Measurement of Cell-Matrix Adhesion at Single-Cell Resolution

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Measurement of Cell-Matrix Adhesion at Single-Cell Resolution Reveals the Functions of Biomaterials for Adherent Cell Culture Sifeng Mao, Qiang Zhang, Haifang Li, Qiushi Huang, Mashooq Khan, Katsumi Uchiyama, and Jin-Ming Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02653 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Analytical Chemistry

Measurement of Cell-Matrix Adhesion at Single-Cell Resolution Reveals the Functions of Biomaterials for Adherent Cell Culture Sifeng Mao,† Qiang Zhang,† Haifang Li,† Qiushi Huang,† Mashooq Khan,† Katsumi Uchiyama,‡ and Jin-Ming Lin*,† †

Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China ‡ Department of Applied Chemistry, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan ABSTRACT: Cell adhesion is essential for cell to maintain its functions, and biomaterials acting as extracellular matrix (ECM) plays a vital role. However, convectional methods for evaluating the functions of biomaterials become insufficient and sometimes incorrect when we give a deeper insight into single-cell researches. In this work, we reported a novel methodology for the measurement of cell-matrix adhesion at single-cell resolution that could precisely evaluate the functions of biomaterials for adherent cell culture. A microfludic device, live single cell extractor (LSCE), was used for cell extraction. We applied this method to evaluate various modified biomaterials. The results indicated that poly(L-polylysine) (PLL) coated glass and fibronection (FN) coated glass slice showed the best biocompatibility for adherent cell culture following by the (3-aminopropyl) triethoxysilane (APTES) coated glass, while piranha solution treated glass slice and octadecyltrichlorosilane (OTS) coated glass showed weak biocompatibilities. Furthermore, APTES, PLL and FN modifications enhanced the cell heterogeneity while the OTS modification weakened the cell heterogeneity compare to the initial piranha solution treated glass. The method not only clarified the cell-matrix adhesion strength at single-cell resolution but also revealed the influences of biomaterials on cell-matrix adhesion and heterogeneity of cell-matrix adhesion for adherent cell culture. It might be a general strategy for precise evaluation of biomaterials.

Cell theory, where cell functions as the basic unit of life, is a cornerstone of biology.1-3 Cell researches provide insights into some of the most fundamental processes in biology and promote us well dissecting life’s mysteries.4-6 Most types of human and animal cells are adherent cells that are closely networked with the matrix.7-9 In both in vivo and in vitro cell researches, the cell matrix and microenvironment are very important to cell behavior.10-12 Cell and matrix jointly construct different tissues.13,14 Adherent cells in suspension will function different from that in adherent state.15,16 Therefore, different biomaterials and modified substrates as matrix will affect cell functions and cell behaviors,17-19 such as metabolism, migration, proliferation and apoptosis. To reconstitute cell matrix and cell microenvironment precisely in vitro, much effort has been made on the development of biomaterials.20,21 Those reported biomaterials have contributed much to in vitro tissue construction and cell behaviors studies.22,23 The biocompatibility and the interaction of the biomaterials with cultured cells are the most important characterizations when they are utilized in cell studies.24 With current methods, it is convenient to clarify the biocompatibility of different biomaterials by measurement of cell viability and cell morphology.25-27 However, those approaches only concern about the status of cells themselves and are incapable for evaluating the influence of biomaterials on the interaction between adhered cells and matrix. In our previous researches28,29 on cells and matrix, cells with good viability and large spreading area did not always hold strong interaction with the matrix. Thus, development of new

