Patterned Thermoresponsive Microgel Surfaces to Control Cell

Jan 7, 2016 - Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Schuster Building, Oxford Road, Manchester, M1...
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Patterned Thermoresponsive Microgel Surfaces to Control Cell Detachment Yongqing Xia,*,† Ying Tang,† Xinlong He,† Fang Pan,‡ Zonyi Li,‡ Hai Xu,† and Jian Ren Lu*,‡ †

Centre for Bioengineering and Biotechnology and the State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Qingdao 266580, China ‡ Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Schuster Building, Oxford Road, Manchester, M13 9PL, United Kingdom S Supporting Information *

ABSTRACT: The aim of this work is to examine how adhered individual cells could detach from the patterned, discontinuous thermoresponsive coating substrate and how different patterns in the form of thermoresponsive squares and gaps would affect cell detachment. Microgels prepared from copolymerization of N-isopropylacrylamide and styrene (pNIPAAmSt) were spin-coated on polyethylenimine (PEI) precoated glass coverslips to form a uniform microgel monolayer; then a surface-moisturized PMDS stamp was used to contact the microgel monolayer at room temperature. The thin layer of water on the PDMS stamp surface worked as an ink to penetrate the microgels so that any microgels in direct contact with the wet stamp surface became swollen and could be peeled away, while uncontacted microgels formed patterns. Using this method, various patterns with different thermoisland diameters and gaps could be fabricated. NIH3T3 fibroblast cells were then cultured on these patterns to study their detachment behavior. It was found that cells could detach not only from these discontinuous thermoresponsive coatings, but also from the patterned surfaces with the thermoresponsive area being as low as 20% of the cell spread area.

1. INTRODUCTION Extensive studies have now reported the growth and detachment of adherent cells from various thermoresponsive surfaces via temperature reduction. This type of approaches has many advantages over conventional cell detachment methods such as lysis of cell matrix structures by proteolytic enzymes or mechanical disaggregation, both of which can damage functional cell membrane proteins or yield low numbers of viable cells.1−5 Poly-N-isopropylacrylamide (pNIPAAm) is a conventional thermoresponsive polymer that has a lower critical solution temperature (LCST) in aqueous conditions around 32 °C. Various forms of pNIPAAm homopolymers, copolymers, and their derivatives have been used as thermoresponsive coatings to exhibit a largely reversible swelling−deswelling transition by temperature modulation ever since the first report of this property from the linear pNIPAAms and their use for harvesting cells in 1990.6 Okano et al. have developed thermoresponsive culture dishes that were created by the covalent grafting of pNIPAAm to ordinary tissue culture dishes using electron beam polymerization (EBP). Thus, cells or cell sheets could be harvested just by temperature reduction.2,4,7−9 Unfortunately, the thicknesses and surface morphologies of the polymer coatings often set the limits beyond which cells could neither adhere on nor detach from the surface.8,10 Many innovative approaches have been developed and refined over © XXXX American Chemical Society

the past few decades to provide better controlled thermoresponsive surfaces for more effective cell and cell sheet recovery.11−17 In spite of different thermoresponsive surfaces fabricated so far, the mechanistic process of cell detachment is commonly regarded as the switch of adhesion/detachment of cells upon the hydrophobic/hydrophilic change of the pNIPAAm incorporated film coating when the temperature is varied across the LCST.18 It has been recently proposed that cell adhesion/detachment on pNIPAAm films or brushes is controlled by the hydration state of the polymer chains, which affects the insertion of adhesion proteins into the brushes, through the change in osmotic pressure that occurs when the pNIPAAm chains go from their collapsed, dehydrated state to their swollen state upon lowering the temperature.19 However, some basic questions are still open in the fabrication of thermoresponsive substrates for the harvesting of cells and cell sheets. For instance, does the thermoresponsive coating have to be continuous? If not, then what would be the critical fraction of coverage by the thermoresponsive coating? We hypothesize that as in the game of tug of war, cell detachment process would be largely featured by the repulsive Received: November 8, 2015 Revised: January 1, 2016

