Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity

Three-dimensional (3D) spheroidal cell cultures are now recognised as better models of cancers as compared to traditional cell cultures. However, established 3D cell culturing protocols and techniques are time-consuming, manually laborious and often expensive due to the excessive consumption of reagents. Microfluidics allows for traditional laboratory-based biological experiments to be scaled down into miniature custom fabricated devices, where cost-effective experiments can be performed through the manipulation and flow of small volumes of fluid. In this study, we characterise a 3D cell culturing microfluidic device fabricated from a 3D printed master. HT29 cells were seeded into the device and 3D spheroids were generated and cultured through the perfusion of cell media. Spheroids were treated with 5-Fluorouracil for five days through continuous perfusion and cell viability was analysed on-chip at different time points using fluorescence microscopy and Lactate dehydrogenase (LDH) assay on the supernatant. Increasing cell death was observed in the HT29 spheroids over the five-day period. The 3D cell culturing microfluidic device described in this study, permits on-chip anti-cancer treatment and viability analysis, and forms the basis of an effective platform for the high-throughput screening of anti-cancer drugs in 3D tumour spheroids.

www.nature.com/scientificreports/ in a 3D structure also experience different mechanical and topographical forces, than those growing on a 2D planar surface 8 . The overall benefit of 3D spheroidal models is that they offer a better understanding of in vivo molecular mechanisms involved in tumour growth and drug resistance 9 . Taken together, 3D spheroids offer a better representation of in vivo environments and microfluidic devices offer convenient and cost-effective platforms for high-throughput screening applications. A common limitation among established 3D cell culturing techniques is the manual effort required to grow and maintain 3D spheroid cultures in non-specialised conventional commercial tissue culturing equipment. An appropriately designed microfluidic platform can improve the success rate of establishing and characterising 3D spheroids by reducing the amount of effort required for cell culturing, administering cytotoxic treatments and evaluating the outcomes of treatment-conveniently within a single device 5,[10][11][12][13][14][15][16] .
In this article, we describe the development and application of a 3D cell culturing microfluidic flow platform and the use of 3D printing to create a master from which polydimethylsiloxane (PDMS) flow chips can be conveniently cast. We demonstrate the platform by culturing 3D spheroids, treating with the anti-cancer drug, 5-Fluorouracil (5-FU) and evaluating cytotoxicity through flow. We show that the 3D cell culturing microfluidic platform we have developed can accommodate the generation of 3D spheroids from cell aggregates 'on-chip' , administer cytotoxic treatment under limited-and continuous perfusion conditions and perform quantitative and qualitative cell viability analysis.

