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Ghosh and Yi
of effective mass transfer [154] . To achieve increased circular orbit [157] . The medium was exchanged weekly
porosity and larger mesopores, the precursor was with fresh liquid NB+S medium. Cultures were kept in
combined with silicon dioxide (SiO ) nanospheres after this state for up to 3 months before being replaced with
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the SiO is removed [154] . The rheological parameters of new semi-solid-to-liquid subculturing.
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SiO -DBSA-TIA inks are identical to those of DBSA- The bioink used to immobilize the rice cells
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TIA inks, indicating that it could be used for bioprinting. contained 12% (w/v) 4-arm polyethylene glycol tetra-
On combination with the SiO nanospheres, the ink acrylate MW 20,000 (PEGTA), with 0.1% (w/v) LAP
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demonstrated shear-thinning and solidifying properties. as the photo initiator. A 10× LAP stock solution and a
Due to a higher solidness, the solidification slopes of concentrated PEGTA solution were made separately in
SiO -DBSA-TIA ink are much slower than those of NB+S medium and vortexed until completely dissolved
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DBSA-TIA ink [148] . In addition, the SiO /TIA mass ratio to prepare the bioink [158] . The LAP stock solution was
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and SiO particle size can be adjusted to create custom added to the PEGTA solution immediately before the
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surface areas and pore size distributions; however, experiments began, and the mixture was vortexed again.
adding excessive amounts of SiO can negatively affect The bioink formulation was prepared for demonstrating
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ink homogeneity, making it impossible for the ink to extrusion by adding nanocellulose crystal powder at
flow. a load of 16 wt% in a solution of 12% (w/v) PEGTA,
Based on the optimized ink formula and taking 50 wt% cells, and 0.1% (w/v) LAP. Experiments were
into account both the surface area as well as rheological carried out in a biosafety cabinet with the bioink extruded
qualities, a well-patterned hierarchical macro- or manually using a 1 mL sterile Luer-lock syringe furnished
mesoporous artificial photosynthetic system can be with an ~840 μm inner diameter tapered tip. To reduce
3D-printed, wherein the leaf structure is obtained through aggregation and assure a healthy exponential phase,
calcination and etching [148] . growing rice cells in the suspension culture were received
Bioprinted 3D leaf structures can provide insights through a 280 μm mesh filter and passaged to fresh growth
into the structure, function, and behavior, such as medium in a shaking flask few days before the start of
gas diffusion of plant leaves, thus contributing to the experiment [155] . Before removing the media on day
advancements in the field of plant biology. 0, 10 – 15 mL cell culture samples were obtained from the
flask and gravity settled in a 15 mL Falcon tube. To obtain
6.2.2. Bioprinting of plant cells for production of a a 50% (w/v) cell loading density, cells were measured as
biodefense agent grams of fresh weight (g FW) and added to the prepared
Transgenic rice cells were immobilized using this bioink bioink solution, or to fresh NB+S growth medium in the
(Oryza sativa) [155] . This is the first report of recombinant case of the liquid suspension culture controls [155] . Using
plant cells being immobilized for the continuous synthesis the Loctite EQ CL30 LED 405 nm Flood System with
of high-value heterologous proteins. The preparation UV LED Flood Controller, cell-laden PEGTA-LAP
of the suspension culture of rice cells is the initial step bioink samples were cured under UV irradiation in a
in this process. Encapsulation was performed using a 6-well plate (3.48 cm diameter, 0.21 cm height, and 2 mL
transgenic O. sativa rice cell line expressing recombinant total volume) by exposing them to high intensity UV light
rice butyrylcholinesterase (rrBChE) . Rice cells were for 10 s. At a 50 mm working distance, the lamp emitted
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−2
cultured and grown in a semi-solid “NB+S” selection a peak irradiance of 1500 mW cm at 405 nm. A working
medium including N6 macronutrients, B5 micronutrients, distance of 21 cm was ensured between the lamp and
and vitamins, 30 g l sucrose, 1.8 g l Gelzan™, 300 mg the sample. To fit into the lip of a 25 mL flask, the cured
−1
−l
l caseinhydrolysate, 250 mg l L-glutamine, 250 mg rice cell-laden PEGTA-LAP hydrogel disks were cut into
−l
−l
l L-proline, 2 mg l 2,4-dichlorophenoxyacetic acid 3 – 4 pieces. The control conditions for liquid suspension
−l
−l
(2,4-D), and 0.02 mg l Kinetin, with 50 mg l geneticin cultures were performed with and without UV curing
−l
−l
as the selection antibiotic [156] . The cells were subcultured (Figure 17).
into liquid sterile NB+S media (without Gelzan™ One gram of FW and 1 mL bioink (or 1 mL media
and geneticin) before the start of the experiments by for liquid suspension culture controls) was used to make
pressing and sieving the calli through a sterile, stainless- cured cell laden PEGTA hydrogels, which were then
steel, 280 m mesh sieve to achieve consistent cell introduced to 9 mL NB+S medium in 25 mL shake flasks.
aggregates [156,157] . Suspension cultures were grown in Flasks were incubated in the dark for up to 14 days at
500 mL or 1 L shake flasks with a 20% working volume 28°C and 140 rpm.
and incubated in the dark at 28°C, 140 rpm in a 19 mm The results suggested that this bioink (polyethylene
glycol-based hydrogel) successfully immobilized
4 The rrBChE promoter is controlled by the metabolically regulated rice transgenic rice cells (Oryza sativa) producing
alpha amylase 3D (RAmy3D) promoter, which induces protein expression
in the absence of a sugar and contains a signal peptide that tags proteins recombinant butyrylcholinesterase, which acts as a
for secretion. prophylactic or therapeutic against cocaine toxicity,
International Journal of Bioprinting (2022)–Volume 8, Issue 4 193