Inexpensive DIY Bioprinting in a Secondary School Setting

ABSTRACT

Bioprinting is a technique that allows custom printing of cell-laden tissue using the principle of three-dimensional (3D) printing. The technique has various applications, ranging from tissue engineering to materials science. Bioprinting is an attractive topic for science, technology, engineering, and math education due to its novelty and interdisciplinary nature. Nonetheless, a basic commercial bioprinter could cost several thousand U.S. dollars. There have been attempts to construct low-cost do-it-yourself bioprinters for research purpose. However, those methods required expertise, uncommon reagents, and professional equipment, making it difficult for teachers and students in secondary schools to replicate. Here, we demonstrate how teachers and students in a secondary school can convert a 3D printer into a bioprinter for conducting a hands-on bioprinting activity using secondary school-available resources. Briefly, an open-source Creality Ender 3 V2 3D printer in a school was converted into a bioprinter using 3D-printed parts and other readily available materials. Cell-laden bioink and support medium were made using school-available reagents. The bioprinter can be easily constructed and operated by teachers and students who do not have prior knowledge in coding and engineering. We used the bioprinter to print a coronary artery model and an algae-laden artificial leaf. The photosynthetic activity of the artificial leaf could be observed and investigated using a hydrogen carbonate indicator. The work described in this paper could make bioprinting available, comprehensible, and enjoyable to secondary school students, opening a door for inexpensive innovative teaching and learning activities using bioprinting in secondary schools.

INTRODUCTION

Recently, research in three-dimensional (3D) bioprinting has grown rapidly (1). Bioprinting allows custom printing of soft living tissue by harnessing the 3D printing technique (2). It has been shown clinically that bioprinting could help repair different types of human organs, including skin, bone, and ear (3). Bioprinting could also enhance drug discovery and allow development of new material (4, 5).

Given the great promise of bioprinting in future research and medicine, it is desirable to introduce bioprinting to secondary school students. Early exposure to bioprinting allows students to acknowledge the latest development of biotechnology, which might inspire them to become future professionals. The novelty and interdisciplinary nature of bioprinting technology makes it an attractive content for science, technology, engineering, and math (STEM) education. In addition, if bioprinting becomes available in secondary schools, cell-laden tissues could be produced on demand as teaching materials to facilitate the teaching of structure and function concepts in biology. However, unlike 3D printing of plastic, bioprinting is uncommon among secondary schools due to its high cost. A basic bioprinter might cost several thousand U.S. dollars, hindering the incorporation of bioprinting into secondary school STEM curricula (6). Researchers in the field have demonstrated ways to make do-it-yourself (DIY) bioprinters (7–9). However, those DIY bioprinters were mainly designed for research purposes, and the construction and operation of them requires uncommon reagents, professional equipment, and advanced engineering skills.

To enable exploration of bioprinting for concept clarification and experimentation in a secondary school setting, here we showcase how a Creality Ender 3 V2 3D printer can be converted into a bioprinter at low cost, using 3D-printed parts and readily available materials (Fig. 1). The DIY bioprinter prints by extruding bioink into a support medium layer-by-layer (10). The bioink contains living cells, culture medium, and sodium alginate, while the support medium contains calcium ions for solidifying the bioink and at the same time preventing the solidified bioink from collapsing during printing (10). We demonstrate how bioink and support medium could be prepared using reagents available in secondary schools. Teachers and students together used the bioprinter to print a coronary artery model and an algae-laden artificial leaf. The print quality of our bioprinter is sufficient for teachers to illustrate the rationale of bioprinting to students. Finally, the photosynthetic activity of the algae-laden artificial leaf was assessed using a hydrogen carbonate indicator, and this enabled students to observe the bioactivity of the bioprinted construct.

FIG 1.

Components of the DIY bioprinter, converted from a Creality Ender 3 V2 3D printer. (A) A bio-extruder unit that is made up of 3D-printed parts, stepper motor, syringe, screws, and bearing. (B) A plastic tube connecting the bio-extruder and the bio-printhead. (C) The bio-printhead is made up of 3D-printed parts, 100-mm 22-gauge blunt-end needle and a luer lock adaptor. (D) Support medium on a petri dish for solidifying and supporting the bioprinted structure.

