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Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture
 
Hiro-taka Masuda1,2, Seiichiro Ishihara2, Ichiro Harada3, Takeomi Mizutani2, Masayori Ishikawa4, Kazushige Kawabata2, and Hisashi Haga2
1Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan
2Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan
3Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
4Department of Medical Physics, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
BioTechniques, Vol. 56, No. 4, April 2014, pp. 172–179
Full Text (PDF)
Supplementary Material
Abstract

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

Glass plates are frequently used as cell culture substrates in cell biology experiments. For example, to study localization of specific molecules by immunofluorescence staining, a cover glass is typically used as a substrate because it is sufficiently thin (approximately 0.15 m) and transparent to allow observation of cells using microscopes with 60× or 100× objectives.

Extracellular matrix (ECM) proteins such as collagen, fibronectin, and laminin are major components of connective tissues and regulate cellular phenotypes (1). For culturing cells, glass plates are often coated with ECM proteins that cells bind using adhesive transmembrane molecules such as integrins (2), thus increasing the efficiency of cell adhesion. Previously, the effects of ECM molecules on cellular behavior were investigated by culturing the cells on glass plates coated with different ECM molecules. For example, epithelial cells on a laminin-coated glass plate had a more spread morphology than cells on a fibronectin-coated glass plate (3), probably due to uniform and robust coating of the glass with ECM molecules to a degree sufficient to affect cellular phenotype. However, the fate of glass-bound ECM proteins during tissue culture has not yet been determined.

ECM-coated glass treated with silane reagents has been utilized as a cellular substrate for observation of focal adhesions (4), which helps in capturing cellular dynamics (5). Furthermore, glass treated with silane reagents has been used in ECM-patterned substrates (6). However, these studies did not show the stability of the ECM on silanized glass. Silane reagents such as (3-aminopropyl)triethoxysilane are aminosilane compounds possessing free amino groups that can bind to free hydroxyl groups, such as those found on glass surfaces (7). When glass bonds with (3-aminopropyl) triethoxysilane, the surface of the glass then presents amino groups that can bind to proteins (8).

Method summary

We established an improved method for coating extracellular matrix (ECM) proteins on a glass surface by using (3-aminopropyl) triethoxysilane. Under cell culture conditions, the ECM on the silanized glass was more uniform and stable than that on non-treated glass.

Here we prepared a glass plate treated with (3-aminopropyl)triethoxysilane (silanized glass), coated the glass with ECM proteins, and observed the ECM proteins on the glass under cell culture conditions. We mainly used Madin- Darby canine kidney (MDCK) cells in this study because MDCK cells have been reported to adhere to ECM molecules such as laminin and fibronectin (9, 10). We expected that a silanized glass prepared in this manner would be more uniformly and stably coated with ECM protein than non-treated glass (NT glass) under cell culture conditions. Materials and methods Cell culture

Madin-Darby Canine Kidney (MDCK) and A431 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (BIST-TEC, Equitech Bio Inc., Kerrville, TX), and 1% antibiotics (penicillin, streptomycin, and amphotericin B; Invitrogen, Carlsbad, CA). T84 cells were cultured in a 1:1 mixture of DMEM/Nutrient F-12 Ham (Sigma) supplemented with 10% FBS (Biological Industries, Beit Haemek, Israel), 1% antibiotics, 1% MEM non-essential amino acid solution (Sigma), and 4 mM l-glutamine (Sigma). The cells were incubated at 37°C in a humidified incubator with 5% CO2. Silanization and coating of glasses

We prepared a silanized cover glass for cell culture. First, we soaked a cover glass (Matsunami Glass Ind., Ltd., Kishiwada, Japan) in saturated KOH in isopropanol solution and incubated it overnight at room temperature. The glass was washed three times with distilled water and dried. We then soaked the glass in 0.33% (3-aminopropyl)triethoxysilane (Sigma) in toluene overnight at room temperature. For optimization of the (3-aminopropyl)triethoxysilane concentration, we prepared 0.0033%–33% (3-aminopropyl) triethoxysilane in toluene. The glass was washed three times with toluene and dried. A glass ring with a radius of 8.0 mm was attached to the cover glass with glue to prepare a glass dish. The dish was sterilized with UV light overnight at room temperature. The glass dish was filled with laminin-111 (Trevigen, Gaithersburg, MD) in DMEM or fibronectin (Roche, Mannheim, Germany) in phosphate-buffered saline (PBS) and incubated overnight at 4°C. After incubation, the glass dish was washed with PBS and used for cell culture. Water contact angle measurement

