Sponge-like Scaffolds for Colorectal Cancer 3D Models: Substrate-Driven Difference in Micro-Tumors Morphology

Abstract

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.

Chitosan versus Carboxymethyl Chitosan Cryogels: Bacterial Colonization, Human Embryonic Kidney 293T Cell Culturing and Co-Culturing

“…This might be one reason for the faster reaching overgrowth phase by the 7th day and the inhibition of cell activity. Similar observations we reported for HCT 116 cells in CH-BD cryogel [ 31 ]. CMC cryogels promoted cell–cell interactions and spheroid growth due to the lack of cell–substrate interactions, which dominated in chitosan cryogels.…”

“…Since the lower molecular weight of the polymer, lower polymer and cross-linker concentrations, and higher freezing temperatures are known to promote formation of larger pores [ 19 , 32 ], we have selected low molecular weight chitosan and CMC, and set the polymer concentrations to 3% and the cryogelation temperature to −10 °C. Prior to thawing, the solutions were kept frozen longer than was required for the chemical cross-linking (7 days for CMC and 12 days for chitosan) to improve mechanical properties of cryogels, as was demonstrated for hyaluronic acid cryogels [ 32 ] and corroborated in our experiments [ 31 ].…”

“…Figure 1 shows that, in comparison with earlier reported BDDGE-cross-linked materials [ 31 ], chitosan and CMC cryogels cross-linked with PEGDGE under selected here conditions had larger average pore size (235 ± 32 and 232 ± 50 µm, respectively) and larger swelling capacity. The interconnected porous structure of all materials allowed free water outflow under compression and reabsorption after removing the pressure.…”

“…Type and degree of cross-linking are among the major factors which determine the porosity, swelling, and mechanical properties of cryogels and, thus, significantly affect their performance as scaffolds for cell culturing. Taking into account that large pores are beneficial for nutrient and metabolite transport and provide more space for the cellular spheroid’s growth, scaffold fabrication conditions were selected to obtain mechanically stable, elastic, and permeable cryogels with larger pore sizes in comparison with materials earlier reported by our group for HCT 116 cell culturing [ 31 ]. Since the lower molecular weight of the polymer, lower polymer and cross-linker concentrations, and higher freezing temperatures are known to promote formation of larger pores [ 19 , 32 ], we have selected low molecular weight chitosan and CMC, and set the polymer concentrations to 3% and the cryogelation temperature to −10 °C.…”

“…For the comparison with cryogels cross-linked with BDDGE [ 31 ], we have selected as a cross-linker PEGDGE with Mn~500. This was expected to allow the fabrication of chitosan and CMC cryogels with a broader variation of elasticity, swelling, and pore size distribution to distinguish the effects of polymer nature and the scaffold’s physical characteristics on bacterial adhesion/colonization and 3D cell culturing.…”