- Email: [email protected]
A microfluidic platform for multi-size 3D tumor culture, monitoring and drug resistance testing
Sensors & Actuators: B. Chemical 292 (2019) 111–120
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A microfluidic platform for multi-size 3D tumor culture, monitoring and drug resistance testing ⁎
Wenming Liua,b, , Meilin Suna, Boxiu Luc, Mingming Yanb, Kai Hana, Jinyi Wangb,
T
⁎
a
Departments of Biomedical Engineering and Pathology, School of Basic Medical Science, Central South University, Changsha, Hunan, 410013, China Department of Chemistry, College of Chemistry and Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, China c Department of Magnetic Resonance Imaging, People's Hospital of Qihe County, Qihe, Shandong, 251100, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Microfluidics Multi-Size 3D tumors Drug resistance Large scale On-Chip orthogonal Analysis
The establishment of a miniaturized platform capable of microscale control for sequential, coordinated, and throughput biomimetic tumor manipulation and analysis offer a high degree of biological and clinical relevance to cancer therapy and pharmaceutical research. In this study, we developed a microfluidic system with wellestablished microwell arrays and simple double-layer composition that allows massive fabrication of biomimetic multi-size 3D tumors and their simultaneous manipulation and orthogonal analysis. Serial manipulations including cell localization, array-like self-assembly/cultivation, and dynamic analysis of different types of 3D tumors, were accomplished in the microfluidic device. We demonstrated that the microfluidic platform is stable and throughput cell trapping and tumor generation with quantity uniform. Furthermore, on-chip monitoring and throughput analysis of tumor phenotypes and responses to culture and different chemotherapies were also achieved in the device. The microfluidic advancement offers a new methodological approach for the development of high-performance and multifunctional 3D tumor systems and for tissue-mimicking cancer research and therapy evaluation. This microfluidic platform, which has the capability of multiple control in time and space, holds great potential for applications in the fields of tumor biology, tissue engineering, and clinical medicine.
1. Introduction Cancer is a major public health problem worldwide. According to the latest statistical presentation from the World Health Organization (WHO), cancer is expected to rank as the first or second leading cause of human death in over 90 countries [1]. Statistical data displays that there are an estimated increase of new cancer cases from 14.1–18.1 million between 2012 and 2018, as well as cancer deaths from 8.2–9.6 million [1,2]. Thus, the establishment of an effective biomimetic tumor system for meticulously exploring tumor pathology and therapy has been the subject of much interest and extensive research. Three-dimensional (3D) tumor culture has been considered to be an improved in vitro model to better simulate the biological properties (e.g., complex cellular organization and interactions, chemical and phenotypical gradients related to nutrient, proliferation, metabolism and viability) of actual tumors in vivo, and to overcome the negative effect of unnatural adhesion and arrangement of conventional two-dimensional (2D) cell monolayer cultures [3–5]. When anticancer drug responses have been directly compared between 2D and 3D culture models, different chemosensitivity between the two models can be represented as either
⁎
enhanced sensitivity or greater resistance. A broad consensus has appeared that in vitro 3D tumor cultures provide more appropriate preclinical models to evaluate the drug efficacy for solid tumors, and more reliable and meaningful therapeutic readouts reduces the potential risks to patients [6]. Over the past several decades, many research groups have attempted to fabricate in vivo-like 3D tumors using hanging drop, anti-adhesive surface, spinner flask, and porous scaffold for heterophenotypic investigation and therapy evaluation [7–10]. However, the applications of these methods have been impeded owing to their drawbacks of being cumbersome, time consuming, labor-intensive, nonuniform tumor size, and low generation yield. To drive the widespread implementation of 3D tumor models in routine tumor analysis and antitumor programs, new simple and popularized methods for the scalable, controllable, and throughput production of 3D tumors with uniform characteristics are required. In recent years, microengineering progresses have generated a series of striking capabilities in performing precise manipulations of mammalian cells with microscale resolution [11–13]. For example, microfabrication and micropatterning methods have been employed to perform controllable localization, orientation, and organization of cells to
Corresponding authors. E-mail addresses: [email protected] (W. Liu), [email protected] (J. Wang).
https://doi.org/10.1016/j.snb.2019.04.121 Received 21 January 2019; Received in revised form 24 April 2019; Accepted 24 April 2019 Available online 26 April 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Sigma-Aldrich (MO, USA). Cell culture medium, fetal bovine serum (FBS), CellTracker Green CMFDA and TRITC-phalloidin were purchased from the Gibco/Life Technologies (CA, USA). Bleomycin (BLM) and Tirapazamine (TPZ) were purchased from Melonepharma Co., Ltd (Dalian, China). All solvents and other chemicals of analytical reagent grade were purchased from local commercial suppliers, unless otherwise stated. All solutions were prepared using ultra-purified water supplied by a Milli-Q system (Millipore®).
produce a large quantity of uniformly prepared 3D tumors, contributing remarkable insights into 3D tumor functions and activities [14,15]. Furthermore, microfluidics is known to be a promising platform for tumor biology because of their satisfactory performance in the spatiotemporal control of fluidic perfusion and tumor cells, in the imitation of tissue microenvironments, and in effective sequential manipulation [16,17]. Tumor-on-a-chip in polydimethylsiloxane (PDMS) provides an optical window into biological events such as tumor development, anticancer drug delivery and therapy [18]. So far, many microfluidic advances technologically based on microdroplets, microgels, microwell arrays, microrotational flow, and 3D acoustic tweezers [19–23], have more or less provided some extremely attractive tumor manipulation features, like simple and controllable process, homogeneous tumor size, mass tumor generation, and high throughput analysis. Nevertheless, onchip localization, culture, formation, and analysis of 3D tumors in a simple, precisely controllable, parallel, and throughput manner for expanding the application of microfluidics in oncotherapy and drug discovery have not been well-achieved. Moreover, it is worth mentioning that the size of tumor tissue in vivo has been reported to exhibit a noticeable relevance to drug resistance due to its impacts on cellular organization/composition/activities and molecule transportation/delivery in 3D tumors [24,25]. Correspondingly, 3D tumors formed in a single device are totally of the same size using most microfluidic methods mentioned above [21,26]. Recently, several research scholars have brilliantly generated tumors with different sizes using micro-funnels in a four-layer device [27]. Microfluidic construction of large scale 3D tumor system with both appropriate functional unit design and simple device fabrication is still under development. Meanwhile, there has been very few microfluidic progress for quantitative and orthogonal analysis of tumor responses to dose sequencing of anticancer drug in the single device. Therefore, the establishment of facile tumor-on-a-chip with the capacity for simultaneous, throughput (i.e., massive), and controllable manipulation of multi-size 3D tumors and their evaluation involving multi-concentration drug resistance remains largely out of reach. In this study, we present a microfluidic approach for simultaneous and massive production of multi-size 3D tumors, as well as monitoring of tumor responses in different fluidic microenvironments during the cultivation and multiple anticancer drug treatments in a single microfluidic device with well-designated microwell arrays and simple doublelayer composition. Microfluidic cell localization at different flow rates and loading times in the established device was evaluated systematically. We demonstrated that this microfluidic device was able to perform quantity-homogeneous cell trapping and size-similar 3D tumor production in each type of microwells. The phenotypic characteristics of different 3D tumors associated to their aggregation, growth, and shape as well as viability and cytoskeleton structure during the culture process were optically/fluorescently recorded and quantitatively analyzed. Based on the robust microfluidic multi-size 3D tumor construction, a proof-of principle application for throughput and orthogonal drug resistance evaluation was accomplished using two antitumor drugs (bleomycin and tirapazamine) with various concentrations and two tumor cell lines (human hepatocellular liver carcinoma HepG2 cells and human glioma U251 cells).
2.2. Device fabrication The microfluidic device was fabricated using soft lithography [17,28,29] with two SU8-2025 master molds (i.e., the fluidic mold and the microwell mold) on two respective silicon substrates. Degassed PDMS was poured onto the molds to make device after all molds were exposed trimethylchlorosilane vapor for 3 min. PDMS mixture with a ratio of 10:1 (RTV 615 A : B) was used to yield a 3 mm thick fluidic layer of the device, whereas PDMS with a ratio of 20:1 was used to obtain a 1 mm thin microwell layer. After the fluidic and microwell layers were cured in an oven at 80 °C for 15 and 25 min respectively, the layers were peeled off from the respective molds. Inlet and outlet holes were punched in the PDMS piece of fluidic layer using a hole puncher for access of cell samples and reagents, as well as waste exclusion. The fluidic layer was trimmed, cleaned, and then aligned onto the cleaned microwell layer. After baking at 80 °C for 12 h, the assembled and bonded layers were trimmed again to fabricate the final device. The PDMS device was ready for use after baking at 80 °C for 48 h for sufficient bonding [17]. 2.3. Cell preparation Human hepatocellular liver carcinoma (HepG2) and human glioma (U251) commonly used as model cell lines for human cancer research were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Before use, the cells were harvested by trypsinization with 0.25% trypsin (Gibco) at 37 °C. Trypsinization was stopped upon the addition of fresh supplemented DMEM, and cell suspensions were centrifuged at 1000 rpm for 3 min. The cells were then resuspended in supplemented DMEM for use. 2.4. Microfluidic 3D tumor cultivation and drug resistance testing The microfluidic device was first sterilized with UV light for 2 h, rinsed with autoclaved phosphate-buffered saline (PBS, 0.01 M, pH 7.4), and specifically treated with a sterilized solution of Pluronic F127 (an anti-cell adhesive reagent, 20 mg/mL in water) for 3 h at room temperature (RT) followed by a PBS rinsing. Next, the cells were selectively prestained using CellTracker Green CMFDA (10 μM in DMEM) for fluorescence-visualized cell tracking before the seeding process according to the manufacturer’s instructions (Gibco), and were loaded into the chambers by flowing the suspension (5.0 × 106 cells/mL, 3–20 μL/min) using a normal syringe pump (Longerpump, LSP04-1 A). The cell seeding was kept for 1–3 min to optimize the cell trapping into the microwells in the device. The cell counting was immediately carried out after the trapping process using the single-layer optical focusing, multilayer imaging and analysis. After cell localization, the chambers in the device were rinsed with fresh supplemented DMEM at a slow flow rate (1 μL/min). For microfluidic 3D tumor culture and formation, the device was then placed at 37 °C in a humidified atmosphere with 5% CO2. The supplemented DMEM culture medium was refreshed every 12 h in the device for at least 4 d. In addition, finite element analysis was conducted using ESI-CFD software (V2010.0, ESI CFD Inc., Huntsville, AL, U.S.A.) to evaluate the flow profile in the microfluidic
2. Materials and methods 2.1. Materials and reagents Polydimethylsiloxane (PDMS) prepolymer (RTV 615 A) and curing agent (RTV 615 B) were purchased from Momentive Performance Materials (Waterford, NY, USA). Surface-oxidized silicon wafers were purchased from Kaihua Shunchen Electronic Technology Ltd. (Zhejiang, China). SU-8 2025 photoresist and developer were obtained from Microchem (Newton, MA, USA). Pluronic F127, Hoechst 33258, fluorescein diacetate (FDA) and propidium iodide (PI) were obtained from 112
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
tumor production) of cell samples and throughput analyses of tumor responses in different fluidic microenvironments with different anticancer drug stimulations, a series of sequential microfluidic performances typically including cell loading, cell trapping, rinsing, tumor cultivation and formation, on-chip tumor analysis need to be performed in the single device. The microfluidic device contained eight parallel fluidic units and culture chambers, as well as six parallel multi-size microwell lines in each chambers. This allows the repeatable production of 3D tumors with different sizes in the single chamber and massive 3D tumor production in multiple chambers for parallel and throughput manipulation/analysis in the single device.
