Green bioprinting: extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds

Abstract

Plant cell cultures produce active agents for pharmaceuticals, food and cosmetics. However, up to now process control for plant cell suspension cultures is challenging. A positive impact of cell immobilization, such as encapsulation in hydrogel beads, on secondary metabolites production has been reported for several plant species. The aim of this work was to develop a method for bioprinting of plant cells in order to allow fabrication of free-formed three-dimensional matrices with defined internal pore architecture for in depth characterization of immobilization conditions, cell agglomeration and interactions. By using extrusion-based 3D plotting of a basil cell-laden hydrogel blend consisting of alginate, agarose and methylcellulose (alg/aga/mc), we could demonstrate that bioprinting is applicable to plant cells. The majority of the cells survived plotting and crosslinking and the embedded cells showed high viability and metabolic activity during the investigated cultivation period of 20 d. Beside its compatibility with the plant cells, the novel alg/aga/mc blend allowed fabrication of defined 3D constructs with open macropores both in vertical and horizontal direction which were stable under culture conditions for several weeks. Thus, Green Bioprinting, an additive manufacturing technology processing live cells from the plant kingdom, is a promising new immobilization tool for plant cells that enables the development of new bioprocesses for secondary metabolites production as well as monitoring methods.

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

“…Another approach demonstrated that the addition of 0.9% agarose dramatically increased the zero-shear viscosity of a blend of 3% alginate and 3% mc resulting in improved shape fidelity (obtained horizontal macropores did not collapse in scaffolds with 20 layers). 35 In this blend, mc contributed positively by its shear-thinning behaviour, whereas alginate-agarose alone could not be printed. This biopolymer blend was developed for Green Bioprinting with plant cell cultures.…”

“…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.…”

“…This biopolymer blend was developed for Green Bioprinting with plant cell cultures. 35 Bioprinting of plant cells is a promising manufacturing method for secondary metabolite production in industrial pharmaceutical processes. 35 Recently, the biological response of cells to the 3% alginate-9% mc blend could be significantly enhanced by dissolving the two biopolymers in human blood plasma.…”

“…35 Bioprinting of plant cells is a promising manufacturing method for secondary metabolite production in industrial pharmaceutical processes. 35 Recently, the biological response of cells to the 3% alginate-9% mc blend could be significantly enhanced by dissolving the two biopolymers in human blood plasma. Whereas the good printability of the blend combination was maintained ( printing of centimetre-scaled complexly shaped constructs with more than 50 layers was demonstrated), the proteins of the blood plasma significantly increased the cell viability and allowed spreading of osteoprogenitor cells within the bioink.…”

Plant-Derived Biomaterials: A Review of 3D Bioprinting and Biomedical Applications

“…Despite their promise as bioinks for tissue engineering, further characterization of the toxicological properties, immunogenicity, and in vivo durability of these materials needs to be elucidated to determine their validity over widely used synthetic materials such as polylactic acid and polycaprolactone (Pariente et al, 2001). Many of the plant-based materials are being translated from the food industry, where their bioactivity, rheological properties, and facile gelation have garnered significant interest in bioprinting (Seidel et al, 2017;Vancauwenberghe et al, 2017). However, many of the crosslinking methods, especially those involving high temperatures, harsh chemical conditions and ultraviolet radiation, warrant further adaptation to ensure biocompatibility, and cell viability is preserved if these methods are to be used for tissue bioprinting purposes.…”