Article pubs.acs.org/Biomac
Thermoresponsive Microgel Films for Harvesting Cells and Cell Sheets Yongqing Xia,† Xinlong He,† Meiwen Cao,† Cuixia Chen,† Hai Xu,*,† Fang Pan,‡ and Jian Ren Lu*,‡ †
State Key Laboratory of Heavy Oil Processing and the Centre for Bioengineering and Biotechnology, China University of Petroleum, East China, Qingdao, 266555, 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: This work reports the formation of thermoresponsive poly(N-isopropylacrylamide-co-styrene) (PNIPAAmSt) microgel films and their use for cell growth and detachment via temperature stimuli. Thermoresponsive surface films can be conveniently produced by spin-coating or drop-coating of PNIPAAmSt microgel dispersions onto substrates such as glass coverslips, cell culture plates, and flasks, making this technique widely accessible. The thickness, stability, and reversibility of the PNIPAAmSt films coated on silicon wafers with respect to temperature switching were examined by spectroscopic ellipsometry (SE) and atomic force microscopy (AFM). The results unraveled the direct link between thermoreversibility and changes in film thickness and surface morphology, showing reversible hydration and dehydration. Under different coating conditions, well-packed microgel monolayers could be utilized for effective cell recovery and harvesting. Furthermore, cell adhesion and detachment processes were reversible and there was no sign of loss of cell viability during repeated surface attachment, growth, and detachment, showing a mild interaction between cells and thermoresponsive surface. More importantly, there was little deterioration of the packing of the thermoresponsive films or any major loss of microgel particles during reuse, indicating their robustness. These PNIPAAmSt microgel films thus open up a convenient interfacial platform for cell and cell sheet harvesting while avoiding the damage of enzymatic cleavage.
1. INTRODUCTION In cell culturing, enzymes such as trypsin are usually used to detach cells from the cell culture substrate surfaces. After trypsinization treatment, however, critical cell surface proteins such as ion channels, growth factor receptors, and cell-to-cell junction proteins are often damaged. Cells with damaged extracellular matrix (ECM) proteins may suffer from low viability and altered differentiation behavior.1−4 To detach cells efficiently and to minimize the impact of enzymatic and chemical treatments on cells, a variety of synthetic surface coatings with stimuli responsive adhesion properties have been developed.5−9 To date, the most promising responsive surfaces for cell detachment are realized using surfaces grafted with thermoresponsive polymers such as poly(N-isopropylacrylamide) (PNIPAAm).10−16 PNIPAAm exhibits a lower critical solution temperature (LCST) in water with a sharp phase transition point in the physiologically relevant range around 32 °C. For this reason, PNIPAAm is frequently used as backbone to prepare thermoresponsive surfaces or surface coatings for cell growth and detachment. Under normal culture conditions at 37 °C, the PNIPAAm culture surface is relatively hydrophobic and cells attach, spread, and proliferate in a manner similar to their growth on typical © 2013 American Chemical Society
tissue culture dishes. However, upon temperature reduction below the polymer’s LCST, the PNIPAAm surface becomes hydrophilic and swells, forming a hydrated layer between the dish surface and the cultured cells, causing spontaneous cell detachment without the need for enzymatic or mechanical treatments, thus, avoiding cell damage. Linear polymers containing PNIPAAm and related derivatives are the most common materials used to prepare thermoresponsive surfaces.10,17−21 Compared with cross-linked thermoresponsive macroscopic hydrogels, the linear ones have faster volume changes and are more popular. Complex and elaborate polymerization procedures such as electron beam irradiation,1,3,6,10−12,22 plasma treatment,23,24 and living radical grafting25−27 are usually performed to covalently graft the linear PNIPAAm polymers onto tissue culture surfaces. However, the thickness of the grafted polymer layers must be controlled, for it was reported that the thicknesses of the linear PNIPAAm coatings are critical for successful cell adhesion and proliferation.25,28,29 In addition to thickness-dependent therReceived: July 4, 2013 Revised: August 10, 2013 Published: August 26, 2013 3615
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coverslips, that is, hydrophilic with the contact angle under 20° and weakly negative charged. 2.2. Preparation and Characterization of PNIPAAmSt Microgels. PNIPAAmSt microgels were prepared by surfactant-free precipitation polymerization, as described elsewhere.38 Typically, 3 g of NIPAAm (2.65 × 10−2 mol), 1 g of styrene (9.6 × 10−3 mol), and 0.041 g of the cross-linking agent MBA (2.7 × 10−4 mol) were added in 190 mL of water. The reaction mixture was then 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, 0.16 g of an initiator (APS) was dissolved in 10 mL of (oxygen free) water and added to the flask to initiate polymerization. The reaction was continued for 4 h in a nitrogen environment by continuous purging. Following the synthesis, the microgels were purified by dialysis (cutoff 11000 Da) for 3 days against water with frequent water changes until the dialysate conductivity was less than 1 μS/cm. After purification, the yield was found to be 87%. The actual percentage of polystyrene after polymerization was about 20.2 wt % (21.6 mol %), as determined from elemental