device. For anticancer drug resistance evaluation, assays of on-chip chemotherapy were conducted using two representative anticancer drugs, i.e., BLM and TPZ. The supplemented DMEM media containing anticancer drug with different concentrations (50–300 μM) were introduced into the respective microfluidic chambers in the single device to stimulate tumors. The anticancer drug-contained supplemented DMEM was refreshed every 12 h in the device and the drug treatment was kept for at least 3 d. The untreated 3D tumors in the chambers were used as controls. 2.5. Cell staining
3.2. Microfluidic cell localization Cell viability was determined by using an FDA/PI staining protocol. After removing the supplemented medium from the device and rinsing with PBS, the tumors in the microwells were incubated with FDA/PI (10 μg mL–1 of each in PBS) solution for 10 min at 37 °C, followed by a final rinse with PBS. For cytoskeleton visualization, 3D tumors in the device were fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min at RT. The fixed tumors were then incubated with 100 nM TRITC-phalloidin in PBS solution for 30 min. Nuclei were counterstained using H33258 (0.5 μg mL–1 in PBS) staining solution.
Precise and quantitative cell organization determines the robustness and controllability of a microfluidic platform for 3D tumor manipulation and on-chip analysis [30]. For an effective cell localization (Fig. 2A and B), the hydrodynamic influence on cell trapping was first estimated in the microfluidic devices. We initially performed finite-element modeling analyses of the device to simulate the velocity profiles of different flow fields (3 μL/min and 10 μL/min). The theoretical calculations (Fig. 2C) show that the high flow condition induced more powerful hydrodynamic microenvironments than low flow rate in both microchambers and microwells, which implies that low flow field shold be considered for cell/tumor manipualtion with shear-stress as low as possible. The calculated results also indicate that the perfusion was equal in the different regions of the chamber. Experimentally, two types of tumor cells (HepG2 and U251) were used and fluorescently labelled using CellTracker Green staining to clearly visualize their existence in the device. Both flow rate and cell loading time were considered for comprehensive investigation of cell localization. The cell suspension at different flow rates ranged from 3 to 20 μL/min was introduced into the Pluronic F127-pretreated channels, chambers and microwells over different time periods ranging 1–3 min. Pluronic F127, is highly effective in preventing protein adsorption and cell adhesion on PDMS substrate [31]. Pluronic F127 treatment bestowed high inhibition of biological cue accessibility on the inner surfaces of the device. The tumor cells along with the flow were trapped naturally in the microwells depending on the gravity settlement and mechanical capture. The residual cells at the non-specific trapping regions in the Pluronic F127-pretreated chambers were readily removed by a rinsing process using the fresh medium flow. Very few cells remained in the non-trapping region of the chambers in tests with different tumor cell types (Fig. 2 and Fig. S2). The expectation of cell preservation as many as possible in the microwells necessitated a very gentle rinse (1 μL/min) during this process. After the trapping process, the cell counting was quickly performed based on the single-layer optical focusing, multilayer imaging and analysis. In fact, the trapped cells in the microwells just formed two or three cell layers being easy to quantify. The quantitative trapping results display that the number of captured cells in different microwells fluctuated as the flow rate increased during a specific loading time. In detail (Fig. 2D), the number of captured cells had a general tendency to increase at low flow rates (3 − 10 μL/min) and then decrease at high flow rates (i.e., over 10 μL/ min), negatively affecting the cell trapping efficiency. The results suggest that the fluidic conditions at low flow conditions supplied greater cell trapping and settlement in the microwells than high flow rates. Moreover, the correlation between the loading time and the trapping efficiency shows that the number of captured cells increased at loading time from 1 to 3 min, which seems to offset the negative effect of high flow condition on cell localization. Notably, the results also indicate that the maximum number of tumor cells was captured after 3 min loading at low flow rates (5 − 10 μL/min). The typical number of captured cells was proportional to the diameter of microwells (100-μm: 20 − 30 HepG2 cells; 200-μm: 100 − 150 cells; 300-μm: 250 − 350 cells; 400-μm: 450 − 550 cells; 500-μm: 600 − 800 cells; 600-μm:
2.6. Microscopy and image analysis A confocal laser scanning microscope (Olympus, FV1000) and an inverted microscope (Olympus, CKX41) with a charge-coupled device camera (Olympus, DP72) and a mercury lamp (Olympus, U-RFLT50) were used to acquire bright-field and fluorescence images. Software Image-Pro® Plus 6.0 (Media Cybernetics, Silver Spring, MD) and SPSS 12.0 (SPSS Inc.) were employed to perform image analysis (i.e., cell counting, tumor size and fluorescence measuring) and data statistical analysis, respectively. Values were represented as means ± SD, and statistical comparisons of means were made using one-way analysis of variance (ANOVA). For all statistical tests *P < 0.05 was used as the criterion for statistical significance. 3. Results and discussion 3.1. Microfluidic design and operation principle Our research purposes focused on three parts: (1) to establish a facile-fabricated and well-operated microfluidic system which efficiently realize cell localization in size-diverse microwells at the same time in the single chambers of a device; (2) to accomplish a simultaneous selfassembly of 3D tumors with different sizes in the single chambers and massive 3D tumor cultivation/production in the single device; (3) to perform parallel/throughput multiconcentration-involved chemotherapy assays and orthogonal (i.e., multiple tumor sizes and drug concentrations) evaluation of anticancer drug resistance in multi-size 3D tumors in the single device. As such, the PDMS microfluidic device in the present study was simply composed of two layers (Fig. 1 and Fig. S1), i.e., the fluidic layer and the microwell layer sequentially from top to bottom. The fluidic layer contained eight individual chambers (60 μm in height), which were parallel arranged for cell/tumor manipulation and analysis in the device. Forty-nine micropillars were set in each chamber to prevent common chamber subsidence. Inlets and outlets were used to perform cell sample loading, chamber purging, and waste exclusion. In the microwell layer, eight groups of microwells (35 μm in height) corresponded to eight chambers in the fluidic layer (Fig. 1A and B). Each group of microwells with various diameters (100–600 μm) were specifically designated in a 6 × 6 geometry to form an array and applied for controllable cell trapping, localization, and multi-size tumor cultivation/formation. To complete multiple manipulations (i.e., cell trapping and 3D 113
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 1. Microfluidic platform for multi-size 3D tumor production. (A) Microfluidic device with eight parallel chambers (multi-size microwell array in each chamber). (B) Schematic presentation of microwell array with versatile diameter designs (100–600 μm) (C) Schematic diagram of 3D tumor culture operations.
900 − 1200 cells). The captured U251 cells was quantitatively more than HepG2 cells in the microwells with the same diameter, probably due to the size variance of two cell types. The optimized fluidic manipulating conditions could be applied for performing controllable and efficient cell manipulations for the production of 3D tumor with different sizes.
