PMMA Hybrid 3D Cell Culture Microfluidic Platform for the

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A Paper/PMMA Hybrid 3D Cell Culture Microfluidic Platform for the Study of Cellular Crosstalk Kin Fong Lei, Chih-Hsuan Chang, and Ming-Jie Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b03021 • Publication Date (Web): 29 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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ACS Applied Materials & Interfaces

A Paper/PMMA Hybrid 3D Cell Culture Microfluidic Platform for the Study of Cellular Crosstalk Kin Fong Lei 1, 2, 3, *, Chih-Hsuan Chang 1, Ming-Jie Chen 1

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Graduate Institute of Medical Mechatronics, Chang Gung University, Taoyuan, 333 Taiwan 2

Department of Mechanical Engineering, Chang Gung University, Taoyuan, 333 Taiwan

3

Department of Radiation Oncology, Chang Gung Memorial Hospital, Linkou Branch, 333 Taiwan

* Corresponding author: Kin Fong Lei Mailing address: 259 Wen-Hwa 1st Road, Kweishan, Taoyuan, 333 Taiwan E-mail: [email protected] Tel: +886-3-2118800 ext 5345

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ACS Paragon Plus Environment


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Abstract Studying cellular crosstalk is important for understanding tumor initiation, progression, metastasis, and therapeutic resistance. Moreover, a 3D cell culture model can provide a more physiologically meaningful culture microenvironment. However, studying cellular crosstalk in 3D cell culture model involves tedious processing. In this study, a paper/polymethylmethacrylate (PMMA) hybrid 3D cell culture microfluidic platform was successfully developed for the study of cellular crosstalk. The platform was a paper substrate with culture microreactors placed on a PMMA substrate with hydrogel-infused channels. Different types of cells were directly seeded and cultured in the microreactors. Aberrant cell proliferation of the affected cells was induced by secretions from transfected cells, and the proliferation ratios were investigated using a colorimetric method. The results showed that the responses of cellular crosstalk were different in different types of cells. Moreover, neutralizing and competitive assays were performed to show the functionality of the platform. Additionally, the triggered signaling pathways of the affected cells were directly analyzed by a subsequent immunoassay. The microfluidic platform provides a simple method for studying cellular crosstalk and the corresponding signaling pathways in a 3D culture model.

Keywords: Paper-based microfluidics; 3D Cell culture; Cellular crosstalk; Cell-based assays; Neutralizing assays; Competitive assays.

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1. Introduction In

cancer

research,

investigating

cellular

crosstalk

in

the

tumor

microenvironment has become attractive for understanding tumor initiation, progression, metastasis, and therapeutic resistance [1-4]. For example, the interactions between cancer and immune cells are known to be associated with many cellular and molecular mechanisms that mediate tumor escape from natural immune surveillance [5, 6]. Further, it was reported that cells infected by Epstein-Barr virus activate the production of cytokines, such as interleukin-6 (IL-6) and IL-10 [7, 8]. Aberrant cell proliferation of neighboring cells was induced and thus allowed progressively aggressive clones to arise [9]. Therefore, investigating the cellular crosstalk that regulated the tumor microenvironment can provide important targets for effective therapeutic strategies. In cell culture techniques, a conventional mono-culture system in which cells are cultured in Petri dish or multi-well microplate may not permit researchers to evaluate the interactions between different types of cells. To study cellular crosstalk, a co-culture system was established using Transwell inserts [10]. The insert is a culture well with a permeable membrane at the bottom. By applying the insert in a standard cylindrical culture well, upper and lower culture wells are constructed but separated by the membrane. Two types of cells can be cultured and physically separated within the same medium. This approach permits paracrine communication but prevents direct cell contact. The proliferation, viability, and gene and protein expression of both cell types can be analyzed by using conventional bio-assays. The co-culture system using Transwell inserts was adapted for various studies of cellular crosstalk [11-13]. These studies were based on a 2-dimensional (2D) culture model in which the cells were seeded and cultured as a monolayer on a solid surface. The major advantages of the 2D culture model are its simplicity of operation and observation and 3



