DOUBLE-LAYER HEPATOCYTE TUMOR CO-CULTURE USING HYDROGEL FOR DRUG EFFECTIVITY AND SPECIFICITY ANALYSIS L. K. Chin1, K. Q. Luo2, W. Park3, M. Je3, J. Tsai3, D. L. Kwong3 and A. Q. Liu1*

1School of Electrical & Electronic Engineering, Nanyang Technological University, SINGAPORE 639798 2School of Biomedical & Chemical Engineering, Nanyang Technological University, SINGAPORE 637457

3Institute of Microelectronics, A*STAR, SINGAPORE 117658

ABSTRACT This paper presents the formation of double-layer

cellular tumor spheroid for the co-culture of hepatocyte (HepG2 cancer liver cell and LO2 normal liver cell) using alginate hydrogel. With the microfluidic chip, double-layer cellular tumor spheroid can be formed by the hydrogel droplet to mimic the in vivo environment in which the cancer cell is surrounded by normal cell. A microchip with concentration gradient generation and tumor spheroid trapping site is designed for anti-cancer drug analysis. INTRODUCTION

Hydrogels are commonly used for cell encapsulation and has been a common tool to form tumor spheroids [1 – 2]. Conventionally, hydrogel tumor spheroids are formed by hanging drops [3 – 4]. However, it has limited control over the droplet size and content.

Previously demonstrated in vitro tumor spheroid formation schemes are either single cellular spheroid or multicellular spheroid [5 – 6]. However, for tumor spheroid under in vivo environment, the tumor cells

are encapsulated by normal cells as shown in Fig. 1. By using this model for anticancer drug analysis, the drug effectiveness and specificity can be better analyzed. This model can be easily realized using microfluidic technology, which provides a robust way in liquid and sample manipulations for different applications [7 – 8].

Recently, droplet microfluidics has emerging as an attractive research area to realize digital and compartmentalized microfluidic systems [9 – 10]. It consists of a continuous phase and a dispersed phase, which are immiscible. Droplets or plugs of the dispersed phase can be formed in the microchannel via a hydrofocusing structure [11] or a T-junction [12] based on flow instabilities and dynamics. Multiphase flow in microfluidic system has been designed to encapsulate cancer cells in hydrogel droplet and form a tumor spheroid [13 – 14]. Hydrogel droplets can be formed using a flow-focusing mechanism with highly uniform diameters (< 5%) [15]. By employing microfluidic technology, the droplet size and content can be easily controlled.

In this paper, two microfluidic chips are designed, one for the formation of double-layer alginate hydrogel droplet where the cancer cells are encapsulated in the core and the external cladding layer is formed with normal cells; and another for the trapping of the formed hydrogel droplets where the cells in the hydrogel droplets are cultured as tumor spheroids and subsequent anti-cancer drug analysis can be performed.

DESIGN AND FABRICATION

The schematic illustration of the double-layer cellular alginate droplet generation in the microfluidic environment is shown in Fig. 2(a). A 5-inlet junction is designed in which HepG2 cell (cancer liver cell) is injected in the central channel, LO2 (normal liver cell) is injected from the two side branches such that the HepG2 cells are focused in the center by the LO2 cells, and mineral oil is injected in the two outer side branches to shape the cell flow streams into plugs. At the downstream, a sudden height expansion structure is designed to transform the cell plugs into cell droplets. Subsequently, the cell droplets are transferred to a calcium ion bath for gelation. After gelation, the double-layer cellular hydrogels are injected into the microchip as shown in Fig. 2(b) for trapping and culturing.

Figure 1: Schematic illustration of double-layer cellular tumour spheroid for drug analysis. Cancer cells are surrounded by normal cells where both cells are encapsulated in an alginate hydrogel. The alginate hydrogel facilitates the formation of tumor spheroid when the cells divide, and also the diffusion of culture medium and drug molecules.

Cancer cells

Normal cells

Drug molecules

Alginate hydrogel

978-1-4673-0325-5/12/$31.00 ©2012 IEEE 808 MEMS 2012, Paris, FRANCE, 29 January - 2 February 2012

The hydrogels are trapped in the microchannel with the trapping site. Each trapping sites consists of a circular pit, several micro-pillars at one pole and guided rail. When a single hydrogel droplet is flowing in the microchannel, the guided rail will guide it into the circular pit to increase the trapping efficiency. As the hydrogel droplet is flowing into the circular pit, the micro-pillars can block the hydrogel droplet from flowing downstream and the hydrogel droplet will be trapped in the circular pit. The circular pit can avoid the hydrogel droplet from escaping during medium exchange or vibration. Tumor spheroids are formed in the hydrogel after 3-day culture and the tumor spheroids can be tested with anti-cancer drugs, such as Etoposide, at different concentrations generated by the on-chip gradient generator.

