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Protein-Substrate Adhesion in Microcontact Printing Regulates Cell Behaviors Shuhuan Hu, Ting-Hsuan Chen, Yanhua Zhao, Zuankai Wang, and Raymond H. W. Lam Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02935 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 6, 2018
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Protein-Substrate Adhesion in Microcontact Printing
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Regulates Cell Behaviors
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Shuhuan Hua*, Ting-Hsuan Chenab, Yanhua Zhaoa, Zuankai Wangab and Raymond
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H.W. Lamab*
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a
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Kong, Hong Kong;
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b
Department of Mechanical and Biomedical Engineering, City University of Hong
City University of Hong Kong Shenzhen Research Institute, Shenzhen, China;
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* Correspondence should be addressed to S Hu (email:
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[email protected]; Tel: +852-3442-7174) or RHW Lam (email:
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[email protected]; Tel: +852-3442-8577; Fax: +852-3442-0172).
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Abstract
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Microcontact printing (μCP) is widely used to create patterns of biomolecules
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essential for studies of cell mechanics, migration, and tissue engineering. However,
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different types of μCPs may create micropatterns with varied protein-substrate
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adhesion, which may change cell behaviors and pose uncertainty in result
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interpretation. Here, we characterize two μCP methods for coating extracellular
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matrix (ECM) proteins (stamp-off and covalent-bond) and demonstrate for the first
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time the important role of protein-substrate adhesion in determining cell behaviors.
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We found that, as compared to cells with weaker traction force (e.g. endothelial cells),
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cells with strong traction force (e.g. vascular smooth muscle cells) may delaminate the
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ECM patterns, which reduced cell viability as a result. Importantly, such ECM
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delamination was observed on patterns by stamp-off, but not on the patterns by
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covalent-bond. Further comparisons on the displacement of the ECM patterns
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between the normal VSMCs and the force-reduced VSMCs suggested that cell
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traction force plays an essential role in this ECM delamination. Together, our results
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indicated that μCPs with insufficient adhesion may lead to ECM delamination and
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cause cell death, providing a new insight for micropatterning in cell-biomaterial
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interaction on biointerfaces.
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Keyword
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Microcontact printing, protein, substrate, extracellular matrix, adhesion, micropattern,
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traction force, smooth muscle cell
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Introduction
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Microcontact printing (μCP) is a widely used biofabrication to coat or self-assemble
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macromolecules such as deoxyribonucleic acids, extracellular matrix (ECM) proteins
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and antibodies with precisely defined micropatterns on planar substrates 1-3. Over the
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past two decades, μCP has become a fundamental tool for cell research, drug
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screening and tissue engineering 4-7. In particular, μCP has enabled detailed studies on
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subcellular motility and cytoskeleton mediated by well-defined adhesion sites
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confined by the ECM proteins. For example, it has been reported that μCP can
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achieve collagen VI stripes with different spacing distances to regulate chondrocytes
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behaviors for development of cartilage replacement 8. Recently a new microcontact
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printing technique called Pattern on Topography (PoT) was developed to examine the
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synergistic regulating effects of biophysical and biochemical cues to the
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cardiomyocytes.9
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The working principle of μCP mostly relies on the cell adhesion to ECM protein,
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which is essential for many physiological activities. At the cell level, cell adhesion
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regulates apoptosis, mitogenesis, cell differentiation, and cell migration 10-15. At the
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tissue/organ level, the adhesive property of ECM is required for tissue integrity 16-18
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and was highlighted by many genetic and autoimmune diseases, such as muscular
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dystrophies 19. Clinical practices have revealed the effectiveness of using immobilized
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ECM proteins in the central nervous system to repair nerves 20. However, increasing
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evidences suggest that cells do not passively attach to ECM. Instead, this cell-ECM
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adhesion is a two-way mechanical interaction mediated by focal adhesions (FAs) 21-23
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and cytoskeletal networks 24-27. That is, the mechanical signals may transduce from
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ECM to cells and reorganize the cytoskeleton networks. As a result, the cellular force
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is then iteratively changed, causing a disruption of the constitution and structure of
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ECM 28-31.
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Early μCP methods involved binding of molecules on a substrate through chemical
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reactions such as metal-alkanethiolate bonding 32-33, silanization 34 and carbodiimide
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crosslinking schemes 33. More recently, researchers have implemented μCP using the 3 ACS Paragon Plus Environment
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non-covalent bindings to achieve the more rapid and flexible micropatterns.35-36 These
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μCP procedures usually include polymer surface activation, either oxygen plasma
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bombardment 2 or ultraviolet-ozone irradiation 37. However, different μCP methods
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may create micropatterned ECM protein with varied protein-substrate adhesion. As a
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result, the cell traction force is resisted in different levels, leading to changes of
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cytoskeleton organization and regulation of cell behaviors, but it is rarely addressed.
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In this paper, we analyze cell behaviors growing on ECM protein micropatterns
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coated with two different µCP methods (stamp-off and covalent-bond). The stamp-off
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patterning relies on the direct molecular adsorption while the covalent-bond scheme
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applies intermediating molecules with the higher binding strength by linking both
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ECM proteins and substrates with covalent bonds. We seed cells onto the substrates
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and inspect the role of protein-substrate adhesion in the two-way cell-ECM
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interactions. On the ECM side, we observe any changes in the protein patterns
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induced by the seeded cells. On the other side, we examine whether the cell
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characteristics such as morphology, viability, and cytoskeleton formation are different
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on the micropatterns coated with the different µCP methods.
