Page 198 - IJB-8-4

A Review on Bioinks and their Application in Plant Bioprinting
mM calcium chloride solution. Calcium crosslinking was for bioprinting with plant cells (3 wt%) . At a temperature
[27]
used to crosslink the alginate gels, which were then kept in of 120°C, alginic acid sodium salt, agarose, and sucrose
10 mM calcium chloride solutions. These gels gradually (3 wt%; as nutrients) were dissolved in deionized water.
turned dark green when submerged in water and lit. To This mixture and mc powder were autoclaved individually
show photosynthetic activity, the undamaged “logpiles” for 20 min at 121°C. After allowing the mc to cool to
were gently agitated in a flask of water and the dissolved room temperature, the mixture was added to the alginate/
O concentration of the water was measured. The O level agarose blend (alg/aga), stirred to form a homogeneous
2
2
climbed above that of water saturated with air during the plotting paste, and incubated for 2 h to allow the mc to
lighted period, and then fell as the light was turned off swell. At room temperature, the rheological properties
overnight. The alginate logpiles broke up over several of alg/aga and alg/aga/mc were measured using a
weeks, either due to bacterial action or simply because rotary rheometer with a plate/plate measurement device
the algae consumed the alginate. In some samples, white (30 mm, h = 0.1 mm). The viscosity was measured for
−1
fungal or bacterial proliferation was visible [135] . 300 s at a constant shear rate of 10 s . Shear thinning
Photosynthetic algae often create antimicrobial experiments were carried out by increasing the shear rate
−1
compounds to defend their colonies against infection by from 0 to 50 s over a period of 600 s (increments of
−1
other species, as well as symbiotic bacteria that help the 0.08 s ) and corresponding viscosity was determined.
algae to reproduce. Every experiment was carried out in triplicates.
Cell cultures are heavily reliant on specific For plant cell bioprinting, an in vitro cell culture
formulations that offer the nutrients required for cell of basil (Ocimum basilicum L. var. purpurascens Benth.
multiplication. This work shows that photosynthetic “Cinnamon Basil”) was used. The initial callus culture
algae can be printed and grown in biopolymer gels, was obtained by transforming a sterile shoot culture with
although it was stated that a synthetic gel may be a better Agrobacterium tumefaciens C58 (wild type). From the
substitute for the long-term study of stable “tissues.” obtained callus, a basil suspension culture was established
As cells can die while their chlorophyll stays intact, by transferring 2 g of biomass (fresh weight) into 50 mL
there was no evidence that the cells in the synthetic gels MS medium with an initial sucrose content of 3 wt% and
were alive in the dark. Chlorophyll can photodegrade, a pH value adjusted to 5.7 ± 0.1 before sterilization. The
hence the presence of the green color under light for basil suspension culture was kept in 50 mL MS medium
an extended period of time indicates that the cells were (250 mL Erlenmeyer flasks) with weekly passaging by
alive and produced chlorophyll, even when they were not transferring 20 vol% of the cell culture to 80 vol% fresh
multiplying [135] . The cells multiplication in the alginate MS medium. All cultivation in this study was carried out
gels can be observed by the change in color from light to in the dark on an orbital rotary shaker (110 rpm, 20 mm
dark green. shaking diameter) at room temperature. Plant cells were
As synthetic gels are denser than alginates, the collected from the MS medium on day 7 after passaging
difficulty in proliferation can possibly be attributed using a glass frit filter (pore size 160 – 250 μm) for
to the toxicity of one of the components or diffusional bioprinting. One gram of concentrated biomass was
limits. Plant cells are frequently provided with glucose gently mixed into 10 mL of prepared hydrogel paste. The
when cultivated in the laboratory, making photosynthesis cell-laden hydrogel was then printed, and the resulted
unnecessary as an energy source. scaffolds were crosslinked.
For growth, cells also require a source of nitrogen Bioscaffolder 3.1 was used for extrusion-based 3D
and phosphorus, as well as numerous minerals. plotting. The hydrogel pastes with the plant cells were
Different cells could also be combined into a gelling discharged using a conic dosing needle with an internal
−1
matrix and shaped into porous structures utilizing 3D diameter of 610 m, plotting speed of 8 – 10 mm s , and
[27]
printing. dosing pressure of 80 – 100 kPa .
A summary of above discussed bioinks is provided To achieve grid-like structures, successive layers
in Table 4. were rotated by 90°. The scaffolds were plotted into
6-well plates in an air-filled plotting environment .
[27]
6. Application of bioinks within the field of After plotting, the scaffolds were incubated in 0.1 M
plant bioprinting CaCl solution containing 3 wt% sucrose for 10 min to
2
crosslink the alginate. The scaffolds were then washed
6.1. Natural bioink twice for 1 min with MS medium (Murashige and Skoog
medium with vitamins) to prepare plant cell cultures. The
6.1.1. Green bioprinting: Fabrication of plant cells
dry weight of the inoculum was determined through the
A hydrogel blend (alg/aga/mc) containing alginate (alg, gravimetric analysis of reference samples (n = 9) after
2.8 wt%), agarose (aga, 0.9 wt%), and mc was developed 48 h of drying at 60°C. Using 3D plotting and a novel

190 International Journal of Bioprinting (2022)–Volume 8, Issue 4