assembly (Fig. 3). Tumor cells in the microwells tended to aggregate together within the first culture period (0.5 d) after cell trapping and the tumor compaction typically occurred after cultivation for 1 d. Comparatively, this compacting process seemed to be more obvious in U251 than HepG2 tumors. The assembled tumor cells in the microwell gradually became a tumor with a clear and discoidal appearance. The tumor size increased daily along with the cultivation (Fig. 4 and Fig. S3), and microfluidic manipulation produced multiple types of 3D tumor array in the device. Meanwhile, viability characterization using FDA/PI double fluorescence staining (i.e., green/red for live/dead cells) at the end-point of the culture confirmed that the cells in multi-size 3D tumors in the microwells were totally viable (Fig. 4A, Figs. S3 and S4). There were scarcely any dead cells in the tumors and typically a fluorescence gradient from the tumor center to the edge. Most of the cells with highly bright fluorescence were distributed in the peripheral rim of 3D tumors in different microwells (especially > 200 μm). The formed 3D tumors grew and touched the ceiling of chamber. The result suggests that cells close to the curved surface of tumors were easier to access various substances (e.g., culture nutrients and drugs in the fluidic microenvironment) than cells adjacent to the top and bottom of tumors. Accordingly, most cells with high viability were located at the curved surface of tumor. Further, it is known that FDA serves as a viability probe that measures both enzymatic activity and cell-membrane integrity [34]. FDA is taken up by cells which convert the non-fluorescent FDA into the fluorescent metabolite fluorescein by intracellular esterase-associated hydrolysis. Hence, these results imply that high cell activities like metabolism and proliferation mainly occurred in the
3.3. Multi-size 3D tumor production Based on the microfluidic cell localization mentioned above, the establishment of 3D tumors (HepG2 and U251) was further conducted in the device. The formation of 3D tumors in vitro is normally on account of cell self-assembly and they are usually maintained on an anticell adhesive surface (e.g., agarose) or under a limited liquid environment (e.g., droplet) [7,32]. In this part of the study, we selected Pluronic F127 as a candidate anti-cell adhesion molecule to prevent cell adhesion on the microwell surface and promote the self-assembly of tumor cells in the device. We also avoided the potential effects of flowinduced shear stress on tumor formation and cellular activities. According to the previous numerical simulation of flow fields in our device (Fig. 2C), a lower velocity of medium flow (1 μL/min) was utilized for culture nutrient refreshing to minimize the flow effects as much as possible on the cell activity in 3D culture. The maximum shear stress in each chamber and even microwell was below 0.02 dyn cm−2. This is significantly lower than the minimum level (0.7 dyn cm−2) of microfluidic flow demonstrated to affect cell growth [33]. During the 4-d cultivation, bioinert modification of the device entirely inhibited tumor cells from attaching to the surface of microwells and directed them self114
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 2. Microfluidic cell localization in the microwells with different diameters. (A) Optical image of HepG2 cell trapping array. (B) Fluorescence image of the trapped cells corresponding to (A). The cells were prestained using CellTracker Green for green fluorescence visualization. (C) Simulated flow conditions in the device at different flow rates (3 and 10 μL/min). (D) Quantification of cell trapping in different microwells at various flow rates (3, 5, 7, 10, and 20 μL/min) and different loading times (1, 2, and 3 min) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
individual range of diameters in various microwells (100-μm: 78–86 μm tumors; 200-μm: 160–180 μm tumors; 300-μm: 240–280 μm tumors; 400-μm: 330–370 μm tumors; 500-μm: 380–460 μm tumors; 600-μm: 460–540 μm tumors) was totally over 80% after 2 days of culture. Microfluidic U251 tumor cultivation exhibited similar achievements (Fig. S3) as the former (Fig. 4C). In addition, high repeatability and reproducibility of multi-size 3D tumor production (Fig. S6) suggest that the microfluidic device enabled controllable, parallel, and massive generation of 3D tumors with uniform geometry. Multi-size 3D tumors could be array-like fabricated in the single device, which is indispensable for convenient, parallel, and throughput evaluation of various tumor-associated events like tumor chemotherapy and targeted drug delivery.
outer zone (especially the curved surface) of the 3D tumors with various sizes. In vitro tumors with diameter of over 150 μm had been demonstrated to be similar to in vivo tumor tissue with diffusion limitation to many molecules, especially O2 meaning hypoxia [3,5,35]. Inefficient mass transportation also results in metabolic waste accumulation and causes cell quiescence inside these cultured tumors, which subsequently increase the resistance to chemotherapy [4,5]. In this part of experiment, the results indicate that the functional gradient-like cell phenotypes from the outer into the center of tumors were successfully simulated in the microfluidic device. Such 3D tumors are thought to reflect the in vivo pathology of tumors more realistically than 2D cell cultures [26]. Additionally, on-chip actin filament visualization of cells using TRITC-phalloidin staining presented that the cultured 3D tumors had intact cytoskeletons and a high degree of cell organization with no intercellular spaces between cells (Fig. S5). The size uniformity of the arrayed tumors statistically benefits throughput on-chip tumor analysis. Further, the size distribution of 3D tumors in culture was quantified for accurate analysis of tumor production (Fig. 4C). The percentage of multi-size HepG2 tumors with the
3.4. Anticancer drug resistance in multi-size 3D tumors One important application of 3D tumors cultured in vitro is to conduct preclinical screening for determine anticancer drug efficacy in drug discovery and tumor therapy. An forceful explanation is 3D tumors 115
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 3. Multi-size 3D tumor production in the microfluidic platform. HepG2 and U251 tumors with different sizes were gradually formed during the 4 days cultivation in the respective microwells.
Fig. 4. Evaluation of tumor cultivation in the device. (A) Cell viability in multi-size HepG2 tumors. Live/dead (green/red) cells were visualized by FDA/PI staining. (B) HepG2 tumor growth in the microwells with different diameters (100–600 μm). (C) Size distributions of multi-size HepG2 tumors cultured for 2 d in the microwells with different diameters (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 116
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 5. Microfluidic chemotherapy in multi-size 3D HepG2 tumors. Two anticancer drugs (BLM and TPZ) with various concentrations (50, 100, 200, and 300 μM) were used for evaluating the chemosensitivity of tumors with different sizes. The drug stimulation was kept for 3 d and the control culture was set accordingly. Live/ dead (green/red) cells were visualized by FDA/PI staining (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
117
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
present more biomimetic than 2D monolayer cultured tumor cells [36]. The presentation of hypoxic regions in most 3D tumors increases the resistance to therapeutic treatments. Hypoxic diversity and structural complexity of 3D tumors with different diameters make themselves different responses to the clinical chemotherapy [18,37]. For orthogonal evaluation of the tumor chemosensitivity, we stimulated multisize 3D tumors using the respective chemotherapeutic drugs (BLM and TPZ) with various concentrations ranging from 50 to 300 μM in the parallel microfluidic chambers in the single devices. Two anticancer drugs have different mechanisms of action and require different oxygen conditions for effective tumor therapy (BLM with normoxic tumor killing and TPZ with hypoxic tumor killing) [38,39]. The aforementioned results suggest that drug molecules can reach the tumor tissue basically from all around, especially from the curved side. For the specific type of either HepG2 or U251 tumors, the multiple concentration-related parallel chemotherapy, as well as the control assay could be performed in the single device because of its parallel fluidic design. The drug treatment on different tumors was maintained for 3 d in the microfluidic device. The optical recording and fluorescent characterization of tumors and their viability were applied for the further quantitative evaluation of chemosensitivity of multi-size 3D tumors. The experimental results were as shown in Figs. 5 and 6. In general, the higher anticancer drug concentration caused a more significant effect of chemotherapy in 3D tumors (Fig. 5 and Figs. S7–S10). The chemotherapy (i.e., the percentage of cell death) analysis was based on the PI staining for dead cell identification and quantification of fluorescence distribution. Most cell deaths happened in the curved side of tumors due to the spatially prior cell-drug contact. The quantitative comparison reveals that the size of tumors could positively impact their drug resistance (Fig. 6). For examples, more than 45% cells in the smallest HepG2 tumors in the 100-μm microwells were dead after 3 days of BLM treatment (300 μM), whereas less than 30% cells in the bigger tumors in the 500 and 600-μm microwells were killed after the same treatment (Fig. 6A). Meanwhile, TPZ-treated HepG2 tumors exhibited similar tendency of viability responses, but their chemotherapy especially in bigger tumors was more effective than the BLM stimulation (Fig. 6B). We explained that this result was probably owing to the hypoxia-selective toxicity of TPZ in bigger tumors, which had more hypoxic tumor cells than smaller tumors. In addition, BLM treatment on U251 tumors induced much higher apoptosis than HepG2 tumors in the microwells with same diameter (Fig. 6C). Significantly, treatment using a high concentration (300 μM) of BLM was able to kill over 65% and 50% cells in U251 tumors in the 100 and 500-μm microwells respectively. It is noteworthy that there were about 10% cell death in the control cultures of U251 tumors. Most of them were clearly located in the center of multi-size tumors in the microwells (particular 500 and 600 μm), implying that long-term microfluidic cultivation of large size of 3D tumors might generate in vivo-like complicated tissue structure consisting of a necrotic core, an inner quiescent cell layer and an outer proliferating zone. All of the results demonstrate that the size of tumor besides their tissue composition and 3D organization determines chemotherapeutic resistance in vitro. The therapeutic and chemosensitive evaluation confirmed the applicability and feasibility of microfluidics for multi-size 3D tumor culture-based throughput analysis, which would be extremely useful for the investigation of many tumor-associated pathophysiological and preclinical anticancer events.
Fig. 6. Therapeutic efficacy of anticancer drugs with different concentrations (50, 100, 200, and 300 μM)) in multi-size 3D tumors. (A) BLM treatment in HepG2 tumors in the microwells with different diameters (100–600 μm). (B) TPZ treatment in HepG2 tumors in different microwells. (C) BLM treatment in U251 tumors in different microwells. Chemotherapy corresponds to the percentage of cell death.
4. Conclusions In summary, we developed a facile, massive, and controllable production process for simultaneously generating multi-size biomimetic 3D tumors using a simple-fabricated and well-established microfluidic system. We also presented an experimental demonstration of its applicability and feasibility in studying orthogonal chemotherapy and drug resistance of multi-size tumors with parallel and throughput, i.e., 4 days of tumor culture, 3 days of chemotherapeutic assay/detection, and
at least 288 (tumors)×4 (cell localization, tumor formation/growth, tumor size uniformity, tumor characterization after culture or drug stimulation) results per total assay time in the single device. On-chip manipulation including cell localization, array-like tumor formation, and on-line analysis of multiple types of 3D tumors can be completed smoothly in the system. We verified that the stable microfluidic 3D 118
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
tumor operations was partly based on a systematic optimization of tumor cell localization in various fluidic conditions. Meanwhile, highly efficient multi-size tumor formation with quantitative-uniformity was experimentally proven in single microfluidic devices. Optical monitoring of response dynamics for the quantitative culture and anticancer evaluation was shown to be very powerful in the PDMS device. The results support the importance of size and composition of 3D tumors with a complex tissue microenvironment while studying oncotherapy and chemosensitivity. We envision that this microscale and throughput microfluidic approach with simple, robust, and long-term manipulative properties is potentially valuable for biological/medical researchers to better perform assays that investigate tumorigenesis and anticancer drug resistance. We believe that this type of platform would greatly promote the development of miniaturized tools for integrated biological analysis with biomimetic, microscale, and high-throughput capabilities.
