell collection ratio and morphology of the collected cells. Following a cooling stimulus (0°C) for 20 min, the number of collected cells was increased by 50% compared to that collected after trypsin treatment without pipetting from the metal culture substrate. Following the cooling stimulus, cells were observed by fluorescence microscopy and scanning electron microscopy; the cell filopodia were shrunken compared to non-cooling-stimulated cells. Furthermore, the combination of collagenase and cooling stimulation resulted in the collection of a comparable number of cells as that obtained using only trypsin. Thus, cell proliferation was improved compared to that following trypsin treatment. Therefore, this method can be applied for culturing cells that are susceptible to trypsin damage.

1. [1] R. Langer and J. P. Vacanti, “Tissue Engineering,” Science, Vol.260, pp. 920-926, 1993.
2. [2] M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson, “Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation,” N. Engl. J. Med., Vol.331, pp. 889-895, 1994.
3. [3] H. G. Chambers, “Osteochondral Autologous Transplantation Was More Effective Than Microfracture for Osteochondritis Dissecans in Children Younger than Eighteen Years,” J. Bone Joint Surg. Am., Vol.92, p. 1998, 2010.
4. [4] R. I. Freshney, “Culture of animal cells: a manual of basic technique and specialized applications,” 6th ed., Wiley-Blackwell, pp. 211-212, 2010.
5. [5] H. Hirai, R. Umegaki, M. Kino-oka, and M. Taya, “Characterization of Cellular Motions through Direct Observation of Individual Cells at Early Stage in Anchorage-Dependent Culture,” J. Biosci. Bioeng., Vol.94, pp. 351-356, 2002.
6. [6] R. Gräbner, U. Till, and R. Heller, “Flow Cytometric Determination of E-selectin, Vascular Cell Adhesion Molecule-1, and Intercellular Cell Adhesion Molecule-1 in Formaldehyde-Fixed Endothelial Cell Monolayers,” Cytometry Res., Vol.40, pp. 238-244, 2000.
7. [7] Y. Wu, J. Wu, D. Y. Lee, A. Yee, L. Cao, Y. Zhang, C. Kiani, and B. B. Yang, “Versican Protects Cells from Oxidative Stress-induced Apoptosis,” Matrix Biol., Vol.24, pp. 3-13, 2005.
8. [8] K. T. Piercy, R. L. Donnell, S. S. Kirkpatrick, B. L. Mundy, S. L. Stevens, M. B. Freeman, and M. H. Goldman, “Effect of Harvesting and Sorting on β-1 Integrin in Canine Microvascular Cells,” J. Surg. Res., Vol.100, pp. 211-216, 2001.
9. [9] J. P. Revel, P. Hoch, and D. Ho, “Adhesion of Culture Cells to Their Substratum,” Exp. Cell Res., Vol.84, pp. 207-218, 1974.
10. [10] F. Rico, C. Chu, M. H. Abdulreda, Y. Qin, and V. T. Moy, “Temperature Modulation of Integrin-Mediated Cell Adhesion,” Biophys. J., Vol.99, pp. 1387-1396, 2010.
11. [11] M. J. Lydon and R. C. Hughes, “Fibronectin Synthesis and Surface Expression is Correlated with Cell Morphology and Adhesiveness in a Cold-sensitive, G1-defective Mustant of CHO Cells,” Exp. Cell Res., Vol.135, pp. 347-354, 1981.
12. [12] Y. Kurashina, T. Hamano, S. Miyata, J. Komotori, and T. Koyama, “Proliferation of Calf Chondrocyte on StainlessSteel Surfaces with Different Microtopography,” J. Japan Inst. Met. Mater., Vol.78, pp. 170-176, 2014.
13. [13] M. F. Underhill and C. M. Smales, “The Cold-shock Response in Mammalian Cells: Investigating the HeLa Cell Cold-shock Proteome,” Cytotechnology, Vol.53, pp. 47-53, 2007.
14. [14] J. C. Adams, “Cell-matrix contact structures,” Cell Mol. Life Sci., Vol.58, pp. 371-392, 2001.
15. [15] P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramco, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale Architecture of Integrin-based Cell Adhesions,” Nature, Vol.468, pp. 580-586, 2010.
16. [16] R. M. Ezzell, W. H. Goldmann, N. Wang, N. Parasharama, and D. E. Ingber, “Vinculin Promotes Cell Spreading by Mechanically Coupling Integrins to the Cytoskeleton,” Exp. Cell Res., Vol.231, pp. 14-26, 1997.
17. [17] C. Bakolitsa, D. M. Cohen, L. A. Bankston, A. A. Bobkov, G. W. Cadwell, L. Jennings, D. R. Critchley, S. W. Craig, and R. C. Liddington, “Structural Basis for Vinculin Activation at Sites of Cell Adhesion,” Nature, Vol.430, pp. 583-586, 2004.
18. [18] C. Grashoff, B. D. Hoffman, M. D. Brenner, R. Zhou, M. Parsons, M. T. Yang, M. A. McLean, S. G. Sligar, C. S. Chen, T. Ha, and M. A. Schwartz, “Structural Basis for Vinculin Activation at Sites of Cell Adhesion,” Nature, Vol.466, pp. 263-266, 2010.
19. [19] F. Boccafoschi, C. Mosca, M. Bosetti, and M. Cannas, “The Role of Mechanical Stretching in the Activation and Localization of Adhesion Proteins and Related Intracellular Molecules,” J. Cell Biochem., Vol.112, pp. 1403-1409, 2011.
20. [20] G. Sagvolden, I. Giaever, E. O. Pettersen, and J. Feder, “Cell adhesion force microscopy,” Proc. Natl. Acad. Sci. USA, Vol.96, pp. 471-476, 1999.
21. [21] T. Inoue and H. Osatake, “A New Drying Method of Biological Specimens for Scanning Electron Microscopy: The t-Butyl Alcohol Freeze-drying Method,” Arch. Histol. Cytol., Vol.51, pp. 53-59, 1988.
22. [22] H. Fujie, K. Oya, Y. Tani, K. Suzuki, and N. Nakamura, “Stem Cell-Based Self-Assembled Tissues Cultured on a Nano-Periodic-Structured Surface Patterned Using Femtosecond Laser Processing,” Int. J. of Automation Technology, Vol.10, pp. 55-61, 2016.
23. [23] M. Mizutani, R. Honda, Y. Kurashina, J. Komotori, and H. Ohmori, “Improved Cytocompatibility of Nanosecond-Pulsed Laser-Treated Commercially Pure Ti Surfaces,” Int. J. of Automation Technology, Vol.8, pp. 102-109, 2014.
24. [24] Y. Kurashina, K. Takemura, S. Miyata, J. Komotori, and T. Koyama, “Effective cell collection method using collagenase and ultrasonic vibration,” Biomicrofluidics, Vol.8, p. 054118, 2014.