Cell culture substrates were prepared on German glass coverslips (15 mm, Carolina Biological Supply, Burlington, NC) as described previously1,8,9
according to the protocol of Pelham and Wang,10,11
with the following modifications. Gels were prepared with 7.5% acrylamide and bisacrylamide (bis) ranging from 0.02 to 1.0%. The elastic moduli of the gels were 500, 1000, and 5000 Pa, to model the elastic modulus range found normally in neural tissues.3
was used as a control to model typical cell culture conditions. To obtain a wider range of substrate stiffnesses in the evaluation of substrate stiffness versus cell stiffness, cells also were cultured on gels with a stiffness gradient, which ranged from 300 to 20,000 Pa.13
The strategy for creating stiffness gradients has been reported previously.13
Briefly, gradient generators, for production of gradient gels, made from polydimethyl siloxane (PDMS) microfluidic channels were fabricated using standard photolithography techniques.14,15
The stiffness of polyacrylamide gels was tuned by varying the concentration of bisacrylamide at a fixed acrylamide concentration.16
Three solutions with the same acrylamide (Bio-Rad, Hercules, CA) concentration but different N,N-methylene-bisacrylamide (Bio-Rad) concentrations were injected into the gradient generator. Each solution had an acrylamide concentration of 8% and a 2,2-diethoxyacetophenone (Sigma 227,102, St. Louis, MO) photoinitiator concentration of 0.5%. The bisacrylamide concentrations of the three inlets were: 0.02%, 0.02%, and 1%. During development of the technique, fluorescein (Sigma F6377) was added to the 0.02% bis-acrylamide solution to evaluate the gradient of bisacrylamide concentration upon polymerization. The solutions then were driven through the microfluidic channels by syringe pumps (Harvard Apparatus, Holliston, MA) at the same flow rate of 8 mL/hour Once the flow in the outlet channel reached a steady state, a UV light was shined on the outlet region for 8 minutes. The syringe pumps were stopped after the outlet region was exposed to UV light for 10 seconds. Peeling off the PDMS gradient generator results in the gel being adherent to the activated coverslip. The resulting gel, 1.8 mm wide and 2 cm long, was immersed immediately in PBS buffer for 12 hours to remove unreacted photoinitiators. Once the gradient polyacrylamide gels were fabricated, the stiffness across the gel was characterized using AFM. The gradual transition in bis-acrylamide concentration, which correlates with gel stiffness, also was evaluated by fluorescence.
Final gel thickness after polymerization was ≈100 μm. A bifunctional cross-linker (2 mM sulfo-SANPAH [Pierce, Rockford, IL] in 50 mM HEPES, pH 8.2) was used to ligate laminin or collagen to the polyacrylamide gels. Laminin (50 μg of 1 mg/mL natural mouse laminin in 5.95 mL HEPES buffer, Invitrogen, Grand Island, NY) or collagen (0.1 mg/mL rat-tail collagen, BD Bioscience, San Diego, CA) was applied to the gels and incubated for 2 hours at 37°C for cross-linking. Gels were rinsed once with Eagle's minimal essential medium (EMEM), and incubated overnight in EMEM in a humidified 37°C incubator. Three hours before plating the cells, EMEM was removed and replaced with culture medium.
The elastic modulus of each polyacrylamide mixture was confirmed using a Perkin Elmer DMA 7e dynamic mechanical analyzer. Each mixture was polymerized in the rheometer. The shear storage modulus G, corresponding to the elastic resistance of the gels, was determined from the shear stress in phase with an oscillatory (1 rad/s) shear strain of 2% maximal amplitude, by standard techniques.
A conditionally immortalized mouse Müller cell line, ImM107
was used in these experiments. The ImM10 cell line was isolated from the retinas of P10 mice that were heterozygous for the “immortomouse” transgene (H-2Kb-tsA58) that encodes an interferon gamma (IFN-γ) inducible, temperature sensitive SV40 large T antigen.17
When grown at 33°C, cells are immortalized by induction of the T antigen with IFN-γ at 50 U/mL final concentration in the media. Following withdrawal of IFN-γ, T-antigen production is stopped, and shifting the cells to 39°C inactivates the remaining, temperature sensitive T-antigen, thereby releasing cells from immortalization.
ImM10 cells were plated at 10,000 cells/well in a 24-well plate, and cultured at 5.5% CO2
in growth medium composed of 500 mL Neurobasal media, 10 mL of B27 supplements, 5 mL of 200 mM L-glutamine, with 2% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin antibiotics (all from Invitrogen). When IFN-γ was required, 500 mg of mouse recombinant IFN-γ (Peprotech, Rocky Hill, NJ) was added to a final concentration of 50 U/ml. The cells were maintained with IFN-γ in the media, at 33°C. All experiments were performed in non-immortalizing conditions without IFN-γ, at 39°C. Cells were studied or RNA was harvested after 21 days. This length of time was selected to allow the cells to accommodate to the substrate upon which they were plated. It has been noted previously that Müller cells that had been cultured for at least 12 days modified their expression profile, up-regulating expression of GFAP, Sox2, CyclinD3, Ceruloplasmin, and Nestin, and down-regulating glutamine synthase, Kip1, Kir4.1, and Acquaporin-1.18
In addition, changes in contractility of Müller cells were noted previously on the time scale of 3 weeks.19
The cells were not confluent at the time of examination and were at passage 10 through 21.