Results
The microfluidic device described in this study utilises the non-cell adherent properties of the PDMS flow chip located between two layers of Poly(methyl methacrylate) (PMMA), to generate 3D spheroids (Fig. 1A) 17 . Placing a cell seeding port directly above the cell culturing wells was vital to improving the efficiency of introducing cells to the wells. The port was added after it was identified that perfusing a HT29 cell suspension through the device to seed the wells was not effective ( Fig. 2A,B). Seeding cells through direct deposition via the cell seeding port, results in 3D spheroids forming in 100% of the wells, as compared to 68% using flow-driven cell seeding (p = 0.02) (Fig. 2C). An equivalent number of HT29 cells, (mean = 377 cells, min. = 361 cells, max. = 386 cells), were deposited into each well to generate 3D spheroids (Fig. 2D). The central region of the PDMS flow chip was designed with a 5 × 5 array of concave-shaped spheroid culturing wells (Fig. 1B). The concave-shaped wells were designed to aid in the cell seeding process, by assisting cells in initially forming cell aggregates, which is vital for successfully generating 3D spheroids (Fig. 3).
As depicted in Fig. 4A, after seeding the HT29 cells into the wells of the PDMS flow chip, cell aggregates formed within a day (Day 0) and subsequent culturing under static conditions, resulted in 3D spheroids forming by Day 2. The HT29 spheroids then continued to mature in density, roundness in spheroidal structures and grew in overall size. A 148% (p < 0.0001), 89% (p < 0.0001) and 58% (p < 0.0001) increase in spheroid volumes were found between Day 2 versus Day 4, Day 4 versus Day 7 and Day 7 versus Day 10 respectively (Fig. 4B). Following 10 days of culture, scanning electron microscopy confirmed the spheroidal structures of the HT29 spheroids (Fig. 4C).
Using the microfluidic platform, further experiments were carried out to highlight the (1) administration of an anti-cancer drug to 3D HT29 spheroids and (2) evaluate cytotoxicity induced by the anti-cancer drug through the microfluidic device. For limited-perfusion experiments, a single syringe pump was connected to the microfluidic devices and was used to drive flow (Fig. 5A,B). Briefly, 3D HT29 spheroids were generated on chip and subjected to 5-FU treatment (500 µM) through perfusion. After 24 h under non-flow static conditions, cell viability analysis showed that 5-FU treated HT29 spheroids were 70% less viable, as compared to untreated spheroids (p = 0.0001) (Fig. 5C). Fluorescent imaging highlighting cell death confirmed cytotoxicity in 5-FU treated spheroids, as indicated by the uptake of propidium iodide and lack of dye uptake in untreated HT29 spheroids (Fig. 5D). Quantifying propidium iodide fluorescence also confirmed cytotoxicity in 5-FU treated spheroids with no toxicity detected in untreated spheroids (p < 0.0001) (Fig. 5E). In addition, a 20% reduction in Hoechst 33342 fluorescence in treated spheroids, as compared to untreated spheroids (p = 0.028) indicated the loss and shedding of cells following 5-FU treatment (Fig. 5E).
Microfluidic device-cultured HT29 spheroids were also subjected to 5-FU treatment (200 µM) through continuous perfusion. The microfluidic devices were connected in parallel to reagent-loaded syringes through a peristaltic pump (Fig. 6A,B). Cell viability was monitored over 5 days and a time dependant reduction in cell viability was observed (p < 0.0001) (Day 0 = 100% cell viability and Day 5 = 15% cell viability, p < 0.0001) (Fig. 6C). Fluorescent imaging confirmed results of the LDH assay, as indicated by the increasing uptake of propidium iodide over time (Fig. 6D). In addition, quantifying propidium iodide fluorescence also showed significant cytotoxicity over the 5 days (Day 0 vs. Day 5: p < 0.0001) (Fig. 6E). A 16% reduction in Hoechst 33342 fluorescence was also observed at Day 5, as compared to Day 0, indication the loss and shedding of cells (p = 0.0003) (Fig. 6E).

