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.
Keywords: 3D culturing; carboxymethyl chitosan; chitosan; cryogel; stiffness.