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a

(A) Schematic demonstration of the surface patterning process based on thermoresponsive microgels to study cell detachment behavior. First, bare cover glass was immersed in PEI solution and then rinsed with water to form an adsorbed PEI layer. Second, fluorescent dye encapsulated pNIPAAmSt microgels spin-coated on PEI coated surface at 1000 rpm for 60 s. Third, thermo-islands were fabricated by a water-treated PDMS stamp using the negative μCP method. Fourth, NIH 3T3 cells were cultured on the patterned surface to spread and grow at 37 °C. Last, after 48 h cell culturing, the culturing vessel was taken out and placed under the ambient temperature, and the DMEM culture was replaced by the cold one to observe cell detachment behavior. (B) A typical topographical surface of the PDMS stamp, with a range of diameters of the round dip areas with different gaps between them. A thin layer of water on the stamp surface was used to transfer the microgels it contacted, and uncontacted microgels in the dip areas were left to form patterns. (C) Each island was comprised of pNIPAAmSt microgels distributed randomly during surface coating with the packing density controlled by the coating conditions, as described previously.24,25

studies have laid useful foundation for us to study how key patterning features affect thermoresponsive cell detachment. Whitesides et al. have developed the microcontact printing technique for studying cell-interface interactions.23 Their work revealed that the geometry of surface patterns controls cell responses and that the “cell-spreading area”, i.e., the area covered by the cell, has a dominant role in deciding its fate, apoptosis, or growth. To find out when and how the repulsive force can overcome the adhesive force, a series of micropatterned surfaces were fabricated in this work using the microcontact printing technique. Thus, a semiquantitative methodological study on cell detachment from a set of micropatterned thermoresponsive islands on the cell adhesion background could be performed, just as shown in Scheme 1. Thermo-islands in the form of thermoresponsive squares were composed of poly(N-isopropylacrylamide-co-styrene) (pNIPAAmSt) microgels, as we have previously reported. These microgels are easy to synthesize and also easy to coat onto different substrate surfaces.24,25 They can support cell attachment and growth and induce cell detachment upon temperature drop over a wide range of surface microgel coverage, with rather little influence of microgel composition and film thickness. These advantageous features make them attractive for further development toward practical applications. The

force coming from the swelling of the pNIPAAm coating and the opposing adhesive force between cell and substrate. When the repulsive force is larger than the adhesive force, cells could detach from the surface. Several patterned thermoresponsive surfaces have previously been reported for the study of cell and cell sheet detachment. Tsai et al. used pNIPAAm microgels to fabricate different microgel patterned strips on polystyrene substrate via dip coating.20 When fibroblasts were seeded on the patterned surfaces, the cells preferred to adhere and proliferate on various microgel covered strips, became confluent on the whole surface, and then detached as cell sheet upon temperature reduction. Okano group fabricated stripe-polyacrylamide patterns on a linear pNIPAAm surface through photolithography.21 When NIH-3T3 cells were cultured on the patterned strips, results showed that the detachment of adhered NIH-3T3 single cells and cell sheets could be accelerated upon temperature drop compared to the detachment on a conventional pNIPAAm surface. Mandal et al. designed micropatterned antiadhesive pNIPAAm brushes (70−80 nm) via deep UV photolithography.22 The adhesive patterns, combined with the temperature-dependent swelling properties of pNIPAAm, make the polymer brushes work as a microactuator to activate cell detachment upon temperature-reduction below 32 °C. These B

