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A Review on Bioinks and their Application in Plant Bioprinting
(HPMC) solution at various weight ratios (3:7, 4:6, 5:5, 5.2.2. Post-printing determination of algal cell stability
and 6:4), hereafter referred to as silk to HPMC ratios. Alginate, a methylcellulose (mc) hydrogel-based bioink,
Three doses of horseradish peroxidase (HRP) were then was chosen for this experiment [116] . For algal cell,
added to the ink mixture at volumes of 60, 120, and 180 Chlamydomonas reinhardtii strain cc125 was chosen.
units/mL [114] . Alginate is an algae-derived natural polymer,
Then, 3D models were first designed with 3Ds whereas mc is a polymer made up of several linked glucose
MAX and printed using the synthesized algae-based silk molecules. First, 3 – 4 g of alginic acid sodium salt was
hydrogel structures (Figure 12). For printing, the ink was added to 100 mL of deionized water. On a heated plate
extruded into a medium containing 0.01% w/w hydrogen with a magnetic stirrer, the alginate solution was swirled
peroxide (H O ), which is generally used to make ink at 900 rpm for 5 h. Once the alginate was heated to 90°C,
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without microalgae while deionized water is used and 6 g of mc powder was added, and then the solution was
for making ink with microalgae, to start cross-linking sterilized in an autoclave for 1 min at 121°C. Algal cells
immediately after printing. The concentration of H O derived from TAP-algae (Tris-acetate-phosphate algal
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in the solution was set to facilitate efficient gel cross- solution) were introduced to the alginate, followed by a
linking while ensuring maximal cell survival. When the mc solution, then the solution was cooled until it reached
microalgal silk hydrogel had sufficiently cross-linked, room temperature [116] . The concentration of algal cells in
the H O solution was replaced with microalgal media to the prepared bioink was 150,000 cells/mL.
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facilitate cell proliferation [114] . After the preparation of the bioink, the printing
The long-term cell survival and performance of process began (Figure 13). After printing, the samples were
these systems allow them to be employed for a variety of immersed in 100 mM calcium chloride (CaCl ) solution for
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purposes, including O replenishment and carbon dioxide 4 min for crosslinking [117] . Cells in printed structures are
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reduction with the goal of a greener, healthier indoor immobilized, and algal cells as those used here can grow
[27]
environment [114] . indefinitely in this state . As a result, bound algal cells
This strategy was successfully used to host can multiply more rapidly, creating a higher cell density,
microalgae, producing a microalgal silk bioink with and generate more metabolites per cell than cells dispersed
[28]
mechanical properties and gelation kinetics suitable for in a liquid medium or less viscous bioink .
3D printing. The silk hydrogels offered a supportive The effects of extrusion pressure and needle diameter
environment for the long-term growth and photosynthetic on the number of algae cells in printed samples were
explored in this study, leading to the following findings:
activity of encapsulated microalgae. The proliferation of i. A higher extrusion pressure reduced the number
microalgae was observed for more than 4 weeks, with of algae cells in printed samples; this pattern was
a sustained photosynthetic activity for at least 90 days. observed 3 and 6 days after printing.
This stability, long-term functionality, and printability
of the support material poses potential environmental 2 For TAP algal cell preparation, 100 ml of tris-acetate-phosphate (TAP)
benefits [114] . culture media was prepared in a 150 ml flask. Cells of the frequently used
C. reinhardtii algae strain cc125 were streaked from a petri dish and added
to the flask. To maintain sterility, the addition was performed in a biosafety
A
B cabinet. The flask containing the TAP-algae solution was shaken for 72 h.
The shaker was set to 100 rpm and kept at 22°C under light bulbs to allow
the algae cells to grow. Thereafter, in a fresh flask containing 100 ml of
liquid TAP medium, 10 ml of the TAP-algae solution was added, yielding
a 110 ml TAP-algae solution. The TAP-algae solution was then transferred
into a new flask and exposed to light for 24 h to allow algal cell proliferation.

Figure 12. Silk/HPMC ink concrete mixtures in 3D printing.
(A) A diagram depicting the printing method of silk/HPMC
ink combination with microalgae. (B) 3D printed constructions
(a square-based pyramid and a bar spanning two conical-shaped
pillars) with silk to HPMC ratio of 6:4 and a 180 unit/mL HRP
ink mixture. The 3ds MAX designs are seen in the insets. Scale
bar: 1 cm. Reprinted with permission from ACS Biomater. Sci.
Eng. 2019, 5, 9, 4808–4816. Copyright 2019 American Chemical
Society [114] . Figure 13. Bioink preparation procedure.

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