analysis, and the product was referred to as PNIPAAmSt microgels. PNIPAAmSt microgels at several other polystyrene fractions were also prepared and their film forming properties briefly assessed by washing and drying following the drop-coating procedure (see below), and the one with 20.2 wt % of polystyrene turned out to be the best film forming candidate. It was subsequently used in this work for more systematic characterization. Pure poly(N-isopropylacrylamide) microgels were prepared in the same process as above and referred to as PNIPAAm microgel. The hydrodynamic diameters and thermoresponsive behavior 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 dispersed in UHQ water or Dulbecco’s modified Eagle’s medium (DMEM) was heated steadily and the microgel size determined every 2 °C by letting the microgel dispersion equilibrate at each temperature for 10 min. At each temperature, 5 consecutive runs were performed and each run composed of 15 individual radius measurements using a 10 s integration time. The ζ-potentials of the PNIPAAmSt microgels were measured as an indicator of microgel charges using the same Malvern Zetasizer instrument. The samples were prepared in the same way as prepared for size analysis and each mobility value is 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 dispersion, deposited on a 400mesh carbon-coated copper grid and dried under the infrared lamp before observation. 2.3. Preparation and Characterization of PNIPAAmSt Microgel Films. Spin-Coating. Spin-coated PNIPAAmSt microgel films were fabricated by initially depositing a 200 μL aliquot of the microgel dispersion (concentration from 0.5−1.0 wt %) onto the glass coverslip (at zero rotation), followed by spinning up to a rate of 1000 rpm for 300 s. All microgel-coated coverslips were annealed in an oven at 120 °C for 2 h and then sterilized under mild UV light for 2 h prior to cell culture experiments. For film morphology and thickness evaluation by atomic force microscopy (AFM) and spectroscopic ellipsometry (SE), PNIPAAmSt films were prepared on optically flat silicon wafers using the same coating procedures as described above. Drop-Coating. A 200 μL aliquot of PNIPAAmSt microgel dispersion (0.1−0.5 wt %) was deposited on the well surfaces of a 96-well cell culture plate. The dispersion was then maintained for 30 min before the excess dispersion removed. The plate was left to dry at 60 °C for 2 h. The microgel film coated wells were then immersed in pure water for at least 12 h, with the water being replaced every 3 h. The plate was dried at 60 °C again. The plate was then sterilized under mild UV light for 2 h prior to cell culture experiments. For cell culture glass flasks (75 mL), only 0.5 wt % microgel dispersion was used to coat the substrate. All coated glass flasks were annealed at 120 °C for at least 2 h. The six-well plates were also coated similarly but the
moresponsive changes, the outer surface structure and composition could also be affected by the layer thickness, making it highly challenging to control during surface grafting. PNIPAAm-containing microgels are colloidal particles consisting of an internal polymer cross-linked network, which can swell in water or other polar solvents in response to temperature changes. Microgels represent an intermediate between linear polymers and macroscopic hydrogels, thus combining some of the advantages of both, such as rapid phase transition, robustness, and having an easily tunable composition. The synthesis, characterization, and applications of stimuli-responsive microgels have been extensively studied over the past few years,30−33 but little effort has been devoted to exploring the use of PNIPAAm-containing microgel films to control cell adhesion and detachment.34,35 We have recently developed a novel approach for the rapid thermoresponsive detachment of adherent cells from CaCO3 mineralized poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAAmAc) microgel films.35 The use of microgel films for cell recovery offers two advantages: (i) the thickness of the microgel film can be more easily adjusted by tuning the size and composition of microgel particles in response to temperature changes than most of the current approaches based on surface polymerization; (ii) the extent of hydration within the microgel film can be controlled by the voids between the microgels and the microgels themselves. Water is easily permeated to the interface between the attached cells and the microgel surface and, thus, accelerates the hydration of the PNIPAAm segments, enabling the adhered cells to detach from the surface more rapidly. Previous studies have indicated that linear PNIPAAm films generated by surface grafting may not be optimal for cell attachment and growth due to their rather hydrophilic nature.36,37 Incorporation of monomers containing hydrophobic moieties could thus tune the amphiphilic nature of the thermoresponsive polymers. In this work, we have used styrene bearing monomer to optimize the amphiphilicity of the microgels. Microgel films are fabricated on different cell culture substrates through drop-coating or spin-coating. The structure of the microgel films was characterized with atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). Cell growth and cell detachment were monitored using phase contrast microscopy. Quantitative assessment of cell growth on our microgel films was achieved through the standard MTT assay. These studies have enabled us to examine how these easy-to-form microgel films can be utilized to benefit the harvesting of cells and cell sheets.