biotechnologies for precisely evaluating the interaction between cells and biomaterials is still a vital issue. Single-cell analysis has become more fascinating among different methods for cell researches.30-36 The single-cell researches focus on cell to cell interaction,37,38 single-cell metabolism,39-41 whole-genome sequencing analysis42-45 from single-cells and cell-cycle dynamics of single-cells46. More and more scientists have jumped and are jumping into singlecell analysis and development of biomaterials for single-cell analysis. On one hand, how to collect live single-cells from tissue samples while keeping their viability becomes an essential issue. On the other hand, how to evaluate the functions of new biomaterials for cell adherence and cell activities is essential for scientists in materials science. Therefore, we aim to develop new methodology to meet those requirements in single-cell biology and materials science. Herein, we established a novel strategy that was capable of measuring cell-matrix adhesion at single-cell resolution, for precise evaluation of functions of biomaterials for adherent cell culture. Various functionalized glass surfaces as biomaterials were prepared using chemical modifications or physical modifications. The feasibility of the method for measurement of cell-matrix adhesion was demonstrated. We used our established method to explore the compatibilities of those biomaterials for adherent cell culture, and the interactions between those biomaterial substrates and cells cultured on them were uncovered at single-cell resolution.

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EXPERIMENTAL SECTION Design and fabrication of LSCE. The geometry of the LSCE were designed by using Adobe Illustrator software (Adobe Systems Incorporated, USA), and a photo mask was generated. The PDMS chip was fabricated using softlithography and replica molding techniques as previous report. Wafer (company) was firstly washed and dried, then was coated with an SU-2050 negative photoresist (Microchem, USA) using a spin coater. Then, it was soft baked on hotplate. After cooled down to room temperature, the wafer was exposed to UV light and developed by developing solution (Microchem, USA). The wafer mold was hard baked on hotplate. A 10:1 weight mixture PDMS prepolymer and curing agent (Sylgard 184, Dow corning, USA) was poured on the wafer mold. The wafer mold with mixture was placed in the oven at 80 ℃ for more than 2 h or overnight at room temperature after well deaerated. Finally, PDMS was peeled off from the wafer mold, and connection holes were punched before the PDMS replica was irreversibly sealed with another piece of PDMS replica with channels by oxygen plasma (PDC-32G, Harrick Plasma, Ithaca, NY) treatment. Operation System. The XYZ stage (Sigma KOKI Co., Ltd.) served as the microfludic chip probe holder. Cell samples were placed on The XY stage of microscopy (Leica DMI 4000 B, Wetzlar, Germany). In all of the experiments, the pen was maintained at its position while the postion of cell samples were controlled by the XY stage of microscopy. The injection and aspiration apertures were connected to two syringes (Hamilton, Graubunden, Switzerland) that were controlled by two individual pumps (Hamilton, Pennsylvania, USA). A petri dish (Corning, New York, USA) with cell sample, filled with cell culture medium, was placed on the XY stage of a microscope. Then, the microfludic chip probe was immersed in the cell culture medium and placed perpendicular to the surface of petri dish. The LSCE was fixed on an XYZ stage that functioned as a positioner. The XYZ stage contain three dividing rulers to monitor distance change in X, Y or Z directions. The resolution of the rulers on our XYZ stage was 5 um. To well address the gap in experiments, we moved down the LSCE by controlling the XYZ stage to make the LSCE to touch the substrate where gap was Zero. Then, we raised the LSCE in Z direction by using the positioner with a desired distance (50 um), resulting in a gap of 50 um between pen end and substrate. Flow Confinement. A solution containing 0.25% trypsin and 0.02% EDTA, as well as 1 μg/ml fluorescein for visualization, was used to monitor the leakage from the working region. The leakage was evaluated by calculating ratio between the fluorescent intensity in microjet and that in surrounding area. Different gaps and aspiration flow rates were applied to evaluate the leakage. COMSOL Multiphysics Simulation. Computational fluid dynamics simulations are commonly used in control of microfluidics.47-48 Comsol Multiphysics 5.3 (Comsol) was used to carry out 3-D simulations on a six-core, 64-bit computer (Dell) with 32 GB of RAM. The geometry of the tool was set as same as that in the experiment. Two 50 μm × 80 μm apertures separated at a distance length of 100 μm. The gap was set as 50 μm. Injection flow rate was 10 μL/min and the aspiration flow rate was 50 μL/min. The injection solution