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the availability of further genetic and proteomic assays. Over the past decade, biologists proposed to encapsulate cells in polymeric scaffold materials for culturing cells in a 3-dimensional (3D) environment [14-16]. This approach was referred to as the 3D culture model, and it is generally believed that this model provides a more physiologically meaningful culture technique that bridges the gap between cell culture and living tissue [17]. These 3D cell-based assays can closely predict the cellular functions of living tissues by moving cells from monolayers to a 3D environment. Although some microfluidic-based tools were demonstrated with a 3D cell culture model [18], studying cellular crosstalk in a 3D culture model still involved tedious processing. Additionally, cells that are encapsulated in a 3D scaffold are difficult to use for conducting further genetic and proteomic assays. Recently, paper-based microfluidics have been proposed as a new class of analytical tools for remote environments [19]. By patterning the paper substrate, aqueous solution can be manipulated within the patterned barriers [20, 21]. A number of rapid diagnostic applications have been demonstrated by paper-based microfluidics and a paper hybrid platform based on various detection techniques [22-29]. Paper-based microfluidics have the advantages of simplicity, inexpensiveness, and conservation of samples and reagents. Moreover, because a paper substrate is constructed by a reticulated structure, a 3D space can be maintained with well-defined dimensions. In vitro 3D cell culture was conveniently achieved by culturing hydrogel-encapsulated cells on a paper substrate [30-33]. By stacking multiple layers of paper substrates, oxygen and nutrient gradients were achieved to mimic tissue- and organ-level functions. The purpose of hydrogel and stacking papers was to create gradients in these studies. However, to study the molecular expressions of cells, hydrogel removal is required and may reduce the efficiency of the processes for RNA and protein extraction. Our previous study reported on culturing cells on a paper 4


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substrate without hydrogel encapsulation [34, 35]. Stimulation of chemokines could be directly applied to cells and a subsequent immunoassay was demonstrated to study the cell phosphorylation and signaling cascades. This gel-free, paper-based cell culture model was suitable for studying the molecular expression levels of cells. In this approach, a paper/polymethylmethacrylate (PMMA) hybrid 3D cell culture microfluidic platform was developed for the study of cellular crosstalk of hepatocyte-derived carcinoma cells in a 3D culture model. Studying the cellular crosstalk in hepatocellular carcinoma (HCC) is very important because the growth, migration, and gene expression of these cells were reported to be regulated by paracrine cytokines [36-38]. Moreover, to better mimic the in vivo structure and functions of the liver, 3D cell culture was adopted to maintain liver-specific functions in the in vitro studies [39-41]. The platform consisted of a paper substrate with culture microreactors and a PMMA substrate with diffusion channels. Different types of cells were directly seeded in the microreactors without hydrogel encapsulation. Then, the paper substrate was placed on the PMMA substrate with hydrogel-infused channels. Secretions from cells could diffuse through the channel and affect other neighboring cells. In this study, cells transfected with the pTT3-EGF-bio-His plasmid were cultured in the center microreactor. Epidermal growth factor (EGF) secreted from the transfected cells affected the cells cultured in the side microreactors. Aberrant cell proliferation of the affected cells was induced, and the proliferation ratio was investigated by using a colorimetric method. The results showed that the responses of cellular crosstalk were different in different types of cells. Moreover, neutralizing and competitive assays were also demonstrated using the platform. The phosphorylation of epidermal growth factor receptor (EGFR) was directly analyzed on the paper substrate by a subsequent immunoassay to further confirm the triggered signaling pathway of the affected cells. This novel analytical tool provided a simple method for 5



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studying cellular crosstalk and the corresponding signaling pathways in a 3D culture model.

2. Materials and methods 2.1 Chemicals and reagents EGF and pTT3-EGF-bio-His plasmid were purchased from Addgene. The primary antibodies for mouse anti-β-actin, mouse anti-GAPDH, rabbit anti-EGF, rabbit anti-EGFR, and rabbit anti-phospho-EGFR were purchased from Proteintech Group, Inc. Secondary antibodies conjugated to gold nanoparticles were purchased from Sigma. A gold enhancement solution for the development of gold nanoparticles was purchased from Nanoprobes, Inc. All experiments were performed at room temperature (22-25°C) unless stated otherwise.

