A RELEASABLE CELL SEPARATION PLATFORM USING TEMPERATURE-RESPONSIVE POLYMERS Li-In Wang1, Yu-Shuan Chen2, Judy M. Obliosca1, Pen-Cheng Wang1, Ging-Ho Hsiue2, and Fan-Gang Tseng1,3,* 1
Engineering and System Science Department, National Tsing Hua University, Taiwan 2 Chemical Engineering Department, National Tsing Hua University, Taiwan 3 Division of Mechanics, Research Center for Applied Science, Academia Sinica, Taiwan * Tel: 886-3-5715131-34270, Fax: 886-3-5720724, E-mail:
[email protected] ABSTRACT This paper to demonstrate temperature-controlled hydrophobic interaction as a method for capturing and releasing cells. Protein-based cell microarray is a powerful tool used in both clinical diagnostics and fundamental researches. However, the cell of interest after screening could not be released from cell microarray chip for further studies. This research presents the idea of combining antibody-antigen interaction and cell sorting system to improve the specificity. The surface was fabricated by grafting thermo-responsive polymer to PDMS (Polydimethylsiloxane), and the concept of capture and release cell were proved by polystyrene beads. The platform can be optimized and applied to provide further collection of cells of interest from protein-based cell microarray. KEYWORDS: hydrophobic interaction, cell separation, N-isopropylacrylamide INTRODUCTION In a protein-based cell microarray, protein such as antibodies were immobilized on the surface through covalent bond, and the composition of cell mixture and characteristic of surface proteins could be determined in single assay within several minutes[1, 2]. However, the cell of interest after screening could not be released from cell microarray chip for further studies. Temperature dependent hydrophobic-hydrophilic changeable surface which is grafted by pNIPAAm (Poly Nisopropylacrylamide) have been used for controllable capture and release of proteins in microfluidic devices[3]. However, no study so far have been demonstrate the concept of using hydrophobic interaction for capture and release cells. This research presents the idea of utilizing thermo-responsive polymer to change surface hydrophobicity for capturing and releasing cells. It also seeks to combine antibody-antigen interaction and cell sorting system to improve the specificity. CONCEPT Poly N-isopropylacrylamide is a temperature sensitive hydrogel, which have been extensively studied in grafting on many kinds of surfaces due to its unique properties, such as its lower critical solution temperature (LCST) around 32-33̓ C in aqueous solution, and the anti-fouling characteristic in room temperature[4, 5]. In this paper, pNIPAAm is used to modify surface of PDMS as a platform for cell capturing and releasing, and polystyrene beads are conjugated with antibodies for cell recognition. The concept is illustrated in Figure 1. At temperature above the LCST of pNIPAAm, the surface is suitable for cell capturing. When temperature is switched to lower temperature, the surface property changed and cells could be released from surface.
Figure 1: Concept of the releasable surface by using thermoresponsive polymer. EXPERIMENTAL The releasable platform was fabricated by UV grafting poly N-isopropylacrylamide (figure 2). PDMS was first treated with oxygen plasma to create free radical on the surface, followed by the exposure to air to create peroxide groups. UV light was provided to start the free radical polymerization of NIPAAm monomer solution. The pNIPAAm surface was fabricated by using 6.25% NIPAAm monomer as reaction mixture. We fabricated the other kind of polymer surface by using monomers of NIPAAm and AAM(acrylamide), with a ratio of NIPAAm:AAm=9:1. After the UV-grafting reaction, the surfaces were rinsed with deionized water and vacuum dried before further experiment. The measurement of surface contact angle was performed with sessile drop method. The surfaces were incubated in a moist container for six hours followed by contact angle measurement. The polystyrene bead (PSB)capture and release experiment was performed to test the surface hydrophobicity. The surface was first immersed in 0.01% polystyrene beads and covered with a coverslide for 30 minutes at 37̓C. The surface was then rinsed briefly with 37̓C deionized water and 978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
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then the number of PSB remaining on the surface was counted as an indication of capture ability. The release steps were carried out by washing the surface with 25̓C deionized water for 15 minutes and sonicating for 30 seconds. The number of PSB remained on the surface was then counted. We then modified polystyrene beads with antibodies for cell recognition. Neu(9G6):sc-08, a surface marker for human breast cancer cells[6], were used to labeled surface antigen of BT474 cells (breast cancer cell line). Then polystyrene bead conjugated to fluorescent secondary antibody were mixed with labeled BT474 cells. The cells were observed under microscope before applying to pNIPAAm surface.