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Figure 1. (a) Fluorescent image of FITC-Na entrapped pNIPAAmSt microgels; (b) TEM image of pNIPAAmSt microgels; (c) Plot of hydrodynamic diameters of FITC-Na entrapped pNIPAAmSt microgels in aqueous dispersion as a function of temperature; (d) Zeta (ζ) potential distribution of the pNIPAAmSt microgels at 20 °C (three repeated cycles). encapsulated microgels was prepared by adding FITC-Na (5 mg) into the solution before polymerization started. Microgel Characterization. The hydrodynamic diameters and thermoresponsive behaviors of the resulting pNIPAAmSt microgels were characterized by dynamic light scattering (DLS, Zetasizer Nano instrument from Malvern Instruments Ltd., with the detector positioned at the scattering angle of 173°) in the temperature range of 20−50 °C. The microgel dispersion was heated steadily and the microgel size determined every 2 °C by letting the microgel dispersion equilibrate at each temperature for 10 min. The ζ-potential of the pNIPAAmSt microgels was measured as an indicator of microgel charges using the same Malvern Zetasizer instrument. The sample was prepared in the same way as prepared for size analysis, and each mobility value was the average of 100 runs. Transmission electron microscopy (TEM, JEM-2100UHR, JEOL) was also used to characterize the synthesized microgels. Specimens for TEM imaging were taken from diluted dispersions, deposited on a 400 mesh carboncoated copper grid and dried under the infrared lamp before observation. Scanning electron microscopy (SEM, S-4800, Hitachi) was used to help examine the resulting microgel films, with sample surfaces coated with a thin Au layer to increase the contrast and quality of the images. 2.3. Patterning pNIPAAmSt Microgel Surface via Microcontact Printing. Freshly cleaned glass coverslips were first immersed in 1.0 wt % PEI solution (pH = 5) and then washed with water and dried with nitrogen. The microgel monolayer was obtained by spin-coating (1000 rpm) 1.0 wt % microgel dispersion onto the PEI-precoated glass coverslip. The fabricated PDMS stamp was treated with plasma to make the surface hydrophilic, and then put over a cup of hot water to condense a thin layer of water on its surface. When the surface was moist (i.e., the condensed water layer began to evaporate from the edge of the surface), the PDMS stamp was immediately used to contact the microgel film for few seconds. The PDMS stamp was then carefully peeled away and the surface pattern formed. The patterns were subsequently immersed in deionized water for 48 h to release superfluous florescent dye, before they were heated to 120 °C for 2 h for sterilization. The morphologies of the patterned surfaces were monitored by both inverted fluorescence microscopy (Leica, DMI3000, Germany) and scanning electron microscopy (SEM, S4800, Hitachi). Patterns were named in the format of a-b, where “a”

results from this work revealed that when the total thermoresponsive area is even as low as 20%, cell detachment could still occur via temperature stimuli, thus, demonstrating great potential for their technological exploitation.

2. MATERIALS AND METHODS 2.1. Materials. All the chemicals used in this work were obtained from Sigma-Aldrich. N-Isopropylacrylamide (NIPAAm) was purified by recrystallization from a toluene/hexane mixture (1:3) and dried in vacuum. Styrene (St) was purified by distillation under reduced pressure, ammonium persulfate (APS) was purified by recrystallization from water. N,N-Methylene bis(acrylamide) (MBA) was used as received. The molecular weight of polyethylenimine (PEI) used was 750 kDa. All water used in this experiment was processed by Milli-Q system (Milli-Q Advantage A10 Water System Production Unit). Cell culture plates (six-well plates, from Corning) were used as received. Glass coverslips (20 × 20 mm2) were immersed into piranha solution (H2O2/H2SO4 = 1:3 by volume) at 90 °C for 1 h, followed by abundantly rinsing with tap water and UHQ water. The freshly cleaned glass was hydrophilic with the contact angle under 20° and carried weakly negative charges. PDMS stamps were commissioned by CapitalBio Corporation (China), and all stamps used had smooth surfaces with round pits with a depth of about 500 nm, the pit diameters of 3, 5, and 10 μm, and gaps of 3, 5, and 10 μm, respectively. 2.2. Preparation of pNIPAAmSt Microgels. All pNIPAAmSt microgels were prepared by surfactant-free precipitation polymerization, as previously described.12 After 1.500 g NIPAAm, 0.041 g MBA, and 0.500 g styrene were added in 190 mL of water, the reaction mixture was transferred to a four-necked, round-bottom flask equipped with a condenser and a nitrogen inlet, and then heated to 70 °C under a gentle stream of nitrogen. After 1 h, 0.120 g of an initiator (APS) was dissolved in 10 mL of water (oxygen-free) and added to the flask to initiate polymerization. The reaction was continued for 4 h while keeping the reaction in a nitrogen environment by continuous N2 purging. Following the synthesis, the microgels were purified by three centrifugation cycles at 10000 rpm for 60 min, with removal of the supernatant and redispersion between each cycle. Rhodamine B encapsulated microgels was prepared as previously reported,26 and sodium fluorescein-5-isothiocyanate (FITC-Na) C

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Figure 2. SEM images of (a) thermoresponsive microgel monolayer formed on PEI-precoated glass coverslip using spin coating; (b) microgel patterns formed by μCP, the insets are magnified images.