2. MATERIALS AND METHODS 2.1. Materials. All the chemicals were obtained from Sigma Aldrich and used as supplied. N-Isopropyl acrylamide (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, and ammonium persulfate (APS) was purified by recrystallization from water. N,N-Methylenebisacrylamide (MBA) was used as received. All water used in this experiment was processed by a Milli-Q system (Milli-Q Advantage A10 Water System Production Unit). Cell culture plates and flasks were also used as received. Silicon wafers (cuts in 15 × 20 mm2) from Compart Technology, U.K., and 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 silicon wafer surface bears a native oxide layer of 15 ± 3 Å. The oxide surface has similar physiochemical properties to freshly cleaned glass 3616
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Figure 1. (a) TEM image of PNIPAAmSt microgels, with the preparation concentration being 0.05%; (b) Plot of hydrodynamic diameters of PNIPAAmSt and PNIPAAm microgels in aqueous dispersion as a function of temperature, the arrows indicate that the volume phase transition temperature of the PNIPAAmSt and PNIPAAm microgels are about 28 and 30 °C, respectively; (c) Size distribution of the PNIPAAmSt microgels at 20 °C; (d) Size distribution of the PNIPAAmSt microgels at 38 °C; (e) Hydrodynamic diameters of PNIPAAmSt microgels in DMEM and pure water plotted as a function of temperature, with the microgels becoming aggregated above 30 °C when under DMEM. adsorbed or deposited thin film on an optically flat surface can be deduced. The measurement processes were performed following the protocols as previously reported.21 In brief, the measurement was first carried out in air and then in water using a specially designed liquid cell. The liquid cell facilitating the measurements at the solid/water interface had a pair of fused quartz plates (0.2 mm thick) as windows and the incoming and exiting beams were aligned perpendicular to the windows. Note that prior to the measurement, the oxide layer of the silicon wafer was characterized and its thickness and composition were assumed to remain unchanged subsequently. The (ψ,Δ) spectra were recorded as a function of temperature, and the microgel film was allowed to equilibrate at each temperature for ∼1 h prior to the measurement. The process was recycled and repeated to examine the extent of film reversibility in its expansion and contraction. The experimental data were analyzed using the DeltaPsi II software provided by Jobin-Yvon to give the thickness of the adsorbed microgel film and its variation with temperature. 2.4. Cell Culture. The glass coverslips spin-coated with the PNIPAAmSt microgel film were sterilized for 2 h by UV light and then transferred into six-well tissue culture plates for subsequent use. NIH 3T3 cells (2 × 104 per well) were seeded uniformly on the coverslips spin-coated with PNIPAAmSt microgels under different concen-
amount of the PNIPAAmSt microgel dispersion had to scale up. For film stability checking, some film coating was made with a preadsorption of cationic polyethyleneimine (PEI) to mediate the binding of the microgel particles onto substrates. Film Characterization. AFM images of dried PNIPAAmSt microgel films coated on silicon wafers were obtained from a Nanoscope IVa system (Digital Instruments, Santa Barbara, CA, U.S.A.) in the tapping mode under laboratory conditions. Images of 10 × 10 μm2 were captured at a scan rate of 1 Hz and a resolution of 512 × 512. The most representative AFM images for each sample were selected from at least four different positions to obtain sufficient statistics. Scanning electron microscopy (SEM, S-4800, Hitachi) was used to help examine microgel films coated on TCPS cell culture plates and glass coverslips, with sample surfaces coated with a thin Au layer to increase the contrast and quality of images. The thermoresponsive changes of the microgel films were determined using a variable angle spectroscopic ellipsometry (SE, Jobin-Yvon UVISEL). The principle of the technique is described elsewhere.39,40 It measures the change of the state of polarization of light reflected at the interface, which is usually expressed as two ellipsometric angles ψ and Δ. By using the model-dependent fitting procedure, the thickness (δ), and the refractive index (n) of an 3617
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Figure 2. AFM images of the microgel deposited film surfaces obtained by spin-coating under different concentrations of PNIPAAmSt microgels at (a) 0.5, (b) 0.75, and (c) 1.0 wt % and (d) the cross-section analysis performed as marked in (a). These images were obtained after the samples have been annealed at 120 °C for 2 h. trations and cultivated in DMEM medium containing 10% FBS at 37 °C and 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. The same procedure was used for PNIPAAmSt microgels drop-coated 96-well cell culture plates, except that the cell density reduced to 5 × 103 cell/well. Cell growth and cell detachment were monitored using a phase contrast inverted microscope (Leica, DMI3000, Germany). Quantitative assessment of cell growth on the microgel films was achieved through the standard MTT assays. To aid cell detachment, 4 °C fresh DMEM was added and the cell detachment process was then observed at ambient temperature (about 20 °C).
