Primary Human Pancreatic Cancer Cells Cultivation in Microfluidic Hydrogel Microcapsules for Drug Evaluation

Abstract

Chemotherapy is an essential postoperative treatment for pancreatic cancer, while due to the lack of effective drug evaluation platforms, the therapeutic outcomes are hampered by tumor heterogeneity among individuals. Here, a novel microfluidic encapsulated and integrated primary pancreatic cancer cells platform is proposed for biomimetic tumor 3D cultivation and clinical drug evaluation. These primary cells are encapsulated into hydrogel microcapsules of carboxymethyl cellulose cores and alginate shells based on a microfluidic electrospray technique. Benefiting from the good monodispersity, stability, and precise dimensional controllability of the technology, the encapsulated cells can proliferate rapidly and spontaneously form 3D tumor spheroids with highly uniform size and good cell viability. By integrating these encapsulated tumor spheroids into a microfluidic chip with concentration gradient channels and culture chambers, dynamic and high-throughput drug evaluation of different chemotherapy regimens could be realized. It is demonstrated that different patient-derived tumor spheroids show different drug sensitivity on-chip, which is significantly consistent with the clinical follow-up study after the operation. The results demonstrate that the microfluidic encapsulated and integrated tumor spheroids platform has great application potential in clinical drug evaluation.

Keywords: drug evaluation; hydrogel; microcapsule; microfluidics; pancreatic cancer.

Figure 1

Schematic of tumor peripheral circulation of pancreatic tumor and hydrogel microcapsules integrated microfluidic chip for drug evaluation. a) The design of the tumor spheroids‐encapsulated hydrogel microcapsules. b) The microcapsules‐integrated chip for drug evaluation.

Figure 2

Preparation and regulation of the CMC/ALG microgels. a) The real‐time image showing the generation of CMC/ALG droplets. b) Uniform core–shell hydrogel microcapsules observed under a stereomicroscope. c) Fluorescent images of microcapsules generated under different flow rate ratios. The ALG and CMC precursor solutions were mixed with fluorescent polystyrene nanoparticles L4655 (green) and L3280 (red), respectively. d) SEM images of i) a freeze‐dried microcapsule and ii) the surface of the shell and iii) core. e) Overall size distributions of the CMC/ALG microcapsules under the parameters listed in the chart. f) Core size distributions of the CMC/ALG microcapsules under the parameters listed in the chart. g) Shell thickness distributions of the CMC/ALG microcapsules under the parameters listed in the chart. Scale bars are 500 µm in (a, b), 200 µm in (c), 200 µm in (d,i), and 10 µm in (d,ii, d,iii).

Figure 3

Generation and characterization of tumor spheroids encapsulated in the hydrogel microcapsules. a) Schematic of the extraction of primary pancreatic cancer cells and the preparation of cell‐laden microcapsules through microfluidic electrospray. b) Bright‐field images of primary pancreatic tumor cells after encapsulation in the microcapsules at 1, 3, 9, and 11 d. c) Live/dead cell staining of tumor spheroids in the microcapsules at days 1, 3, 9, and 11. d) Quantitative analysis of cell proliferation of tumor spheroids at days 1, 3, 5, 9, and 11 through CellTiter‐Glo 3D cell viability assay. Lum refers to the luminescent signal indirectly reflecting the amount of adenosine triphosphate (ATP) and the number of cells (n = 6 for each group). e) Quantitative analysis of cell viability of tumor spheroids at days 1, 3, 9, and 11 (n = 6 for each group). f) Size distribution of tumor spheroids at day 11 (n = 100 for each group). Data are shown as mean ± SD. Scale bars are 50 µm in (b, c).

Figure 4

a) Photograph of the microfluidic concentration gradient generator. b) Optical microscopic picture of the chip gradient generator. c) Light microscopy image of the spheroids culture chamber. d) Optical images of the tumor spheroids‐integrated microfluidic chip. e) Confocal laser‐scanning image of a microcapsule‐encapsulated tumor spheroid in the culture chamber with live/dead cell staining. f) Quantitative analysis of cell viability of the tumor spheroids at days 1, 2, and 3 after seeded into the cell culture chamber (n = 30 for each group). g) HE staining image of a pancreatic tumor spheroid. SEM images of h) a whole pancreatic tumor spheroid and i) the magnified image showing the surface structure of the tumor spheroid. Scale bars represent 2 mm in (b, c), 50 µm in (e), 50 µm in (g), 50 µm in (h), and 5 µm in (i).

Figure 5

a) Immunofluorescent images of pancreatic tumor spheroid for CD44 and MUC1. b) Immunofluorescent images of pancreatic tumor spheroid for MUC5AC and CD133. c) The fluorescence images of tumor spheroids staining with Ki67 and CK19. a–c) Scale bars are 20 µm.

Figure 6

Response of pancreatic tumor spheroids to antineoplastic agents. a) Numerical simulation of the drug concentration gradient formed in the microfluidic concentration gradient generator. b) Schematic of the drug solution flowing through the cell culture chamber. c) Numerical simulation of drug permeating into a microcapsule under the flow (x‐axis was the direction of fluid flow). The unit of scale bar was mol m⁻³ (in (a) and (c)). Response of three patient‐derived tumor spheroids to d) 4 h Gem treatment, e) 48 h S‐1 treatment, and f) 4 h GS (combination of Gem and S‐1) and 44 h S‐1 treatment on‐chip (n = 30 for each group). There was no statistical difference between PDA01 and PDA03 in regimen 6. Computed tomography data of the PDA02 patient in the g) preoperative, h) postoperative, and i) tumor metastasis (at the fifth chemotherapy cycle) stage.