was assumed to be water with a density of 999.7 kg/m3 and a viscosity of 0.001 Pa·s. The simulations were run under steady-state conditions with the flow boundary conditions at the edges of the microfluidic chip probe perimeter sides set as open boundaries (equal to atmospheric pressure). Different injection and aspiration flow rates were applied in the simulation while kept the ratio constant (QA/QI = 5). Biomaterials Preparation and Characterizations. Glass slides were modified with hydroxyl group (OH) by immersing the glass into piranha solution (15 mL concentrated sulfuric acid and 5 mL hydrogen peroxide, Beijing Chemical Works, China) for 1 h. OTS-Toluene (1% v/v, J&K, China) and APTES-Toluene (1%, v/v, J&K, China) were respectively poured onto the OH modified glass and then shook incessantly for 1h to convert OH to amino group (NH2) and alkyl group (C18). The OH modified glass slides were immersed into 0.01% PLL (Sigma, USA) solution and 250 μg/mL FN solution (Invitrogen, USA), incubated for 1 h to prompt the adsorption of biomolecules to the glass, then washed with PBS for 3 times and utilized immediately. All biomaterials surface were analyzed by X-ray photoelectron spectroscopy (XPS, PHI Quantera II, Ulvac-Phi Inc., Japan) and Atomic force microscope (AFM, Dimension, Bruker, German) before and after modifications. Adherent Cell Culture on Biomaterials. U87 cells was purchased from Cancer Institute & Hospital of the Chinese Academy of Medical Science (Beijing, China). Cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. U87 cells were maintained in minimal essential medium (MEM, Corning, USA) with Earle’s Salts and Lglutamine supplemented with 10% fetal bovine serum (FBS, Corning, USA), nonessential amino acids, 100 units/mL penicillin, and 100 units/mL streptomycin. Cells were maintained in Petri dishes for 2−3 days prior to commencing the experiments. All the experiments were carried out when the cells were in the exponential growth phase. Cells were detached from the Petri dishes with 0.25% trypsin, resuspended in cell culture medium, seed onto various biomaterials at a final density of ∼1 × 104 cells/cm2. Cells were maintained on the biomaterials for at least 6 h for cell adherence prior to cell-matrix adhesion measurement experiments. Cell-Matrix Adhesion Measurement at Single-Cell Resolution. Solution containing 0.25% trypsin and 0.02% EDTA (Corning, USA), as well as 1 μg/ml fluorescein for visualization, was used to detach cells adhering to the biomaterials. We first well adjusted the gap and aspiration flow. Then, we positioned single cell right under the working zone by moving XY stage and the detaching process started immediately. The U87 cells cultured in a biomaterials surface were covered with fresh cell culture medium. The LSCE was dipped into the cell culture medium and placed perpendicular to the petri dish. A solution containing 0.25% trypsin and 0.02% EDTA was injected for cell extraction. The gap was defined as Zero when the bottom surface of the LSCE just touched the substrate. Then, the LSCE was raised using the position holder to realize a gap of 50 μm. The flow of both pumps for injection (10 μL min -1) and aspiration (50 μL min-1) were continued during the cell extraction process. The position of the device was held static. We moved the target cell to the right position by the XY stage of the microscope manually.


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Analytical Chemistry

Figure 1. Single cell-matrix adhesion measurement for precise evaluating functions of biomaterials. (A) The operation system for cell adhesion measurement. (B) LSCE device for single-cell extraction. (C) The zone of fluorescein solution at the surface underneath the LSCE. (D) A photograph of the device filled with red dye. (E) Mechanism of cell-matrix adhesion. (F) The process of cell detachment and dominent impact factors.