2.2 Cell culture and transfection Three cancer cell lines of Huh7, Hep-G2, and BM-1 were used in this study. They were kindly provided by Prof. I-Chi Lee, Prof. Kwang-Huei Lin, and Dr. Jenny Liu at Chang Gung University, Taiwan, respectively. Huh7 and Hep-G2 were well-differentiated hepatocyte-derived carcinoma cell lines. They are the commonly used human hepatocarcinoma cell lines for the study of HCC [36, 42, 43]. BM-1 cells were derived from a bone marrow metastatic biopsy. Culture medium was Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco-RBL Life Technologies) and antibiotic/antimycotic (100 U/mL of penicillin G sodium, 100 mg/mL of streptomycin, and 0.25 mg/mL of amphotericin B; Gibco-BRL Life Technologies). The cells were amplified by a standard cell culture technique and trypsinized using 0.05% trypsin for 3 min, centrifuged at 1000 rpm for 5 min, and re-suspended in the medium for further experiments. The number of cells was counted 6


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by an automated cell counter (Model: Countess II FL; Invitrogen). To study the cellular crosstalk, an artificial model was established with a cell transfection technique. Cells (cell line: Huh7) were seeded on 6-well microplates at a cell number of 105 cells/well (30-40% confluence) on the day before transfection. The pTT3-EGF-bio-His plasmid were used for cell transfection by Lipofectamine LTX and PLUS Reagents (Invitrogen) according to the manufacturer’s Lipofection protocol.

2.3 Fabrication of the paper/PMMA hybrid microfluidic platform The paper/PMMA hybrid microfluidic platform consisted of a paper substrate with culture microreactors and a PMMA substrate with diffusion channels. The fabrication process of the paper substrate with microreactors was based on previous publication [20]. Five microreactors arranged in a cross shape were printed on a filter paper (cat no.: 1004-917; Whatman) with a solid wax printer (Model: ColorQube 8570; Xerox Co.). In our design, there was 1 circular microreactor (10 mm in diameter) in the center and 4 square microreactors (5×5 mm2) on the neighboring sides. An illustration of the arrangement of 5 microreactors is shown in Fig. S1(a) in the supplementary materials. Then, the paper substrate was heated by a hot plate at 100 °C for 10 min. The wax was melted and infiltrated the entire thickness of the paper substrate. Aqueous solutions could be trapped within the microreactors by the wax barriers. The paper substrate was cooled and used immediately or was stored at room temperature for later use. Alternatively, diffusion channels on a PMMA substrate were fabricated by a CNC engraving machine (Model: EGX-400; Roland). An illustration of the diffusion channels on the PMMA substrate with dimensions is shown in Fig. S1(b) in the supplementary materials. The paper and PMMA substrates 7



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were sterilized by ultraviolet light overnight before use. A photograph of the paper/PMMA hybrid 3D cell culture platform is shown in Fig. S2 in the supplementary materials.

2.4 Procedure of studying cellular crosstalk by using the paper/PMMA hybrid microfluidic platform The gel-free, paper-based cell culture model was adopted to study cellular crosstalk in a 3D environment. Before cell seeding, paper-based microreactors were moistened by 5 µL of phosphate-buffered saline (PBS) containing 1 µg/mL collagen (Sigma) to enhance the suitability of culture substance. Different types of cells in a seeding cell number of 6000 were directly applied to the corresponding paper-based microreactors without hydrogel encapsulation. The initial seeding cell number was optimized, and the optimization is discussed in Fig. S3 in the supplementary materials. Because the cells were not encapsulated in hydrogel, cells should anchor the paper filters through collagen to avoid being washed away from the paper substrate. Then, the paper substrate was transferred to a 37 °C, humidified, and 5% CO2 incubator (Model: 370; Thermoscientific) for 40 min for cell seeding. Meanwhile, agarose hydrogel (0.5% (w/v) agarose powder (Sigma) mixing with culture medium) was loaded in the diffusion channels of the PMMA substrate. After cell seeding, the paper substrate was placed on the hydrogel-infused channels of the PMMA substrate with proper alignment. The objective of having the hydrogel was to maintain the wettability of the microreactors and provide nutrients to the cells. Additionally, the hydrogel was a medium of diffusing molecules between microreactors when studying cellular crosstalk. During the culture course, the hydrogel was not replaced or refreshed. Finally, the paper/PMMA hybrid microfluidic platform was placed in the incubator to study cellular crosstalk. The procedure for studying cellular crosstalk by 8


