細胞冷凍是一種廣泛應用在各領域的細胞保存方法，能夠提供人們在培養細胞與保存細胞上極大的幫助。在細胞冷凍技術尚未出現前，細胞在需要保存時會有難度，但現今細胞冷凍技術已經日趨成熟，大幅降低了使用細胞保存的門檻。目前已經有學者嘗試找到比較適合使細胞從常溫狀態到冷凍狀態的方法，為了讓細胞冷凍的過程中，可能使細胞死亡的因素能夠被大幅降低，例如：細胞膜內形成冰晶、細胞脫水。在細胞冷凍的過程中，確定細胞膜通透性和冷凍保護劑等參數對於研究冷凍保存細胞的存活率是很重要的。為了分析這些未知參數，本研究使用一種利用曝光顯影技術製作的簡易微灌注系統，可以不借助外力將實驗用細胞固定在攝影機所對準的觀察位置。在不同環境溫度時灌注不同濃度的磷酸鹽緩衝生理鹽水，細胞體積會在灌注過程產生變化，對影像信息進行處理和分析，推估細胞膜的通透性。 

Cell cryopreservation is a widely used biotechnology, providing people great help in culturing and preserving cells, which had been a challenging task until the cryopreservation technology gradually became mature. Scientists had, through experiments, found methods suitable for revising cells from normal temperature to frozen state. These methods, such as the formation of ice crystals in the cell membrane and cell dehydration, could cause less death of cells. In the process of cell cryopreservation, it is essential to determine the cell membrane permeability and cryoprotectant parameters for studying the survival rate of cryopreserved cells. In order to analyze these unknown parameters, this study uses a simple micro-perfusion system made by soft lithography, which can fix the experimental cells in the observation position pointed by the camera without external force. By perfusing different concentrations of phosphate buffered saline, the volume of the cells will change. Then by using ImageJ to analyze the image information, these unknown parameters can therefore be predicted.

參考文獻
[1]. Polge, C. Smith, AU. & Parkes, A.S. 1949, “Revival of spermatozoa after vitrification and dehydration at low temperatures,” Nature, vol. 164, issue. 4172, pp.666-666.
[2]. Toner, M. Cravalho, E. G. & Karel, M. 1990, “Thermodynamics and Kinetics of Intracellular Ice Formation during Freezing of Biological Cells,” Journal of Applied Physics, vol. 67, no. 3, pp. 1582-1593.
[3]. Toner, M. Cravalho, E. G. Stachecki, J. Fitzgerald, Tomlkins, R. G. Yarmush, M. L. & Armant, D. R. 1993, “Nonequilibrium Freezing of One-Cell Mouse Embryos Membrane Integrity and Development Potential,” Biophysical Journal, vol. 64, no.6, pp.1908-1921.
[4]. Shannon L. Stott, Chia-Hsien Hsu, Dina I. Tsukrov, Min Yu, David T. Miyamoto, Belinda A. Waltman, S. Michael Rothenberg, Ajay M. Shah, Malgorzata E. Smas, George K. Korir, Frederick P. Floyd Jr., Anna J. Gilman, Jenna B. Lord, Daniel Winokur, Simeon Springer, Daniel Irimia, Sunitha Nagrath, Lecia V. Sequist, Richard J. Lee, Kurt J. Isselbacher, Shyamala Maheswaran, Daniel A. Haber, and Mehmet Toner. 2010, “Isolation of circulating tumor cells using a microvortex-generating herringbone- chip,” PNAS October 26, 107 (43) 18392-18397.
[5]. Giovanna. Vona, Abdelmajid. Sabile. 2000,” Isolation by Size of Epithelial Tumor Cells: A New Method for the Immunomorphological and Molecular Characterization of Circulating Tumor Cells,” The American Journal of Pathology, Volume 156, Issue 1, January, Pages 57-63.
[6]. Jacques. Izard, Ronald J. Limberger 2003,” Rapid screening method for quantitation of bacterial cell lipids from whole cells,” Journal of Microbiological Methods
Volume 55, Issue 2, November, Pages 411-418.
[7]. Pape WJ, Pfannenbecker U, Hoppe U 1987,” Validation of the red blood cell test system as in vitro assay for the rapid screening of irritation potential of surfactants”, Molecular Toxicology, 01 Sep 1987, 1(4):525-536.
[8]. Boris Rubinsky, 2003, “Principles of Low Temperature Cell Preservation “Heart Failure Reviews volume 8, pp. 277–284.
[9]. J. E. LOVELOCK & M. W. H. BISHOP. 1959, “Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide, “Nature volume 183, pp. 1394–1395.
[10]. C. POLGE, A. U. SMITH & A. S. PARKES, 1949, “Revival of Spermatozoa after Vitrification and Dehydration at Low Temperatures “Nature volume 164, pp. 666.
[11]. Lyndsey A. Withers, Patrick J. King. 1979, “A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L, “Plant Physiology Vol. 64, Issue 5 , pp. 675.
[12]. Prehoda RW,” Suspended Animation. Philadelphia”, Chilton Book Co, pp.211.
[13]. Franks F ed,” Water: A Comprehensive Treatise,” Plenum Press,
[14]. Rall W, Fahy G,” Ice free cryopreservation of mouse embryos at minus 196 Celsius by vitrification,” Nature, pp.573–575.
[15]. Gilmore, J. A. Mcgann, L. E. Liu, J. Gao, D. Y. Peter, A. T. Kleinhans, F. W. & Critser, J. K. 1995, “Effect of Cryoprotectant Solutes on Water Permeability of Human Spermatozoa,” Biology of reproduction, vol. 53, no. 5, pp. 985-995.