Figure 3. (a) Florescent images of pattern 10−10 before cell culture; (b) bright field images of cells cultured on the pattern after 48 h; (c) florescent images of the pattern with cells. The images in the top line were taken from the microgel surface pattern encapsulated with Rhodamine B and the images in bottom line were taken from the microgel surface pattern encapsulated with FITC-Na. Scale bars = 50 μm. The difference in (c) shows the instability of the Rhodamine B incorporated pattern. denotes the diameter of thermoresponsive islands (μm) and “b” the gap between them (μm). 2.4. Cell Culture. Following sterilization at 120 °C in an oven for 2 h, the glass coverslips with different pNIPAAmSt microgel patterns were transferred into six-well tissue culture plates for subsequent use. NIH 3T3 cells (2 × 104 per well) were seeded uniformly on the surface patterned coverslips and cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37 °C and under 5% CO2. All the experiments were undertaken in triplicates and fresh and warm culture medium (preheated to 37 °C to avoid any possible cell detachment due to temperature drop) was used to replace the old medium every other day. After 48 h, the culturing plates were moved out and cold fresh DMEM was used to replace the warm culture. The cell detachment process was then observed at ambient temperature (about 20 °C) in real time. 2.5. Data Analysis. At least three individual samples were used for averaging the detachment dynamics, and each sample included at least 200 individual cells. The projected areas of cells adhering on microgel patterned surfaces (Aspread) were gained using free software Image-J (freely available at http://www.nih.gov), and at least 50 spreading cells were randomly selected to determine the average project area.

were prepared to study their effect on cell attachment and detachment behaviors as a monolayer, and it was observed that microgels with the ratio of NIPAAm: St at 3:1 was optimal on cell detachment, consistent with the outcome from our previous study.25 Using the same polymerization composition and procedure, microgels were also prepared with fluorescein dyes (FITC-Na and Rhodamine B) incorporated noncovalently as stains for convenient characterizations. The structure and thermoresponsivity of the microgels are very much similar to those of the previous microgels. The bear negative zeta potential is about −13.3 ± 0.3 mV. Figure 1 shows the typical size and shape of microgels stained by FITC (Figure 1a), as viewed under TEM (Figure 1b). They display similar changes of hydrodynamic diameters with temperature (Figure 1c) and surface zeta potential (Figure 1d) to the microgels prepared previously were used. 3.2. Patterns. The thermoresponsive microgels based patterns were prepared by the negative microcontact printing method (μCP),26 after the monolayer of thermoresponsive microgels was spin-coated on the PEI-precoated glass coverslip. It was found that the microgels were not closely packed under the low concentration of 1.0 wt % and electrostatic repulsion must work between microgels to keep them apart (Figure 2a). After being treated with PDMS stamp, round patterns were

3. RESULTS AND DISCUSSION 3.1. Microgel Preparation and Characterization. At the start of this work, a series of poly(N-isopropylacrylamidestyrene) microgels (pNIPAAmSt) with different weight ratios D

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Figure 4. Images of NIH 3T3 cells on typical patterns (as marked on top of the figure) after incubation at 37 °C for 48 h (top line) and after cooling the culture plate down to the room temperature about 20 °C for 60 min (bottom line). Scale bars: 50 μm. The results showed that cells could only detach from patterns such as 10:10 and 5:5, but detachment is poor from patterns such as 3:5.

surface patterns to study if there exists a “critical” thermoresponsive area for the detachment of individual cells. On the basis of the work from Whitesides et al., we first explored the surface patterns with the largest gap of 10 μm by analyzing the attachment and spreading of NIH 3T3 cells. The gap distances between the thermoresponsive squares were smaller than the width of a cell, thus, cells could spread over at least one thermoresponsive island. Through studying individual cell detachment on various patterns, we hope to find the critical thermoresponsive areas for the cells to detach. Figure 4 shows typical cell morphologies on the representative patterns at high (37 °C) and low (20 °C) temperatures after 48 h cell growth. For all patterns studied, cells could attach and proliferate on the surfaces with no obvious differences in their viability (cell shapes and numbers). After temperature was reduced and kept at the ambient temperature about 20 °C for 60 min, cells became round only on certain patterns, such as 10−10 and 5− 5, remained elongated on pattern 3−5, and became partly round on pattern 5−10. It seems difficult to relate the cell responses to the different surface patterns formed. However, the combinations of surface patterns produced different fractions of thermoresponsive microislands for cells to sit on. Table 1 lists the fractions of thermoresponsive microislands formed for all surface patterns studied together with the cell detachment observations where cell detachment ratio meant the ratio of cells that became round with no obvious sign of stretching or elongation in shape