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using the platform is shown in Scheme 1. Secretions from cells could diffuse through the hydrogel-infused channel and affect the cells in neighboring microreactors. The proliferation ratio of cells in microreactors was investigated by using a conventional bio-assay at the preset schedule. Moreover, because cells were cultured in paper substrate, the phosphorylation of kinases and transcription factors could be analyzed by a subsequent immunoassay.

2.5 Quantification of cell proliferation in the paper-based microreactor To investigate cell proliferation in a paper-based environment, cell proliferation in the paper-based microreactor was quantified by a conventional bio-assay (WST-1 assay; Roche Applied Science). WST-1 assay is a colorimetric assay for cell proliferation, cell viability, and cytotoxicity. It is a stable tetrazolium salt (with a slight red color) and is cleaved to formazan (yellow-orange color) by cellular enzymes. The increase in the overall activity of mitochondrial dehydrogenases leads to an increase in the amount of formazan dye that was formed. The color intensity of the formazan dye solution directly correlated to the number of metabolically active cells. At the preset schedule for quantification, 2 µL of WST-1 reagent was pipetted into the microreactor and incubated at 37 °C for 1 h. Then, the number of viable cells directly correlated to the color intensity of the microreactor, i.e., the yellow-orange-colored product of formazan. The colorimetric signal was captured by a flatbed scanner, and the gray level was quantified using the ImageJ computer software. The intensity was defined as the gray level minus the gray level of the background. The proliferation ratio was defined as the intensity at the successive time point divided by the intensity at 0 h.

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2.6 Study of cell phosphorylation in the paper-based microreactor In this study, the cells were cultured in the paper-based microreactor without hydrogel encapsulation. This approach could facilitate the subsequent immunoassay for the study of cell phosphorylation. The methodology was based on our previous study [34, 35]. Briefly, at a preset schedule, cell poration was conducted by applying cold methanol to the microreactors for 20 min. Then, 10 µL of solutions containing primary and secondary antibodies in a 1:100 dilution was pipetted into each microreactor and incubated for 10 min, respectively. The immunoassay result was amplified by applying gold enhancement solution for 20 min. The colorimetric signal of the microreactor was captured by a flatbed scanner, and the gray level was quantified using the ImageJ computer software. The intensity was defined as the gray level minus the gray level of the background.

2.7 Characterization of cell morphology in the paper-based environment Scanning electron microscopy (SEM) was used to characterize the cell morphology in the paper-based environment. The cells were cultured in the paper substrate for 1 and 5 days. Before SEM imaging, the paper substrate was treated with glutaraldehyde (Sigma) for 2 h and then washed by PBS three times for 5 min each. Then, it was dehydrated through serial applications of different concentrations of ethanol from 50% to 100%, immersed in pure ethanol twice, and placed in a critical point drying machine with dry carbon dioxide. Finally, a thin conducting gold layer was coated by vacuum evaporation.

2.8 Characterization of gene expression in the paper-based environment The total RNA of cells from the gel-free paper-based cell culture was extracted 10


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using Trizol reagent (Biokit) according to the supplier’s instruction. cDNA was synthesized by reverse transcription-PCR (RT-PCR; 30 cycles, Oligo(dT)20, cDNA synthesis kit; Invitrogen). The relative quantity of mRNA was determined by real time-PCR (30 cycles, One-step RT-qPCR kit; Kapa Biosystems). The claudin, integrin, laminin, CXCL2, IL-8, and ANGPTL4 mRNA expression levels were examined. The mRNA of GAPDH was used as the internal control. The primer sequences are shown in Table S1 in the supplementary materials.