The microfluidic chips are fabricated using standard soft-lithography techniques [16 – 17]. For the hydrogel

droplet formation microchip, the 5-inlet junction and the sudden height expansion are patterned using a 50-μm and 150-μm thick layer of SU8 master structure, respectively. Then, the mixture of poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning) prepolymer and curing agent (10:1) is poured over the masters, degassed, baked for 2 hours at 75 °C and then peeled off. After manually punching inlets and outlets, the PDMS slabs are exposed to air plasma for 15 s using a corona treater (BD-25, Electro-Technic Products), aligned and bonded. Similarly, the microchip used for tumor spheroid trapping and drug analysis is fabricated using the same process.

HepG2 and LO2 cells are maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin. All cells were cultured in flasks for 3 days prior to microfluidic experiments. Cells were

Figure 2: Schematic illustrations of (a) the chip design to form the double-layer cellular alginate hydrogel droplet, (b) the microchip for tumor spheroid trapping with concentration gradient generation for drug analysis, and (c) zoom view of the trapping site with trapping pillars and guided rail.

Mineral oil

LO2 cells + alginate solution

HepG2 cells + alginate solution

Cell plug

Sudden height expansion

Cell droplet

a

Pillars

Concentration gradient generation

b

c Guided rail

Tumor spheroid

Figure 3: Microphotos of (a) alginate droplet formation, (b) alginate droplet gelation, and (c) double-layer cellular hydrogel, and (d) tumor spheroid trapping.

a LO2 cells

HepG2 cells

LO2 cells

Mineral oil Cell plug

Gelation

c LO2 cells

HepG2 cells

d Guided rail

Pillars

Tumor spheroid

b

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detached from the culture flasks using 0.25% trypsin in PBS. Cell suspensions were prepared at a concentration of 107 cell/mL using DMEM medium mixed with 2.0 wt% alginate (Sigma-Aldrich). For gelation of alginate, 40 mM CaCl2 solution (Sigma-Aldrich) is prepared as the buffer bath. For droplet formation, the continuous phase used in the experiment is mineral oil with 1.0 wt% surfactant Span 80. Mineral oil is chosen due to its biocompatibility and low cytotoxicity. The hydrogel droplets are collected externally in the calcium ion bath and gelled. The gelled droplets are then loaded into the microfluidic chip and trapped in the trapping sites for cell culture.

RESULTS AND DISCUSSIONS

Figure 3(a) shows the microphotos of the formation of double-layer cellular hydrogels. HepG2 and LO2 cells are injected into the core and the cladding of the dispersed phase and a stream of cell plugs are formed at the junction due to the mineral oil stream by shear. Fig. 3(b) shows a group of cellular hydrogel droplets in the calcium ion bath. When the calcium ion is diffused into the hydrogel droplet, the alginate hydrogel is gelled

instantly. Fig. 3(c) shows the formation of the double-layer cellular hydrogel in which the HepG2 cells (red fluorescent) are surrounded by the LO2 cells (green fluorescent). The gelled hydrogel droplets are then loaded into the trapping microchip. Cellular hydrogel droplet is guided into the trapping site by the designed rail and trapped by the pillars as shown in Fig. 3(d). The cellular hydrogel droplets are restricted from escaping by the circular pits.

Viability test is performed using Calcein AM in which live cells are green fluorescent as shown in Fig. 3(a). Calcein AM is a non-fluorescent, hydrophobic compound which can be diffuse into the intact, live cells. The hydrolysis of Calcein AM by intracellular esterases produces calcein, which is a hydrophilic, strongly fluorescent compound. Calcein can be well-retained in the cell cytoplasm. 5 μM of Calcein AM is added to the culture medium and injected into the microchip with cultured tumor spheroids. The microchip is incubated in 37oC for 30 mins and subsequently fluorescence images are captured using confocal microscope.

The proliferation rate in the 3-day culture is shown in Fig. 3(b). The proliferation rate is calculated as the ratio between the number of new cells to the initial number of cells. By ensuring cell proliferation and remains viable, the system can be used to generate double-layer cellular tumor spheroid for anticancer drug analysis. CONCLUSIONS

In conclusion, a microfluidic chip is developed to form double-layer cellular tumor spheroid in alginate hydrogel and a microchip is designed for the culture and drug analysis of the tumor spheroid. HepG2 cells are surrounded by LO2 cells in the hydrogel to mimic the in vivo environment in which cancer cells are surrounded by normal cells. Cell proliferation and viability for the tumor spheroid in alginate hydrogel are investigated. The formed tumor spheroids can be tested with anti-cancer drugs, such as Etoposide. The double-layer cellular tumor spheroid model is a better model for anticancer drug analysis for different tumor spheroids.

ACKNOWLEDGEMENTS

This work is supported by Environmental & Water Industry Development Council of Singapore (MEWR C651/06/171). REFERENCES [1] M. W. Tibbitt and K. S. Anseth, “Hydrogel as

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Figure 4: (a) Microphotos of the formation of tumorspheroid with viability indicator using Calcein AM,and (b) proliferation rate in 3-day culture.

Day 3

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Proliferation rate

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Number of day 1 2 3

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CONTACT *A. Q. Liu, Tel: +65-6790-4336; eaqliu@ntu.edu.sg

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