The water contact angle was measured using the sessile drop method (11), where distilled water (5 L) was dropped on a KOH-treated glass plate or a silanized glass plate at a right angle to the glass surface. The glass was not coated with any ECM molecules. We dropped a total of nine water drops on each of three independently prepared glass plates. Using a stereomicroscope system (VB-G25, Keyence, Osaka, Japan), the drops were imaged at a horizontal angle to the glass surface. The water contact angle was analyzed using a low-bond axisymmetric drop shape analysis plug-in (12) of the ImageJ software. The statistical analysis was performed using Student's t-test. Antibodies and reagents

The following were used as primary antibodies for immunofluorescence staining: anti-laminin-111 L9393 (Sigma), anti-fibronectin 13G3B7 (Developmental Studies Hybridoma Bank; University of Iowa, Iowa City, IA), and anti-paxillin (cat#610052; BD, Franklin Lakes, NJ) and anti-integrin β3 (cat#555752; BD). AlexaFluor 488-, AlexaFluor 546-, or AlexaFluor 594-labeled anti-mouse or rabbit antibodies (Invitrogen) were used as secondary antibodies for immunofluorescence staining. AlexaFluor 488-labeled phalloidin (Invitrogen) was used for F-actin staining. Time-lapse observation

Cells were cultured on each glass dish. Two days after seeding, each dish was filled with culture medium and sealed with silicone grease to prevent the pH of the medium from changing. A phase contrast microscope (TE2005; Nikon Instech., Tokyo, Japan) equipped with a 10× objective and kept at 37°C in an acrylic resin box was used for time-lapse observations. The Image-Pro software (Media Cybernetics Inc., Silver Spring, MD) was used for time-lapse observation by capturing images every 5 min for 12 h. Movies were edited from a series of captured images. Cell velocity was calculated by tracking cells every 30 min using the manual tracking plug-in of the ImageJ software. Statistical analysis was performed using Student's t-test. Immunofluorescence staining

Cells cultured on each glass dish were fixed with 3.6% paraformaldehyde in PBS, permeabilized by 0.5% Triton X-100 in PBS, and blocked with 0.5% BSA (Sigma) in PBS at room temperature. Primary and secondary antibody reactions were performed at 37°C for 1 h or at 4°C overnight. The dilution of the primary and secondary antibodies was 1:250 and that of AlexaFluor 488-labeled phalloidin was 1:200. Fluorescence images were obtained using confocal laser scanning microscopy (C1 confocal imaging system; Nikon Instech). Relative fluorescence intensities were calculated with the ImageJ software. Statistical analysis was performed using Student's or Welch's t-test. Reverse transcription PCR (RT-PCR)

Cells were lysed using ISOGEN (Wako, Osaka, Japan) for RNA extraction, and reverse transcription was performed using a ReverTra Ace qPCR RT kit (TAKARA, Otsu, Japan). PCR was performed using Taq polymerase in ThermoPol Buffer (NEB, Ipswich, MA). The primers were as follows: human GAPDH, forward: 5′-ACCACAGTCCATGCCATCAC-3′, re ver s e: 5′-TCCACCACCCTGTTGCTGTA-3′; fibronectin, forward: 5′-AGGGCTGGAACCGGGCATTG-3′, reverse: 5′-GAGGTGGGCCCAGGTGACA-3′. Results and discussion

To demonstrate that (3-aminopropyl)triethoxysi lane immobi l ization on silanized glass was successful, we performed water contact angle measurement for KOH-treated glass and silanized glass. This method determines the degree of hydrophobicity of the surface, as a higher water contact angle indicates higher hydrophobicity (8). Because the surface of KOH-treated glass, but not that of silanized glass, is negatively charged, when (3-aminopropyl) triethoxysilane is immobilized on silanized glass, the surface of silanized glass should be much more hydrophobic than that of KOH-treated glass. In the present study, the water contact angle of the silanized glass was greater than KOH-treated glass (Supplementary Figure S1, A and B), indicating that silanized glass was more hydrophobic than the KOH-treated glass and that (3-aminopropyl)triethoxysilane immobilization on the silanized glass was successful.