3. Results and discussions 3.1 Characterization of the gel-free paper-based cell culture model To characterize the gel-free paper-based cell culture model, both the cell morphology and gene expression were studied to confirm that the cells were expressing the properties of cultures in a 3D environment. For the study of cell morphology, cells (cell line: Huh7) were cultured on a paper substrate for 1 and 5 days. Cell morphology was investigated by SEM, and the images are shown in Fig. 1(a, b). The cells were successfully seeded on the paper fibers, and spherical morphology was observed after 1 day of culture, as shown in Fig. 1(a). The results showed that cells did not spread on the surface of the paper fibers. In contrast to Huh7 cells that were cultured in tissue culture polystyrene (TCPS) for 1 day, the cells should have spread on the surface. The cell morphologies were shown to differ between cells cultured on a paper substrate and TCPS. Additionally, in Fig. 1(b), cells aggregated and formed spheroid-like structures after 5 days of culture. Compared to Huh7 cells cultured in TCPS, the morphology of cells that were cultured in a paper substrate was completely different. From Fig. 1(a) and 1(b), the cell morphology showed that paper fibers did not provide a 2D curved surface for cell seeding and 11



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attachment. Moreover, the gene expression of cells cultured in the paper substrate for 1 day was investigated, including claudin, integrin, laminin, CXCL2, IL-8, and ANGPTL4. Claudin, integrin, and laminin are important components of the bridges for cell-cell and cell-extracellular matrix (ECM) interactions. CXCL2 (Chemokine ligand 2), IL-8 (Interleukin 8), and ANGPTL4 (Angiopoietin-like 4) play important roles in numerous cancers. The results comparing cells cultured on TCPS and the paper substrate are shown in Fig. 1(c, d). Down-regulation of claudin, integrin, and laminin occurred in the paper-based cell culture. Cell-cell and cell-ECM interactions were suppressed, which indicated that the cells were cultured in a 3D environment. In contrast, CXCL2, IL-8, and ANGPTL4 were up-regulated, and the results implied that the cells that expressed these molecules were more cancerous. These results were re-examined and compared between cells cultured on TCPS and standard 3D culture model (cells encapsulated in hydrogel). The results are shown in Fig. S4 in the supplementary materials. Down-regulation of claudin, integrin, and laminin was found, and CXCL2, IL-8, and ANGPTL4 were up-regulated when cells were cultured in a 3D environment. These results agree with the outcomes of previous studies [44-47]. Together with the results from cell morphology and gene expression, this gel-free paper-based cell culture model cultured cells expressing the properties of cells in a 3D environment. The efficiency of cell seeding on the paper substrate was evaluated. After wetting the paper with collagen, 20 µL of culture medium containing cells (cell line: Huh7) in different numbers, i.e., 0 (control), 3000, 6000, 9000, 12000, and 15000, was pipetted into the microreactors of the paper substrate. After 4 h for cell seeding and stabilization, quantification of the number of viable cells was conducted using a WST-1 assay. The results are shown in Fig. 2(a). Generally, the cell number and 12


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intensity were linearly correlated, with R2 = 0.9126. Because the reagent of the WST-1 reacted with the respiratory chain of cell mitochondria, the cells were viable and functional after cell seeding. In addition, 6000 cells were seeded and cultured in the paper-based microreactors for 4 days. The proliferation ratio was quantified using the WST-1 assay at days 0, 1, 2, 3, and 4, as shown in Fig 2(b). The results revealed that cells proliferated in the paper-based environment for up to 3 days and then plateaued. Because the hydrogel was not replaced or refreshed due to the limited nutrients in the hydrogel, the cells could proliferate for 3 days and then reached a plateau. The results confirmed that the cells were metabolically active for up to 3 days. Furthermore, the detection of housekeeping genes, including actin and GAPDH, was conducted to confirm the proliferation of the cells cultured in the paper substrate. Housekeeping genes were uniformly expressed at relatively constant levels, and they were used as reference points. In this experiment, 6000 cells were seeded and cultured in the paper-based microreactors. At a preset schedule, the cells were fixed, and an immunoassay was directly performed by using the respective primary antibodies for anti-actin and anti-GAPDH. The results of cell proliferation obtained via the detection of housekeeping genes are shown in Fig. 2(c). The outcomes confirmed that the cells proliferated for up to 3 days in the gel-free paper-based cell culture model. The paper substrate provided an appropriate environment for cell culture.

