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Here, we discuss the applicability of (methylenebis(salicylaldehyde)—MbSA) for the fabrication of the stimuli-responsive N-carboxyethylchitosan (CEC) hydrogels with a tunable dissolution rate under physiological conditions. In comparison with non-covalent salicylimine hydrogels, MbSA cross-linking via covalent bis(‘imine clip’) and non-covalent hydrophobic interactions allowed the fabrication of hydrogels with storage moduli > 1 kPa at ten-fold lower aldehyde/CEC molar ratio with the preservation of pH- and amino-acid responsive behavior. Although MbSA-cross-linked CEC hydrogels were stable at neutral and weakly alkaline pH, their disassembly in cell growth medium (Dulbecco’s modified Eagle’s medium, DMEM) under physiological conditions was feasible due to transimination reaction with amino acids contained in DMEM. Depending on the cross-linking density, the complete dissolution time of the fabricated hydrogels varied from 28 h to 11 days. The cytotoxicity of MbSA cross-linked CEC hydrogels toward a human colon carcinoma cell line (HCT 116) and primary human dermal fibroblasts (HDF) was remarkably lower in comparison with CEC-salicylimine hydrogels. Fast gelation, relatively low cytotoxicity, and tunable stimuli-induced disassembly under physiological conditions make MbSA cross-linked CEC hydrogels promising for drug encapsulation and release, 3D printing, cell culturing, and other biomedical applications.

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Macroporous scaffolds (cryogels) for the 3D cell culturing of colorectal cancer micro-tumors have been fabricated by cross-linking chitosan and carboxymethyl chitosan (CMC) with 1,4-butandiol diglycidyl ether (BDDGE) under subzero temperature. Due to the different intrinsic properties and reactivity of CMC and chitosan under the same cross-linking conditions, Young′s moduli and swelling of the permeable for HCT 116 cells cryogels varied in the broad range 3–41 kPa and 3500–6000%, respectively. We have demonstrated that the morphology of micro-tumors can be controlled via selection of the polymer for the scaffold fabrication. Although both types of the cryogels had low cytotoxicity and supported fast cell proliferation, round-shaped tightly packed HCT 116 spheroids with an average size of 104 ± 30 µm were formed in CMC cryogels (Young′s moduli 3–6 kPa), while epithelia-like continuous sheets with thickness up to 150 µm grew in chitosan cryogel (Young′s modulus 41 kPa). There was an explicit similarity between HCT 116 micro-tumor morphology in soft (CMC cryogel) or stiff (chitosan cryogel) and in ultra-low attachment or adhesive culture plates, respectively, but cryogels provided the better control of the micro-tumor’s size distribution and the possibility to perform long-term investigations of drug–response, cell–cell and cell–matrix interactions in vitro.

The potential of chitosan and carboxymethyl chitosan (CMC) cryogels cross-linked with diglycidyl ether of 1,4-butandiol (BDDGE) and poly(ethylene glycol) (PEGDGE) have been compared in terms of 3D culturing HEK-293T cell line and preventing the bacterial colonization of the scaffolds. The first attempts to apply cryogels for the 3D co-culturing of bacteria and human cells have been undertaken toward the development of new models of host–pathogen interactions and bioimplant-associated infections. Using a combination of scanning electron microscopy, confocal laser scanning microscopy, and flow cytometry, we have demonstrated that CMC cryogels provided microenvironment stimulating cell–cell interactions and the growth of tightly packed multicellular spheroids, while cell–substrate interactions dominated in both chitosan cryogels, despite a significant difference in swelling capacities and Young’s modulus of BDDGE- and PEGDGE-cross-linked scaffolds. Chitosan cryogels demonstrated only mild antimicrobial properties against Pseudomonas fluorescence, and could not prevent the formation of Staphylococcus aureus biofilm in DMEM media. CMC cryogels were more efficient in preventing the adhesion and colonization of both P. fluorescence and S. aureus on the surface, demonstrating antifouling properties rather than the ability to kill bacteria. The application of CMC cryogels to 3D co-culture HEK-293T spheroids with P. fluorescence revealed a higher resistance of human cells to bacterial toxins than in the 2D co-culture.

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