Adherent cells are an important class of cells. The in vitro processing, manipulation, and analysis of adherent cells are essential tasks in biomedical research, tissue engineering, toxicology, and biotechnology. Successful protocols not only depend on high levels of efficiency and precision but also on the preservation of the vitality of the cell material. In this context, the non-invasive detachment of adherent cells from synthetic cultivation substrates or scaffolds is crucial for making these cells available for cell-based approaches in the fields mentioned above.
Standard cell detachment by enzymes (e.g., trypsin) results in unspecific digestion of membrane proteins. These destroyed membrane proteins cannot be investigated after detachment (e.g., using patch clamp or FACS) until the cells have replaced them. In contrast, surface-mediated cell detachment by thermoresponsive polymers does not destroy membrane proteins, and is hence usually referred to as “non-invasive” in the literature [1
Thermoresponsive polymer coatings have been identified as a valuable tool for achieving non-invasive control over cell adhesion [2
]. Thermoresponsive polymers exhibit a sharp reversible structural transition upon temperature change around the lower critical solution temperature (LCST). Above the LCST the polymers show a compact conformation, whereas below the LCST the polymers swell in aqueous solutions and exhibit an open, highly-hydrated structure. During recent years, considerable progress has been achieved in exploiting the potential of such coatings for cell cultivation, tissue engineering, and the development of assays. Polymers of choice for cell cultivation applications include poly(N
-isopropylacrylamide) (poly(NiPAm)), polyethylene glycol (PEG) and poly(vinyl methyl ether), as they show an LCST that is a few degrees below the physiological cultivation temperature, at between 26 and 33 °C [2
]. When appropriately immobilised to solid substrates, thermoresponsive polymer coatings mediate protein and cell adhesion at 37 °C. Decreasing the temperature below the LCST triggers non-invasive cell detachment due to polymer hydration, which reduces protein and cell adhesion. The use of such coatings may replace common detachment methods that rely on proteases which impair the vitality of cells and invalidate results obtained from them.
Recently, we have introduced thermoresponsive coatings that are based on microgels. Besides excellent functional properties such as changing hydration, elasticity, and topography depending on temperature (strongly influencing cell adhesion), these coatings possess some features that greatly expand their applicability [10
]. As the size of the microgels is large enough to support firm attachment to many substrate materials, various simple formation methods can be employed for the production of versatile and inexpensive thermoresponsive coatings. Dipping, spin coating, spraying, spotting, and printing all allow the fabrication of highly functional coatings. The latter two methods facilitate local application of the microgels in order to produce defined patterns and structures [11
]. In all cases, the microgels form highly regular monolayers. In addition, adhesion and detachment of cells to the coatings can be easily modulated by introducing microparticles with distinct surface properties into the coatings.
In the following, we present results that highlight the functionality and versatility of thermoresponsive microgel coatings. As an example for demonstrating the ease of integrating these coatings into microfluidic systems, we systematically investigate the detachment of cells as a function of the flow velocity of the medium. The potential of the patterned microgel coating for the development of cell assays is demonstrated in two examples. Finally, we report results on the modulation of the cell adhesion properties by the addition of microparticles into the thermoresponsive microgel coatings.
2. Materials and Methods
Polymer synthesis: The microgel (MZ140) was synthesized through precipitation polymerization as previously described [10
]. In short, N
-isopropylacrylamide (NIPAM; Sigma-Aldrich 97%, Saint Louis, MO, USA) was cross linked by N
’-methylenebis(acrylamide) (BIS; Sigma-Aldrich, 99%) to obtain thermoresponsive microgels. For this purpose, 10.568 mmol NIPAM and 0.98 mmol BIS were dissolved in 150 mL purified water. The solution was heated up to 70 °C under continuous stirring and purged with nitrogen. Subsequently, the reaction was initiated as described previously, and the work-up of the obtained product was performed as in our previous works. The obtained microgels were characterized by PCS [11
Sample preparation: For uniform coating with thermoresponsive microgels, freshly cleaned glass substrates (20 × 20 mm2, Menzel, Braunschweig, Germany) were covered with 200 µL of 1% poly(ethylenimine) solution (PEI, Sigma Aldrich, Steinheim, Germany) for one minute. The supernatant was removed using a spin coater (CPS 20, Semitec, Dresden, Germany) for 20 s with 3000 rpm. Afterwards, 200 µL of a 0.5 wt % microgel suspension was incubated for 30 s on the PEI-modified glass substrate. Finally, the supernatant was again spun for 10 s at 2000 rpm and for 10 s at 3000 rpm.