PROCEDURE

Modification of a commercial 3D printer into a bioprinter

The commercial 3D printer chosen to be modified in this work was a Creality Ender 3 V2 3D printer, which is currently one of the most popular low-cost 3D printers for hobbyists. As shown in Fig. 1, the 3D printer was converted into a bioprinter by using 3D-printed parts: a syringe, plastic tubes, bearing, screws, etc. The conversion was mainly done by replacing the original extruder and printhead with our bio-extruder and bio-printhead. The 3D files for our bio-extruder and bio-printhead are available online in STL format (see Appendix S1 in the supplemental material). The bio-printhead was of our own design, while the bio-extruder was designed by modifying an open-source paste extruder named Spritzstruder, designed by user kitingmare on Thingiverse (278905; licensed under the Creative Commons Attribution-Non Commercial 4.0 International open-source license). The detailed steps for assembling the bioprinter are described in Appendix S2.

Preparation of bioink

To prepare for bioprinting, the syringe in the bio-extruder needs to be filled with bioink. To prepare 100 mL of bioink, dissolve 2 g of sodium alginate in 100 mL of cell culture to obtain bioink with 2% (wt/wt) sodium alginate. Sodium alginate can be polymerized into hydrogel when it encounters calcium ions in the support medium. In our work, we prepared an algae culture by referring to a previously described method (11). Cell culture could be replaced with colored water for testing or demonstration purposes.

Preparation of support medium

The biostructure is printed on a petri dish containing support medium. To prepare 600 mL of support medium, dissolve 4 g of agar in 400 mL of 11 mM CaCl₂ to give 1% (wt/wt) agar in CaCl₂. After solidification, add 100 mL 11 mM CaCl₂ solution to the agar and use a spatula to smash the agar into pieces. Then, transfer the agar solution mix to a blender and blend the mixture for 3 min to generate a Bingham plastic-like slurry. Put the slurry in a 4°C refrigerator and wait for a week to remove the air bubbles, as the presence of air bubbles could undermine print quality. If a centrifuge is available, one can quickly remove the bubbles by centrifuging the slurry at 8,000 rpm for 1 min. The prepared support medium can be stored at 4°C.

Hands-on bioprinting

To prepare 3D models for bioprinting, open-source models can be downloaded on websites like Thingiverse or NIH 3D print exchange. G-code can be generated using a slicing setting, as described in Appendix S3. The G-code could be stored in a micro-SD card and plugged into the bioprinter for printing. Before printing, the syringe in the bio-extruder is filled with bioink and a petri dish filled with support medium needs to be placed at the middle of the build plate. To ensure that the nozzle is completely filled with bioink before printing, one could press the plunger until seeing bioink coming out from the bio-printhead. After that, printing can be initiated. The printing time depends on the volume of the print. For example, printing the coronary artery, as shown in Fig. 2, requires 15 min. After printing, leave the petri dish overnight to ensure complete solidification. The 3D printed biostructure can then be isolated by washing away the support medium carefully in running water. To obtain optimal print quality, see Appendix S4, which outlines questions and answers for troubleshooting.

FIG 2.

Two example constructs printed by the DIY bioprinter. 3D models (A and B) in STL format were used to bioprint a right coronary artery tree (C) and a maple leaf (D). Scale bar, 10 mm. The right coronary artery tree is a test print printed with 2% sodium alginate with red food coloring. The maple leaf was printed with algae culture with 2% sodium alginate.

Bioprinting an algae-laden artificial leaf and assessing its bioactivity

In this bioprinting activity, students downloaded a maple leaf model on Thingiverse (number 25938 by user MakerBot) and printed it with an algae-laden bioink, as shown in Fig. 2 (see Appendix S1 for the STL file). To assess the bioactivity of the bioprinted leaf, it is submerged in 30 mL of hydrogen carbonate indicator (see Appendix S5). Photosynthetic and respiratory activities can be demonstrated by color change of the indicator. Photosynthesis under bright light results in a net consumption of carbon dioxide and turns the indicator purple, while respiration in the absence of light releases carbon dioxide and turns the indicator yellow.

Safety issues

This work does not involve hazardous chemicals or pathogenic microorganisms. To eliminate the risk of overheating during printing, the original heater must be disconnected from the mainboard when assembling the bioprinter (see Appendix S2).

CONCLUSION

This work demonstrates how DIY bioprinting can be done in a secondary school setting by adapting an available 3D printer at low cost. We have showcased how cell-embedded biostructures can be printed with satisfactory quality and how their bioactivity can be observed by students to enhance curiosity, interest, and motivation. We believe that our work could stimulate innovative and inspirational teaching and learning attempts in secondary schools. Not only might this work stimulate the learning of bioprinting in secondary schools, but also it might stimulate creative attempts in enhancing the teaching of structure-function concepts in biology. It is recommended that educators further explore ways to integrate DIY bioprinters into structured curricula.