shown in Figure 1e, the deswelling of the microgel in DMEM is similar to that observed in pure water below 30 °C and DMEM has little influence on the microgel size and its thermosensivity. As the temperature goes above this temperature, the microgel system in DMEM becomes aggregated. The instability could arise from the screening effect to surface charges arising from ion association or binding or flocculating effect due to the presence of other organic components. 3.2. Spin-Coated Microgel Films. Spin-coating is a common and convenient approach to prepare thin layers on solid substrates. The films produced via this method were uniform and reproducible, and the amount of the deposited particles could be easily controlled by the rotation speed or the concentration of the microgel dispersion. With the speed of rotation being fixed at 1000 rpm, the volume of the microgel dispersion at 200 μL, and the rotation duration at 300 s, this study assessed the effect of varying the concentration of the PNIPAAmSt microgel dispersion from 0.5−1.0 wt % on the quality of films coated on silicon wafers or glass coverslips. The coated surfaces were annealed at 120 °C for 2 h. Note that the basic physicochemical properties of glass coverslip and silicon wafer are similar, but the latter has far better smoothness and uniformity. Thus, the basic physical characterizations were made on the silicon wafer surface. Figure 2 shows PNIPAAmSt microgel films formed on silicon wafer prepared from different microgel dispersions after annealing. Clearly, the amount of surface deposited microgel particles increased with increasing bulk concentration, but under the experimental conditions, the microgel particles have not formed a close-packed monolayer. The average particle packing density under the three different bulk concentrations studied were 1.24, 4.92, and 5.75 particles/ μm2, respectively. It is evident that, for all concentrations studied, the microgel particles are well attached to the surface and form uniform microgel films. No secondary particle attachment out of the surface plane was observed, which would cause inhomogeneous surface packing. Figure 2d shows a cross-sectional view of the microgel particle packed surface. The horizontal diameters of the deposited microgel particles are much larger than the vertical ones, suggesting that the microgel particles are flattened as a combined effect of their softness and interaction with the surface. AFM profiling revealed the vertical height (h) to be about 85 ± 5 nm and
3. RESULTS AND DISCUSSION 3.1. Bulk PNIPAAmSt Microgels. The morphology, size, and size distribution of the PNIPAAmSt microgels prepared in this work were first characterized by TEM and DLS. As shown in Figure 1a, these microgels are spherical with narrow size distribution and the average diameter is 183 ± 5 nm, as estimated from the statistic analysis measured from the TEM image. The temperature dependence of the hydrodynamic diameters measured directly from the microgel suspensions by DLS are shown as Figure 1b and the phase transition temperature was determined to be about 28 °C for the PNIPAAmSt microgels, lower than that of the PNIPAAm microgels (30 °C) without the hydrophobic monomer styrene copolymerized. Note that the values used in Figure 1b were the average diameters. As examples, the distributions of the hydrodynamic diameters for these microgel particles at 20 and 38 °C are shown in Figure 1c and d, respectively, resulting in their average values around 630 nm at 20 °C and about 300 nm at 38 °C, showing a clear trend of shrinking as a result of dehydration. More interestingly, the diameters are distributed from 300 to 1000 nm at 20 °C, and the range becomes narrowed from 200 to 500 nm at 38 °C. DMEM is a cell culture medium and contains certain concentrations of amino acids, vitamins, inorganic salts, and additional supplementary components. The electrolyte can cause the screening effect on any surface charges present and can alter the deswelling behavior.41 We thus explored the deswelling of the PNIPAAmSt microgel in DMEM, the cell culture medium used for NIH-3T3 cell culture in this work. As 3618
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Figure 3. (a) Thickness and its matching refractive index plotted against temperature for 1.0 wt % microgel dispersion spin-coated film, measured by heating from 20 to 40 °C; (b) Thickness changes of the film when placed under water at 22 °C, heated to 37 °C, cooled to 22 °C, again heated to 37 °C, and then cooled to 22 °C to complete the two full thermal cycles; (c) AFM image of the film comprised of microgel particles in their swollen state (observed at ambient temperature, about 20 °C); (d) Cross-section analysis performed as shown in (c).