Discussion
Three-dimensional in vitro spheroidal models of cancer are recognised as more accurate representations of the tumour microenvironment as compared to 2D monolayers, and responses to treatment in 3D cultured cells are more reflective of responses observed in vivo 8 . Combined microfluidics and 3D spheroid culturing has allowed conventional laborious 3D cell culturing protocols to be scaled down onto custom micro-fabricated devices. This allows the generation, culturing, treatment and analysis of 3D spheroids to be performed conveniently on-chip through fluid perfusion [18][19][20][21][22][23] , with the additional practical advantages of reduced experimental and consumables costs and reduced manual labour 23 .
The 3D cell culturing microfluidic device described in this paper, builds upon previous work and is based upon the 'non-cell adherent' technique for generating 3D spheroids 10,24-27 . Cells are seeded on a non-adherent  www.nature.com/scientificreports/ SU-8 based microfluidic platforms are also commonly used for 3D cell culturing systems, however the chemical treatment of SU-8 to produce hydrophobic surfaces requires dangerous and toxic chemicals to be used 42 . We employed 3D printing and PDMS casting, which does not require surface treatment or bonding to other materials, making the fabrication of the flow chips inherently simple. The flow chips were cast from a mould designed with concave-shaped spheroid-culturing wells to facilitate the aggregation of cells and assist in the generation of 3D spheroids, as has been described previously for 3D cell culturing platforms 43,44 . Due to the nature of the photocuring method for forming the 3D prints, the master mould is built in layers forming 'steps-like' structures as an approximation to the convex well to make the 3D cell culturing wells (Fig. 7). These structures were nevertheless still successful in generating spheroids in the polygonal concave-shaped wells. Our method for making 3D spheroid culturing chips is effective, yet simple enough to allow biologists and non-microfluidics specialists to produce and operate these chips in their laboratories without the need of a cleanroom or delicate microfabrication/assembly steps. The PDMS-based spheroid culturing chip was designed to accommodate the culture of twenty-five spheroids (Fig. 1B). As a prototype design, we limited culturing to twenty-five spheroids, yet retain reliability and simplicity in generating 3D spheroids, as compared to conventional 96-well plates. However, the advantage of our design is such that the 5 × 5 array of spheroid-culturing wells, could easily be expanded to accommodate the simultaneous culturing of larger numbers of 3D spheroids. A limitation of previously reported 3D cell culturing microfluidic platforms is that they have relied on external 3D cell culturing methods, such as the use of ultra-low attachment plates or the hanging drop method, to generate spheroids off-chip before being introduced into the microfluidic platforms 5,13,35,36,40 . The microfluidic platform described in this study eliminates the need for generating 3D spheroids beforehand and allows non-aggregated cells to be seeded directly into the device, with equivalent numbers of cells seeded into each well (Fig. 2D). We found the seeding of cells through flow, to be inefficient in generating spheroids. For biomedical applications, the use of a pipette to seed cells in an exact location on a microfluidic chip is an advantage when accurately producing spheroids. This user-friendly approach is also compatible in conventional biology laboratories. For our experiments, the human colorectal adenocarcinoma cell line, HT29, was used to generate 3D spheroids. HT29 has been used extensively in 3D cell culturing based studies, and from previous multiple studies, we have found HT29 to form spheroidal cultures with relative ease [45][46][47][48] . As demonstrated in Figs. 2A and 8, we found the microfluidic platform presented here, enables HT29 cells to form uniformly sized spheroidal structures. 3D spheroids grew to a maximum of around 250 μm in diameter, dictated by the diameters of the spheroid-culturing wells. Typically, HT29 3D spheroids with ≥ 300 μm diameters, possess hypoxic and necrotic cores with physiological gradients of cell proliferation (outer rapidly proliferating and inner non-proliferating cells) 49 . It could therefore be postulated, that the HT29 3D spheroids grown in this study did not possess the in vivo like physiological gradients or hypoxic/necrotic cores.
Our device was additionally designed to streamline high-throughput chemosensitivity experiments and improve the overall efficiency of 3D cell culturing. To demonstrate this, we used the chemotherapeutic agent, 5-Fluorouracil, to treat the HT29 spheroids. As 5-FU is currently the primary anti-cancer drug used to treat colorectal cancers 50 , it makes for an excellent model system and demonstrates the relevant real-world application of our device. Following the culturing of 3D spheroids, treatment and analysis could be performed conveniently on-chip, through the perfusion of anti-cancer drugs and other culture media. Fluorescent imaging (qualitative  www.nature.com/scientificreports/ makes use of a single 3D cell culturing microfluidic platform, to administer an anti-cancer treatment and permit subsequent on-chip viability imaging confirmed by an LDH assay. In our study, we demonstrated two different setups for performing chemosensitivity experiments in the microfluidic devices. The first utilised a single syringe pump to drive flow through one device at a time (Fig. 5). This limited-perfusion setup permits simple experiments to be conducted with a single experimental endpoint. However, experiments can also be performed with continuous flow as demonstrated by the second setup utilising a multichannel pump (Fig. 6). An advantage of continuous flow setup is that it better models the physiological flow of systemic circulation in the body. This second setup also enables multiple devices to be run simultaneously in parallel. Each channel is controlled independently, therefore flow through one microfluidic device can be switched off without disrupting flow through the rest of the channels or devices. In addition, the continuous flow setup was designed to be placed into a conventional tissue culturing incubator, permitting prolonged experiments (lasting multiple days) to be performed using existing laboratory infrastructure and environmental standards.

conclusion
In this study, we have developed and evaluated a 3D cell culturing microfluidic device. The device comprises a layer of PDMS with concave 3D cell culturing wells sandwiched between two layers of transparent PMMA, allowing for direct visualization. HT29 colorectal cancer cells in suspension were seeded into the wells through the central port on the top PMMA layer and subsequently formed into 3D spheroids. These spheroids were www.nature.com/scientificreports/ treated through one-time or continuous perfusion of the anti-cancer drug, 5-Fluorouracil, and the resulting cell viability was analysed using fluorescent microscopy and an LDH assay. The two perfusion regimes simulated different physiological flow conditions and enabled the evaluation of distinct methods of administering anti-cancer treatment. Finally, the microfluidic device described here offers a convenient, efficient and quick platform for generating, culturing, treating and analysing 3D cancer spheroids and will facilitate high-throughput screening of anti-cancer drugs on complex 3D in vitro models.