[19] L.F. Yu, M.C.W. Chen, K.C. Cheung, Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing, Lab Chip 10 (2010) 2424–2432. [20] A. Zuchowska, K. Marciniak, U. Bazylinska, E. Jastrzebska, K.A. Wilk, Z. Brzozka, Different action of nanoencapsulated meso-tetraphenylporphyrin in breast spheroid co-culture and mono-culture under microfluidic conditions, Sensor. Actuat. BChem. 275 (2018) 69–77. [21] C. Kim, J.H. Bang, Y.E. Kim, S.H. Lee, J.Y. Kang, On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network, Lab Chip 12 (2012) 4135–4142. [22] H. Ota, R. Yamamoto, K. Deguchi, Y. Tanaka, Y. Kazoe, Y. Sato, N. Miki, Threedimensional spheroid-forming lab-on-a-chip using micro-rotational flow, Sensor. Actuat. B-Chem. 147 (2010) 359–365. [23] K.J. Chen, M.X. Wu, F. Guo, P. Li, C.Y. Chan, Z.M. Mao, S. Li, L. Ren, R. Zhang, T.J. Huang, Rapid formation of size-controllable multicellular spheroids via 3D acoustic tweezers, Lab Chip 16 (2016) 2636–2643. [24] M. Van Glabbeke, J. Verweij, P.G. Casali, A. Le Cesne, P. Hohenberger, I. RayCoquard, M. Schlemmer, A.T. van Oosterom, D. Goldstein, R. Sciot, P.C.W. Hogendoorn, M. Brown, R. Bertulli, I.R. Judson, Initial and late resistance to imatinib in advanced gastrointestinal stromal. Tumors are predicted by different prognostic factors: a European organisation for research and treatment of cancerItalian Sarcoma group-Australasian Gastrointestinal Trials group study, J. Clin. Oncol. 23 (2005) 5795–5804. [25] R.S. Kerbel, A cancer therapy resistant to resistance, Nature 390 (1997) 335–336. [26] W. Lim, H.H. Hoang, D. You, J. Han, J.E. Lee, S. Kim, S. Park, Formation of sizecontrollable tumour spheroids using a microfluidic pillar array (μFPA) device, Analyst 143 (2018) 5841–5848. [27] M. Marimuthu, N. Rousset, A. St-Georges-Robillard, M.A. Lateef, M. Ferland, A.M. Mes-Masson, T. Gervais, Multi-size spheroid formation using microfluidic funnels, Lab Chip 18 (2018) 304–314. [28] Y. Xia, G.M. Whitesides, Soft Lithography, Angew. Chem. Int. Ed. 37 (1998) 550–575. [29] S. Shen, C. Tian, T. Li, J. Xu, S.W. Chen, Q. Tu, M. Yuan, W. Liu, J. Wang, Spiral microchannel with ordered micro-obstacles for continuous and highly-efficient particle separation, Lab Chip 17 (2017) 3578–3591. [30] K. Moshksayan, N. Kashaninejad, M.E. Warkiani, J.G. Lock, H. Moghadas, B. Firoozabadi, M.S. Saidi, N. Nguyen, Spheroids-on-a-chip: recent advances and design considerations inmicrofluidic platforms for spheroid formation and culture, Sensor. Actuat. B-Chem. 263 (2018) 151–176. [31] S. Raghavan, R.A. Desai, Y. Kwon, M. Mrksich, C.S. Chen, Micropatterned dynamically adhesive substrates for cell migration, Langmuir 26 (2010) 17733–17738. [32] S. Breslin, L. O’Driscoll, Three-dimensional cell culture: the missing link in drug discovery, Drug Discov. Today 18 (2013) 240–249. [33] N. Bhattacharjee, N. Li, T.M. Keenan, A. Folch, A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors, Integr. Biol. (Camb) 2 (2010) 669–679. [34] X. Xiao, Z.Y. Han, Y.X. Chen, X.Q. Liang, H. Li, Y.C. Qian, Optimization of FDA-PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa, Phys. Chem. Earth (2002) 36 (2011) 424–429. [35] C.R. Thoma, M. Zimmermann, I. Agarkova, J.M. Kelm, W. Krek, 3D cell culture systems modeling tumor growth determinants in cancer target discovery, Adv. drug deliver. Rev. 69 (2014) 29–41. [36] J. Friedrich, C. Seidel, R. Ebner, L.A. Kunz-Schughart, Spheroid-based drug screen: considerations and practical approach, Nat. Protoc. 4 (2009) 309–324. [37] G. Mehta, A.Y. Hsiao, M. Ingram, G.D. Luker, S. Takayama, Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy, J. Control. Release 164 (2012) 192–204. [38] G. Chowdhury, U. Sarkar, S. Pullen, W.R. Wilson, A. Rajapakse, T. Fuchs-Knotts, K.S. Gates, DNA strand cleavage by the phenazine Di-N-oxide natural product myxin under both aerobic and anaerobic conditions, Chem. Res. Toxicol. 25 (2012) 197–206. [39] L. Wang, W.M. Liu, L. Wang, J.C. Wang, Q. Tu, R. Liu, J. Wang, Construction of oxygen and chemical concentration gradients in a single microfluidic device for studying tumor cell-drug interactions in a dynamic hypoxia microenvironment, Lab Chip 13 (2013) 695–705.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31470971, No. 21375106, No. 31100726), the Fundamental Research Funds for the Central Universities (202045001), and Central South University. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.04.121. References [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (2018) 394–424. [2] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2015) 87–108. [3] F. Hirschhaeuser, H. Menne, C. Dittfeld, J. West, W. Mueller-Klieser, L.A. KunzSchughart, Multicellular tumor spheroids: an underestimated tool is catching up again, J. Biotechnol. 148 (2010) 3–15. [4] S. Sant, P.A. Johnston, The production of 3D tumor spheroids for cancer drug discovery, Drug Discov. Today Technol. 23 (2017) 27–36. [5] R.Z. Lin, H.Y. Chang, Recent advances in three-dimensional multicellular spheroid culture for biomedical research, Biotechnol. J. 3 (2008) 1172–1184. [6] B.A. Justice, N.A. Badr, R.A. Felder, 3D cell culture opens new dimensions in cellbased assays, Drug Discov. Today 14 (2009) 102–107. [7] E. Fennema, N. Rivron, J. Rouwkema, C. van Blitterswijk, J. de Boer, Spheroid culture as a tool for creating 3D complex tissues, Trends Biotechnol. 31 (2013) 108–115. [8] C.T. Kuo, J.Y. Wang, Y.F. Lin, A.M. Wo, B.P.C. Chen, H. Lee, Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array, Sci. Rep. 7 (2017) 4363. [9] P. Benien, A. Swami, 3D tumor models: history, advances and future perspectives, Future Oncol. 10 (2014) 1311–1327. [10] C. Fischbach, R. Chen, T. Matsumoto, T. Schmelzle, J.S. Brugge, P.J. Polverini, D.J. Mooney, Engineering tumors with 3D scaffolds, Nat. Methods 4 (2007) 855–860. [11] S.R. Khetani, S.N. Bhatia, Engineering tissues for in vitro applications, Curr. Opin. Biotechnol. 17 (2006) 524–531. [12] Y. Xie, W. Zhang, L. Wang, K. Sun, Y. Sun, X. Jiang, A microchip-based model wound with multiple types of cells, Lab Chip 11 (2011) 2819–2822. [13] S.P. Zhao, Y. Ma, Q. Lou, H. Zhu, B. Yang, Q. Fang, Three-dimensional cell culture and drug testing in a microfluidic sidewall-attached droplet array, Anal. Chem. 89 (2017) 10153–10157. [14] K.E. Sung, D.J. Beebe, Microfluidic 3D models of cancer, Adv. Drug Deliv. Rev. 79–80 (2014) 68–78. [15] H. Hardelauf, J.P. Frimat, J.D. Stewart, W. Schormann, Y.Y. Chiang, P. Lampen, P. Lampen, J. Franzke, J.G. Hengstler, C. Cadenas, L.A. Kunz-Schughartc, J. West, Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits, Lab Chip 11 (2011) 419–428. [16] Q. Zhang, T. Liu, J. Qin, A microfluidic-based device for study of transendothelial invasion of tumor aggregates in realtime, Lab Chip 12 (2012) 2837–2842. [17] C. Zheng, L. Zhao, G. Chen, Y. Zhou, Y. Pang, Y. Huang, Quantitative study of the dynamic tumor-endothelial cell interactions through an integrated microfluidic coculture system, Anal. Chem. 84 (2012) 2088–2093. [18] K.P. Valente, S. Khetani, A.R. Kolahchi, A. Sanati-Nezhad, A. Suleman, M. Akbari, Microfluidic technologies for anticancer drug studies, Drug Discov, Today 22 (2017) 1654–1670.
Wenming Liu received his Ph.D. degree from Northwest A&F University in 2010. Currently, he is working as a research director at School of Basic Medical Science, Central South University. His research work focuses on the intersection of micro-engineering, microfluidics, and cell biology to develop novel strategies and original platforms for exploring life science. Boxiu Lu obtained her B.S. degree from Taishan Medical University in 2011. She is now working as a staff of medical imaging at People's Hospital of Qihe County, Shandong, China. Her research interests aim at cancer-related biological imaging. Mingming Yan obtained his M.S. degree from Northwest A&F University in 2017. He is currently our lab analyst. His research interests include microfluidics-based cell imaging and real-time analysis. Kai Han obtained his B.S. degree from University of Jinan in 2018. He is studying as a postgraduate student at School of Basic Medical Science, Central South University. His research interests include developing microfluidic tumor cell platforms and their applications.
119
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Meilin Sun obtained her B.S. degree from Yuncheng University in 2018. She is studying as a postgraduate student at School of Basic Medical Science, Central South University. Her research interests include developing microfluidic systems for cell trapping and 3D tumor simulation.
Jinyi Wang received his Ph.D. degree from Lanzhou University in 2002. He is currently a research director at College of Chemistry & Pharmacy, Northwest A&F University. His research interests include constructing the novel and sensitive methods to detect the harmful ions and molecules in the agricultural environment, and constructing the integrated microfluidic systems for life science.