3.2 Study of cellular crosstalk using the paper/PMMA hybrid microfluidic platform Secretions from cells could diffuse through the hydrogel-infused channel and affect the cells in neighboring microreactors. Diffusion of the hydrogel-infused channel was evaluated, and the results are shown in Fig. S5 in the supplementary materials. A study of cellular crosstalk was demonstrated with the paper/PMMA 13



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hybrid microfluidic platform. Different types of cells, i.e., Huh7, Hep-G2, BM-1, and blank (background), were seeded in the side microreactors. Four experimental groups were designed in the center microreactor, including seeding transfected cells (TF), seeding cells without transfection (Non-TF), direct application of 0.5 µg/mL EGF (EGF), and blank (Control), as shown in Fig. 3(a). Cell proliferation was investigated by bio-assay at days 0, 1, 2, and 3. To analyze the proliferation ratio of different types of cells in 4 groups, the colorimetric signals of the microreactors were quantified. The results were re-arranged and plotted in Fig. 3(b). For Huh7, regardless of which EGF was applied directly (in the group of EGF) or produced by transfected cells (in the group of TF) in the center microreactor, it could successfully diffuse through the channel and affect the cells in the side microreactors. Aberrant cell proliferation was induced by the stimulation of EGF compared to the control group. However, the results also indicated that the concentration of EGF produced by the transfected cells was lower than the concentration that was applied directly. Moreover, in the group of Non-TF, the proliferation ratio was shown to be higher than the control group. It was suspected that the cells were stimulated by the autocrine system because Huh7 was cultured in both center and side microreactors. For Hep-G2, a similar situation was found in the EGF and TF groups. The cells were stimulated by EGF, and aberrant cell proliferation was induced. However, the proliferation ratio in the Non-TF group showed no significant difference from the control group. It was suspected that Hep-G2 was not stimulated by the paracrine secreted by Huh7. For BM-1, there was no significant difference among the 4 experimental groups. The results indicated that the proliferation ratio of BM-1 was not affected by EGF or paracrine secreted by Huh7. In addition, a western blotting assay was performed to confirm the results 14


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obtained by the microfluidic platform. Cells were stimulated by direct application of EGF. The phosphorylation of EGFR was investigated, and the results are shown in Fig. 4. In Huh7 and Hep-G2, EGFR was phosphorylated after EGF stimulation. A higher proliferation ratio was induced by the initiation of downstream signaling pathways compared to the group without EGF stimulation. However, in BM-1, EGFR was phosphorylated with or without EGF stimulation. The results revealed that EGF did not affect the phosphorylation of EGFR. This outcome indicated that the proliferation ratio of BM-1 was not affected by EGF.

3.3 Neutralizing and competitive assays on the paper/PMMA hybrid microfluidic platform Neutralizing and competitive assays were conducted on a single paper substrate using the microfluidic platform. The design of the experiment is illustrated in Fig. 5(a). The transfected cells were seeded in the center microreactor. Four side microreactors were used for Huh7 cells only (Positive control), Huh7 cells blocked by 50 µM anti-EGFR antibody for 90 min in advance (Neutralized Huh7; Neutralizing assay), Huh7 cells together with 50 µM of anti-EGF antibody (Huh7+EGF Ab; Competitive assay), and a blank (Negative control). The cell proliferation was investigated by a bio-assay at days 0, 1, 2, and 3, and the proliferation ratios were quantified from the colorimetric signals of the microreactors, as shown in Fig. 5(b). Generally, the proliferation ratios in the groups of neutralized Huh7 and Huh7+EGF Ab were lower compared to the positive control. In the group of neutralized Huh7, because cells were blocked by the anti-EGFR antibody before seeding on the microreactor, EGF secreted from transfected cells could not bind to the EGFR on the cell membrane. The cells were neutralized and could not be stimulated. Therefore, the 15



