||Miniaturized Cell Culture System
||Department of Electrical Engineering
Cell Culture System
CO2 Quantity Control
Cancer has become more and more common in this era; therefore, a lot of assays have been put forward to analyze the viability of cells in these decades. Among those methods, electric analysis is more developed. However, it is an imperfection that electric analysis chips cannot culture cells on chip while facing a long term measurement. In a general cell culturing, temperature, humidity and CO2 partial pressure are factors need to be concerned. This study proposes a miniaturized cell culture system to achieve those conditions. The heating source herein is an electric heating sheet that can produce saturated temperature with a constant input voltage. It also has a thin thickness which aid scale decreasing. Polydimethylsiloxane (PDMS) is used for forming chamber because it is biocompatible and gas-permeable. Instead of raising humidity to reduce evaporation, here use a polymethylmethacrylate (PMMA) cover to isolate the chamber by blocking up the gas paths of PDMS. This action can make the chamber insulated from the atmosphere and prevent the escaping of vapor. Based on that, the pore spaces can be used to control and retain the CO2 quantity. Apply laser confocal microscopy and Van der Waals equation to figure out the porosity and the needed quantity. Then take deionized (DI) water as the carrier for transporting CO2 where the dissolved quantity is calibrated by pH value. This solution will be injected into the chamber and release CO2. After applying this proposed system and a normal incubator to culture cells respectively for several times, there is nearly no obvious difference in cell growth.
LIST OF TABLES VI
LIST OF FIGURES VII
CHAPTER 1 INTRODUCTION 1
1.1 Background and Motivation 1
1.2 Cell Culture 3
1.3 Tiny-scale Device 4
1.4 Polydimethylsiloxane 5
CHAPTER 2 DEVICE DESIGN 7
2.1 Temperature Control 7
2.2 Reduction of Evaporation 13
2.3 CO2 Quantity Control 22
2.3.1 Previous Studies 22
2.3.2 pH Transformation 24
2.3.3 Porosity of PDMS 29
2.3.4 Chamber Parameters 33
CHAPTER 3 RELATIVE WORKS 37
3.1 Chamber Fabrication 37
3.2 System Setup 40
3.3 Materials 42
3.4 Culturing Setup 44
CHAPTER 4 RESULT AND DISCUSSION 47
4.1 CO2 and Evaporation 47
4.2 Comparison Analysis 50
4.2.1 Morphology 50
4.2.2 Counts 52
CHAPTER 5 CONCLUSION AND FUTURE WORK 54
 American Cancer Society Inc., "Cancer Facts & Figures 2013," Atlanta, 2013.
 J. C. Stockert, A. Blázquez-Castro, M. Ca˜nete and R. W. Horobin, "MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets," Acta Histochemica, vol. 114, pp. 785-796, 2012.
 Y. Li, W. Huang, S. Huang, J. Du and C. Huang, "Screening of anti-cancer agent using zebrafish: Comparison with the MTT assay," Biochemical and Biophysical Research Communications, vol. 422, pp. 85-90, 2012.
 R. Scherließ, "The MTT assay as tool to evaluate and compare excipient toxicity in vitro on respiratory epithelial cells," International Journal of Pharmaceutics, vol. 411, pp. 98-105, 2011.
 B. Daniel and M. A. DeCoster, "Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining," Brain Research Protocols, vol. 13, pp. 144-150, 2004.
 T. Gichner, A. Mukherjee and J. Velem´ınsk´y, "DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I," Mutation Research, vol. 605, pp. 17-21, 2006.
 G. K. Srivastava, R. Reinoso, A. K. Singh, I. Fernandez-Bueno, D. Hileeto, M. Martino, M. T. Garcia-Gutierrez, J. M. P. Merino, N. F. Alonso, A. Corell and J. C. Pastor, "Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis," Experimental Eye Research, vol. 93, pp. 956-962, 2011.
 H. L. Yang, C. S. Chen, W. H. Chang, F. J. Lu, Y. C. Lai, C. C. Chen, T. H. Hseu, C. T. Kuo and Y. C. Hseu, "Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata," Cancer Letters, vol. 231, pp. 215-227, 2006.
 Wikimedia Foundation, Inc., "Trypan blue," Wikimedia Foundation, Inc., [Online]. Available: http://en.wikipedia.org/wiki/Trypan_blue. [Accessed 5 8 2013].
 P. M. Goodwin, R. L. Affleck, W. P. Ambroset, J. N. Demas, J. H. Jett, J. C. Martin, L. J. Reha-Krantz, D. J. Semin, J. A. Schecker, M. Wu and R. A. Keller, "Progress Towards DNA Sequencing at the Single Molecule Level," Experimental Technique of Physics, 30 10 1995.
 K. C. Lan and L. S. Jang, "Integration of single-cell trapping and impedance measurement utilizing microwell electrodes," Biosensors and Bioelectronics, vol. 26, pp. 2025-2031, 2011.