To investigate the stability of ECM molecules on glass, we cultured MDCK cells on glass coated with ECM molecules for three days and observed ECM protein immunofluorescence. We used laminin-111 (laminin) because MDCK cells are known to adhere to laminins (9). In the area that was not covered by cells, the fluorescence intensity of the coated laminin was not significantly different between the NT glass and the silanized glass (Figure 1, B and C). In contrast, in the area under the cells, the intensity of laminin on the NT glass was significantly lower than that on the silanized glass (Figure 1, B and C). These findings were obtained for three experimental replicates (Supplementary Figure S2). The results indicate that the coated laminin molecules under the MDCK cells on the silanized glass were more stable than those on the NT glass. In addition, on the KOH-treated glass, the fluorescence intensity of the coated laminin was significantly lower than that on the silanized glass, both in the area not covered by cells and in the area under the cells (Supplementary Figure S3, A and B). Furthermore, the optimal concentration of (3-aminopropyl)triethoxysilane for coating the glass was found to be 0.33%, since at this concentration the silanized glass showed the highest fluorescence intensity of coated laminin without an uneven distribution of laminin, both in the area not covered by and under the cells (Supplementary Figure S3, A and B). These findings demonstrate that the coated laminin molecules under the MDCK cells on the silanized glass are more stable than the coated molecules under cells on the NT or KOH-treated glass.




Figure 1.  Silanized glass stabilizes coated ECM molecules. ( (Click to enlarge)




We next cultured A431 epidermoid carcinoma cells and T84 colon cancer cells on silanized glass or NT glass coated with laminin. The results obtained with A431 cells were similar to those from MDCK cells (Supplementary Figure S4, A and B). In contrast, on the silanized glass the amount of laminin in the area under the T84 cells was significantly lower than that in the area not covered by cells (Supplementary Figure S4, C and D). This result suggests that T84 cells degrade laminin by secreting proteases such as matrix metalloproteinase 7, which has been reported to be secreted by T84 cells (13).

We also investigated fibronectin-coated glass because MDCK cells have been reported to adhere to fibronectin (10). Here, the fluorescence intensity of the coated fibronectin was lower on the NT glass than on the silanized glass, both in the non-cell area and in the area under the cells (Figure 1, D and E). Thus, the silanized glass was successfully coated with fibronectin, whereas the NT glass could not retain fibronectin on its surface. Taken together, these findings indicate that when MDCK cells are cultured on ECM-coated glass, the stability of the ECM molecules is higher on silanized glass than on NT glass.

Next, we investigated whether another type of ECM molecule existed under the MDCK cells on the glass coated with laminin, since MDCK cells are reported to secrete their own ECM (14). We cultured MDCK cells on laminin-coated glass and detected fibronectin by immunofluorescence staining. On the silanized glass coated with laminin, localization of fibronectin was not observed (Figure 2A). In contrast, on the NT glass coated with laminin, dot-like localization of fibronectin was observed under the cells (Figure 2A). The fluorescence intensity of fibronectin under the cells on NT glass coated with laminin was significantly higher than that on silanized glass coated with laminin (Figure 2B), suggesting that on NT glass coated with laminin, MDCK cells remove the originally coated laminin and secrete fibronectin. To investigate whether these differences are regulated at the transcriptional level, we used RT-PCR to detect fibronectin mRNA. Fibronectin mRNA was more highly expressed in cells on NT glass coated with laminin than in cells on silanized glass coated with laminin (Figure 2C). These results show that, on NT glass coated with laminin, MDCK cells express fibronectin at the transcriptional level and secrete the fibronectin under their somata.




Figure 2.  Distribution and expression of ECM proteins and integrin β3 differ between cells cultured on non-treated (NT) glass coated with laminin and cells cultured on silanized glass coated with laminin. (Click to enlarge)




Taken together, these findings indicate that silanized glass coated with laminin should be used as a laminin substrate for cell culture, as the cultured cells did not express fibronectin and stably maintained laminin molecules under their somata. In contrast, NT glass coated with laminin should not be used as a laminin substrate for cellular experiments because the cultured cells remove the original coat of laminin and express fibronectin. It should be noted that a previous study showed that laminin 111 neutralizes HGF stimulation in mammary epithelial organoids (15). Another study reported that HGF stimulation induces fibronectin expression at the transcriptional level in MDCK cells (16). These studies suggest that biochemical signaling activated by the laminin molecule inhibits fibronectin expression via suppression of HGF signaling. Therefore, on silanized glass coated with laminin, MDCK cells may suppress fibronectin expression by preventing HGF signaling, whereas on NT glass coated with laminin, the cells may induce fibronectin expression via HGF signaling.

