(386e) Engineering Cell Adhesion to Thermoresponsive Substrates: Effect of Spreading Coefficient
Thermoresponsive polymers are one class of stimulus-responsive materials that have been widely used in bioengineering applications as “smart bioactive surfaces”, intelligent materials designed to interact specifically and selectively with biological tissues. Currently, poly(N-isopropylacrylamide) (PNIPAM) is the gold standard in stimuli-responsive polymers for bioapplications because it exhibits an aqueous lower critical solution temperature (LCST) of 32oC and is relatively insensitive to environmental factors, such as ion concentration or pH. In traditional cell culture, cells are biochemically released from tissue culture plastic (TCP), disrupting cell-cell interactions and destroying membrane-associated proteins critical for maintaining cell phenotype. Thermoresponsive polymer brushes allow for the gentle detachment of cells from their culture surface by simply changing the temperature below the LCST of the polymer in aqueous media. Under standard culture conditions (37oC, 5% CO2), above the LCST, the thermoresponsive polymer phase-separates from aqueous culture media to form a hydrophobic surface that supports cell growth in confluent monolayers. Switching the temperature below the LCST of the polymer causes the cells to detach as a result of the phase change of the polymer in aqueous media to a miscible, hydrophilic state. Using this thermoresponsive polymer system, cells can be harvested in a manner that preserves cell-cell and cell-matrix interactions and obviates the use of biochemical enzymes, i.e. trypsin.
An alternative to the PNIPAM system has recently been explored by Wischerhoff et al. that offers distinct advantages over PNIPAM: tunable graft density and LCST.1 These thermoresponsive brushes are synthesized from random copolymers of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) (P(MEO2MA-co-OEGMA)) and exhibit aqueous LCST values between 26 and 90°C, which can be precisely adjusted by varying the comonomer composition.2 In this work, we investigated changes in cellular morphology and spreading on thermoresponsive polymers (TRPs) PNIPAM and P(MEO2MA-co-OEGMA) of variable LCSTs compared to biochemical release from TCP and examined the cellular phenotypic response using gene expression analysis for markers of cell adhesion and apoptosis. Results are correlated to the substrate’s spreading coefficient, which is related to the LCST and calculated from contact angle measurements.
P(MEO2MA-co-OEGMA) thermobrushes were fabricated as follows: multilayer structures were assembled on glass slides consisting of an alternating polyelectrolyte multilayer system (PEI/PSS/(PDADMAC/PSS)4/PDADMAC) with a nonlinear growth regime, a single macroinitiator layer, and the polymer brush grafted on the macroinitiator layer via surface-initiated ATRP (atom transfer radical polymerization). LCST was varied by altering the comonomer ratio of MEO2MA to OEGMA between 100:0 (28oC) and 92:8 (37oC). PNIPAM surfaces were purchased from Thermo Scientific (UpCell). Mouse fibroblasts (CCL-1/L929) were cultured on TRPs and TCP and imaged over 48 hours to assess cell spreading. Cells were released mechanically from thermobrushes by incubating at room temperature for 20 minutes, while control cells were harvested from TCP using trypsin, and RNA was isolated and reverse-transcribed to cDNA for gene expression analysis using real time RT-PCR. Axisymmetric drop shape analysis was utilized to measure the contact angle of a water droplet on thermobrushes of variable LCST in decane. Temperature was varied between 20-45oC, with a step size of 2-3oC.
Cell morphology was assessed using phase contrast microscopy at 0, 6, 18, 24, 30, and 48 hours. Images revealed an increase in cell adhesion as a function of spreading coefficient (LCST). Cells cultured on LCST 37oC exhibited significant delays in attachment, even after 24 hours. Cells cultured on PNIPAM did not exhibit any delays in attachment relative to TCP. PNIPAM and P(MEO2MA-co-OEGMA) thermobrush with LCST 31oC showed similar levels of gene expression for adhesion genes FN1, RhoA, Dusp2 and apoptosis genes BCL2 and TRP53, suggesting a role for spreading coefficient, independent of surface chemistry. Further gene analysis using adhesion microarrays suggests cell attachment to TRPs occurs via different mechanisms, as expression levels of different integrins are differentially upregulated as a function of surface chemistry and spreading coefficient. These key molecular signatures, important for engineering cell adhesion to TRPs, will be presented and discussed.
1. Wischerhoff et al. Langmuir 2009, 25, 5949.
2. Lutz, J.-F.; Hoth, A. Macromolecules 2006, 39, 893-896.