- 65 - Developing a real-time fluorescence cell growth monitoring system Jo-Ting Wang 1, Chun-Han Lu 2, Yao-Nan Wang 2, Ko-Tung Chang 1,* 1 Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan 2 Department of Vehicle Engineering, National Pingtung University of Science and Technology, Pingtung, Taiwan *Corresponding author: kotungc@mail.npust.edu.tw Abstract CMOS (Complementary Metal-Oxide-Semiconductor) based real-time fluorescent cells monitoring system can be applied for measuring transfection efficiency or cytotoxicity on pharmacokinetic study. CMOS sensors have many advantages over traditional microscope CCD (Charge Coupled Device) sensors, including smaller physical size, lighter weight, and lower costs. The aim of the study is to confirm the feasibility and efficiency of our newly developed CMOS-based real-time fluorescent cells monitoring system. We grow CFSE (carboxyfluorescein succinimidyl ester) -labeling RAW264.7 cells in vitro and measure the proliferation rate of these cells by our newly developed instrument and by flow cytometry analysis. CFSE is a fluorescent cell staining dye. This reagent is easily uptaken into cells and yields a well-retained fluorescent emission after blue light excitation. When CFSElabelling cells divide, the mean fluorescent intensity degenerates into half. Thereby, we can detect in real-time the distinct generations of proliferating cells and their growth rate by using our new instrument instead of using conventional flow cytometry analysis. Keywords: CFSE, Cell proliferation, RAW264.7, Real-time, Fluorescence cell growth monitoring Introduction Micro-electro-mechanical systems technologies have in advance led to the development of many microfluidic devices in recent years, including micromixers electrophoresis microchips, optical microflow cytometers, coulter counters, micropumps, cell culture chips, and
- 66 - optofluidic microscopes. The integration of microelectromechanical technology with real-time monitoring system for cells cultures was very common in biological sciences. It can apply for measuring cell proliferation, cell migration or cytotoxicity assay on pharmacokinetic study. In recent years, many researches develop novel cell-culture real-time monitoring system. For example, Chang et al. 1 in our laboratory previously developed a multichannel lens-free CMOS sensors and applied it for detection of HepG2 cell growth rate and for the cytotoxicity test of these cells treated with chemotherapeutic drug, cyclophosphamide. 1 Thus, we continually propose a CMOS sensors system that can be used for real-time detection of cell growth with fluorescent illumination. This modified system has many advantages, including a smaller physical size, a lighter weight, and a lower cost. To confirm the feasibility and efficiency of the system, we ll use RAW264.7 cells labeled with the intracellular covalent coupling dye CFSE. This reagent is fluorescence-free until its two acetate side chains are removed by intracellular esterase of live cells to produce bright fluorescent compounds. The compounds covalently bind to intracellular amines, which results in a stable status. 2 CFSE is capable of monitoring distinct generations during cell proliferation by its dye dilution after live cells covalently labeled with a very bright, stable dye. When CFSE-labeling cells divide, the mean fluorescent intensity (MFI) degenerates into half. Every generation of cells appears as a different level of fluorescent emission. In our study, we will compare the growth rate of RAW264.7 cells labelling with CFSE detected by our innovative real-time fluorescence cell growth monitoring system and by conventional flow cytometry analysis. Materials and Methods Cells culture RAW 264.7 cells were culture in 3.5 cm dish with 90% Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, HyClone Laboratories, Inc, UT, U.S) under a humidified 5% CO 2 atmosphere. Labelling procedure with CFSE CellTrace CFSE Cell Proliferation Kit (Invitrogen, NY, USA) was used as described by the manuals. Briefly, adding the dry dimethyl sulfoxide (DMSO) to one vial of CellTrace reagent and mixing well. The RAW264.7 cells were adjusted to 5 10 5 cells in 1
- 67 - ml phosphate buffered saline (J.T Baker, Phillipsburg, NJ, USA). The corresponding volume of CFSE in 5mM was added to the cell suspension to make a final CFSE working solution in 5 M and 0.2 M, respectively. Incubate the cells for 20 minutes at 37 C in dark field. Add 5X volumes of PBS with 5% FBS to the cells and incubate for 5 minutes. Centrifuge and re-suspend the cells in fresh pre-warmed complete culture medium (10% FBS). The cultures were further incubated for 1 to 5 days. Viable cell number counting The number of viable cells was determined by hemocytometer under a light microscope after staining with tryphan blue solution (Sigma-Aldrich, MO, USA) and by CMOS real-time fluorescence monitoring system in bright field, respectively. Fluorescent microscope cell images analysis Put the fluorescent cells with 10 l on a glass slide, cover with cover glass was performed under an Olympus BX50F4 microscope (Olympus, Japan). Detection of mean fluorescence intensity (MFI) by flow cytometry MFI of CFSE-labeling cells was analyzed by flow cytometry (BD FACSAria II) with a 488 nm excitation light source and data were processed by BD FACSDiva Software (Becton, Dickinson and Company, NJ, USA). Detection of mean fluorescence intensity (MFI) by Real-time fluorescence cell growth monitoring system The platform of devices has an overall dimension of 60 80 35 mm 3. The LED blue light sources and CMOS sensor were automatically controlled by using self-written LabVIEW software. The light sources and sensor were briefly turned on at scheduled 1 hour intervals during the cultivation process and were then turned off as soon as the image acquisition process was completed. Statistical analysis All results were collected from at least triplicate experimental groups. Statistical analysis was performed with Prism 5 software (GraphPad Software, Inc, La Jolla, CA, USA).