Because cells on the NT glass coated with laminin removed the laminin molecules and expressed fibronectin, we investigated the localization of integrin β3, which binds to fibronectin but not laminin (2). On the NT glass coated with laminin, dot-like localization of integrin β3 was observed under MDCK cells (Figure 2D). In contrast, on silanized glass coated with laminin, the cells did not show such integrin β3 localization (Figure 2D). Thus, MDCK cells on NT glass coated with laminin remodel the ECM to fibronectin and change the distribution of integrin β3.

To investigate whether the difference in ECM composition between NT glass and silanized glass affects cellular phenotype, we observed the morphological features and migration of MDCK cells using a time-lapse method. On NT glass coated with laminin, the cells showed a cobblestone-like morphology and low migration (Figure 3, A and B). In contrast, on the silanized glass coated with laminin, the cells presented a spread morphology and high migration (Figure 3, A and B), indicating that the ECM composition affected MDCK cell phenotype.




Figure 3.  Motility differences between cells on NT glass and those on silanized glass. (Click to enlarge)




We also compared the phenotype of cells on silanized glass coated with one ECM molecule to that on silanized glass coated with another ECM molecule. Cells on silanized glass coated with laminin showed more spreading morphology and higher migration than those on silanized glass coated with fibronectin (Figure 3, A and B), indicating that laminin substrates induce higher migratory activity in MDCK cells compared to fibronectin substrates.

Pseudopodia, which contain actin fibers and adhesion complexes, play an important role in cell migration (17). To observe adhesion complexes, we focused on paxillin, which is a component of adhesion complexes and a key molecule in cell migration (18). We performed immunofluorescence staining for actin fibers and paxillin. Pseudopodia were defined as protrusions of actin fibers containing paxillin. On silanized glass coated with laminin or fibronectin, the number of pseudopodia in MDCK cells was higher than that on NT glass coated with laminin (Figure 4), suggesting that MDCK cells on silanized glass coated with laminin or fibronectin demonstrate high migration activity by generating pseudopodia. MDCK cells on silanized glass coated with laminin migrated faster than those on silanized glass coated with fibronectin (Figure 3B). However, cells under both conditions showed almost the same number of pseudopodia. Thus, laminin but not fibronectin may produce biochemical signals to promote cell migration independent of the generation of pseudopodia.




Figure 4.  The number of pseudopodia in cells grown on NT glass vs. cells grown on silanized glass. (Click to enlarge)




In summary, silanized glasses can stably retain ECM molecules, creating a useful surface for cell biology experiments that examine the effect of ECM. Cells on NT glass coated with ECM molecules remove the originally coated ECM and secrete another type of ECM. Therefore, NT glass coated with ECM molecules may produce artifactual results due to the remodeling of the ECM by the cells. Stable ECM substrates such as silanized glass coated with ECM should therefore be used in experiments examining the effects of ECM molecules on cells. Author contributions

H.M. designed the research, performed the experiments, analyzed the data, and wrote the manuscript. S.I. designed the research and wrote the manuscript. I.H. established the methods for the experiments. T.M., M.I., and K.K. designed the research. H.H. managed the overall research and wrote the manuscript.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (B) (24390285) and Scientific Research on Innovative Areas (25127701) to H.H., Young Scientists (B) (23770167) and Scientific Research on Innovative Areas (24106502) to T.M., Scientific Research (B) (25287106) to K.K., and Special Expenditures for “Reverse Translational Research from Advanced Medical Technology to Advanced Life Science” to S.I., M.I., and H.H., from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors thank Sawa Kobayashi for technical assistance. I.H. is currently affiliated with the Laboratory for Mechanical Medicine, Locomotive Syndrome Research Institute, Nadogaya Hospital.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Hisashi Haga, Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan. E-mails: [email protected]


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