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results showed that the proliferation ratio was limited during the culture course. Moreover, in the group of Huh7+EGF Ab, the EGF that was secreted from transfected cells was competitively captured by the EGFR on the cell membrane and anti-EGF antibody. Therefore, the cells received less EGF to be stimulated compared to the positive control on days 1 and 2. After day 2, the results showed that the cells were stimulated and the cell proliferation increased. That result might have occurred because the concentration of the anti-EGF antibody became low after day 2. To confirm the signaling pathway of the affected cells that was triggered, a subsequent immunoassay of the detection of phosphorylation of EGFR was performed, as illustrated in Fig. 6(a). After 1 day of culture, a subsequent immunoassay was directly performed in the microreactors of the paper substrate by using the primary antibody of anti-phospho-EGFR. Photograph and the quantified results of the microreactors are shown in Fig. 6(b). The outcomes indicated that EGFR was phosphorylated compared to the negative control. When EGF engages the receptor (EGFR) on the cell membrane, phosphorylation of EGFR is triggered and initiates downstream signaling pathways. The EGFR signaling pathway is one of the most important pathways that regulates growth, survival, proliferation, and differentiation in mammalian cells and has been the target of effective cancer therapies [48, 49]. Moreover, a higher activation level of phosphorylated EGFR was observed in the Huh7 group (positive control) compared to the other groups (Neutralized Huh7 and Huh7+EGF Ab). These results revealed that cell proliferation was suppressed by the neutralizing effects and competition of EGF.

Conclusions A paper/PMMA hybrid 3D cell culture microfluidic platform was developed and 16


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successfully demonstrated in a study of cellular crosstalk. A gel-free paper-based cell culture model was adopted in this platform. The results confirmed that the cells expressed the culture properties in a 3D environment and that the paper substrate provided an appropriate culture environment. Cellular crosstalk was studied by culturing different types of cells in the paper substrate. Aberrant cell proliferation of the affected cells was induced by the secretions from transfected cells, and proliferation ratios were investigated using a colorimetric method. In addition, neutralizing and competitive assays were conducted to demonstrate the functionality of the platform. The phosphorylation of EGFR could be directly analyzed on the paper substrate to further confirm the triggered signaling pathway of the affected cells. The gel-free paper-based cell culture model produced 3D cell culture conveniently and facilitated a direct analysis of cell phosphorylation. Combined with the hydrogel-infused PMMA channel, the paper/PMMA hybrid platform provided a simple method for studying cellular crosstalk and the corresponding signaling pathways in a 3D culture model. Hopefully, this method can accelerate the investigations of cancer and develop more effective therapeutic strategies.

Acknowledgments The authors would like to thank Mr. Chia-Hao Huang for his technical support. This work was supported by the Ministry of Science and Technology, Taiwan (project no. MOST104-2221-E-182-014-MY3) and Chang Gung Memorial Hospital, Linkou Branch, Taiwan (Project no. BMRPC05).

References 1

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Figure captions Scheme 1. Procedure for studying cellular crosstalk by using the paper/PMMA hybrid microfluidic platform. (a) Different types of cells were directly seeded in the microreactors of the paper substrate without hydrogel encapsulation. (b) Hydrogel was loaded in the diffusion channel of the PMMA substrate. (c) The paper substrate was placed on the PMMA substrate with proper alignment. The platform was put in the incubator to study cellular crosstalk.

Fig. 1. Characterization of gel-free paper-based cell culture. (a, b) SEM images of cell morphology after Huh7 cells cultured in a paper substrate for (a) 1 and (b) 5 days, respectively. (c, d) Relative quantity of gene expression between cells cultured on TCPS and the paper substrate. (c) Down-regulation of claudin, integrin, and laminin. (d) Up-regulation of CXCL2, IL-8, and ANGPTL4. The columns represent the mean of at least 3 experiments (n ≥ 3). Error bars represent standard deviations. The results were analyzed by one-way analysis of variance (ANOVA). Statistical significance is indicated as *p

PMMA Hybrid 3D Cell Culture Microfluidic Platform for the

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