was sufficient to fit the measured ellipsometric data, showing the absence of any of the corona layer formed. This observation also implied the absence of any attachment of hydrogel particles forming a secondary layer out of the primary surface layer. Because the primary aim of our SE measurements was to determine the conditions under which the microgel layer swell and deswell, we thus adopted the uniform layer modeling in the subsequent SE data analysis, as did Nerapusri et al.39 In this way, the fitted thickness (δ) should be considered as an apparent thickness of the layer which can be used to indicate the extent of thermal responsive changes. As shown in Figure 3a, the thickness−temperature curve of the adsorbed microgel layer is broadly similar to that of the bulk PNIPAAmSt microgel swelling curve (Figure 1b). Film thickness declines with rising temperature and the accompanying refractive index rises, revealing the steady dehydration of the surface bound microgels due to the expulsion of the water from the interface. The extent of water inside the film could be estimated from the refractive indices49 and for convenience the refractive index of the dry microgel was taken to be 1.48. The ellipsometric measurements show that, upon crossing the LCST, the water volume fraction changes from 0.87 at 22 °C to about 0.70 at 37 °C, and the outcome means that, even in its collapsed state, the microgel still retains a high content of water. The same finding was also reported by Schmidt et al.34 for similar systems. Figure 3b gives the fitted thicknesses at temperatures of 22 and 37 °C when the system was subject to two thermal cycles. In cycle 1, the film thickness was about 289 nm at 22 °C, but dropped to around 140 nm when the temperature increased to 37 °C, showing a drastic shrinkage and also confirming that the microgel film on the surface swells and collapses at the lower and higher temperature, respectively, similar to the bulk
the width at half height (w) to be 295 ± 20 nm. We estimated the dry microgel particle volumes (V) by assuming that they adopted a sphere-segment shape, as reported by Horecha et al.42 From the average volume calculated, the diameter of the corresponding spherical microgel having the same volume could be estimated. In this fashion, the average diameter of the microgel particles in the entirely dry state was determined to be around 184 ± 11 nm. This value is similar to that of 183 ± 8 nm estimated from the parallel TEM measurements. The difference possibly reflected the different extent of flattening and deformation under different experimental conditions and errors associated with the volume estimation. As expected, both values are significantly less than the hydrodynamic diameters of the microgel particles due to their heavy hydration in aqueous solution. 3.2.1. Thermosensitivity. Due to surface confinement, the adsorbed microgel particles may display different switching behavior from those in the bulk. Thus, SE was used to determine if they have the ability to swell and collapse (shrink) with temperature change in water. Hellweg et al. have revealed from small angle neutron scattering (SANS) measurements that the PNIPAAmSt particles formed the core−shell structure in both dilute and concentrated solutions,38 showing that most polymeric fragments are confined in the core with some loose fragments distributed around it as the outer surface corona. In contrast, when deposited onto the substrate surface, the most appropriate ellipsometric model matching the bulk core−shell distribution would be a two-layer, with the inner layer denoting the dense cores and the outer loose layer denoting the loose coronas extending into the bulk solution. Because of the rather strong confinement, it would be unlikely to expect the formation of a corona layer with significant thickness close to the substrate surface. It turned out that a uniform layer model 3619
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Figure 4. (a) Ellipsometric thicknesses of the microgel layer when it was freshly dried (dry state-1), placed under water at 22 °C for 7 days and then fully dried and the thickness measured again (dry state-2); (b) AFM image of the investigated microgel film in the dry state after completing the ellipsometric experiments in water for 7 days. Compared to the images taken before the swelling experiments no obvious changes could be observed, showing a high degree of film stability.
polyethyleneimine (PEI) was commonly used. Thus electrostatic attraction between the PNIPAAm-containing microgels and the cationic polymer is responsible for the stabilization of the layers or films formed.34,40,43−46 In the present work, however, the PNIPAAmSt microgel films are stable on the negatively charged silicon wafer or glass coverslip and their thicknesses showed little difference from those formed on PEIcoated silicon (Figure S2). The results mean that the charges of the substrate do not control the adsorbed amount of particles and their stability, and the electrostatic contribution of the substrate to the microgel adsorption seemed to be less important in comparison to nonelectrostatic interactions such as chain entanglement and hydrophilic affinity associated hydrogen bonding between the acrylamide groups and the substrate. This observation is supported by the work of Schmidt et al.47 In our work, thermal annealing treatment was found to improve film stability. As the annealing temperature was close to the glass transition temperature (Tg) of the copolymer, the heating process helped improve the contact with the substrate and interdigitation between neighboring microgel particles. If the annealing process was neglected, the microgel particles would become more easily removed from the wafer when immersed in water. 3.2.3. Cell Detachment from Spin-Coated Microgel Films. After incubation for 48 h at 37 °C, there was no obvious cell growth difference between the microgel films spin-coated at different bulk concentrations (0.5−1.0 wt %) and bare glass coverslips (left column in Figure 5). This observation suggests that the cells seeded on the thermoresponsive films could adhere and proliferate in manners similar to those seeded on the bare glass coverslips. As the temperature was reduced to around 20 °C, the cells on the microgel films started to shrink and became rounded as the culture cooled down. In contrast, little changes in cell shape or morphology was observed from those seeded on the glass coverslips. The difference was a common sign of detachment from the PNIPAAmSt surfaces during the early stage. It was also noted that some of the single NIH 3T3 cells failed to detach completely from the microgel films because of the absence of intracellular interactions as in