Methods
Designing and fabricating the microfluidic devices. A single microfluidic device consists of a central PDMS chip consisting of a single straight flow channel joining the inlet to outlet, with an array of 3D cell culturing wells located towards the centre. This is sandwiched between two layers of transparent PMMA allowing for direct optimal imaging. Schematic designs for the different parts of the flow device were drawn in SolidWorks (Dassault Systèmes, Vélizy-Villacoublay, France). To create the PDMS flow chips, the inverse design was printed as a reusable master mould on an EnvisionTEC 3D printer (EnvisionTEC GmbH, Gladbeck, Germany) in a photocurable acrylonitrile butadiene styrene (ABS) thermoplastic polymer. The well-forming hemispherical "pim- www.nature.com/scientificreports/ ples" in the master mould are 250 µm in diameter located in 5 × 5 array, on a pitch of 500 µm × 500 µm (Fig. 7). Chips were then cast from the master mould using PDMS with a release agent to aid removal. The PDMS flow chips were fixed between two layers of PMMA. The top layer of PMMA was fabricated with fluid inlet and outlet ports and a cell seeding port in the centre, while the bottom layer of PMMA serves as the device base (Fig. 1). The PMMA layers were machined from cast sheet, with inlet and outlet ports positioned to match the ends of the PDMS chip and machined to take standard UNF 1/4-28 flangeless fittings. An additional port was machined into the top layer of PMMA, located directly above the 3D cell culturing wells. The microfluidic device was fashioned together using four bolts, one in each corner, to hold the assembly together. Inlet and outlet ports were connected to tubing using flangeless fittings and a blanking plug was fitted to the central port to allow direct access to the cell culturing wells (Fig. 1). Following assembly, the microfluidic device was sterilised using a steam and high-pressured autoclave prior to the addition of cells.
Preparation of cancer cells. Culturing 3D tumour spheroids in a microfluidic device. Cells were washed and trypsinised to create a cell suspension of 2 × 10 7 cells/mL in cell medium. 20 μL of cell suspension was deposited into the wells of assembled PDMS chips. The chips were then incubated and maintained under static conditions at 37 °C/5% CO 2 /95% RH. All cell media was changed daily using a pipette. Manual cell counting and acquisition of the images of spheroids was performed using the EVOS FL Imaging System (Life Technologies). For scanning electron microscopy (SEM), spheroids were cultured for 10 days then briefly washed with DPBS, fixed in 2.5% (v/v) glutaraldehyde for 20 min and subjected to increasing gradients of ethanol dehydration (25%, 40%, 60%, 80%, 90% and 100%). The top surface of the PDMS flow chip was then coated with 4 nm of iridium and imaged using a FEI Nova NanoSEM450 scanning electron microscope (Thermo Fisher).  www.nature.com/scientificreports/

5-FU treatment (Limited-perfusion conditions).
Two different flow conditions were used to study chemosensitivity in 3D HT29 spheroids. For the first (limited-perfusion) condition, 48 h after seeding cells, 500 µM 5-FU was prepared in cell media and perfused through the microfluidic device at 20 µL/min using the single-syringe pump. This approach can only accommodate one device at a time. Following the perfusion of 500 µL of 5-FU (20 µL/min) completely through the microfluidic device, lasting 25 min, flow was stopped and the device was detached from the pump and placed back into incubation (37 °C/5% CO 2 /95% RH). The spheroids were incubated with 5-FU under static conditions for an additional 24 h. Evaluating cytotoxicity in 3D tumour spheroids. Lactate dehydrogenase (LDH) assay. Spent cell culture media supernatant was collected from the microfluidic device and stored at − 20 °C until the end of the continuous flow 5-FU treatment period. The LDH cytotoxicity assay was performed using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher) and carried out as per the manufacturer's instructions and guidelines. This method has been previously applied to evaluate the cell viability of tumour tissue specimens cultured on a microfluidic platform [51][52][53] .

5-FU treatment (Continuous perfusion conditions
Fluorescent imaging. 5 μg/mL Hoechst 33342 and 1.3 μg/mL Propidium Iodide was prepared in cell media, and flowed into the device at 20 µL/min. Spheroids were incubated with the dyes for 30 min, washed with DPBS www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.