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

Janne T Koivisto,Christine Gering,Reeja Maria Cherian,Jenny Parraga,Minna Kellomäki,Katriina Aalto-Setälä,Birhanu Belay,Jari Hyttinen,Jennika Karvinen

doi:10.1021/acsami.8b22343

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.

Highlights

The aim of tissue engineering (TE) is to create a new living tissue in vitro using a combination of biomaterial scaffolds, living tissue-specific cells, and biochemical factors.[1]
The cardiomyocyte differentiation was done by modulating Wnt signaling with small molecules, according to the protocol published by Lian et al 2012.41 In short, differentiation was initiated by plating 700 000 human-induced pluripotent stem cell (hiPSC)/well in a Nunc 12-multiwell plate (Thermo Fisher Scientific, USA) in feeder-free condition on Geltrexatom bridge). (b) Periodate oxidation of vicinal diols in gellan gum (GG). (c) Hydrazone cross-linking reaction between gelatin-adipic dihydrazide (ADH)/CDH and GG-CHO. (d) 1H nuclear magnetic resonance (NMR)-spectra of nonmodified gelatin, gelatin-ADH, and gelatin-CDH modifications
To form hydrazone cross-links between GG and gelatin, we hypothesized that hydrazide groups could be introduced to the gelatin backbone to form cross-links with the aldehyde groups generated in the GG molecule (Figure 2)

Summary

INTRODUCTION

The aim of tissue engineering (TE) is to create a new living tissue in vitro using a combination of biomaterial scaffolds, living tissue-specific cells, and biochemical factors.[1]. The most relevant cardiac 3D cell culture systems are engineered heart tissues, the so-called Biowire, 3D bioprinted structures, and even 3D printed organs-onchip.[15−18] All of the above examples use an extracellular matrix (ECM) protein-based hydrogel scaffold, either Matrigel or gelatin methacrylate (GelMA), to support 3D cell culturing. In these studies, the focus is more on cardiomyocyte electrophysiology than on the relationship between the mechanical properties of the material and how cellular mechanotransduction affects the biological response.[19] more emphasis should be placed on the design and mechanical characterization of these soft biomaterial scaffolds. We demonstrate that this rational hydrogel design supports the transition from 2D to 3D without interfering with the cardiomyocyte behavior and furthers the aim toward in vitro 3D hiPSC-derived cardiac disease modeling and drug screening

EXPERIMENTAL SECTION

RESULTS AND DISCUSSION

CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES