3D Printing of Thermo-Responsive Methylcellulose Hydrogels for Cell-Sheet Engineering

Abstract

A possible strategy in regenerative medicine is cell-sheet engineering (CSE), i.e., developing smart cell culture surfaces from which to obtain intact cell sheets (CS). The main goal of this study was to develop 3D printing via extrusion-based bioprinting of methylcellulose (MC)-based hydrogels. Hydrogels were prepared by mixing MC powder in saline solutions (Na2SO4 and PBS). MC-based hydrogels were analyzed to investigate the rheological behavior and thus optimize the printing process parameters. Cells were tested in vitro on ring-shaped printed hydrogels; bulk MC hydrogels were used for comparison. In vitro tests used murine embryonic fibroblasts (NIH/3T3) and endothelial murine cells (MS1), and the resulting cell sheets were characterized analyzing cell viability and immunofluorescence. In terms of CS preparation, 3D printing proved to be an optimal approach to obtain ring-shaped CS. Cell orientation was observed for the ring-shaped CS and was confirmed by the degree of circularity of their nuclei: cell nuclei in ring-shaped CS were more elongated than those in sheets detached from bulk hydrogels. The 3D printing process appears adequate for the preparation of cell sheets of different shapes for the regeneration of complex tissues.

Methylcellulose – a versatile printing material that enables biofabrication of tissue equivalents with high shape fidelity

“…Due to this reason, commercially available mc usually is characterized by its viscosity (of a 2% solution at 20°C) and the molecular weight is a recalculated value, which does not allow drawing conclusions for the distribution of the molecular weight. We found that most studies [32][33][34][35][36][37][38][39][40][41] used an mc with a given viscosity of 4000 mPa s (M n ≈ 86 kDa); 30 these studies have in common to have achieved printing of multiple layers and only limited collapse of predesigned macropores. Other studies 42,43 reported about the use of mc with a given viscosity of 15 mPa s (M n ≈ 14 kDa) and found significant improvements of the printed shape fidelity in presence of mc compared to mc-free controls, but those structures lacked the evidence of multiple layer stacking.…”

“…21 Thus, the stability of printed mc-containing constructs, as well as their degradation behaviour can be controlled by the presence of salts in the (bio-)ink and the cell culture medium. The best printing results of salt-doped mc-inks were achieved by printing in the range of 20-25°C, 39,40 which created the optimum for viscous behaviour of mc in sol form near to the gelation state.…”

“…21,25 MC solutions of a concentration of 8 wt% were prepared in salt-containing solutions and printed in ring-like structures with high shape fidelity (outer diameter 10 mm, inner diameter 6 mm, the obtained printed structures revealed the same dimensions). 39,40 Afterwards, fibroblasts and endothelial cells were seeded on top; non-printed bulk samples acted as controls. Due to the saline mc solution, mc was in gel state at 37°C and thus, the printed and cell-seeded structures were stable in cell culture conditions.…”

“…Due to the saline mc solution, mc was in gel state at 37°C and thus, the printed and cell-seeded structures were stable in cell culture conditions. 21,40 After 20 min incubation at 4°C, it was possible to remove cell layers by dissolving the mc structures. Interestingly, both cell types responded to the printed ring-like structure and displayed a matching morphology, which was not observed by the cells cultured on the bulk hydrogels.…”

Four-Dimensional Bioprinting for Regenerative Medicine: Mechanisms to Induce Shape Variation and Potential Applications

“…Lastly, it is understood that some diseases induce changes in body temperature; thus, thermoresponsive polymers may be a useful tool for diagnostic purposes. 50 With regard to thermoresponsive hydrogels, Cochis et al 52 used methylcellulose for cellsheet engineering. After optimising the printing process parameters, methylcellulose hydrogel rings were extruded for the first time.…”