Abstract: BACKGROUND: Matrigel is the best material for the culture of tumor organoids, but matrigel alone is not enough to simulate the mechanical environment of tumor growth in vitro. Although the introduction of sodium alginate material can improve the stiffness of the hydrogel based on matrigel, its mechanical properties of hydrogel are difficult to maintain stability in long-term culture. 
OBJECTIVE: To introduce a small amount of calcium ions into the medium of breast cancer organoids and to observe its maintenance effect on the long-term mechanical properties of the matrigel-sodium alginate hydrogel. 
METHODS: (1) Sodium alginate composite hydrogels with low, medium, and high stiffness were prepared by introducing different mass concentrations (0, 2.5, and 5 mg/mL) of sodium alginate into the constant mass concentration (5 mg/mL) of matrigel. The mechanical properties of hydrogels were measured regularly by rheometer. (2) Human triple negative breast cancer cells MDA-MB-231 were resuspended in hydrogel pre-gels with different stiffness. After gelling, breast cancer organoid factor medium containing (or without) calcium ions was added for breast cancer organoid culture. At a set time point, rheometer was used to regularly measure the effect of calcium ion introduction on the mechanical properties of hydrogel. The morphologic changes of breast cancer organoids were observed under optical microscope. Rate of breast cancer organoids forming into pellets was calculated on day 13. After 7 days of breast cancer organoid culture, different concentrations of the chemotherapy drug docetaxel (0.1, 1, 10, and 100 nmol/L) were added for intervention for 6 days. Cell viability was detected and the semi-inhibitory concentration of docetaxel, IC50, was calculated.  
RESULTS AND CONCLUSION: (1) The introduction of sodium alginate effectively improved the mechanical strength of the composite hydrogel. (2) With the extension of breast cancer organoid culture time, the mechanical strength of hydrogels decreased. On day 13 of culture, the mechanical properties of medium and high stiffness hydrogels in the culture environment containing calcium ions were significantly higher than those in the culture environment without calcium ions (P < 0.05). There was no significant change in the mechanical properties of low stiffness hydrogels in the two cultures (P > 0.05). In long-term culture (13 days), breast cancer organoids changed from round to spindle shape with the decrease of hydrogel mechanical properties in the medium and high stiffness hydrogel groups. After the introduction of calcium ions, the morphology of breast cancer organoids did not change with the extension of culture time in the two groups. The introduction of calcium ions in the culture environment had no effect on the pellet formation rate of breast cancer organoids in the low stiffness hydrogel group, but could improve the pellet formation rate of breast cancer organoids in the medium and high stiffness hydrogel groups. (3) In the culture environment without calcium ions, the cell viability of breast cancer organoids decreased with the increase of docetaxel concentration, and there was no significant difference in IC50 among the three hydrogel groups (P > 0.05). In the culture environment containing calcium ions, the cell viability of breast cancer organoids decreased with the increase of docetaxel concentration. The cell viability of breast cancer organoids in the medium and high stiffness hydrogel groups was stronger than that in the low stiffness hydrogel group, and the IC50 was higher than that in the low stiffness hydrogel group (P < 0.05). (4) The results showed that the mechanical properties of the matrigel-sodium alginate hydrogel could be maintained by introducing calcium ions into the breast cancer organoid culture system. 

Key words: tumor organoids, breast cancer organoids, sodium alginate, matrigel, composite hydrogel, matrix stiffness, drug responsiveness