RESULTS AND DISCUSSION Device design and operation. The operation system in our experiments was shown in Figure 1A. The designed LSCE, shown as a schematic in Figure 1B, consists of a channel for trypsin solution injection and another channel for solution aspiration. The graphs of channels and tip-channels were detailed in Figure S1. The tip of LSCE contains two parallel microchannels used to create a stable zone of trypsin solution by hydrodynamic confinement for single-cell extraction to measure cell-matrix adhesion, using a simultaneous aspiration flow and injection flow of trypsin solution controlled by syringe pumps (Figure 1A). The bottom end of the pen was placed parallel to the sample surface while both were immersed in solution. The sample were placed on a hotplate to preserve a steady temperature (37 oC as a example), while the temperature was controlled by a temperature controller (Pecon, Wetzlar, Germany). The clearance between the bottom end of the pen and the sample surface is defined as “gap”. A XYZ stage was ultlized as a LSCE holder for adjusting the gap. When the gap is sufficiently small and the aspiration flow rate (QA) is sufficiently larger than the injection flow rate (QI), convection is fast enough to prevent diffusion of molecules of interest out of the region, as demonstrated in the experiment using fluorescein solution (1 μg mL-1) (Figure 1C; where QI = 10 μL/min, QA = 50 μL/min, gap= 50 μm). At the same time, the upper bound of the region also depends on the diffusivity of the molecule transported. The zone size was adjusted to be slightly larger than the size of the cell (10 to 60 μm here) to allow extraction of only a single cell underneath the LSCE tip. A photograph of the LSCE device filled with a red dye was detailed in Figure 1D. In cell researches, cells were usually cultured on biomaterials. Therefore, the strength of cell-matrix adhesion

will significantly affect the behaviors of cells, such as cell spreading, migration, division, metabolism, and interactions with other cells. Usually, cell adhesion is the fundamental to cell behaviors. After cells were loaded on biomaterials, celladhesion molecules (CAMs) on cell surface binded to various components of ECM, mediating cell-matrix adhesion (Figure 1E). Confinement of the trypsin solution to a zone ensures that neighboring cells are not extracted, when allowing the target cell’s capture back into the device (Figure 1B). As detailed in Figure 1F, trypsin digests the CAMs between cell and matrix gradually.49 Meanwihle, flow stream pushes the cell right, and the subatmospheric pressure near the aspiration aperture pulls the cell. The time for achieving cell detachment represents the cell-matrix adhesion strength. Numeric Simulation. As demonstrated in a simulation of flow and species transport equations using COMSOL Multiphysics (Figure 2A), radial flow fields formed between the injection aperture and the aspiration aperture. Here, the geometry and condition were same as that in Figure 1C. Navier-Stoke equations and convection-diffusion equations were used in the simulation. The diffusion coefficient of fluorescein is 500 μm2 s-1. The flow streams and velocity (Figure 2B) indicated the flow direction and velocity distribution. The concentration distribution (Figure 2B) represented molecule of interest in the solution near the cell surface. We observed that the concentration of molecule was higher than that near the boundaries, which indicated molecule diffusion at the interface betwee injected solution and surrounding medium. To define a boundary for molecule of interest, we set 10% of the maximum concentration as the boundary of the fluorescein zone. The maximum (L) and minimum (W) diameter (Figure 2C) of the zone could be further tuned using the ratio of the flow rates (Figure 2D). We



were able to obtained trypsin zones with desirable shapes and sizes.

Figure 2. Numeric Simulation of zone underneath the LSCE on the substrate. (A) Flowstreams and calculated line velocity distribution. (B) The concentration distribution of the molecule of interest in the zone. (C) The boundary (10% of the maximum concentration) of the trypsin zone. (D) Variation of the zone (W/L) with the ratio of aspiration flow rate to injection flow rate. Biomaterials Preparation and Characterizations. Without appropriate cell matrix, cells might lose some essential functions.12 Therefore, this necessitates the search for proper biomaterials that are suitable for cell culture, especially for cell adheision. In order to accomplish this, reliable methodologies should be established for sufficiently revealing the functions of biomaterials for cell adhesion. We prepared various biomaterials, including piranha solution (Caution: piranha is corrosive and must be handled with care) treated glass slice (OH-glass) as the substrate. (3-aminopropyl) triethoxysilane coated glass slice (APTES-glass), octadecyltrichlorosilane coated glass slice (OTS-glass), poly (L-polylysine) coated glass slice (PLL-glass) and fibronectin coated glass slice (FN-glass) were prepared for cell culture