120
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A microfluidic platform for multi-size 3D tumor culture, monitoring and drug resistance testing ⁎
Wenming Liua,b, , Meilin Suna, Boxiu Luc, Mingming Yanb, Kai Hana, Jinyi Wangb,
T
⁎
a
Departments of Biomedical Engineering and Pathology, School of Basic Medical Science, Central South University, Changsha, Hunan, 410013, China Department of Chemistry, College of Chemistry and Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, China c Department of Magnetic Resonance Imaging, People's Hospital of Qihe County, Qihe, Shandong, 251100, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Microfluidics Multi-Size 3D tumors Drug resistance Large scale On-Chip orthogonal Analysis
The establishment of a miniaturized platform capable of microscale control for sequential, coordinated, and throughput biomimetic tumor manipulation and analysis offer a high degree of biological and clinical relevance to cancer therapy and pharmaceutical research. In this study, we developed a microfluidic system with wellestablished microwell arrays and simple double-layer composition that allows massive fabrication of biomimetic multi-size 3D tumors and their simultaneous manipulation and orthogonal analysis. Serial manipulations including cell localization, array-like self-assembly/cultivation, and dynamic analysis of different types of 3D tumors, were accomplished in the microfluidic device. We demonstrated that the microfluidic platform is stable and throughput cell trapping and tumor generation with quantity uniform. Furthermore, on-chip monitoring and throughput analysis of tumor phenotypes and responses to culture and different chemotherapies were also achieved in the device. The microfluidic advancement offers a new methodological approach for the development of high-performance and multifunctional 3D tumor systems and for tissue-mimicking cancer research and therapy evaluation. This microfluidic platform, which has the capability of multiple control in time and space, holds great potential for applications in the fields of tumor biology, tissue engineering, and clinical medicine.
1. Introduction Cancer is a major public health problem worldwide. According to the latest statistical presentation from the World Health Organization (WHO), cancer is expected to rank as the first or second leading cause of human death in over 90 countries [1]. Statistical data displays that there are an estimated increase of new cancer cases from 14.1–18.1 million between 2012 and 2018, as well as cancer deaths from 8.2–9.6 million [1,2]. Thus, the establishment of an effective biomimetic tumor system for meticulously exploring tumor pathology and therapy has been the subject of much interest and extensive research. Three-dimensional (3D) tumor culture has been considered to be an improved in vitro model to better simulate the biological properties (e.g., complex cellular organization and interactions, chemical and phenotypical gradients related to nutrient, proliferation, metabolism and viability) of actual tumors in vivo, and to overcome the negative effect of unnatural adhesion and arrangement of conventional two-dimensional (2D) cell monolayer cultures [3–5]. When anticancer drug responses have been directly compared between 2D and 3D culture models, different chemosensitivity between the two models can be represented as either
⁎
enhanced sensitivity or greater resistance. A broad consensus has appeared that in vitro 3D tumor cultures provide more appropriate preclinical models to evaluate the drug efficacy for solid tumors, and more reliable and meaningful therapeutic readouts reduces the potential risks to patients [6]. Over the past several decades, many research groups have attempted to fabricate in vivo-like 3D tumors using hanging drop, anti-adhesive surface, spinner flask, and porous scaffold for heterophenotypic investigation and therapy evaluation [7–10]. However, the applications of these methods have been impeded owing to their drawbacks of being cumbersome, time consuming, labor-intensive, nonuniform tumor size, and low generation yield. To drive the widespread implementation of 3D tumor models in routine tumor analysis and antitumor programs, new simple and popularized methods for the scalable, controllable, and throughput production of 3D tumors with uniform characteristics are required. In recent years, microengineering progresses have generated a series of striking capabilities in performing precise manipulations of mammalian cells with microscale resolution [11–13]. For example, microfabrication and micropatterning methods have been employed to perform controllable localization, orientation, and organization of cells to
Corresponding authors. E-mail addresses: [email protected] (W. Liu), [email protected] (J. Wang).
https://doi.org/10.1016/j.snb.2019.04.121 Received 21 January 2019; Received in revised form 24 April 2019; Accepted 24 April 2019 Available online 26 April 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Sigma-Aldrich (MO, USA). Cell culture medium, fetal bovine serum (FBS), CellTracker Green CMFDA and TRITC-phalloidin were purchased from the Gibco/Life Technologies (CA, USA). Bleomycin (BLM) and Tirapazamine (TPZ) were purchased from Melonepharma Co., Ltd (Dalian, China). All solvents and other chemicals of analytical reagent grade were purchased from local commercial suppliers, unless otherwise stated. All solutions were prepared using ultra-purified water supplied by a Milli-Q system (Millipore®).
produce a large quantity of uniformly prepared 3D tumors, contributing remarkable insights into 3D tumor functions and activities [14,15]. Furthermore, microfluidics is known to be a promising platform for tumor biology because of their satisfactory performance in the spatiotemporal control of fluidic perfusion and tumor cells, in the imitation of tissue microenvironments, and in effective sequential manipulation [16,17]. Tumor-on-a-chip in polydimethylsiloxane (PDMS) provides an optical window into biological events such as tumor development, anticancer drug delivery and therapy [18]. So far, many microfluidic advances technologically based on microdroplets, microgels, microwell arrays, microrotational flow, and 3D acoustic tweezers [19–23], have more or less provided some extremely attractive tumor manipulation features, like simple and controllable process, homogeneous tumor size, mass tumor generation, and high throughput analysis. Nevertheless, onchip localization, culture, formation, and analysis of 3D tumors in a simple, precisely controllable, parallel, and throughput manner for expanding the application of microfluidics in oncotherapy and drug discovery have not been well-achieved. Moreover, it is worth mentioning that the size of tumor tissue in vivo has been reported to exhibit a noticeable relevance to drug resistance due to its impacts on cellular organization/composition/activities and molecule transportation/delivery in 3D tumors [24,25]. Correspondingly, 3D tumors formed in a single device are totally of the same size using most microfluidic methods mentioned above [21,26]. Recently, several research scholars have brilliantly generated tumors with different sizes using micro-funnels in a four-layer device [27]. Microfluidic construction of large scale 3D tumor system with both appropriate functional unit design and simple device fabrication is still under development. Meanwhile, there has been very few microfluidic progress for quantitative and orthogonal analysis of tumor responses to dose sequencing of anticancer drug in the single device. Therefore, the establishment of facile tumor-on-a-chip with the capacity for simultaneous, throughput (i.e., massive), and controllable manipulation of multi-size 3D tumors and their evaluation involving multi-concentration drug resistance remains largely out of reach. In this study, we present a microfluidic approach for simultaneous and massive production of multi-size 3D tumors, as well as monitoring of tumor responses in different fluidic microenvironments during the cultivation and multiple anticancer drug treatments in a single microfluidic device with well-designated microwell arrays and simple doublelayer composition. Microfluidic cell localization at different flow rates and loading times in the established device was evaluated systematically. We demonstrated that this microfluidic device was able to perform quantity-homogeneous cell trapping and size-similar 3D tumor production in each type of microwells. The phenotypic characteristics of different 3D tumors associated to their aggregation, growth, and shape as well as viability and cytoskeleton structure during the culture process were optically/fluorescently recorded and quantitatively analyzed. Based on the robust microfluidic multi-size 3D tumor construction, a proof-of principle application for throughput and orthogonal drug resistance evaluation was accomplished using two antitumor drugs (bleomycin and tirapazamine) with various concentrations and two tumor cell lines (human hepatocellular liver carcinoma HepG2 cells and human glioma U251 cells).
2.2. Device fabrication The microfluidic device was fabricated using soft lithography [17,28,29] with two SU8-2025 master molds (i.e., the fluidic mold and the microwell mold) on two respective silicon substrates. Degassed PDMS was poured onto the molds to make device after all molds were exposed trimethylchlorosilane vapor for 3 min. PDMS mixture with a ratio of 10:1 (RTV 615 A : B) was used to yield a 3 mm thick fluidic layer of the device, whereas PDMS with a ratio of 20:1 was used to obtain a 1 mm thin microwell layer. After the fluidic and microwell layers were cured in an oven at 80 °C for 15 and 25 min respectively, the layers were peeled off from the respective molds. Inlet and outlet holes were punched in the PDMS piece of fluidic layer using a hole puncher for access of cell samples and reagents, as well as waste exclusion. The fluidic layer was trimmed, cleaned, and then aligned onto the cleaned microwell layer. After baking at 80 °C for 12 h, the assembled and bonded layers were trimmed again to fabricate the final device. The PDMS device was ready for use after baking at 80 °C for 48 h for sufficient bonding [17]. 2.3. Cell preparation Human hepatocellular liver carcinoma (HepG2) and human glioma (U251) commonly used as model cell lines for human cancer research were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Before use, the cells were harvested by trypsinization with 0.25% trypsin (Gibco) at 37 °C. Trypsinization was stopped upon the addition of fresh supplemented DMEM, and cell suspensions were centrifuged at 1000 rpm for 3 min. The cells were then resuspended in supplemented DMEM for use. 2.4. Microfluidic 3D tumor cultivation and drug resistance testing The microfluidic device was first sterilized with UV light for 2 h, rinsed with autoclaved phosphate-buffered saline (PBS, 0.01 M, pH 7.4), and specifically treated with a sterilized solution of Pluronic F127 (an anti-cell adhesive reagent, 20 mg/mL in water) for 3 h at room temperature (RT) followed by a PBS rinsing. Next, the cells were selectively prestained using CellTracker Green CMFDA (10 μM in DMEM) for fluorescence-visualized cell tracking before the seeding process according to the manufacturer’s instructions (Gibco), and were loaded into the chambers by flowing the suspension (5.0 × 106 cells/mL, 3–20 μL/min) using a normal syringe pump (Longerpump, LSP04-1 A). The cell seeding was kept for 1–3 min to optimize the cell trapping into the microwells in the device. The cell counting was immediately carried out after the trapping process using the single-layer optical focusing, multilayer imaging and analysis. After cell localization, the chambers in the device were rinsed with fresh supplemented DMEM at a slow flow rate (1 μL/min). For microfluidic 3D tumor culture and formation, the device was then placed at 37 °C in a humidified atmosphere with 5% CO2. The supplemented DMEM culture medium was refreshed every 12 h in the device for at least 4 d. In addition, finite element analysis was conducted using ESI-CFD software (V2010.0, ESI CFD Inc., Huntsville, AL, U.S.A.) to evaluate the flow profile in the microfluidic
2. Materials and methods 2.1. Materials and reagents Polydimethylsiloxane (PDMS) prepolymer (RTV 615 A) and curing agent (RTV 615 B) were purchased from Momentive Performance Materials (Waterford, NY, USA). Surface-oxidized silicon wafers were purchased from Kaihua Shunchen Electronic Technology Ltd. (Zhejiang, China). SU-8 2025 photoresist and developer were obtained from Microchem (Newton, MA, USA). Pluronic F127, Hoechst 33258, fluorescein diacetate (FDA) and propidium iodide (PI) were obtained from 112
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
tumor production) of cell samples and throughput analyses of tumor responses in different fluidic microenvironments with different anticancer drug stimulations, a series of sequential microfluidic performances typically including cell loading, cell trapping, rinsing, tumor cultivation and formation, on-chip tumor analysis need to be performed in the single device. The microfluidic device contained eight parallel fluidic units and culture chambers, as well as six parallel multi-size microwell lines in each chambers. This allows the repeatable production of 3D tumors with different sizes in the single chamber and massive 3D tumor production in multiple chambers for parallel and throughput manipulation/analysis in the single device.
