Tailoring esophageal tumor spheroids on a chip with inverse opal scaffolds for drug screening
doi: 10.1088/2752-5724/ad5f47
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Abstract: AbstractEsophageal cancer (EC) is characterized by high morbidity and mortality, and chemotherapy has become an indispensable means for comprehensive treatment. However, due to the limitation of the effective in vitro disease model, the development of chemotherapeutic agents still faces great challenges. In this paper, we present a novel tumor spheroid on a chip platform based on inverse opal hydrogel scaffolds to screen chemotherapeutic agents for EC treatment. With the microfluidic emulsion approach, the inverse opal hydrogel scaffolds were generated with tunable and organized pores, which could provide spatial confinement for cell growth. Thus, the suspended KYSE-70 cells could successfully form uniform cell spheroids on the inverse opal hydrogel scaffolds. It was demonstrated that the tumor cell spheroids could recapitulate 3D growth patterns in vivo and exhibited higher sensitivity to the chemotherapy agents compared with monolayer cells. Besides, by employing the scaffolds into a microfluidics to construct esophageal tumor on a chip, the device could realize high-throughput tumor cell spheroids generation and drug screening, indicating its promising role in chemotherapy drug development.
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Key words:
- tumor on a chip /
- inverse opal /
- microfluidics /
- chemotherapy drug screening /
- esophageal cancer
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Figure 1. Schematic of microfluidic chip-based approach for quality assessment and screening of esophageal cancer drugs. A three-dimensional scaffold with inverse opal structure was fabricated using a single-emulsion microfluidic device. The esophageal cancer cells were loaded into the microfluidic chip to construct an esophageal cancer drug screening platform.
Figure 2. Fabrication and characterization of the inverse-opal structured scaffold. (a) Schematic diagram of the preparation of the inverse-opal structured scaffold. (b) The generation of single-emulsion droplet. (c) The self-assemble process of droplet templates: (i) polymerization, (ii) elution. (d) SEM images of dehydrated scaffolds: (i) surface. (ii) cross-section. The scale bar is 100 μm.
Figure 3. Fabrication and characterization of the microfluidic chip. (a) Schematic diagram of the construction of the integrated microfluidic chip. (b) Representative optical image of the entire integrated microfluidic chip. (c)-(d) Representative microscopy optical image of the scaffold in the chamber: (c) surface, (d) cross section. The scale bar is 200 μm.
Figure 4. The formation of the tumor spheroids in the microfluidic chip. (a) Cell spheroid formation process in the PEGDA scaffold in chip. The scale bar is 100 μm. (b) Fluorescence image of the Calcium AM stained KYSE-70 cells in the PEGDA scaffold. The scale bar is 100 μm. (c) Three-dimensional reconstruction image of the DiD stained cell spheroid. The scale bar is 100 μm. (d) Biosafety investigation of the 60% PEGDA hydrogel scaffold. (e) Fluorescence quantification of figure (b).
Figure 5. Evaluation of the sensitivity of the cell spheroids to the chemotherapeutic agents. (a) Fluorescence image of KYSE-70 cells with live/dead stain after the treatment of chemotherapeutic agents (blue: hoechst33342; green: Calcein AM; red: PI). The scale bar is 100 μm. (b) Fluorescence image of KYSE-70 cell spheroids in chip after the treatment of chemotherapeutic agents (blue: hoechst33342; green: Calcein AM; red:PI). The scale bar is 100 μm. (c)-(f) The results of MTT assay performed on 2D cultured KYSE-70 cells and cell spheroids treated with different concentrations of tumor chemotherapeutic agents. (g) Fluorescence quantification of (a). (h) Fluorescence quantification of (b).
Figure 6. Evaluation the cell apoptosis and proliferation of the tumor spheorids. (a) Fluorescence image of KYSE-70 cell with immunofluorescence after the treatment of chemotherapeutic agents. (b) Fluorescence image of KYSE-70 cells in chip with live/dead stain after the treatment of chemotherapeutic agents (blue:DAPI, green:TUNEL, red:Ki67). The scale bar is 100 μm. (c) Fluorescence quantification of (a). (d) Fluorescence quantification of (b). (e)-(g) The level of the tumor markers (CYFRA21-1,SCCAg and CEA) in the effluent liquid were detected.
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