Engineering considerations on extrusion-based bioprinting: interactions of material behavior, mechanical forces and cells in the printing needle.

Systematic analysis of the extrusion process in 3D bioprinting is mandatory for process optimization concerning production speed, shape fidelity of the 3D construct and cell viability. In this study, we applied numerical and analytical modeling to describe the fluid flow inside the printing head based on a Herschel-Bulkley model. The presented analytical calculation method nicely reproduces the results of Computational Fluid Dynamics simulation concerning pressure drop over the printing head and maximal shear parameters at the outlet. An approach with dimensionless flow parameter enables the user to adapt rheological characteristics of a bioink, the printing pressure and needle diameter with regard to processing time, shear sensitivity of the integrated cells, shape fidelity and strand dimension. Bioinks consist of a blend of polymers and cells, which lead to a complex fluid behavior. In the present study, a bioink containing alginate, methylcellulose and agarose (AMA) was used as experimental model to compare the calculated with the experimental pressure gradient. With cultures of an immortalized human mesenchymal stem cell line and plant cells (basil) it was tested how cells influence the flow and how mechanical forces inside the printing needle affect cell viability. Influences on both sides increased with cell (aggregation) size as well as a less spherical shape. This study contributes to a systematic description of the extrusion-based bioprinting process and introduces a general strategy for process design, transferable to other bioinks.

LED-enhanced biosynthesis of biologically active ingredients in callus cultures of Ocimum basilicum.

Ocimum basilicum is a medicinal plant with multiple health benefits including cardiovascular, cancer and diabetics. In the present study, the influences of light emitting diodes (LEDs) were investigated on the accumulation of biologically active ingredients in callus cultures of Ocimum basilicum. Among the various tested treatments optimum levels of Total phenolic content (TPC) was noted in callus culture grown under blue lights as compared to control, while maximum accumulation of Total flavonoid content (TFC) was noted in callus culture grown under red light as compared to control. HPLC analyses showed that highest concentrations of Rosmarinic acid (96.0 mg/g DW) and Eugenol (0.273 mg/g DW) were accumulated in blue light which was 2.46 and 2.25 times greater than control (39.0 mg/g DW, 0.171 mg/g DW), respectively. Chicoric acid (81.40 mg/g DW) optimum accumulation was noted in callus grown under the continuous white light, which was almost 4.52 times greater than control. Anthocyanins content were also analyzed, the highest amount of Peonidin (0.127 mg/g DW) and cyanidin (0.1216 mg/g DW) were found in callus culture grown under red light. These findings suggest that application of LED's is a promising strategy for enhancing production of biologically active ingredients in callus cultures Ocimum basilicum.

A new method for non-invasive biomass determination based on stereo photogrammetry.

A novel, non-destructive method for the biomass estimation of biological samples on culture dishes was developed. To achieve this, a photogrammetric system, which consists of a digital single-lens reflex camera (DSLR), an illuminated platform where the culture dishes are positioned and an Arduino board which controls the capturing process, was constructed. The camera was mounted on a holder which set the camera at different title angles and the platform rotated, to capture images from different directions. A software, based on stereo photogrammetry, was developed for the three-dimensional (3D) reconstruction of the samples. The proof-of-concept was demonstrated in a series of experiments with plant tissue cultures and specifically with calli cultures of Salvia fruticosa and Ocimum basilicum. For a period of 14 days images of these cultures were acquired and 3D-reconstructions and volumetric data were obtained. The volumetric data correlated well with the experimental measurements and made the calculation of the specific growth rate, µ max, possible. The µ max value for S. fruticosa samples was 0.14 day-1 and for O. basilicum 0.16 day-1. The developed method demonstrated the high potential of this photogrammetric approach in the biological sciences.

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

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.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

In vitro rosmarinic acid accumulation in sweet basil (Ocimum basilicum L.).

Leaf-derived suspension cultures of sweet basil, Ocimum basilicum L. accumulated rosmarinic acid up to 10 mg g(-1) dry wt, a value up to 11 times higher than in callus cultures or in leaves of donor plants. Immobilized cells accumulated less than 15 microg rosmarinic acid g(-1).