device. For anticancer drug resistance evaluation, assays of on-chip chemotherapy were conducted using two representative anticancer drugs, i.e., BLM and TPZ. The supplemented DMEM media containing anticancer drug with different concentrations (50–300 μM) were introduced into the respective microfluidic chambers in the single device to stimulate tumors. The anticancer drug-contained supplemented DMEM was refreshed every 12 h in the device and the drug treatment was kept for at least 3 d. The untreated 3D tumors in the chambers were used as controls. 2.5. Cell staining
3.2. Microfluidic cell localization Cell viability was determined by using an FDA/PI staining protocol. After removing the supplemented medium from the device and rinsing with PBS, the tumors in the microwells were incubated with FDA/PI (10 μg mL–1 of each in PBS) solution for 10 min at 37 °C, followed by a final rinse with PBS. For cytoskeleton visualization, 3D tumors in the device were fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min at RT. The fixed tumors were then incubated with 100 nM TRITC-phalloidin in PBS solution for 30 min. Nuclei were counterstained using H33258 (0.5 μg mL–1 in PBS) staining solution.
Precise and quantitative cell organization determines the robustness and controllability of a microfluidic platform for 3D tumor manipulation and on-chip analysis [30]. For an effective cell localization (Fig. 2A and B), the hydrodynamic influence on cell trapping was first estimated in the microfluidic devices. We initially performed finite-element modeling analyses of the device to simulate the velocity profiles of different flow fields (3 μL/min and 10 μL/min). The theoretical calculations (Fig. 2C) show that the high flow condition induced more powerful hydrodynamic microenvironments than low flow rate in both microchambers and microwells, which implies that low flow field shold be considered for cell/tumor manipualtion with shear-stress as low as possible. The calculated results also indicate that the perfusion was equal in the different regions of the chamber. Experimentally, two types of tumor cells (HepG2 and U251) were used and fluorescently labelled using CellTracker Green staining to clearly visualize their existence in the device. Both flow rate and cell loading time were considered for comprehensive investigation of cell localization. The cell suspension at different flow rates ranged from 3 to 20 μL/min was introduced into the Pluronic F127-pretreated channels, chambers and microwells over different time periods ranging 1–3 min. Pluronic F127, is highly effective in preventing protein adsorption and cell adhesion on PDMS substrate [31]. Pluronic F127 treatment bestowed high inhibition of biological cue accessibility on the inner surfaces of the device. The tumor cells along with the flow were trapped naturally in the microwells depending on the gravity settlement and mechanical capture. The residual cells at the non-specific trapping regions in the Pluronic F127-pretreated chambers were readily removed by a rinsing process using the fresh medium flow. Very few cells remained in the non-trapping region of the chambers in tests with different tumor cell types (Fig. 2 and Fig. S2). The expectation of cell preservation as many as possible in the microwells necessitated a very gentle rinse (1 μL/min) during this process. After the trapping process, the cell counting was quickly performed based on the single-layer optical focusing, multilayer imaging and analysis. In fact, the trapped cells in the microwells just formed two or three cell layers being easy to quantify. The quantitative trapping results display that the number of captured cells in different microwells fluctuated as the flow rate increased during a specific loading time. In detail (Fig. 2D), the number of captured cells had a general tendency to increase at low flow rates (3 − 10 μL/min) and then decrease at high flow rates (i.e., over 10 μL/ min), negatively affecting the cell trapping efficiency. The results suggest that the fluidic conditions at low flow conditions supplied greater cell trapping and settlement in the microwells than high flow rates. Moreover, the correlation between the loading time and the trapping efficiency shows that the number of captured cells increased at loading time from 1 to 3 min, which seems to offset the negative effect of high flow condition on cell localization. Notably, the results also indicate that the maximum number of tumor cells was captured after 3 min loading at low flow rates (5 − 10 μL/min). The typical number of captured cells was proportional to the diameter of microwells (100-μm: 20 − 30 HepG2 cells; 200-μm: 100 − 150 cells; 300-μm: 250 − 350 cells; 400-μm: 450 − 550 cells; 500-μm: 600 − 800 cells; 600-μm:
2.6. Microscopy and image analysis A confocal laser scanning microscope (Olympus, FV1000) and an inverted microscope (Olympus, CKX41) with a charge-coupled device camera (Olympus, DP72) and a mercury lamp (Olympus, U-RFLT50) were used to acquire bright-field and fluorescence images. Software Image-Pro® Plus 6.0 (Media Cybernetics, Silver Spring, MD) and SPSS 12.0 (SPSS Inc.) were employed to perform image analysis (i.e., cell counting, tumor size and fluorescence measuring) and data statistical analysis, respectively. Values were represented as means ± SD, and statistical comparisons of means were made using one-way analysis of variance (ANOVA). For all statistical tests *P < 0.05 was used as the criterion for statistical significance. 3. Results and discussion 3.1. Microfluidic design and operation principle Our research purposes focused on three parts: (1) to establish a facile-fabricated and well-operated microfluidic system which efficiently realize cell localization in size-diverse microwells at the same time in the single chambers of a device; (2) to accomplish a simultaneous selfassembly of 3D tumors with different sizes in the single chambers and massive 3D tumor cultivation/production in the single device; (3) to perform parallel/throughput multiconcentration-involved chemotherapy assays and orthogonal (i.e., multiple tumor sizes and drug concentrations) evaluation of anticancer drug resistance in multi-size 3D tumors in the single device. As such, the PDMS microfluidic device in the present study was simply composed of two layers (Fig. 1 and Fig. S1), i.e., the fluidic layer and the microwell layer sequentially from top to bottom. The fluidic layer contained eight individual chambers (60 μm in height), which were parallel arranged for cell/tumor manipulation and analysis in the device. Forty-nine micropillars were set in each chamber to prevent common chamber subsidence. Inlets and outlets were used to perform cell sample loading, chamber purging, and waste exclusion. In the microwell layer, eight groups of microwells (35 μm in height) corresponded to eight chambers in the fluidic layer (Fig. 1A and B). Each group of microwells with various diameters (100–600 μm) were specifically designated in a 6 × 6 geometry to form an array and applied for controllable cell trapping, localization, and multi-size tumor cultivation/formation. To complete multiple manipulations (i.e., cell trapping and 3D 113
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 1. Microfluidic platform for multi-size 3D tumor production. (A) Microfluidic device with eight parallel chambers (multi-size microwell array in each chamber). (B) Schematic presentation of microwell array with versatile diameter designs (100–600 μm) (C) Schematic diagram of 3D tumor culture operations.
900 − 1200 cells). The captured U251 cells was quantitatively more than HepG2 cells in the microwells with the same diameter, probably due to the size variance of two cell types. The optimized fluidic manipulating conditions could be applied for performing controllable and efficient cell manipulations for the production of 3D tumor with different sizes.