To alter the surface properties of thermoresponsive microgel coatings, carboxylated polystyrene beads (Fluoresbrite® YG Carboxylate (0.20 µm), Polysciences Europe GmbH, Hirschberg an der Bergstrasse, Germany) were mixed with the microgel suspension before the surface immobilization process mentioned above. To this end, a 0.6 wt % microgel suspension and a 0.6 wt % polystyrene bead (PS beads) suspension were prepared and both were mixed according to the following volume ratios: 1:0; 200:1; 100:1; 50:1; 5:1, and 0:1. To simplify the nomenclature of the various mixing ratios, we herein use a concentration indication in percent which is related to mixing volumes. It should be noted that the densities of microgel and PS beads are different, and thus the percentages are rough approximations. Thus, 0% is equivalent a ratio that did not contain PS beads (1:0), and the percentages of 0.5%, 1%, 2%, 20%, and 100% are equivalent to ratios of 200:1, 100:1, 50:1, 5:1, and 0:1, respectively. For visualisation of the fluorescent PS bead distribution on the surface, a confocal laser scanning microscope (510 Meta, Zeiss, Oberkochen, Germany) equipped with an argon laser and a 63 × / 1.4 oil immersion objective was employed. For image acquisition, the pinhole was set to 1 Airy unit (image slice of approximately 0.7 µm).
For generating patterned coatings on cyclo olefin polymer substrates (COP ibiTreat, ibidi, Planegg, Germany), a nano-plotter (NP2.1, GeSiM, Großerkmannsdorf, Germany) equipped with a piezo dispenser (Nano-Tip A, GeSiM, Germany) was employed. Circular spots with individual volumes of 300 pL were dispensed in a grid of 353 µm. The overall area of spots was 1 cm2. To generate lines, the spotting grid was decreased in one dimension to 190 µm and individual spots were dispended using a suspension concentration of 0.35 wt %. During the spotting process, the liquid was completely evaporated. Afterwards, new spots were placed between the previously positioned spots to generate lines, resulting in a final spotting distance of 95 µm. The other dimension was increased to establish a line distance of 600 µm.
Cell culture: L929 mouse fibroblasts (ACC 2, DSMZ, Germany) were cultivated in DMEM containing HEPES (25 mM), FCS (10%), penicillin–streptomycin (1%), and L-glutamine (2 mM, all Biochrom, Germany). CHO-K1 cells (ACC 110, DSMZ Germany) were cultivated in Ham’s F12 supplemented with FCS (10%) and penicillin– streptomycin (1%, all Biochrom, Berlin, Germany) at 37 °C and 5% CO2.
Shear force assay: The microsystems were incubated with cell medium overnight. During this time, air bubbles occurred, and were flushed out of the system with additional medium. Then, 2 × 106 L929 mouse fibroblasts mL−1 were injected via the side channel and cultivated without additional medium supply for one day in an incubator. After 30 min under microscopic observation at ~22 °C, flow rates of up to 2000 µL min−1 were applied using a 1-mL glass syringe (ILS, Stützerbach, Germany) operated by a syringe pump (SP230IWZ, WPI, Hitchin, UK). The average flow velocity across the channel cross section was calculated by dividing the volume flux (volume supplied by the syringe pump per time) by the channel cross sectional area (A = 0.045 mm2). In order to exclusively measure the effect of the physical shear force and to avoid any influence from biological or chemical changes (shear history), the cells should be exposed to every velocity step as quickly as possible. For practical reasons (the syringe pump being operated manually), a shear time of 10 s was selected. Thus, every 10 s, the flow velocity was increased stepwise until all cells were rinsed off the surface or the maximum pump velocity was reached.
Quantification of the shear force: The hydrodynamic shear force F
acting on a spherical cell in contact with the channel bottom was numerically derived using the program Comsol Multiphysics 4.3a for any of the chosen flow velocities according to our estimations in previously published work [12
Cell migration assay: For the cell migration assay, two kinds of patterns were used. The substrates coated with microgel spots were placed in petri dishes and 3 × 104 CHO-K1 cells cm−2 were seeded. The COP substrates coated with microgel lines were stuck in microfluidic channels (Sticky-Slide IV 0.4, ibidi, Germany) and 2.5 × 104 L929 cells were seeded in the microchannel. After one day of cell culture at 37 °C, the samples were cooled to 22 °C for 30 min. Afterwards, the cells located on the microgel were rinsed off in a petri dish using a 1-mL Eppendorf pipette and in the microchannels with a 10-mL syringe. All cell migration observations were performed with a fully automated set-up (Cell-R, Olympus, Hamburg, Germany) equipped with a 10 × / 0.3 objective and an incubation chamber (Air Conditioning Unit, Evotec, Hamburg, Germany).
Cell adhesion assay: To observe the cell adhesion on the substrates coated with microgel and PS beads, the samples were placed in a six-well plate and 2 × 104 L929 cells cm−2 were seeded in each well. Immediately after seeding, the samples were placed under the microscope at 37 °C for recording a time lapse film. The delay until the time lapse acquisition started was approximately five minutes. The percentage of cells which changed their morphology from a round to a spread state over one hour was analysed. Subsequently, cell detachment from the surfaces upon temperature decrease was investigated. To this end, the samples were cooled to 22 °C for 30 min after one day of cell culture. Then, the percentage of cells which reduced the cell surface contact area from a spread to a round state was determined. Finally, the samples were rinsed using a 1-mL Eppendorf pipette.