Mechanically Biomimetic Gelatin-Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes

Abstract

To promote the transition of cell cultures from 2D to 3D, hydrogels are needed to biomimic the extracellular matrix (ECM). One potential material for this purpose is gellan gum (GG), a biocompatible and mechanically tunable hydrogel. However, GG alone does not provide attachment sites for cells to thrive in 3D. One option for biofunctionalization is the introduction of gelatin, a derivative of the abundant ECM protein collagen. Unfortunately, gelatin lacks cross-linking moieties, making the production of self-standing hydrogels difficult under physiological conditions. Here, we explore the functionalization of GG with gelatin at biologically relevant concentrations using semiorthogonal, cytocompatible, and facile chemistry based on hydrazone reaction. These hydrogels exhibit mechanical behavior, especially elasticity, which resembles the cardiac tissue. The use of optical projection tomography for 3D cell microscopy demonstrates good cytocompatibility and elongation of human fibroblasts (WI-38). In addition, human-induced pluripotent stem cell-derived cardiomyocytes attach to the hydrogels and recover their spontaneous beating in 24 h culture. Beating is studied using in-house-built phase contrast video analysis software, and it is comparable with the beating of control cardiomyocytes under regular culture conditions. These hydrogels provide a promising platform to transition cardiac tissue engineering and disease modeling from 2D to 3D.

Keywords: 3D hydrogel; compression testing; gelatin; gellan gum; hiPSC-derived cardiomyocytes.

Figure 1

Beating pattern of cardiomyocyte aggregate in F4-CDH hydrogel as an example of the BeatView analysis. This is the same aggregate as in Video S8. Graph (a) shows regular beating rhythm; (b) shows the breakdown of a single beat into relaxed state (1) and contracting (2) and relaxing (3) movements.

Figure 2

(a) Chemical modification of gelatin carboxylic groups with hydrazide molecules ADH (provides 10-atom bridge) and CDH (provides 5-atom bridge). (b) Periodate oxidation of vicinal diols in GG. (c) Hydrazone cross-linking reaction between gelatin-ADH/CDH and GG-CHO. (d) ¹H NMR-spectra of nonmodified gelatin, gelatin-ADH, and gelatin-CDH modifications. The arrows highlight the appearance of aromatic protons in gelatin-ADH and gelatin-CDH spectra after the coupling reaction of CDH and ADH with 4-hydroxybelzandehyde. Chemical modification was successful based on the appearance of extra peaks.

Figure 3

Degradation profiles of the tested hydrogels incubated with collagenase for 56 h. Values represent the mean and standard deviation. Sigmoidal curve fits were applied to the data.

Figure 4

Representative stress–strain curves of the different hydrogel compositions and the rabbit heart tissue. (a) All representative curves with stress range 0 to 40 kPa. (b) Extended graph with the stress scale up to 450 kPa to highlight the similarities between F5-CDH and rabbit heart tissue. (c) Compressive moduli of the hydrogels compared to the rabbit heart. (d) Fracture strain and strength measured by compression testing. The y-axis on the left and the dark gray bars show the fracture strain relative to the original sample height. The y-axis on the right and the light gray bars show the fracture strength. In (c) and (d), n = 5; * = significantly different from other formulations at p < 0.05.

Figure 5

Representative images of Live/Dead stained fibroblast cell cultures in all tested hydrogel formulations and both 2D and 3D culture conditions at the 3-day and 7-day time points. The 3D cultures were imaged in the middle of the hydrogel. Green indicates live cells and red indicates dead cells. Rows (a), (c), (e), and (g) are with lower magnification, and the scale bar length is 1000 μm; rows (b), (d), (f), and (h) are with higher magnification with a scale bar length of 200 μm.

Figure 6

Measured fibroblast viability percentage based on amount of live cells compared to amount of all cells, 4× magnification images. Error bars represent mean values ± standard deviation, n ≥ 3.

Figure 7

Bright-field OPT visualization of fibroblast cell culture under 3D hydrogel condition. (a) Single projection image of F3-ADH hydrogel, with highly elongated cells highlighted with arrows, (b) 3D reconstruction of the previous giving a view of the whole sample, (c) single projection image of negative control F7-SPD hydrogel, and (d) 3D reconstruction of the previous.

Figure 8

Microscope images of hiPSC-derived cardiomyocyte aggregates cultured under hydrogel conditions: (a) F1-ADH, (b) F3-ADH, (c) F4-CDH, and (d) F5-CDH. The scale bar length is 200 μm.

Figure 9

(a) Quantitative RT-PCR validation of cardiac specific genes expressed in hiPSC-derived cardiomyocytes cultured in the gelatin coating control, F5-CDH and F3-ADH. Shown are the expression levels of cardiac type troponin T2 (TNNT2), α-actinin 2 (ACTN2), and Myosin binding protein C (MYBPC3), relative to the housekeeping gene GAPDH. Standard deviations are from three biological replicates, each done in technical triplicate in qPCR. (b–d) Immunocytochemical staining of hiPSC-derived cardiomyocytes using red for TNNT2, green for ACTN2, and blue for DAPI. (b) 2D control on gelatin coating. (c) Aggregate 3D culture in F3-ADH. (d) Aggregate 3D culture in F5-CDH. The density of cell aggregate in F5-CDH slightly prevents antibody and fluorescent light penetration, causing blurriness in the image.