microgel particles. However, a slight decrease in thickness was observed during cycle 2, with the thickness of ∼263 nm at 22 °C and ∼121 nm at 37 °C. This may arise from the lack of a sufficient time for the swelling and deswelling to reach equilibration. It was found that if significantly longer time was allowed for these processes, e.g., over 10 h then better reproducibility would occur, for example, the thickness at 22 °C could restore close to 290 nm again. We applied liquid AFM to characterize these microgel particles in the swollen state at the room temperature of about 20 °C, as shown in Figure 3c. From the section profile of a swollen particle (Figure 3d), its vertical height (h) and the width at half height (w) were determined to be 230 and 564 nm, respectively. Using the method as described earlier, its equivalent spherical diameter in the swollen state can be calculated to be around 406 nm, lower than the hydrodynamic diameter measured by DLS in the bulk (630 nm). 3.2.2. Stability. The stability of the spin-coated films was assessed by monitoring variations in film thickness during one week of immersion in water at room temperature. As shown in Figure 4a, little variation in the thicknesses of the microgel film (prepared from 1.0 wt % of the microgel dispersion) was observed, suggesting its good stability. After 7 days, the microgel film was taken out and dried, followed by the SE and AFM characterizations. In comparison with its initial dried state, there was no appreciable variation in the SE thickness and the packing density was 5.98 particles/μm2 (Figure 4b), indicating that the microgel film was very stable and no desorption of microgel particles from the surface occurred. During the PNIPAAm-containing microgel synthesis, ammonium peroxodisulfate was used as initiator to start the polymerization, and the microgels obtained thus had a negative ξ-potential. In our work, the surface potential of the PNIPAAmSt microgels in water was about −8.4 mV (Figure S1). Because the PNIPAAm-containing microgels were negatively charged, most published studies have been devoted to obtaining the microgel monolayer or multilayer films by layer-by-layer techniques using a positively charged molecule as a mediating agent. A positively charged polymer, such as 3620
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Figure 5. Phase contrast microscopy images of NIH 3T3 cells 48 h after incubation at 37 °C and 30 min after cooling the surface to 20 °C. NIH 3T3 cells were seeded on (A) 0.5, (B) 0.75, and (C) 1.0 wt % PNIPAAmSt microgel films prepared by spin-coating on glass coverslips and (D) bare glass coverslip which was used as control. All scale bars are 100 μm.
the case of the fully grown cell sheets where the detached region could help lift the loosely attached region off. In any case, the rounded cells could be removed from the surface by gentle rinsing with the culture medium. Thus, the rounded cells were regarded as detached cells. After about 30 min at the ambient temperature, 66 and 76% of adhered cells became rounded on the 0.5 and 0.75 wt % microgel films, respectively, in contrast to over 90%, as observed on the 1.0 wt % microgel film. Gentle rinsing and pipetting of culture medium against the loosely attached cells could in majority of cases help remove the cells, with the final percentage of the total cell removal over 90% in all cases. Clearly, the difference from the observed cell detachment arose from different PNIPAAmSt microgel film properties and the 1.0 wt % microgel film worked better as the
thermoresponsive surface for the detachment of the NIH-3T3 cells. In contrast, there was little morphological variation for the cells grown on bare glass coverslips with respect to such a temperature change and gentle pipetting, showing little influence of these treatments to their binding to the substrate. Time lapse images were taken to show more detailed morphological changes during the detachment of individual cells on the 1.0 wt % microgel film spin-coated on glass coverslip. As shown in Figure 6, the spread and elongated cells contracted gradually with cooling and a more rounded form emerged. In this instance, the NIH-3T3 cells could be detached completely from the PNIPAAmSt microgel film in some 10 min upon the cold treatment, faster than the detachment from the commercially available thermoresponsive UpCell culture dishes. 3621
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Figure 6. (A) Images to show detachment of individual 3T3 cells from a 1.0 wt % PNIPAAmSt film spin-coated onto glass coverslip (20 mm ×20 mm) upon cooling to ambient temperature. Images are taken sequentially from left to right. The 0 min means the cultured six-well plate was taken out of the incubator and observed by microscopy at room temperature; (B) The corresponding higher magnification views of single cells retracting as they were detaching from the spin-coated PNIPAAmSt film upon cooling. The red lines are indicative of shrinks of typical skeletal filaments and their stretches with medium cooling.
