Development of an in vitro 3D tumor model to study therapeutic efficiency of an anticancer drug

Abstract

The importance and advantages of three-dimensional (3D) cell cultures have been well-recognized. Tumor cells cultured in a 3D culture system as multicellular tumor spheroids (MTS) can bridge the gap between in vitro and in vivo anticancer drug evaluations. An in vitro 3D tumor model capable of providing close predictions of in vivo drug efficacy will enhance our understanding, design, and development of better drug delivery systems. Here, we developed an in vitro 3D tumor model by adapting the hydrogel template strategy to culture uniformly sized spheroids in a hydrogel scaffold containing microwells. The in vitro 3D tumor model was to closely simulate an in vivo solid tumor and its microenvironment for evaluation of anticancer drug delivery systems. MTS cultured in the hydrogel scaffold are used to examine the effect of culture conditions on the drug responses. Free MTS released from the scaffold are transferred to a microfluidic channel to simulate a dynamic in vivo microenvironment. The in vitro 3D tumor model that mimics biologically relevant parameters of in vivo microenvironments such as cell-cell and cell-ECM interactions, and a dynamic environment would be a valuable device to examine efficiency of anticancer drug and targeting specificity. These models have potential to provide in vivo correlated information to improve and optimize drug delivery systems for an effective chemotherapy.

Figure 1

Overall schematic of fabrication of a hydrogel scaffold containing microwells and formation of MTS consisting of MCF-7 cells. (A) A master template was pressed into the gelatin solution mixed with HCl and glutaraldehyde followed by warming at 60 °C to allow cross-linking. Upon removal of the master template, the hydrogel scaffold containing microwells was rinsed prior to cell seeding. (B) MCF-7 cells suspended in 50% Matrigel solution were transferred onto the hydrogel scaffold. After MTS were formed, Matrigel encapsulating MTS were released from the hydrogel scaffold.

Figure 2

Overall schematic of a microfluidic channel and experimental setup. (A) Schematic of fabrication of a microfluidic channel. (B) Design and bright field image of the microfluidic channel. The black arrow shows the direction of medium flow. Scale bar: 300 µm. (C) Schematic of experimental setup of the microfluidic channel.

Figure 3

Phase contrast microscopic images of microwells in the hydrogel scaffold and MCF-7 seeded in the hydrogel scaffolds. (A) The PDMS master template containing circular micropatterns of 50 µm diameter. (B) Cross-linked hydrogel scaffold containing 50 µm diameter microwells. (C) The MTS after 1 day of culture in the hydrogel scaffold and (D) image of the MTS in a microwell after 3 days of culture. (E) Free MTS released from the scaffold after 3 days of culture. Scale bar: 50 µm.

Figure 4

Characterization of the MTS. (A) MCF-7 cells cultured in a scaffold-free environment. (B–F) MTS grown in the hydrogel scaffold containing microwells: (A) SEM image of the MCF-7 cells depicting morphological differences in a loosely aggregated form; (B) SEM image of the free MTS after 3 days of culture; (C and D) confocal fluorescence and bright field overlay images of the MTS revealing the tight packing of the cells (plasma membranes stained with CellMask shown in red); (E and F) optically sectioned immunofluorescence images of the MTS showing scattered signals of α6-integrin (E) and signals of E-cadherin between cells (F). Scale bars: (A and B) 10 µm and (C) 50 µm.

Figure 5

Confocal fluorescence images of MTS 24 h after treated with DOX micelles (0.5 µM DOX equivalent). (A) Accumulation of DOX micelles were observed on the surface of MTS. (B) An overlay image of both bright field and fluorescence images. Scale bar: 50 µm.

Figure 6

Confocal fluorescence images of viability/cytotoxicity assay of the MTS after DOX-HCl treatment in the static condition. (A) Most cells were viable shown in green. The interior of the MTS appeared to be darker since the cells were not on the same focal plane. (B) The white arrow indicating a dead cell. (C) A composite image of both bright field and fluorescence images (black arrow indicating the stained dead cell). Scale bar: 50 µm.

Figure 7

Bright-field image of the MTS in the microfluidic channel. (A) The MTS in Matrigel prior to DOX-HCl treatment. (B) Real-time, enlarged views of the MTS are outlined in a white dotted box in (A) during 24 h of treatment with DOX-HCl. The MTS appeared to lose its spheroidal shape over time. Scale bar: 50 µm.

Figure 8

Bright field images of the MTS in the microfluidic channel. (A) The MTS in Matrigel prior to DOX micelle treatment at 25 µM DOX equivalent concentration. (B and C) Enlarged views of the MTS outlined in white and black dotted box in (A), respectively, during 24 h of treatment with DOX micelles. Scale bar: 100 µm.