formed on the substrate (Figure 2). Note that some boundary areas of the patterned objects might become damaged from the peeling process, showing the limitation of this approach. It is, however, easy to manipulate, and the main patterned feature is highly reproducible. The patterns could be formed in areas about mm2, sufficiently large for us to study cell performance. The stability of pNIPAAmSt microgel patterns deposited on glass coverslip was examined after thermal treatment for sterilization. The patterned surfaces were immersed in water at room temperature. Results showed that the patterns formed on bare glass coverslip could not keep stability in water, and some of the patterns would disappear in just 1 day. This is because the microgel layer was discontinuous and it was easy for water to penetrate into the contact interface between microgels and the glass coverslip. To enhance the interaction between the microgels and substrate, positively charged PEI (polyethylenimine) was coated on the bare glass coverslips. Results showed that even when the surfaces were not closely packed with microgels, the patterns formed were still stable in water for more than 3 days, the longest period of the assessment. The improvement could arise from the electrostatic attraction between the negatively charged microgels and positively charged PEI surface and chain entanglements between them. Rhodamine B was used as the first fluorescent dye to stain the microgels in this study. However, after cells were cultured on the dye-stained surface for 48 h, cells could be observed under the same emitting wavelength (Figure 3, top line), and the patterns became blurred. They could not be observed clearly. These observations indicate that Rhodamine B stained microgels were inappropriate for such a study. When FITC-Na was chosen as the fluorescent dye, cells could be observed under the bright field, with no disturbance to the fluorescent patterned background. Thus, in the following work, FITC-Na stained microgels were used to fabricate a series of patterns, as exemplified further in Figure SI1. 3.3. Temperature-Induced Cell Detachment. As already indicated earlier, Whitesides et al. have used surface patterning for studying cell−interface interactions.23 They revealed that the “cell-spreading area” instead of the “cell-ECM contact area” has a dominant role in controlling cell apoptosis or growth. To find out the minimal fraction of the thermoresponsive surface area covered by the cell that can trigger its detachment upon temperature drop, we designed a series of thermoresponsive

Table 1. Cell Detachment (NIH 3T3 Fibroblasts) Behavior on Different Surface Patterns patterned structure

area ratioa

10−3 10−5 5−3 3−3 10−10 5−5 3−5 5−10 3−10

0.46 0.35 0.31 0.20 0.20 0.20 0.11 0.09 0.04

cell detachment ratio 0.87 0.70 0.88 0.88 0.83 0.90 0.08 0.09 0.11

± ± ± ± ± ± ± ± ±

0.03 0.07 0.10 0.08 0.11 0.06 0.02 0.02 0.02

a

Area ratio means the ratio of the total area of thermo-islands to the total substrate area a cell covers on each surface pattern studied.

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Figure 5. Time lapse imaging tracked the transition of individual cells upon cold treatment. Three cells were followed over the period of 60 min. The numbers marked to each cell over the period marked the contractions of their lengths. Cells transformed from flattened elongated shape to a more rounded form.