Figure 2: Schematic illustration of grafting process. RESULTS AND DISCUSSION The grafted pNIPAAm can be characterized by FT-IR and contact angle measurement. The contact angle of pNIPAAm-g-PDMS were 46.5̓ at temperature lower than LCST and 66.9̓ at temperature above LCST (Figure 3). The temperature dependent hydrophobicity change occurred at around 32-36̓C. The results indicated that grafting process was able to change the surface property of PDMS. The addition acrylamide (AAM), a hydrogel that has similar structure to NIPAAm, shifts the surface to lower contact angle region due to the hydrophilic nature of AAM. The temperature dependent hydrophobicity change of pNIPAAm-pAAm-g-PDMS occurred at broader range of temperature, 30-36̓C. 800
120
37°C 80 60 40
66.9 47.8
46.5
28.2
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1st Capture (37к) 600 (Normalized)
contact angle (degree)
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Number of PS beads/mm2
104.7 98.0
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1st Release (25к) 2nd Capture (37к)
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2nd Release (25к)
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0 pNIPAAm:AAm 9:1
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Figure 4. pNIPAAm-g-PDMS surface was able to capture polystyrene beads at 37к by hydrophobic interaction and release the beads at 25к due to surface hydrophobicity change.
Figure 3. Contact angle of three surfaces at temperature below and above LCST of pNIPAAm.
Figure 4 demonstrates the capture and release of fluorescence tagged polystyrene beads by temperature-dependent surface hydrophobicity change. The capture steps were performed at 37̓C, while the release steps were carried out by washing the surface with 25̓C deionized water. The number of beads constantly captured by the surface at 25̓C were around 100. At 37̓C, hydrophobic pNIPAAm-g-PDMS surface were able to capture around 400 more beads that the surface at 25̓C, and the difference in the number of captured beads can be released when temperature is lowered. pNIPAAm-g-PDMS surface has successful performance for two cycles of temperature change, while pNIPAAm-AAM-gPDMS surface showed change of surface property at the second cycle. The polystyrene beads modified with antibodies were used for cell recognition. After the antibody reaction, the cells were observed under microscope before applying to pNIPAAm surface. In figure 5, the beads were able to bind to the surface of the cells through antibody recognition.
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Figure 5. The binding of antibody-conjugated polystyrene beads to BT474 cell surface were observed under microscope. (A)Phase contrast image of BT474 cells. (B) fluorescent image of conjugated polystyrene beads. (C) merged images. CONCLUSION In this paper we presented the idea that utilizing temperature-controlled hydrophobic interaction for capture and release cell. Successfulness of grafting process were proved by using temperature sensitive contact angle change and swelling ratio change, and the concept of capture and release cell were proved by polystyrene beads. This platform displays the concept of a releasable cell biochip and can be optimized and applied to provide further collection of cells of interest from protein-based cell microarray. ACKNOWLEDGEMENTS This work was supported by National Science Council, NSC 98-2120-M-007-001 and NSC98-2627-M-007-003. REFERENCES [1] L. Belov, O. de la Vega, C. G. dos Remedios, S. P. Mulligan, and R. I. Christopherson, "Immunophenotyping of leukemias using a cluster of differentiation antibody microarray," Cancer Research, vol. 61, pp. 4483-4489, Jun 1 2001. [2] I. K. Ko, K. Kato, and H. Iwata, "Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays," Biomaterials, vol. 26, pp. 4882-4891, Aug 2005. [3] D. L. Huber, R. P. Manginell, M. A. Samara, B. I. Kim, and B. C. Bunker, "Programmed adsorption and release of proteins in a microfluidic device," Science, vol. 301, pp. 352-354, Jul 18 2003. [4] M. Ebara, J. M. Hoffman, P. S. Stayton, and A. S. Hoffman, "Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and 'smart' polymers," Radiation Physics and Chemistry, vol. 76, pp. 1409-1413, Aug-Sep 2007. [5] P. S. Curti, M. R. de Moura, W. Veiga, E. Radovanovic, A. F. Rubira, and E. C. Muniz, "Characterization of PNIPAAm photografted on PET and PS surfaces," Applied Surface Science, vol. 245, pp. 223-233, May 30 2005. [6] D. J. Slamon, G. M. Clark, S. G. Wong, W. J. Levin, A. Ullrich, and W. L. McGuire, "Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene," Science, vol. 235, pp. 17782, Jan 9 1987. CONTACT *Fan-gang Tseng, tel: +886-3-5715131-34270, Fax: 886-3-5720724, E-mail:
[email protected]
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