- 68 - P-value less than 0.05 was considered as statistically significant. *P < 0.05; **P < 0.01 and ***P < 0.001 represent significant difference compared with zero hour. Apoptosis was analysed in the spleen and bone marrow cells by flow cytometry. Results and Discussion Growth curve of RAW 264.7 cells labeled with 0.2 M CFSE A growth curve illustrated 0.2 M CFSE-labeling RAW 264.7 cells proliferation rate measured by hemocytometer. The cell doubling time (DT) was calculated as follows: DT (Doubling time) = t [log2 / (lognt-logn0)]. A statistical analysis of the results shows the cell DT is approximately 11.8 hours (Fig. 1). Figure 1. Growth rate curve of RAW 264.7 cells. Initial seeding of 5 10 5 cells/ml RAW 264.7 cells labeling with 0.2 μm CFSE were grown from 0-120 hours and the cell counts were calculated by hemacytometer (N=3). *P < 0.05; **P < 0.01 and ***P < 0.001 compared to zero hour.
- 69 - Fluorescent microscope images analysis of RAW 264.7 cells labeled with 0.2 M CFSE We observed RAW 264.7 cells in fluorescent microscope. The fluorescent cells degenerated sharply and can t be detected by fluorescent microscope after 24 hours in culture (Fig. 2). Figure 2. Raw 264.7 labeled with 0.2 μm CFSE showed intensive green fluorescence at the beginning of culture. After 24 hours cultivation the fluorescence was barely detected by fluorescent microscope with 200x magnification. (A) 0 hour (B) 24 th hour (left: bright field, right: dark field with fluorescence) Detection of mean fluorescence intensity (MFI) of RAW 264.7 cells by flow cytometry We used MFI value measured by flow cytometry to track the distinct generation of proliferating RAW 264.7 cells. The MFIs was 103545 for day 0, 3910 for day 1, 798.3 for day 2, 144.3 for day 3, 68.3 for day 4, and 54.3 for day 5 (Fig 3A). Cells generations were identified according to fluorescent intensity (magenta, blue, orange, green, red, gray) from day 0 to day 5 (Fig 3B). When CFSE-labelling cells divide, the mean fluorescent intensity degenerates into half. Thus, a statistical analysis of cells doubling time (DT) was around 10.3 hours (Fig. 3C).
- 70 - Figure 3. MFIs of CFSE-labeled RAW 264.7 cells (0.2 M) measured by flow cytometry. (A) Representative data of CFSE-labeling RAW 264.7 cells mean fluorescent intensity (MFI) at days 0 to 5 by FACS analysis. (B) Cells generations were identified according to fluorescent intensity (magenta, blue, orange, green, red, gray) from day 0 to day 5 (N=3) (C) Mean fluorescent intensity (MFI) was degenerated by FACS analysis in CFSE-labeled RAW 264.7 cells from day 0 to 5 cultivation. (N=3) Detection of mean fluorescence intensity (MFI) of RAW 264.7 cells by CMOS Realtime fluorescence cell growth monitoring system We observed CFSE (5 M)-labeling RAW 264.7 cells growing in CMOS-based realtime fluorescent cells monitoring system. The fluorescence degenerated from day 0 to day 5 (Fig. 4) cultivation. Statistics of CFSE-labeling-RAW 264.7 cell proliferation rate were measured by CMOS-based images in Figure 5A. CFSE-labeling RAW 264.7 cells
- 71 - fluorescent intensity were degraded from day 0 to day 5 (Figure 5B) cultivation. The DT of RAW 264.7 cells was calculated as following formula: MFI= fluorescent intensity / pixel. The DT was around 16.4 hours after statistical analysis of the CMOS-based image data. Figure 4. CMOS-based images of CFSE-labeled RAW 264.7 cells on Day 0 to 4 cultivation (A to E). (left: bright field, right: 5 M CFSE-labeled cells in dark field with fluorescence emission). Figure 5. CMOS-based image collected from NI VISION program. Cell count in bright field was recorded as pixel dot and fluorescence intensity was recorded in dark field by blue light excitation. (A) RAW 264.7 cells growth curve. (B) Fluorescence intensity of CFSE-labeled RAW 264.7 cells
- 72 - Conclusion The study of Sakagami H et al. (2009) mentioned that RAW 264.7 cells grew very fast, with an approximate doubling time of 11 hours 3. Thus, we confirmed our results calculated by hemocytometer, flow cytometry, and the CMOS-based real-time fluorescent cells monitoring system were almost identical. Hence, our innovative CMOS-based real-time fluorescence cell growth monitoring system is accurate and easy-to-use for the study on biological science compared to conventional flow cytometry analysis. Base on our results, we propose a low-cost platform for real-time cell growth monitoring system. The statistical calculation of doubling time of CFSE-labeled RAW 264.7 cells were 11.8 hours by hemocytometer, 10.3 hours by flow cytometry analysis, and 16.4 hours CMOS-based real-time fluorescent cells monitoring system, respectively. In the future, we ll minor modify the system and applied it for the study on biological science. References Chang, Ko Tung, Chang, Yu Jen, Chen, Chia Ling, Wang, Yao Nan., 2015. Multichannel lens free CMOS sensors for real time monitoring of cell growth. Electrophoresis, 36(3): 413-419. Parish, Christopher R. 1999. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunology and cell biology, 77(6): 499-508. Sakagami, Hiroshi, Kishino, Kaori, Amano, Osamu, Kanda, Yumiko, Kunii, Shiro, Yokote, Yoshiko, Oizumi, Takaaki. 2009. Cell death induced by nutritional starvation in mouse macrophage-like RAW264. 7 cells. Anticancer research, 29(1): 343-347.