Figure 6. NIH 3T3 cell detachment kinetics on different patterns. (A) The effects of gaps on cell detachment dynamics with the same thermo-island diameters fixed at 10, 5, and 3 μm; (B) The effects of thermo-island diameters on cell detachment dynamics with the same gaps fixed at 3, 5, and 10 μm. The results show that increase in thermo-island diameter or decrease in the gap distance favors cell detachment due to the increase in the fraction of the coverage of the thermoresponsive area under the cells.

against those that showed no sign of detachment. The data show that the critical fraction of the thermoresponsive area existed at about 20% and that only above this value could cell detachment occur. After NIH3T3 cells were seeded on the 10−10 pattern and incubated for 48 h, time lapse microscopy was used to monitor the detachment of individual cells (Figure 5). This sequential imaging tracked the retraction of the cells with time after cooling, and cells transformed from their flattened and elongated shape gradually to a rounded form. It should be noted that some cells could not achieve complete detachment, but they could be removed by gently pipetting the medium against the surface. In some rare occasions, cells attached

entirely along a gap, they would not contract upon cold treatment because the substrate was PEI-coated glass coverslip (Figure SI2). This proved that the micropatterned thermoresponsive area (green area) can really be used for cell detachment manipulation. To further investigate the effects of the diameters of thermoislands and the gaps between them on cell detachment kinetics, we have also followed the time course of cell detachment on the patterns with the same diameters of thermo-islands but varied gap distances (Figure 6A), or on the patterns with the same gap distances but varied diameters of thermo-islands (Figure 6B). When the diameter of thermo-islands is 10 μm, cells could detach from all patterns, the gap distances have no F

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Figure 7. (a, b) Images of cells spread on 5−5 and 10−10 surface patterns. White outlines indicate cell boundaries. (c) Spread cell area and total thermoresponsive island area under a given cell would affect contraction after cold treatment. (d) Cell detachment kinetics on patterns with equal ratios of thermoresponsive areas.

and 3−3), the cell detachment kinetic processes were similar, just as shown in Figure 7d. As shown in Table 1, six out of the nine patterns studied enabled cell detachment, with the percentage of over 70% of cells detached. The three patterned surfaces that could not cause cell detachment had the area ratios below 0.2. In most studies reported, the thermoresponsive coatings are continuous, and changes in polymeric hydrophobicity with temperature force cell detachment from substrate surface. Changes in the surface energy of the polymer layer alone may not account for the changes of adhesion, as cell metabolism also affects cell detachment. Our previous studies showed that microgel film surfaces could repel and detach cells at temperatures below VPTT (volume phase transition temperature). The cell detachment mechanism reported here is however quite different from what has been described previously, because it involves not only a thermoresponsive area but also a cell adhesive region. Cells detach from thermoresponsive coatings just as in the game of tug of war, encompassing interfacial adhesion and expulsion. This work has revealed that even when the thermoresponsive coating in the form of thermo-islands is only 20% of the adhesion background, cells could still detach from the patterned surface successfully, as demonstrated visually in Scheme 2.

obvious influence on cell detachment. When the diameter of thermo-islands decreases to 5 μm, cells could detach from patterns with gaps of 3 and 5 μm, but only partly detach from the pattern of the gaps of 10 μm. For patterns with the thermoisland diameter of 3 μm, cells could only detach from those with the gap distance of 3 μm, and could not detach from the patterns with bigger gap distances such as 5 and 10 μm. In contrast, cell detachment on patterns with varied thermo-island diameters but with the same gap distance also shows similar trends (Figure 6B). When the gap distance is 3 μm, cells could detach from all patterns studied, with no obvious differences of detachment kinetics. When the gap distance increases to 5 μm, cells could detach from patterns with thermo-island diameters of 10 and 5 μm, respectively, and the larger thermo-island diameter, the faster the process and more cells detached. When the gap distance is 10 μm, cells could only detach from patterns with island diameters of 10 μm or more (data not shown). To calculate the ratios of thermo-island area (Athermo) to the spread cell area (Aspread) before cold treatment, we tried to observe Aspread on patterns of 3−3, 5−3, 5−5, 10−3, 10−5, 10− 10, 3−5, 5−10, and 3−10, using software Image-J, which could be analyzed from the bright field images, as they offered clear morphological boundaries (Figure 7a,b). Aspread values revealed that, for patterns 10−10 and 10−5, having the same thermoresponsive island sizes but different gaps, cells appeared to have a larger Aspread on the substrate with the narrower gap (Figure 7c). The ratios of Athermo to Aspread appear to support this trend. From the cell detachment work after the cold treatment, the experimental values of Athermo/Aspread were found and listed in Table 1. These values are close to the theoretical ones calculated from the fractional occupancy of each type of patterned circles to the entire surface area. For the patterns with equal ratios of thermo-island area (such as 10−10, 5−5,