 C. C. Wang, K. C. Lan, M. K. Chen, M. H. Wang and L. S. Jang, "Adjustable trapping position for single cells using voltage phase-controlled method," Biosensors and Bioelectronics, vol. 49, pp. 297-304, 2013.
 Z. R. Xu, C. G. Yang, C. H. Liu, Z. Zhou, J. Fang and J. H. Wang, "An osmotic micro-pump integrated on a microfluidic chip for perfusion cell culture," Talanta, vol. 80, pp. 1088-1093, 2010.
 M. Marimuthu and S. Kim, "Pumpless steady-flow microfluidic chip for cell culture," Analytical Biochemistry, vol. 437, pp. 161-163, 2013.
 K. Z. "kowska, A. Stelmachowska, R. Kwapiszewski, M. Chudy, A. Dybko and Z. B. zka, "Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip," Biosensors and Bioelectronics, vol. 40, pp. 68-74, 2013.
 S. P. Forry and L. E. Locascio, "On-chip CO2 control for microfluidic cell culture," Lab Chip, vol. 11, pp. 4041-4046, 2011.
 R. S. Shapiro and L. E. Cowen, "Thermal Control of Microbial Development and Virulence: Molecular Mechanisms of Microbial Temperature Sensing," mBio, vol. 3, pp. 1-6, 2012.
 V. A. Robert and A. Casadevall, "Vertebrate Endothermy Restricts Most Fungi as Potential Pathogens," The Journal of Infectious Diseases, vol. 200, pp. 1623-1626, 2009.
 A. Casadevall, "Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connection?," Fungal Genetics and Biology, vol. 42, pp. 98-106, 2005.
 N. G. A. MANZ and H. M. WIDMER, "Miniaturized Total Chemical Analysis Systems: a Novel Concept for Chemical Sensing," Sensors and Actuators, pp. 244-248, 1990.
 J. W. Gardner, V. K. Varadan and O. O. Awadelkarim, Microsensors, MEMS, and Smart Devices, Wiley, 2001.
 P. Harms, Y. Kostov and G. Rao, "Bioprocess monitoring," Current Opinion in Biotechnology, vol. 13, p. 124–127, 2002.
 P. Liepold, H. Wieder, H. Hillebrandt, A. Friebel and G. Hartwich, "DNA-arrays with electrical detection: A label-free low cost technology for routine use in life sciences and diagnostics," Bioelectrochemistry, vol. 67, pp. 143-150, 2005.
 M. Ionescu, B. Winton, D. Wexler, R. Siegele, A. Deslantes, E. Stelcer, A. Atanacio and D. Cohen, "Enhanced biocompatibility of PDMS (polydimethylsiloxane) polymer films by ion irradiation," Nuclear Instruments and Methods in Physics Research B, vol. 273, pp. 161-163, 2012.
 S. G. Charati and S. A. Stern, "Diffusion of Gases in Silicone Polymers: Molecular Dynamics Simulations," Macromolecules, vol. 31, pp. 5529-5535, 1998.
 N. Szita, A. Zanzotto, P. Boccazzi, A. J. Sinskey, M. A. Schmidt and K. F. Jensen, "Monitoring of Cell Growth, Oxygen and pH in Microfermentors," Micro Total Analysis Systems, vol. J, pp. 7-9, 2002.
 T. C. Merkel, V. I. Bondar, K. Nagai, B. D. Freeman and I. Pinnau, "Gas Sorption, Diffusion, and Permeation in Poly(dimethylsiloxane)," Journal of Polymer Science: Part B: Polymer Physics, vol. 38, pp. 415-434, 2000.
 Wikimedia Foundation, Inc., "Thermal conductivities of the elements (data page)," [Online]. Available: http://en.wikipedia.org/wiki/Thermal_conductivities_of_the_elements_(data_page). [Accessed 17 2 2014].
 S. A. Stern, "The “Barrer” Permeability Unit," JOURNAL OF POLYMER SCIENCE, vol. 6, 1968.
 Wikimedia Foundation, Inc., "Carbonic acid," [Online]. Available: http://en.wikipedia.org/wiki/Carbonic_acid. [Accessed 6 5 2014].
 J. Bear, Dynamics of Fluids in Porosity Media, New York: American Elsevier Publishing Company, 1972, pp. 15-22.
 Wikimedia Foundation, Inc., "Van der Waals constants (data page)," [Online]. Available: http://en.wikipedia.org/wiki/Van_der_Waals_constants_(data_page). [Accessed 23 10 2013].
 ATCC, "B16-F10 ATCC ® CRL-6475™ Mus musculus skin melanoma," [Online]. Available: http://www.atcc.org/products/all/CRL-6475.aspx. [Accessed 28 5 2014].
 Wikimedia Foundation, Inc., "Extracellular matrix," [Online]. Available: http://en.wikipedia.org/wiki/Extracellular_matrix. [Accessed 19 6 2014].