One-step generation of droplet-filled hydrogel microfibers for 3D cell culture using an all-aqueous microfluidic system

doi:10.3724/SP.J.1123.2023.06008

Abstract

Abstract:

Hydrogel microfibers, which are characterized by flexible mechanical properties, a uniform spatial distribution, large surface areas, and excellent biocompatibility, hold great potential for various biomedical applications. However, the fabrication of heterogeneous hydrogel microfibers with high cell-loading capacity and the ability to carry multiple components via an environmentally friendly method remains challenging. In this study, we developed a novel pneumatic pump-assisted all-aqueous microfluidic system that enables the one-step fabrication of all-aqueous droplet-filled hydrogel microfibers with unique morphologies and adjustable configurations. By designing a pump-valve cycling system and selecting two immiscible fluids with stable water interfaces (dextran and polyethylene glycol), we successfully fabricated alginate microfibers with equidistantly arranged droplets through the ionotropic gelation reaction between sodium alginate and calcium chloride. The droplet size, interdroplet spacing, and microfiber dimensions could be flexibly controlled by adjusting the flow rates of the inner-phase, middle-phase, and outer-phase inlets. The results showed that the system enabled the high-throughput in situ formation of functional three-dimensional cell spheroids. The generated cell spheroids exhibited excellent cell viability and drug-testing functionality, indicating their potential applications in cell cultures. The developed technique offers strong support for future biomedical research and applications, and provides a new approach for the preparation of multifunctional hydrogel microfibers for materials science, tissue engineering, and drug testing.

Key words: all-aqueous microfluidic system, droplet-filled hydrogel microfibers, cellular carriers

CLC Number:

O658

ZHAO Mengqian, LIU Haitao, ZHANG Xu, GAN Zhongqiao, QIN Jianhua. One-step generation of droplet-filled hydrogel microfibers for 3D cell culture using an all-aqueous microfluidic system[J]. Chinese Journal of Chromatography, 2023, 41(9): 742-751.

Figures/Tables 6

Fig. 1 Schematic diagrams of the microfluidic system for generating all-aqueous droplet-filled hydrogel microfibers a. configuration of the microfluidic chip: the injection unit, the droplet generation unit, fiber generation unit, the collection unit and the procedure of the pneumatic valve for the generation of droplet. Dextran (DEX), poly(ethylene glycol) (PEG) with alginate (NaA), and PEG with calcium chloride (CaCl2) were introduced to the system through inner-phase, middle-phase, and outer-phase inlets, respectively. b. procedures for droplet and fiber generation. c. schematic illustration of the reaction of NaA and CaCl2 within the microfibers.

Fig. 2 Generation and morphological characterization of the all-aqueous droplet-filled hydrogel microfibers a. bright-field image of the flows in the droplet generation unit and fiber generation unit; b. macroscopic (i), microscopic (ii), and schematic (iii) images of the all-aqueous droplet-filled hydrogel microfibers; c. fluorescence and bright-field images of fluorescein isothiocyanate-labeled sodium alginate (FITC-labeled NaA) collected in the all-aqueous droplet-filled hydrogel microfibers; d. SEM images of the (i and ii) side and (iii) cross-sectional views of the freeze-dried microfibers. The valve cycle was fixed to 0.4 s, and the inner-phase, middle-phase, and outer-phase flow rates were set to 0.6, 5.5, and 300 μL/min, respectively, for all collected fibers shown in the figure.

Fig. 3 Characterization and distribution maps of different-shaped droplets encapsulated in the alginate microfibers a. morphology of different types of droplet-filled and hollow fibers; b. distribution of fiber morphologies formed at different inner-phase and middle-phase flow rates.

Fig. 4 Bright-field images of droplet-filled hydrogel microfibers and statistical diagrams of the droplet diameter, droplet distance, and microfiber width under the different (a) inner-phase, (b) middle-phase, (c) outer-phase flow rates **p<0.01; ***p<0.001. Quantitative analyses of the droplet diameter, droplet distance, and microfiber width were performed on at least 20 samples.

Fig. 5 Encapsulation and formation of 3D cell spheroids in the all-aqueous droplet-filled hydrogel microfibers a. schematic diagram of the encapsulation of tumor cells in the all-aqueous droplet-filled hydrogel microfibers and formation of 3D cell spheroids; b. bright-field image of continuous fibers loaded with multiple tumor spheroids; c. bright-field images of tumor spheroids in the droplets 1, 3, 5, and 7 d after loading and the corresponding statistical analysis of the tumor spheroid diameter (n=3); d. fluorescence images of the live/dead staining experiment and fluorescence quantitative analysis to assess the viability of cells within the tumor spheroids 1, 3, 5, and 7 d after loading (n=3).

Fig. 6 Characterization of the drug response of A549 cells in the spheroids a. bright-field and fluorescence images of the live/dead staining of A549 cells in 2D cultures treated with different concentrations of cisplatin (0.1-1000 μmol/L) for 24 h; b. the corresponding dose-response curve and IC50 value of cisplatin in A549 cells cultured in 2D plate (n=3); c. bright-field and fluorescence images of the live/dead staining of A549 tumor spheroids in all-aqueous droplet-filled hydrogel microfibers treated with different concentrations of cisplatin (0.1-1000 μmol/L) for 24 h; d. the corresponding dose-response curve and IC50 value of cisplatin in A549 cells cultured in the all-aqueous droplet-filled hydrogel microfibers (n=3).

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