AFM measurements were performed according to a published protocol.9
Briefly, Müller cells on substrates were rinsed with phosphate buffered saline (PBS) and placed in serum-free Dulbecco's modified Eagle's medium (DMEM). AFM measurements were done with a DAFM-2X Bioscope (Veeco, Woodbury, NY) mounted on an Axiovert 100 microscope (Zeiss, Thornwood, NY) using silicon nitride cantilevers (196 μm long, 23 μm wide, 0.6 μm thick) with a pyramidal tip (when the cell is indented by 1 μm, the area of the indentation projected on the sample plane is 1.6 μm2
) for indentation. The spring constant of the cantilever, calibrated by resonance measurements, was typically 0.06 N/m (DNP; Veeco). To quantify cellular stiffness, the first 600 nm of tip deflection from the horizontal (Δd
) were fit with the Hertz model modified for a cone:20,21
where k and Δz are the bending rigidity and the vertical indentation of the cantilever, E is the Young's modulus, α is the cone tip angle, and ν the Poisson ratio. Young's modulus is the inverse ratio between the strain (δz/z) applied to the material and the resulting stress. The Poisson ratio is defined as the ratio of compressional strain in the direction normal to the applied stress and the extensional strain in the direction of the applied stress, and is taken to be 0.5 for all samples. To determine cell stiffness, AFM measurements were made on single cells by indenting on three positions of the peripheral cell body within a period of approximately 30 minutes. The average elastic modulus for each condition was calculated by averaging the three measurements for each cell followed by averaging all obtained values. At very large indentation, it is possible that the tip can rupture the cell membrane. However, small indentations will not lead to cell rupture. For this reason, we only indented the Müller cells by approximately 1 μm, where the possibility of the tip penetrating the cell membrane is very low. Publications by the investigators and others previously have used this AFM system reproducibly to obtain data. Moreover, we also monitored the indentation process using an optical microscope, and did not observe changes in cell morphology after the AFM measurements.
It has been shown previously that AFM measurements will be influenced by the underlying substrate when the indentation is larger than 10% of the sample thickness.22
Therefore, it is likely that the stiffness will be influenced by the substrate stiffness if the AFM indentations were taken over the cell lamelipodia, whose thickness often is less than 1 μm. Being aware of this issue, and specifically seeking to avoid the influence of the substrate, we obtained measurements on the peri-nuclei regions of the cells. The cell thickness in these regions is on the order of a few micrometers, significantly larger than the thickness of a lamelipodium. To verify the appropriateness of this technique, we plotted AFM cantilever deflection as a function indentation as well as cell stiffness as the indentation depth increases from 200 nm to 1 μm. The cell stiffness values did not vary significantly with the indentation depth (see Supplementary Material, and , http://www.iovs.org/content/53/6/3014/suppl/DC1
Figure 1. Photomicrographs of rhodamine-phalloidin stained Müller cells, with nuclear counterstaining, on laminin-coated substrates. Cells grown on laminin-coated glass (A) and 5000 Pa substrates (B) display prominent stress fibers. Cells on 1000 Pa (C (more ...)
Figure 2. Images (from left to right) of Müller cells cultured on collagen-coated plastic, and collagen-coated gels of stiffness 1500 and 500 Pa. Cells were stained with Texas Red-Phalloidin with nuclear counterstaining. Note the transformation of cell (more ...)
To evaluate cell morphology, the cells were fixed with 4% paraformaldehyde and prepared for dual immunofluorescence and epifluorescence microscopy. Cells undergoing rhodamine-phalloidin (Invitrogen) staining were treated according to a standard protocol23
with nuclear counterstaining. Cells were imaged using an inverted Olympus microscope (IX71; Olympus, Tokyo, Japan) equipped with a monochrome, cooled CCD digital camera (Rolera-XR; Q-Imaging, Surrey, BC, Canada). Images were quantified for average spread area using NIH Image, with at least 10 cells per high-powered field analyzed. Propagation was measured in triplicate using a hemocytometer and Trpan blue stain. Less than 1% of cells counted were non-viable.
Gene expression was analyzed by quantitative qRT-PCR using the Stratagene MX3005P real-time PCR system, and the SABiosciences Extracellular Matrix and Adhesion Molecules real-time PCR array (which is composed of 85 genes important for cell-cell and cell-matrix interactions plus 5 housekeeping genes for normalization, controls for genomic DNA contamination, and positive and negative controls, Valencia, CA). Total RNA was isolated from cells using affinity columns (RNeasy; Qiagen, Valencia, CA). Briefly, cells were lysed in a guanidine hydrochloride buffer, loaded onto affinity columns, washed, and eluted in RNAse free water (RNeasy; Qiagen). Samples were quantified by 260 nm absorbance using spectrophotometry (Nanodrop, Wilmington, DE) and stored at −80°C. First strand cDNA was synthesized using reverse transcriptase (RT2 First Strand Kit; SABiosciences) according to the manufacturer's instructions. The RT2 kit uses a combination of random hexamers and oligo-dT to prime the reverse transcriptase reaction, and uses a proprietary procedure to eliminate any contaminating genomic DNA from the reactions. Master mix, containing 12.5 μL cDNA was added to each well of a PCR array (Extracellular Matrix and Adhesion Molecules PCR Array, PAMM-013; SABiosciences). Cycling conditions followed manufacturer's recommendations and performed on a real-time PCR instrument (Mx3005p; Stratagene, Santa Clara, CA). Web-based analysis software (SABiosciences) was used to analyze the results of the experiments. The proteins of greatest interest in this study are secreted and, thus, the protein cannot be obtained purely from cell lysates. Also, the proteins are known to degrade rapidly, producing broad bands on Western Blotting. For this reason, mRNA was quantified as an indirect measure of protein production.
Descriptive statistics, including mean, median, and standard deviation, were calculated. Linear regression was used to analyze AFM elastic modulus measurements. ANOVA was used to assess the relationship between the genes expressed.