Stiffness-Modulation of Collagen Gels by Genipin-Crosslinking for Cell Culture

Abstract

The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.

Keywords: YAP; cancer cells; cell culture; cell morphology; collagen gel; differentiation; extracellular matrix; genipin; mesenchymal stromal cells; stiffness.

Figure 1

(A) The schematic procedure of preparing genipin-mixed collagen gels for cell culture. HEPES: N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, DMEM: Dulbecco’s modified Eagle’s medium, FBS: fetal bovine serum. (B) Young’s moduli of the 0, 0.01, 0.05, 0.1, 0.5, 1, or 10 mM genipin-mixed collagen gels. Mean ± S.D. N = 3 experiments.

Figure 2

(A) Cell morphology of H1299 cells on the 0, 0.01, or 10 mM genipin-mixed collagen gels or collagen-coated glass substrates. Scale bar = 100 μm. (B) Cell area of H1299 cells shown in (A). Mean ± S.D. N = at least 23 cells in 3 independent experiments. p value was calculated using Welch’s t-test with Bonferroni correction.

Figure 3

(A) Fluorescent staining of nuclei and YAP of H1299 cells on the 0, 0.01, or 10 mM genipin-mixed collagen gels or collagen-coated glass substrates. Scale bar = 50 μm. (B) YAP localization of H1299 cells shown in (A). Mean ± S.E. N = at least 84 cells in 4 independent experiments. p value was calculated using Welch’s t-test with Bonferroni correction.

Figure 4

(A) Cell morphology of mouse mesenchymal stromal cells on the 0 or 10 mM genipin-mixed collagen gels or collagen-coated plastic substrates with smooth muscle-differentiation condition. Scale bar = 100 μm. (B) qPCR of ACTA2 and ACTG2 in (A). S18 was used as an internal control. Mean ± S.D. N = 3 independent experiments. * Statistical significance determined with 95% confidence interval with Bonferroni correction.

Figure 5

(A) Cell morphology of mouse mesenchymal stromal cells on the 0 or 10 mM genipin-mixed collagen gels or collagen-coated plastic substrates with adipogenic condition. Scale bar = 100 μm. (B) qPCR of CEBPA and PPARG in (A). S18 was used as an internal control. Mean ± S.D. N = 3 independent experiments. * Statistical significance determined with 95% confidence interval with Bonferroni correction.

Figure 6

Stiffness-modulation of genipin-mixed collagen gels with the indicated genipin concentrations. The reported stiffness of brain, lung, liver, and tumor is also shown [6,9].