[16]. Andrea K. Bryan, 2012,” Continuous and Long-Term Volume Measurements with a Commercial Coulter Counter,”
[17]. Han, X. Luo, D. Cui, X. Heimfeld, S. & Gao, D. Y. 2007, “A modified differential scanning calorimetry method for determining water transport properties in biological cells during the freezing process,” Cell Preservation Technology, vol. 5, no. 1, pp. 25-32.
[18]. Luo, D. Han, X. He, L. Cui, X. Cheng, S. Lu, C. Liu, J. & Gao, D.Y. 2002, “A modeified differential scanning calorimetry for determination of cell volumetric change during the freezing process,” CryoLetters, vol. 23, pp. 229-236.
[19]. Devireddy, R. V. Bischof, J. C. 1998, “Measurement of water transport during freezing in mammalian liver tissue – the use of differential scanning calorimetry,” Journal of Biomechanical Engineering, vol. 12, pp.559-569.
[20]. Shinsuke. Sekia, F. W. Kleinhansab, Peter Mazur. 2008,” Intracellular ice formation in yeast cells vs. cooling rate: Predictions from modeling vs. experimental observations by differential scanning calorimetry,” Cryobiology Volume 58, Issue 2, Pages 157-165.
[21]. Hsiu-Yang Tseng. 2011, “A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures, Biopreservation and Biobanking VOL. 9, NO. 4 pp.355-362.
[22]. Mazur, P. 1984, “Freezing of Living Cells – Mechanisms and Implications,” American Journal of Physiology, vol. 14, no. 3, pp. C125-C142.
[23]. Lovelock, J. E. 1953, “The Haemolysis of Human Red Blood-Cells by Freezing and Thawing,” Biochimica et biophysica acta, vol. 10, no. 3, pp. 414-426.
[24]. P. Acker, L. E,” Membrane damage occurs during the formation of intracellular ice,” Cryo Lett., 22 (2001), pp. 241-254.
[25]. J.P. Acker, L.E. McGann,” Innocuous intracellular ice improves survival of frozen cells,” Cell Transplant., 11 (2002), pp. 563-571.
[26]. P. Mazur,” Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing,” J. Gen. Physiol., 47 (1963), pp. 347-369.
[27]. P. Mazur,” The role of cell membranes in the freezing of yeast and other single cells,” Ann. N. Y. Acad. Sci., 125 (1965), pp. 658-676.
[28]. R.E. Pitt, P.L. Steponkus,” Quantitative analysis of the probability of intracellular ice formation during freezing of isolated protoplasts,” Cryobiology, 26 (1989), pp. 44-63.
[29]. Richell. C. Prickett, Leah. A. Marquez-Curtis, Janet A. W. Elliott, Locksley E. McGann, 2015,“ Effect of supercooling and cell volume on intracellular ice formation, “ Cryobiology Volume 70, Issue 2, April 2015, Pages 156-163.
[30]. Jacques Loeb 1904,” The Recent Development of Biology,” Science, New Series, Vol. 20, No. 519 pp. 777-786.
[31]. Hugo Fricke 1925,” The electric capacity of suspensions with special reference to blood,” J Gen Physiol, pp.137–152.
[32]. E. Gorter, F. Grendel, 1925,” On bimolecular layers of lipoids on the chromocytes of the blood,” J Exp Med. pp.439–443.
[33]. F. S. Sjöstrand. Ebba. Andersson-Cedergren M. M. Dewey, 1958,” The ultrastructure of the intercalated discs of frog, mouse and guinea pig cardiac muscle,” Journal of Ultrastructure Research Volume 1, Issue 3, Pages 271-287.
[34]. Danko D. Georgiev, Glazebrook, 2007,” Subneuronal processing of information by solitary waves and stochastic processes,” CRC Press, pp.17-1–17-41.
[35]. Green, K. & Downs, S. 1976, “Corneal membrane water permeability as a function of temperature,” Investigative Ophthalmology, vol. 15, no. 4, pp. 304-307.
[36]. Noiles E. E, Bailey J. L. & Storey B. T. 1995, “The temperature dependence in the hydraulic conductivity, Lp, of the mouse sperm plasma membrane shows a discontinuity between 4 and 0 °C,” Cryobiology, vol. 32, pp. 220 – 238.
[37]. David E. Pegg. 2020, “Principles of Cryopreservation, “Cryopreservation and Freeze-Drying Protocols pp 39-57.
[38]. Matsuoka, S. Hibara, A. Ueno, M. & Kitamori, T. 2006, “Supercooled micro flows and application for asymmetric synthesis,” Lab on a chip, 6, pp. 1236-1238.
[39]. P. Acker, L. E,” Membrane damage occurs during the formation of intracellular ice,” Cryo Lett., 22 (2001), pp. 241-254.
[40]. J.P. Acker, L.E. McGann,” Innocuous intracellular ice improves survival of frozen cells,” Cell Transplant., 11 (2002), pp. 563-571.
[41]. Gao, D. Y. Mcgrath, J. J. Tao, J. Benson, C. T. Critser, E. S. & Critser, J. K. 1994, “Membrane-transport properties of mammalian oocytes – a micropipette perfusion technique,” Journal.
[42]. Mazur, P. Leibo, S. P. & Chu, E. H. Y. 1972,” A two-factor hypothesis of freezing injury,” Exp. Cell Res. 71, pp. 345-355.
[43]. Mazur, P. 1990, “Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos,” Cell biophysics, vol. 17, no. 1, pp. 53-92.
[44]. Hugh Davson, J.F. Danielli. 1970,“ The Permeability of Natural Membranes,” Cambridge University Press, Cambridge, UK.
[45]. B. Lucke, M. McCutcheon. 1932, “The living cell as an osmotic system and its permeability to water,” Physiol. Rev. (12) (1932), pp. 68-138.