Scheme 1. Illustration of Cell Detachment from the PNIPAAmSt Microgel Film
Figure 7. SEM images of PNIPAAmSt microgels spin-coated on a glass coverslip from 1.0 wt % dispersion. (a) before cell culture and (b) after cell detachment, showing that the surface underwent some minor changes but remained largely intact.
Rapid cell detachment is thought to be essential for retaining the biological functions of detached cells. When adhered cells are kept at low temperature for a lengthy time, the cold environment may damage cells and their functions. Several attempts have been carried out to accelerate cell sheet detachment and recovery to prevent damage.5,6,11,17 In this work, the microgel particles formed a nonclose packed film by spin-coating, and water can easily penetrate into the film and
It should be commented that some of the cells appeared to be round under culturing conditions before temperature was dropped, evident from Figures 5 and 6A. As can be seen from Figure 6 under the magnified condition, stretching and swirling of the microfilaments are extensive for the round and stretched cell bodies. These hallmarks of cell attachment are similar to those observed on bare glass coverslips. 3622
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Figure 8. (a) NIH 3T3 cells cultured on microgel particle film spin-coated glass coverslip, (b) two days after nonenzymatic detachment from microgel film upon cooling to the ambient temperature. (c) The cells in the medium were reseeded on the same coated surface for 3 days. (d) When cold-treated again, the cell sheet could be observed to roll up, indicating that the ECM is undisturbed.
common substrates used in laboratories for cell culturing and harvesting, and spin-coating is not suitable for making microgel particle coating on the bottom of these substrates. In this part of studies, we seek to develop a one-step simplistic method for thermoresponsive culture plate/flask coating that can be easily adopted for cell harvesting. As an example, SEM images were taken (results not shown) to reveal that the microgel films drop-coated on the TCPS plate were highly uniform, but the surface packing density could vary from edge to center. This was quite different from the microgel films spin-coated on silicon wafer or glass coverslip. This phenomenon could have occurred during the final stage of drying and could be remedied by careful rinsing to remove residual particles left in the solvent. The above experiments were undertaken in the six-well plates using NIH-3T3 cells. To check the versatility of the dropcoating method, the same experiments were repeated using the 96-well plates, coated with 0.1−0.5 wt % PNIPAAmSt dispersions by the same method. After 48 h, the cells in the well plate were cooled down with the rest of the procedure being kept the same as that undertaken on the spin-coated microgel films. The results showed that under the same conditions the films prepared by the 0.5 wt % microgel dispersion showed the best performance in cell attachment, growth and detachment (Figure S4). Thus, in the following experiments, the 0.5 wt % microgel dispersion was used for preparing the thermoresponsive films by drop-coating method. The viability of cells grown on the PNIPAAmSt microgel drop-coated TCPS (0.5 wt %) was compared with that grown on the bare TCPS (positive control). The microscopic images showed that after 48 h of seeding, most of the NIH 3T3 cells attached and spread on both the microgel-coated film and bare TCPS (Figure S5a,b), showing no visual difference. Cell viability from the MTT results revealed similar absorbance on both the PNIPAAmSt microgel film and the positive control (Figure S5c). It is useful to note that TCPS surface is nonsmooth compared to the optically flat silicon wafer surface. This explains why some regions of the microscopic images can
hydrate the microgel particles upon cooling, just as illustrated in Scheme 1. This type of film morphology can easily uptake and expel water in response to temperature changes and help accelerate cell detachment. After detachment, the same well plate was then put into the incubator again to assess the viability of the detached cells and the integrity of the film. The results revealed no observable difference between growth and detachment when the same coated surface was used four times (Figure S3). This suggests that the coated microgel films were stable and did not cause any adverse effects on cell adhesion and growth. To further confirm the stability of the microgel film in the process of cell detachment, SEM was used to reveal its morphology. Compared with the surface before cell culture (Figure 7a), almost all microgel particles still remained on the surface of the glass coverslip after cell detachment (Figure 7b). However, loss of microgel particles did occur in some small areas, though such changes did not affect the entire cell culturing. Because the above repeated culturing using the cells that had been grown and removed through temperature changes between 37 °C and the ambient temperature, it was necessary to check the viability of the cells carefully. This was done by observing cell attachment and detachment and also by MTT assays. No difference was observed within 5−10% experimental errors, indicating that the detachment process did not affect any cellular functions. The same growth and detachment process was also applied by growing cell sheets with more cells seeded at the beginning and the culturing lasted for 3 days. When the confluent cells were cooled down again, the cell sheet could be observed rolling up, indicating minimal disturbance upon detachment to harvest cells in the form of cell sheets (Figure 8). 3.3. Drop-Coated PNIPAAmSt Microgel Films. Dropcoating is another method to prepare thermoresponsive films. Its key advantage is the combination of simplicity and versatility, as it can be used to fabricate films on almost any kind of substrates. Cell culture plates and flasks are the most 3623
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Figure 9. (a−d) Detachment process of a NIH-3T3 cell sheet from the microgel film surface drop-coated from the 0.5 wt % microgel dispersion onto the well bottoms of a 96-well TCPS plate. The detachment was aided by gently pipetting the culture medium using 100 μL pipet tip at the pipetting frequency about once per second: (a) 0, (b) 20, (c) 40, and (d) 60 min. (e) A higher magnified image of a section of the cell fragments showing individual cells; (f) Live/dead fluorescent staining of a freshly detached NIH 3T3 cell sheet fragment demonstrating that almost all cells are alive (green) with few dead cells (red).