(Figure 3A). The details of modifications were included in the experimental section in the Supporting Information (Figure S2). Measurements of cell-matrix adhesion on those biomaterials were then carried out to evaluate the functions of those biomaterials for cell adhesion. To characterize the surficial characters of the biomaterials, XPS analysis was utilized to investigate the surficial structure on various biomaterials and to demonstrate the successful modification. Compare to the XPS analysis of OH-glass, an obvious increase of the core level of C1s belonging to Carbon was found on the OTS-glass (Figure 3B), so that the successful immobilization of OTS was confirmed. After the OH-galss was treated by APTES solution, a remarkable C1s belonging to Nitrogen in XPS analytis indicated the successful immobilization of APTES (Figure 3C). In the same manner, the successful modifications of PLL (Figure 3D) and FN (Figure 3E) were demonstrated, respectively. AFM analysis was also carried out to demonstrate the various modifications. The the height sensor results (Figure S3) of various modified glasses showed remarkable difference from that of the OH-glass. The amplitude error results from AFM also demonstrated the difference between the glasses before (Figure S4A) and after OTS (Figure S4b), APTES (Figure S4C), PLL (Figure S4D) and FN (Figure S4E) modifications. Cell-Matrix Adhesion Measurement for Evaluating Functions of Biomaterials for Adherent Cell Culture. U87 cells were cultured on the various biomaterials (APTES-glass for example) with an appropriate cell density (about 1 x 104 cell/cm2). After cell adhered U87 cells were cultured on the various biomaterials (APTES-glass for example) with an appropriate cell density (about 1 x 104 cell/cm2). After cell adhered on the biomaterials, the sample was placed underneath the LSCE. Both the sample and LSCE were immersed in cell culture medium. The gap was adjusted to be 50 μm by adjusting the XYZ stage (Supplementary Figure 2). The injection flow rate of trypsin flow and aspiration flow rate were set as 10 and 50 μL/min, respectively. The position of the cell sample was fine-tuned to make a target single-cell right at the clearance between the two apertures using the XY stage


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Analytical Chemistry Figure 3. Biomaterials preparation and characterizations. (A) Biomaterials preparation and scheme for cell-matrix adhesion. XPS analysis for (B) OTS, (C) APTES, (D) PLL, (E) FN coated glass from OH-glass. on the microscope (Figure 4A-i). Measurement of cell-marix cells spreading area on various biomaterials. (D) Distribution adhesion was immediately initiated.The process of cell-matrix of extracting time for single-cells on various biomaterials. adhesion measurement was detailed in Supplementry Video In conventional methods,50 the cell spreading is a vital S1 (The video speed was 20 x of the original recording). factor to evaluate the functions of biomaterials and their During all the experiments, working temperature was kept at biocompatibilities. Cell spreading areas on four of the five 37 ℃ using the heating plate on the microscope. Trypsin kinds of biomaterials showed little difference, although those dissolved the ECM and CAMs that located on cell surface on APTES-glass appeared significantly larger (Figure 4C). from the cell edge (Fig. 1b). Cell gradually left the substrate The results indicated that APTES glass owned the highest and shrank simultaneously (Figure 4A-ii). As time passed, part biocompatibility while the other four held comparable of the cell folded over remained part towards the flow biocaompatibilities. Bsed on the comparison of cell spreading direction (Figure 4A-iii). Finally, the cell-matrix adhesion was area, the order of compatibilies of various biomaterials was completely overcome and the whole cell left away from the displayed as follows: APTES-glass > FN-glass > OH-glass substrate and was drawn back into the right aperture (Figure OTS-glass > PLL-glass (Figure 4C). On one hand, the cell 4A-iv). The extracting time for the whole process represented spreading area on various biomaterials excepted APTES-glass the strength of the cell-matrix adhesion between this singleshowed slight difference. On the other hand, the order was not cell and substrate. As a result, the cell-matrix adhesion at consistent with reported compatibilies of those biomaterails.5153 single-cell resolution was clarified. By repeating the process, In this case, it was difficult to determine which kind of the cell-matrix adhesion at different points were measured by biomaterial was better for adherent cell culture. The extracting single-cells one by one. conventional methods became powerless to distinguish those Besides trypsin, flow shear stress also has remarkable biomaterials, and the results became insufficient and influences on the extracting time (Figure 4B). We increased inaccurate. the injection and aspiration flow rates simultaneously while By using our method to measure the cell-matrix adhesion, keeping the ratio constant. The extracting time revealed the results (Figure 4D) indicated that PLL-glass and FNsignificant decreases (P-value below 0.05) when the injection coated glass slice showed the best biocompatibility for flow rate increased from 4 to 8 μL/min (P = 0.023) and then to adherent cell culture following by the APTES-glass, which 10 μL/min (P = 0.001), while no significance was found when were consistent with reported findings.51-53 Piranha solution the injection flow rate increased from 10 to 15 μL/min (P = treated glass slice and OTS-glass showed weak 0.254). Thus, we measured the cell-matrix adhesion strength biocompatibilities (Figure 4D). Therefore, the presented between adherent U87 cells and various biomaterials under the showed a more precise and reliable evaluation of biomaterials condition of QI = 10 μL/min, QA = 50 μL/min. In this work, for adherent cell culture. Besides compatibility, influence on we aim to distinguish the differences between different cell heterogeneity was also a dominent parameter to evaluate samples. So, excessive decrease on extracting time is not the function of biomaterials for cell researches. The coefficient benefit for observe this differences. In the future, we can of variation (CV) of extracting time was calculated to reveal reduce the extracting time by increasing the trypsin the cell heterogeneity of cell-matrix adhesion. The results concentration to meet the requirements of high-throughput implied that the OTS modification weakened the heterogeneity assay. while APTES, PLL and FN modifications enhanced the heterogeneity compare the initial OH-glass (Figure 4D). On various biomaterials, the ascending order of cell heterogeneity of cell-matrix adhesion was displayed as follow: FN-glass (CV=0.82) > PLL-glass (CV=0.70) > APTES-glass (CV=0.64) > OH-glass (CV=0.51) > OTS-glass (CV=0.45) (Table S1). Although there are several approaches for cell adhesion measurement, none of them enabled the measurement at single-cell resolution and the estimation of the individual differences between different single-cells. The presented method was capable of uncovering not only the compatibility of the biomaterials, but also the influences of them on cell heterogeneity.