assembly (Fig. 3). Tumor cells in the microwells tended to aggregate together within the first culture period (0.5 d) after cell trapping and the tumor compaction typically occurred after cultivation for 1 d. Comparatively, this compacting process seemed to be more obvious in U251 than HepG2 tumors. The assembled tumor cells in the microwell gradually became a tumor with a clear and discoidal appearance. The tumor size increased daily along with the cultivation (Fig. 4 and Fig. S3), and microfluidic manipulation produced multiple types of 3D tumor array in the device. Meanwhile, viability characterization using FDA/PI double fluorescence staining (i.e., green/red for live/dead cells) at the end-point of the culture confirmed that the cells in multi-size 3D tumors in the microwells were totally viable (Fig. 4A, Figs. S3 and S4). There were scarcely any dead cells in the tumors and typically a fluorescence gradient from the tumor center to the edge. Most of the cells with highly bright fluorescence were distributed in the peripheral rim of 3D tumors in different microwells (especially > 200 μm). The formed 3D tumors grew and touched the ceiling of chamber. The result suggests that cells close to the curved surface of tumors were easier to access various substances (e.g., culture nutrients and drugs in the fluidic microenvironment) than cells adjacent to the top and bottom of tumors. Accordingly, most cells with high viability were located at the curved surface of tumor. Further, it is known that FDA serves as a viability probe that measures both enzymatic activity and cell-membrane integrity [34]. FDA is taken up by cells which convert the non-fluorescent FDA into the fluorescent metabolite fluorescein by intracellular esterase-associated hydrolysis. Hence, these results imply that high cell activities like metabolism and proliferation mainly occurred in the
3.3. Multi-size 3D tumor production Based on the microfluidic cell localization mentioned above, the establishment of 3D tumors (HepG2 and U251) was further conducted in the device. The formation of 3D tumors in vitro is normally on account of cell self-assembly and they are usually maintained on an anticell adhesive surface (e.g., agarose) or under a limited liquid environment (e.g., droplet) [7,32]. In this part of the study, we selected Pluronic F127 as a candidate anti-cell adhesion molecule to prevent cell adhesion on the microwell surface and promote the self-assembly of tumor cells in the device. We also avoided the potential effects of flowinduced shear stress on tumor formation and cellular activities. According to the previous numerical simulation of flow fields in our device (Fig. 2C), a lower velocity of medium flow (1 μL/min) was utilized for culture nutrient refreshing to minimize the flow effects as much as possible on the cell activity in 3D culture. The maximum shear stress in each chamber and even microwell was below 0.02 dyn cm−2. This is significantly lower than the minimum level (0.7 dyn cm−2) of microfluidic flow demonstrated to affect cell growth [33]. During the 4-d cultivation, bioinert modification of the device entirely inhibited tumor cells from attaching to the surface of microwells and directed them self114
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 2. Microfluidic cell localization in the microwells with different diameters. (A) Optical image of HepG2 cell trapping array. (B) Fluorescence image of the trapped cells corresponding to (A). The cells were prestained using CellTracker Green for green fluorescence visualization. (C) Simulated flow conditions in the device at different flow rates (3 and 10 μL/min). (D) Quantification of cell trapping in different microwells at various flow rates (3, 5, 7, 10, and 20 μL/min) and different loading times (1, 2, and 3 min) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
individual range of diameters in various microwells (100-μm: 78–86 μm tumors; 200-μm: 160–180 μm tumors; 300-μm: 240–280 μm tumors; 400-μm: 330–370 μm tumors; 500-μm: 380–460 μm tumors; 600-μm: 460–540 μm tumors) was totally over 80% after 2 days of culture. Microfluidic U251 tumor cultivation exhibited similar achievements (Fig. S3) as the former (Fig. 4C). In addition, high repeatability and reproducibility of multi-size 3D tumor production (Fig. S6) suggest that the microfluidic device enabled controllable, parallel, and massive generation of 3D tumors with uniform geometry. Multi-size 3D tumors could be array-like fabricated in the single device, which is indispensable for convenient, parallel, and throughput evaluation of various tumor-associated events like tumor chemotherapy and targeted drug delivery.
outer zone (especially the curved surface) of the 3D tumors with various sizes. In vitro tumors with diameter of over 150 μm had been demonstrated to be similar to in vivo tumor tissue with diffusion limitation to many molecules, especially O2 meaning hypoxia [3,5,35]. Inefficient mass transportation also results in metabolic waste accumulation and causes cell quiescence inside these cultured tumors, which subsequently increase the resistance to chemotherapy [4,5]. In this part of experiment, the results indicate that the functional gradient-like cell phenotypes from the outer into the center of tumors were successfully simulated in the microfluidic device. Such 3D tumors are thought to reflect the in vivo pathology of tumors more realistically than 2D cell cultures [26]. Additionally, on-chip actin filament visualization of cells using TRITC-phalloidin staining presented that the cultured 3D tumors had intact cytoskeletons and a high degree of cell organization with no intercellular spaces between cells (Fig. S5). The size uniformity of the arrayed tumors statistically benefits throughput on-chip tumor analysis. Further, the size distribution of 3D tumors in culture was quantified for accurate analysis of tumor production (Fig. 4C). The percentage of multi-size HepG2 tumors with the
3.4. Anticancer drug resistance in multi-size 3D tumors One important application of 3D tumors cultured in vitro is to conduct preclinical screening for determine anticancer drug efficacy in drug discovery and tumor therapy. An forceful explanation is 3D tumors 115
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 3. Multi-size 3D tumor production in the microfluidic platform. HepG2 and U251 tumors with different sizes were gradually formed during the 4 days cultivation in the respective microwells.
Fig. 4. Evaluation of tumor cultivation in the device. (A) Cell viability in multi-size HepG2 tumors. Live/dead (green/red) cells were visualized by FDA/PI staining. (B) HepG2 tumor growth in the microwells with different diameters (100–600 μm). (C) Size distributions of multi-size HepG2 tumors cultured for 2 d in the microwells with different diameters (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 116
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Fig. 5. Microfluidic chemotherapy in multi-size 3D HepG2 tumors. Two anticancer drugs (BLM and TPZ) with various concentrations (50, 100, 200, and 300 μM) were used for evaluating the chemosensitivity of tumors with different sizes. The drug stimulation was kept for 3 d and the control culture was set accordingly. Live/ dead (green/red) cells were visualized by FDA/PI staining (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
117
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
present more biomimetic than 2D monolayer cultured tumor cells [36]. The presentation of hypoxic regions in most 3D tumors increases the resistance to therapeutic treatments. Hypoxic diversity and structural complexity of 3D tumors with different diameters make themselves different responses to the clinical chemotherapy [18,37]. For orthogonal evaluation of the tumor chemosensitivity, we stimulated multisize 3D tumors using the respective chemotherapeutic drugs (BLM and TPZ) with various concentrations ranging from 50 to 300 μM in the parallel microfluidic chambers in the single devices. Two anticancer drugs have different mechanisms of action and require different oxygen conditions for effective tumor therapy (BLM with normoxic tumor killing and TPZ with hypoxic tumor killing) [38,39]. The aforementioned results suggest that drug molecules can reach the tumor tissue basically from all around, especially from the curved side. For the specific type of either HepG2 or U251 tumors, the multiple concentration-related parallel chemotherapy, as well as the control assay could be performed in the single device because of its parallel fluidic design. The drug treatment on different tumors was maintained for 3 d in the microfluidic device. The optical recording and fluorescent characterization of tumors and their viability were applied for the further quantitative evaluation of chemosensitivity of multi-size 3D tumors. The experimental results were as shown in Figs. 5 and 6. In general, the higher anticancer drug concentration caused a more significant effect of chemotherapy in 3D tumors (Fig. 5 and Figs. S7–S10). The chemotherapy (i.e., the percentage of cell death) analysis was based on the PI staining for dead cell identification and quantification of fluorescence distribution. Most cell deaths happened in the curved side of tumors due to the spatially prior cell-drug contact. The quantitative comparison reveals that the size of tumors could positively impact their drug resistance (Fig. 6). For examples, more than 45% cells in the smallest HepG2 tumors in the 100-μm microwells were dead after 3 days of BLM treatment (300 μM), whereas less than 30% cells in the bigger tumors in the 500 and 600-μm microwells were killed after the same treatment (Fig. 6A). Meanwhile, TPZ-treated HepG2 tumors exhibited similar tendency of viability responses, but their chemotherapy especially in bigger tumors was more effective than the BLM stimulation (Fig. 6B). We explained that this result was probably owing to the hypoxia-selective toxicity of TPZ in bigger tumors, which had more hypoxic tumor cells than smaller tumors. In addition, BLM treatment on U251 tumors induced much higher apoptosis than HepG2 tumors in the microwells with same diameter (Fig. 6C). Significantly, treatment using a high concentration (300 μM) of BLM was able to kill over 65% and 50% cells in U251 tumors in the 100 and 500-μm microwells respectively. It is noteworthy that there were about 10% cell death in the control cultures of U251 tumors. Most of them were clearly located in the center of multi-size tumors in the microwells (particular 500 and 600 μm), implying that long-term microfluidic cultivation of large size of 3D tumors might generate in vivo-like complicated tissue structure consisting of a necrotic core, an inner quiescent cell layer and an outer proliferating zone. All of the results demonstrate that the size of tumor besides their tissue composition and 3D organization determines chemotherapeutic resistance in vitro. The therapeutic and chemosensitive evaluation confirmed the applicability and feasibility of microfluidics for multi-size 3D tumor culture-based throughput analysis, which would be extremely useful for the investigation of many tumor-associated pathophysiological and preclinical anticancer events.
Fig. 6. Therapeutic efficacy of anticancer drugs with different concentrations (50, 100, 200, and 300 μM)) in multi-size 3D tumors. (A) BLM treatment in HepG2 tumors in the microwells with different diameters (100–600 μm). (B) TPZ treatment in HepG2 tumors in different microwells. (C) BLM treatment in U251 tumors in different microwells. Chemotherapy corresponds to the percentage of cell death.
4. Conclusions In summary, we developed a facile, massive, and controllable production process for simultaneously generating multi-size biomimetic 3D tumors using a simple-fabricated and well-established microfluidic system. We also presented an experimental demonstration of its applicability and feasibility in studying orthogonal chemotherapy and drug resistance of multi-size tumors with parallel and throughput, i.e., 4 days of tumor culture, 3 days of chemotherapeutic assay/detection, and
at least 288 (tumors)×4 (cell localization, tumor formation/growth, tumor size uniformity, tumor characterization after culture or drug stimulation) results per total assay time in the single device. On-chip manipulation including cell localization, array-like tumor formation, and on-line analysis of multiple types of 3D tumors can be completed smoothly in the system. We verified that the stable microfluidic 3D 118
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
tumor operations was partly based on a systematic optimization of tumor cell localization in various fluidic conditions. Meanwhile, highly efficient multi-size tumor formation with quantitative-uniformity was experimentally proven in single microfluidic devices. Optical monitoring of response dynamics for the quantitative culture and anticancer evaluation was shown to be very powerful in the PDMS device. The results support the importance of size and composition of 3D tumors with a complex tissue microenvironment while studying oncotherapy and chemosensitivity. We envision that this microscale and throughput microfluidic approach with simple, robust, and long-term manipulative properties is potentially valuable for biological/medical researchers to better perform assays that investigate tumorigenesis and anticancer drug resistance. We believe that this type of platform would greatly promote the development of miniaturized tools for integrated biological analysis with biomimetic, microscale, and high-throughput capabilities.