conditions (Figure 9f). To further check the stability of the microgel surfaces coated from drop-coating, the viability of cells was checked following repeated attachment and detachment processes as described previously. No sign of different behavior in cell attachment or detachment was observed (Figure S6). To check the generality of this procedure, drop-coating was extended to the cell culture flasks which are also widely used for expanding cell stocks. We have attempted to coat the flask (the bottom surface area of 37.5 cm2) using 0.5 wt % PNIPAAmSt microgel dispersion, with the bare flask used as control. After 3 days, the cells grown on the microgel particle coated film surface and in the control flask were compared through visual observation under microscopic imagining and MTT assays (Figure S7). The results showed that the cell viability from the two different surfaces were almost identical.
sometimes appear to be out of focus (e.g., Figure S5a,b). There is, however, no evidence to link such nonsmoothness to cell growth and detachment. Although drop-coating is convenient, it yet has to perform well in cell detachment. It was found that films drop-coated from the 0.5 wt % microgel particle dispersion started to detach as soon as the culture temperature dropped to below 28 °C. It was also observed that cells could persist on some patches. The detachment could be assisted by gentle pipetting culture medium against the attached cell regions. With the help of pipetting, over 90% cells could be detached from the 0.5 wt % microgel dispersion drop-coated films in about 2−3 min if the culture medium was replaced by the 4 °C cold medium, achieving the fastest cell detachment ever reported so far using the thermoresponsive surfaces. Throughout this work, it was observed that both spin-coated and drop-coated surfaces could lead to the harvesting of cells or small cell fragments easily, especially with the help of gentle pipetting. In the area of tissue regeneration, it would be useful to generate sizable and viable cell sheets which could work as the starting materials for tissue fabrication. When the cell layer was completely confluent, DMEM at 4 °C was added to the cell layer, the cell sheet began detaching from drop-coated PNIPAAmSt microgel film immediately following the cold treatment. Under most cases, the cell sheet started detaching from the edge, which then moved toward the center. During the current detachment process, however, the cell sheets could easily end up to fold and overlay upon themselves, resulting in a cell sheet pile or lump. This process could benefit from better control and manipulation as elaborated by Okano et al.48 Figures 9a−d showed a typical process of cell sheet detachment. In this case, the whole sheet could not spontaneously detach from the surface completely, but by gently pipetting the culture medium against the anchoring sheet region, the whole cell sheet could detach from the well within a few minutes. Note also that strong pipetting against the surface could also remove some of the microgel particles. A higher magnified image of a section of this sheet revealed individual cells within the sheet (Figure 9e). Live−dead staining assay using calcein and propidium iodide showed that the peeled cell sheet’s viability was the same as under standard culture
4. CONCLUSIONS We have developed robust and reliable technical procedures to fabricate thermoresponsive surfaces for cell harvesting from water-based microgel particle dispersions. The thermoresponsive surfaces can be conveniently produced by spin-coating or drop-coating on substrates, which are widely used in laboratory, such as glass coverslips, cell culture plates, and flasks. The technical procedures as developed enabled the formation of microgel particle monolayer films with good reproducibility, thus, ensuring consistent performance in cell growth and detachment. Similar charge and physiochemical properties between substrates and microgel particle surfaces could undermine the stability, but the particles could still be irreversibly anchored, suggesting that other interactions were dominant. Individual cells, small cell fragments, and even the whole cell sheets can be detached from the microgel particle coated film surfaces within minutes. This technical advantage will enable the widespread use of thermoresponsive substrates for harvesting of cells and cell sheets, avoiding the complication of enzymatic cleavage.
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ASSOCIATED CONTENT
S Supporting Information *
Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. 3624
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-532-86981569 (H.X.); +44-161-3063926 (J.R.L.). E-mail:
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[email protected] (J.R.L.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Natural Science Foundation of China (#20804057), Natural Science Fund of Shandong Province (#Q2008B03), the U.K. Engineering and Physical Sciences Research Council (EPSRC), and the Royal Society (London). The authors also thank to Dr. Colin Grant for his valuable comments on AFM data analysis.
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