# Development of Injectable Thermosensitive Chitosan-Based Hydrogels for Cell Encapsulation
###### Abstract  
The three-dimensional complexity of the native extracellular matrix (ECM) suggests switching from 2D to 3D culture systems for providing the cells with an architecture more similar to the physiological environment. Reproducing the three-dimensionality in vitro can guarantee beneficial effects in terms of cell growth, adhesion, proliferation, and/or their differentiation. Hydrogels have the same tailorable physico-chemical and biological characteristics as ECM materials. In this study, we propose a thermoresponsive chitosan-based hydrogel that gels thanks to the addition of organic and inorganic salt solutions (beta-glycerolphosphate and sodium hydrogen carbonate) and is suitable for cell encapsulation allowing obtaining 3D culture systems. Physico-chemical analyses showed that the hydrogel formulations jellify at physiological conditions (37 \({}^{\circ}\)C, pH 7.4), are stable in vitro up to three weeks, have high swelling ratios and mechanical stiffness suitable for cellular encapsulation. Moreover, preliminary biological tests underlined the pronounced biocompatibility of the system. Therefore, these chitosan-based hydrogels are proposed as valid biomaterials for cell encapsulation. stimuli-responsive hydrogel; natural polymers; cell encapsulation; in vitro degradation; swelling ratio; injectable hydrogel; 3D matrices; biocompatibility +
Footnote †: journal: Applied Physics Letters  
studies showed the importance of 3D architectures in many cellular processes, such as cell migration and differentiation [4; 5]. 3D culture systems were successfully proposed as disease models, useful to evaluate in vitro the effects of drugs or build tissue analogous for regenerative medicine and tissue engineering applications [6; 7]. In recent years, the role of biomaterials has gone beyond the conventional vision of a passive support for cell growth [8]. The increasing knowledge of the wide range of interactions between matrices and cells drove scientific interest toward the development of biomaterials able to mimic spatial-temporal signals of the ECM [9]. The biomaterial, besides meeting the fundamental requirements of biocompatibility, biodegradability, and porosity, must also be able to interact with specific cell types, for example displaying ad hoc mechanical properties and \"active\" surface characteristics capable of guiding the desired cellular processes [10; 11]. The similarity between the composition of the ECM and some natural or synthetic polymers is the basis of the development of so-called hydrogels used in 3D culture systems. A hydrogel is defined as \"a three-dimensional hydrophilic polymer network capable of absorbing and retaining large amounts of water or biological fluids\" [12]. Based on their material of origin, hydrogels can be classified as natural or synthetic [13], and divided into two further groups based on the type of crosslinking, chemical or physical, which influences hydrogel physical properties and the irreversible or transient nature of polymer chain interaction [14; 15]. Moreover, stimuli-responsive hydrogel systems can be designed by integrating specific functional groups, responsive to different stimuli such as pH, temperature, magnetic or electric field, and bioactive molecules [16]. Among them, the physiological body temperature acting as a trigger for inducing the sol-gel transition appears to be a very promising strategy, especially for biomedical uses. They can swell in situ under physiological conditions and provide the advantage of being conveniently administered. Typically, aqueous solutions of hydrogels used in biomedical applications are liquid at room temperature (RT) (injectable hydrogel) and gel at physiological temperature [17]. In this regard, injectable hydrogels have important properties such as being minimally invasive for the body as well as able to retain large quantities of water.