[19] L.F. Yu, M.C.W. Chen, K.C. Cheung, Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing, Lab Chip 10 (2010) 2424–2432. [20] A. Zuchowska, K. Marciniak, U. Bazylinska, E. Jastrzebska, K.A. Wilk, Z. Brzozka, Different action of nanoencapsulated meso-tetraphenylporphyrin in breast spheroid co-culture and mono-culture under microfluidic conditions, Sensor. Actuat. BChem. 275 (2018) 69–77. [21] C. Kim, J.H. Bang, Y.E. Kim, S.H. Lee, J.Y. Kang, On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network, Lab Chip 12 (2012) 4135–4142. [22] H. Ota, R. Yamamoto, K. Deguchi, Y. Tanaka, Y. Kazoe, Y. Sato, N. Miki, Threedimensional spheroid-forming lab-on-a-chip using micro-rotational flow, Sensor. Actuat. B-Chem. 147 (2010) 359–365. [23] K.J. Chen, M.X. Wu, F. Guo, P. Li, C.Y. Chan, Z.M. Mao, S. Li, L. Ren, R. Zhang, T.J. Huang, Rapid formation of size-controllable multicellular spheroids via 3D acoustic tweezers, Lab Chip 16 (2016) 2636–2643. [24] M. Van Glabbeke, J. Verweij, P.G. Casali, A. Le Cesne, P. Hohenberger, I. RayCoquard, M. Schlemmer, A.T. van Oosterom, D. Goldstein, R. Sciot, P.C.W. Hogendoorn, M. Brown, R. Bertulli, I.R. Judson, Initial and late resistance to imatinib in advanced gastrointestinal stromal. Tumors are predicted by different prognostic factors: a European organisation for research and treatment of cancerItalian Sarcoma group-Australasian Gastrointestinal Trials group study, J. Clin. Oncol. 23 (2005) 5795–5804. [25] R.S. Kerbel, A cancer therapy resistant to resistance, Nature 390 (1997) 335–336. [26] W. Lim, H.H. Hoang, D. You, J. Han, J.E. Lee, S. Kim, S. Park, Formation of sizecontrollable tumour spheroids using a microfluidic pillar array (μFPA) device, Analyst 143 (2018) 5841–5848. [27] M. Marimuthu, N. Rousset, A. St-Georges-Robillard, M.A. Lateef, M. Ferland, A.M. Mes-Masson, T. Gervais, Multi-size spheroid formation using microfluidic funnels, Lab Chip 18 (2018) 304–314. [28] Y. Xia, G.M. Whitesides, Soft Lithography, Angew. Chem. Int. Ed. 37 (1998) 550–575. [29] S. Shen, C. Tian, T. Li, J. Xu, S.W. Chen, Q. Tu, M. Yuan, W. Liu, J. Wang, Spiral microchannel with ordered micro-obstacles for continuous and highly-efficient particle separation, Lab Chip 17 (2017) 3578–3591. [30] K. Moshksayan, N. Kashaninejad, M.E. Warkiani, J.G. Lock, H. Moghadas, B. Firoozabadi, M.S. Saidi, N. Nguyen, Spheroids-on-a-chip: recent advances and design considerations inmicrofluidic platforms for spheroid formation and culture, Sensor. Actuat. B-Chem. 263 (2018) 151–176. [31] S. Raghavan, R.A. Desai, Y. Kwon, M. Mrksich, C.S. Chen, Micropatterned dynamically adhesive substrates for cell migration, Langmuir 26 (2010) 17733–17738. [32] S. Breslin, L. O’Driscoll, Three-dimensional cell culture: the missing link in drug discovery, Drug Discov. Today 18 (2013) 240–249. [33] N. Bhattacharjee, N. Li, T.M. Keenan, A. Folch, A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors, Integr. Biol. (Camb) 2 (2010) 669–679. [34] X. Xiao, Z.Y. Han, Y.X. Chen, X.Q. Liang, H. Li, Y.C. Qian, Optimization of FDA-PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa, Phys. Chem. Earth (2002) 36 (2011) 424–429. [35] C.R. Thoma, M. Zimmermann, I. Agarkova, J.M. Kelm, W. Krek, 3D cell culture systems modeling tumor growth determinants in cancer target discovery, Adv. drug deliver. Rev. 69 (2014) 29–41. [36] J. Friedrich, C. Seidel, R. Ebner, L.A. Kunz-Schughart, Spheroid-based drug screen: considerations and practical approach, Nat. Protoc. 4 (2009) 309–324. [37] G. Mehta, A.Y. Hsiao, M. Ingram, G.D. Luker, S. Takayama, Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy, J. Control. Release 164 (2012) 192–204. [38] G. Chowdhury, U. Sarkar, S. Pullen, W.R. Wilson, A. Rajapakse, T. Fuchs-Knotts, K.S. Gates, DNA strand cleavage by the phenazine Di-N-oxide natural product myxin under both aerobic and anaerobic conditions, Chem. Res. Toxicol. 25 (2012) 197–206. [39] L. Wang, W.M. Liu, L. Wang, J.C. Wang, Q. Tu, R. Liu, J. Wang, Construction of oxygen and chemical concentration gradients in a single microfluidic device for studying tumor cell-drug interactions in a dynamic hypoxia microenvironment, Lab Chip 13 (2013) 695–705.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31470971, No. 21375106, No. 31100726), the Fundamental Research Funds for the Central Universities (202045001), and Central South University. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.04.121. References [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (2018) 394–424. [2] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2015) 87–108. [3] F. Hirschhaeuser, H. Menne, C. Dittfeld, J. West, W. Mueller-Klieser, L.A. KunzSchughart, Multicellular tumor spheroids: an underestimated tool is catching up again, J. Biotechnol. 148 (2010) 3–15. [4] S. Sant, P.A. Johnston, The production of 3D tumor spheroids for cancer drug discovery, Drug Discov. Today Technol. 23 (2017) 27–36. [5] R.Z. Lin, H.Y. Chang, Recent advances in three-dimensional multicellular spheroid culture for biomedical research, Biotechnol. J. 3 (2008) 1172–1184. [6] B.A. Justice, N.A. Badr, R.A. Felder, 3D cell culture opens new dimensions in cellbased assays, Drug Discov. Today 14 (2009) 102–107. [7] E. Fennema, N. Rivron, J. Rouwkema, C. van Blitterswijk, J. de Boer, Spheroid culture as a tool for creating 3D complex tissues, Trends Biotechnol. 31 (2013) 108–115. [8] C.T. Kuo, J.Y. Wang, Y.F. Lin, A.M. Wo, B.P.C. Chen, H. Lee, Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array, Sci. Rep. 7 (2017) 4363. [9] P. Benien, A. Swami, 3D tumor models: history, advances and future perspectives, Future Oncol. 10 (2014) 1311–1327. [10] C. Fischbach, R. Chen, T. Matsumoto, T. Schmelzle, J.S. Brugge, P.J. Polverini, D.J. Mooney, Engineering tumors with 3D scaffolds, Nat. Methods 4 (2007) 855–860. [11] S.R. Khetani, S.N. Bhatia, Engineering tissues for in vitro applications, Curr. Opin. Biotechnol. 17 (2006) 524–531. [12] Y. Xie, W. Zhang, L. Wang, K. Sun, Y. Sun, X. Jiang, A microchip-based model wound with multiple types of cells, Lab Chip 11 (2011) 2819–2822. [13] S.P. Zhao, Y. Ma, Q. Lou, H. Zhu, B. Yang, Q. Fang, Three-dimensional cell culture and drug testing in a microfluidic sidewall-attached droplet array, Anal. Chem. 89 (2017) 10153–10157. [14] K.E. Sung, D.J. Beebe, Microfluidic 3D models of cancer, Adv. Drug Deliv. Rev. 79–80 (2014) 68–78. [15] H. Hardelauf, J.P. Frimat, J.D. Stewart, W. Schormann, Y.Y. Chiang, P. Lampen, P. Lampen, J. Franzke, J.G. Hengstler, C. Cadenas, L.A. Kunz-Schughartc, J. West, Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits, Lab Chip 11 (2011) 419–428. [16] Q. Zhang, T. Liu, J. Qin, A microfluidic-based device for study of transendothelial invasion of tumor aggregates in realtime, Lab Chip 12 (2012) 2837–2842. [17] C. Zheng, L. Zhao, G. Chen, Y. Zhou, Y. Pang, Y. Huang, Quantitative study of the dynamic tumor-endothelial cell interactions through an integrated microfluidic coculture system, Anal. Chem. 84 (2012) 2088–2093. [18] K.P. Valente, S. Khetani, A.R. Kolahchi, A. Sanati-Nezhad, A. Suleman, M. Akbari, Microfluidic technologies for anticancer drug studies, Drug Discov, Today 22 (2017) 1654–1670.
Wenming Liu received his Ph.D. degree from Northwest A&F University in 2010. Currently, he is working as a research director at School of Basic Medical Science, Central South University. His research work focuses on the intersection of micro-engineering, microfluidics, and cell biology to develop novel strategies and original platforms for exploring life science. Boxiu Lu obtained her B.S. degree from Taishan Medical University in 2011. She is now working as a staff of medical imaging at People's Hospital of Qihe County, Shandong, China. Her research interests aim at cancer-related biological imaging. Mingming Yan obtained his M.S. degree from Northwest A&F University in 2017. He is currently our lab analyst. His research interests include microfluidics-based cell imaging and real-time analysis. Kai Han obtained his B.S. degree from University of Jinan in 2018. He is studying as a postgraduate student at School of Basic Medical Science, Central South University. His research interests include developing microfluidic tumor cell platforms and their applications.
119
Sensors & Actuators: B. Chemical 292 (2019) 111–120
W. Liu, et al.
Meilin Sun obtained her B.S. degree from Yuncheng University in 2018. She is studying as a postgraduate student at School of Basic Medical Science, Central South University. Her research interests include developing microfluidic systems for cell trapping and 3D tumor simulation.
Jinyi Wang received his Ph.D. degree from Lanzhou University in 2002. He is currently a research director at College of Chemistry & Pharmacy, Northwest A&F University. His research interests include constructing the novel and sensitive methods to detect the harmful ions and molecules in the agricultural environment, and constructing the integrated microfluidic systems for life science.
120