CONCLUSIONS

Figure 4. Cell-matrix adhesion measurement for evaluating of biomaterials. (A) Cell-matrix adhesion measurement by extracting single-cell from the biomaterials surface. (B) The influence of flow rates on extracting time. *P < 0.05, **P > 0.05, one-sided Student’s t-test. (C) Distributions of single-

We have established a novel methodology for cell-matrix adhesion measurement at single-cell resolution that could precisely evaluate the functions of biomaterials for adherent cell culture. When cell spreading area failed to evaluate the biomaterials, our methods showed precise evaluation and the results were consistent with the reported findings. Moreover, the presented method successfully uncovered the influences of various biomaterials on cell heterogeneity of cell-matrix adhesion. The results indicated that the conventional methods for evaluating biomaterials based on cell spreading and



viability were not sufficient and sometimes incorrect. The established method was not only a useful tool for single-cell analysis, but also a general strategy for precise evaluation of biomaterials that open a new way for scientists in materials science to evaluate their developed materials.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Device fabrication, biomaterials preparations, AFM characterizations of biomaterials and Statistical analysis (PDF) Single-cell extraction for measurement of cell-matrix adhesion (AVI)

AUTHOR INFORMATION Corresponding Author * E-mail: 62792343.

[email protected].

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Author Contributions J.-M.L conceived and supervised the project. S.M. and J.-M.L. designed the study and discussed the results; S.M. and Q.Z. conducted the experiments and performed the analyses; S.M. and Q.Z. carried out the numerical simulation and date analysis. S.M., Q.Z. and Q.H. provided key technical expertise with instrumentation, protocols and reagents. S.M. wrote the manuscript with contributions from J.-M.L., H.L. and K.U.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We acknowledge Beijing Natural Science Foundation (2184106), National Natural Science Foundation of China (Nos. 214350002, 21727814 and 21621003) and China Postdoctoral Science Foundation (2017M620733).

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We present a novel methodology for cell-matrix adhesion measurement at single-cell resolution that could precisely evaluate the functions of biomaterials for adherent cell culture. We applied this method to evaluate various modified biomaterials.

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Measurement of Cell-Matrix Adhesion at Single-Cell Resolution

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