4. CONCLUSION A series of circular micropatterns of thermoresponsive islands of varied diameters and gaps on PEI-precoated glass coverslip was fabricated, and NIH 3T3 cells were cultured on the patterned surfaces to investigate cell detachment behavior by temperature reduction. Compared with continuous thermoresponsive coatings, this work has demonstrated that cells could detach from discontinuous thermoresponsive coatings which G

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(10) Fukumori, K.; Akiyama, Y.; Yamato, M.; Kobayashi, J.; Sakai, K.; Okano, T. Acta Biomater. 2009, 5, 470−476. (11) Hong, Y.; Yu, M.; Weng, W.; Cheng, K.; Wang, H.; Lin, J. Biomaterials 2013, 34, 11−18. (12) Xia, Y.; He, X.; Cao, M.; Chen, C.; Xu, H.; Pan, F.; Lu, J. R. Biomacromolecules 2013, 14, 3615−3625. (13) Schmidt, S.; Zeiser, M.; Hellweg, T.; Duschl, C.; Fery, A.; Möhwald, H. Adv. Funct. Mater. 2010, 20, 3235−3243. (14) Zahn, R.; Thomasson, E.; Guillaume-Gentil, O.; Vörös, J.; Zambelli, T. Biomaterials 2012, 33, 3421−3427. (15) Pan, G.; Guo, Q.; Ma, Y.; Yang, H.; Li, B. Angew. Chem., Int. Ed. 2013, 52, 6907−6911. (16) Liu, X. L.; Wang, S. T. Chem. Soc. Rev. 2014, 43, 2385−2401. (17) Liu, H.; Liu, X.; Meng, J.; Zhang, P.; Yang, G.; Su, B.; Sun, K.; Chen, L.; Han, D.; Wang, S.; Jiang, L. Adv. Mater. 2013, 25, 922−927. (18) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16, 297−303. (19) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506−5511. (20) Tsai, H.-Y.; Vats, K.; Yates, M. Z.; Benoit, D. S. W. Langmuir 2013, 29, 12183−12193. (21) Kumashiro, Y.; Matsunaga, T.; Muraoka, M.; Tanaka, N.; Itoga, K.; Kobayashi, J.; Tomiyama, Y.; Kuroda, M.; Shimizu, T.; Hashimoto, I.; Umemura, K.; Yamato, M.; Okano, T. J. Biomed. Mater. Res., Part A 2014, 102, 2849−2856. (22) Mandal, K.; Balland, M.; Bureau, L. PLoS One 2012, 7, e37548. (23) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425−1428. (24) Xia, Y.; Gu, Y.; Zhou, X.; Xu, H.; Zhao, X.; Yaseen, M.; Lu, J. R. Biomacromolecules 2012, 13, 2299−2308. (25) Xia, Y.; He, X.; Cao, M.; Wang, X.; Sun, Y.; He, H.; Xu, H.; Lu, J. R. Biomacromolecules 2014, 15, 4021−4031. (26) Peng, J.; Zhao, D.; Tang, X.; Tong, F.; Guan, L.; Wang, Y.; Zhang, M.; Cao, T. Langmuir 2013, 29, 11809−11814.

Scheme 2. Cell Detachment Behavior from Thermoresponsive Microgel Patterned Surface

were patterned on cell-adhesive substrate. Semiquantitative calculation data demonstrated that cells could detach from the patterned surfaces when the thermoresponsive area is just about 20% of the cell spread area. The size-dependent behavior of cell detachment on micropatterned substrates has potential applications in pertinent fields such as cell chips and biomodeling.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01507. Supplementary results on the characterization of various patterned microgel surfaces and the detachment responses of cells within the adhesive gaps against temperature drop (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-532-86983455. E-mail: [email protected]. *Tel.: +44-161-3063926. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Natural Science Fund of Shandong Province (ZR2015BM013) and the Fundamental Research-Funds for the Central Universities (14CX02121A). We thank UK Engineering and Physical Sciences Research Council (EPSRC) and Innovative UK for financial support.

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REFERENCES

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DOI: 10.1021/acs.biomac.5b01507 Biomacromolecules XXXX, XXX, XXX−XXX