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Experimental Section
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ECM Patterning Techniques
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We adopted two μCP techniques (stamp-off and covalent-bond) with different levels
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of the protein-substrate adhesion. Polydimethylsiloxane (PDMS), the widely-used
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biocompatible polymeric material (Dow Corning, Midland, MI)38, is used as the
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substrate. All the PDMS substrates were casted from a
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trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma-Aldrich, St. Louis, MO) treated
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flat silicon wafer with a monomer-curing agent weight ratio of 10:1 after baking at 80
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ºC for >20 hr. In this work, we selected the fibronectin conjugated with fluorescent
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molecules (Alexa Fluor 488 protein label kit, Thermo Fisher Scientific, Waltham, MA)
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as the ECM protein for visualizing the coated protein patterns using the selected μCP
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methods.
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The first μCP method (stamp-off) utilized a microstructured stamp to create
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patterns by removing ECM protein from some regions on a fibronectin-adsorbed
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PDMS substrate 35. As shown in Fig. 1a, we prepared a PDMS stamp (10:1
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monomer-curing agent ratio) with microstructures of a ‘negative’ pattern based on the
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traditional soft lithography 39. The PDMS stamp was prepared using photolithography
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of SU-8 photoresist (Microchem) on a silicon wafer followed by silanization of
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trichloro(1H,1H,2H,2H-perfluorooctyl)silane. The PDMS stamp was further treated
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with oxygen plasma (PDC001, Harrick Plasma, Ithaca, NY) to enhance its molecular
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adhesion to fibronectin. To coat fibronectin on the PDMS substrate, we pipetted 50
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μg/ml of the fluorescent fibronectin in water to cover the PDMS substrate for 2 hr in
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order to facilitate the protein adsorption. After blow-drying with compressed air, the
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microstructured PDMS stamp was placed onto the flat fibronectin-coated PDMS for
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20 s. As the plasma-treated PDMS stamp had a stronger molecular adhesion,
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fibronectin on the contact surfaces between the stamp and the substrate were removed
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after the stamp was peeled off 35, 40-41. We then applied a 0.1 % surfactant (pluronic
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F127; Sigma-Aldrich) on the substrate for 15 min to prevent unexpected cell-substrate 5 ACS Paragon Plus Environment
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attachment in the uncoated regions and rinsed the substrate with distilled water. The second µCP method (covalent-bond) applied intermediate molecules for
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achieving covalent bonds between the protein coating and the substrate for stronger
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protein-substrate adhesion (Fig. 1b). An ester based protein immobilization was
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adapted to bind the fibronectin molecules onto the PDMS substrate through covalent
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bonding 42 (Fig. 2a). Briefly, we treated the PDMS substrate with oxygen plasma,
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following by applying 4 % (v/v) 3-mercaptopropyl trimethoxysilane (MPTMS; Gelest,
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Morrisville, PA) in ethanol to cover the PDMS substrate for 45 min. The substrate was
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washed with ethanol and then covered by 0.28 % (w/v) N-γ-maleimidobutyryloxy
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succinimide ester (GMBS; Thermo Fisher Scientific) in ethanol for another 15 min.
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On the other side, fibronectin was applied onto a ‘positive’ microstructured stamp. We
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then placed the stamp onto the chemically treated substrate for 20 s. After peeling of
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the stamp, we applied pluronic F127 and rinsed the substrate to avoid any unexpected
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cell attachment.
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Surface Characterization
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X-ray photoelectron spectroscopy (XPS) experiments were conducted using an XPS
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facility (PHI Model 5802 with a monochromatic Al Kα X-ray source at 1486.6 eV;
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Physical Electronics, Chanhassen, MN) with a pressure of 10-10 mBar and a resolution
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of 0.1081 eV. An attenuated total reflectance Fourier transform infrared spectroscopy
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(ATR-FTIR, Perkin Elmer 1600, Perkin Elmer, Waltham, MA) together with a zinc
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selenide crystal was used to characterize the covalent bonds between molecules. An
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ellipsometer with a resolution of 0.1 nm (Jobin Yvon-PZ2000; Horiba Scientific,
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Edison, NJ) using a He–Ne laser (632.8 nm) was used to measure the thickness of the
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protein layer. The adhesion force were determined by the Universal Testing Machine
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(UTM-EZ-LX, Shimadzu Scientific Instruments, Columbia, MD). Immediately before
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the test, the moving plate (diameter: 8 mm) was treated with underwater glue
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(JH-5553; Jin Hong Glues, Hangzhou, China). The speed of the moving plate was set
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to 10 mm•min-1 to obtain the load-displacement curve. The applying load was set as
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10 N and the contact time between the sample and the moving plate surface was 5 min. 6 ACS Paragon Plus Environment
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In addition, we measured surface hydrophobicity and infrared spectra of the
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fibronectin-coated substrates in different fabrication stages. The surface
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hydrophobicity was measured using a drop-shape analyzer (DSA100, Kruss,
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Hamburg, Germany).
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Cell Culture
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Primary human vascular smooth muscle cells (VSMCs; Lonza, Walkersville, MD)
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were cultured in the standard medium kit (SmGM-2™ BulletKit™, Lonza). Primary
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human umbilical vein endothelial cells (VECs; Lonza) were cultured in the standard
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medium kit (EGM™-2 BulletKit™, Lonza). NIH/3T3 fibroblasts (3T3; ATCC,
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Manassas, VA) were cultured in the Dulbecco’s modified Eagle’s medium (DMEM;
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Life Technologies, Carlsbad, CA) supplemented with 10% Bovine Serum, 100
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units/mL penicillin and 1 % L-glutamine. The cells were cultured in 5% CO2
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humidified cell culture incubator at 37 °C. The cells were trypsinized and subcultured
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once their population reached >80% confluence. Only the cells with a passage number
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